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Secretion of exosomes through the potential endolysosomal pathway in Rab3DKO and Non-Obese Diabetic murine models
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Secretion of exosomes through the potential endolysosomal pathway in Rab3DKO and Non-Obese Diabetic murine models
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Content
Secretion of exosomes through the potential endolysosomal pathway in Rab3DKO and Non-
Obese Diabetic murine models
by
Xiaoyang Li
A Thesis Presented to the
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF PHARMACY AND
PHARMACEUTICAL SCIENCES
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2023
Copyright 2023 Xiaoyang Li
ii
Acknowledgments
I would like to express my heartfelt gratitude to my Principal Investigator, Dr. Sarah
Hamm-Alvarez. I would also like to thank Dr. Maria C. Edman-Woolcott. They provided me with
endless guidance and helped me through this thesis project. They have been very supportive and
very patient in giving me time to learn everything from scratch. Their commitment to my academic
growth is deeply appreciated.
I also thank my committee members, Dr. Yong (Tiger) Zhang, and Dr. Curtis Okamoto,
for their help in reviewing and editing my thesis.
I would like to thank Dr. Shruti Singh Kakan for her friendship and support. Her patience
and all the insightful suggestions on my research helped me through my project.
I would also like to thank Minchang Choi for his friendship, constant encouragement,
endless guidance, and valuable insights which taught me a lot in study and life.
I am also grateful to Karolina Kutnik for her friendship and advice on my academic work
and research.
In addition, I would like to show appreciation to all my colleagues, Cindy Toscano, Sara
Abdelhamid, Carlos Delgado, and Shiyu (Summer) Bian in Dr. Sarah Hamm-Alvarez’s lab for
their friendship, and support during my two-year journey.
Finally, I want to thank my parents for always believing in me, supporting me, and their
unconditional love, on my way to growth. Their encouragement has been the driving force along
this journey. I would never have the chance to pursue higher education without them.
Table of Contents
Acknowledgments ........................................................................................................................... ii
List of Figures ................................................................................................................................ iii
List of Tables ...................................................................................................................................v
Abbreviations ................................................................................................................................. vi
Abstract ......................................................................................................................................... vii
Chapter One .....................................................................................................................................1
1. Introduction ..........................................................................................................................1
1.1. Sjögren’s Syndrome .............................................................................................................1
1.2. Lacrimal Gland & Rab3D-enriched secretory pathway .......................................................1
1.3. Exosome biogenesis .............................................................................................................3
1.4. Exosomes in SS ...................................................................................................................5
1.5. The male Non-Obese Diabetic (NOD) mouse – a murine model of SS ..............................5
1.6. Aims and hypothesis ............................................................................................................6
1.6.1. Aim 1: Exosomes as a biomarker for exploring the “secondary” pathway .........................6
1.6.2. Aim 2: Validation of predicted target of increased miR-375-3p in Rab3DKO tears ..........6
Chapter Two .....................................................................................................................................7
2. Materials and Methods .........................................................................................................7
2.1. Mice .....................................................................................................................................7
2.2. Tear collection .....................................................................................................................7
2.3. Saliva collection ...................................................................................................................8
2.4. Serum collection ..................................................................................................................9
2.5. Exosome isolation ..............................................................................................................10
2.6. ZetaView Nanoparticle Tracking Analysis ........................................................................13
2.7. BCA ...................................................................................................................................13
2.8. Western blotting .................................................................................................................14
2.8.1. Tissue .................................................................................................................................14
2.8.2. Tears ...................................................................................................................................16
2.9. Cell culture .........................................................................................................................16
2.10. Cell Transfection ................................................................................................................16
2.11. Cornea isolation .................................................................................................................18
2.12. RNA isolation ....................................................................................................................18
2.12.1. Tissue .................................................................................................................................18
2.12.2. Cell RNA ...........................................................................................................................20
2.13. RT-qPCR ...........................................................................................................................20
2.14. NanoDrop
TM
Spectrophotometer .......................................................................................23
Chapter Three .................................................................................................................................24
3. Results ................................................................................................................................24
3.1. The expression of CIP2A at mRNA level is higher in Rab3DKO female LG ..................24
3.2. The expression of CIP2A at mRNA level in mouse corneas .............................................26
3.3. CIP2A Western Blotting ....................................................................................................26
3.4. The CIP2A functional assay ..............................................................................................28
3.4.1. Protein Tyrosine Kinase 9 (PTK9) gene expression ..........................................................28
3.4.2. miR-375-3p transfection efficiency ...................................................................................29
3.4.3. The CIP2A expression after miR-375-3p mimic transfection ...........................................29
3.5. ZetaView Nanoparticle Tracking Analysis (ZV-NTA) .....................................................30
3.5.1. Tear exosomes in Rab3DKO and C57/BL6 mouse tears ...................................................31
3.5.2. Saliva exosomes in Rab3DKO and C57/BL6 mouse saliva ..............................................32
3.5.3. Tear Exosomes in NOD and BALB/C mouse tears ...........................................................32
3.5.4. Serum Exosomes in male NOD mice ................................................................................34
3.6. Western Blotting of exosomal protein markers .................................................................35
3.6.1. Alix Tear Western Blotting ................................................................................................35
3.6.2. TSG101 Tear Western Blotting .........................................................................................36
3.6.3. CD63 LG Western Blotting ...............................................................................................37
3.6.4. CD81 LG Western Blotting ...............................................................................................38
3.6.5. CD9 LG Western Blotting .................................................................................................39
3.6.6. LAMP2 LG Western Blotting ............................................................................................40
Chapter Four ..................................................................................................................................41
4. Discussion ..........................................................................................................................41
Chapter Five ...................................................................................................................................45
5. Conclusion .........................................................................................................................45
References ......................................................................................................................................47
iii
List of Figures
Figure 1. Biogenesis of Exosomes. ................................................................................................. 4
Figure 2. Tear Collection Procedure. .............................................................................................. 8
Figure 3. Saliva Collection Procedure ............................................................................................ 9
Figure 4. Exosome Isolation ......................................................................................................... 11
Figure 5. Amicon Ultra-10k Centrifugal Filters ........................................................................... 12
Figure 6. miRNA Mimic Transfection Protocol ........................................................................... 17
Figure 7. RNA Purification Procedure .......................................................................................... 19
Figure 8. The CIP2A expression at the mRNA level in LG from Rab3DKO and C57/BL6
mice. .............................................................................................................................................. 25
Figure 9. CIP2A expression at the mRNA level in the cornea from Rab3DKO and C57/BL6
mice. .............................................................................................................................................. 26
Figure 10. CIP2A Western Blotting of male Rab3DKO and C57/BL6 LG from 12-16 weeks
mice. .............................................................................................................................................. 27
Figure 11. miRNA mimic transfection validation experiments. ................................................... 28
Figure 12. The CIP2A expression after 48 h, 72 h incubation. .................................................... 30
Figure 13. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from Rab3DKO
and C57/BL6. ................................................................................................................................ 31
Figure 14. Saliva Exosome ZetaView Nanoparticle Tracking Analysis results, from
Rab3DKO and C57/BL6. .............................................................................................................. 32
Figure 15. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from NOD and
BALB/C. ....................................................................................................................................... 33
iv
Figure 16. Tear and serum Exosome ZetaView Nanoparticle Tracking Analysis results from
male NOD. .................................................................................................................................... 34
Figure 17. Western Blotting analysis of Alix in tears of male and female NOD and BALB/C,
aged 12-14 weeks. ......................................................................................................................... 35
Figure 18. Western Blotting analysis of TSG101 in tears of male and female NOD and
BALB/C, aged 12-14 weeks. ........................................................................................................ 36
Figure 19. Western Blotting analysis of CD63 in LG from male NOD and BALB/C mice,
aged 12-14 weeks. ......................................................................................................................... 37
Figure 20. Western Blotting analysis of CD81 in LG of male NOD and Balb/c, aged 12-14
weeks. ............................................................................................................................................ 38
Figure 21. Western Blotting analysis of CD9 in LG of male NOD and Balb/c, aged 12-14
weeks. ............................................................................................................................................ 39
Figure 22. Western Blotting analysis of LAMP2 in LG of male NOD and Balb/c, aged 12-14
weeks. ............................................................................................................................................ 40
v
List of Tables
Table 1. iZON qEVoriginal 35 nm Size Exclusion Column Fraction collected. .......................... 12
Table 2. Primary antibodies. ......................................................................................................... 15
Table 3. Secondary antibodies ...................................................................................................... 15
Table 4. Qiagen primer list ........................................................................................................... 20
Table 5. TaqManTM primer list ................................................................................................... 21
Table 6. miRNA mimic list ........................................................................................................... 21
Table 7. miCURY LNA Reverse Transcription reaction setup per sample .................................. 22
Table 8. Reverse Transcription cycling setup ............................................................................... 22
Table 9. TaqManTM Reverse Transcription reaction setup per sample ....................................... 22
Table 10. Reverse Transcription cycling setup ............................................................................. 22
vi
Abbreviations
EV: Extracellular Vesicle ILV: Intraluminal Vesicle miRNA: Micro RNA
MVB: Multivesicular Body
ILV: Intraluminal Vesicle
SS: Sjögren's Syndrome
SV: Secretory Vesicle
CIP2A: Cancerous Inhibitor of Protein Phosphatase 2A
CTSS: Cathepsin S
Rab3DKO: Rab3D Knock Out
NOD: Non-Obese Diabetic
RA: Rheumatoid Arthritis
SLE: Systemic Lupus Erythematosus
MGD: Meibomian gland disease
LE: Late Endosomes
LG: Lacrimal Gland
LGAC: Lacrimal Gland Acinar Cell
NTA: Nanoparticle Tracking Analysis
BCA: Bicinchroninic Acid
WB: Western Blot
HCE-T: Human Corneal Epithelial Transformed
vii
Abstract
Sjögren’s Syndrome (SS) is a chronic, inflammatory disorder characterized by inflammation of
the lacrimal gland (LG) and the salivary gland (SG) leading to dysfunction, with the most seen
manifestations being dry eyes and dry mouth. Exosomes are nano-sized, cell-derived vesicles that
play an important role in delivering a variety of biomolecules such as proteins, and lipids as well
as nucleic acids to different cells in the body. Increased evidence shows that these bioactive
cargoes in exosomes facilitate intercellular communication by transmitting phenotypic
information to other cells under both physiological and pathological conditions. Rab3D is a
GTPase that regulates exocytosis in exocrine epithelial cells in association with mature secretory
vesicles. It is well-acknowledged that most proteins are secreted through the Rab3D-regulated
secretory pathway in lacrimal gland acinar cells (LGAC). We then came up with a hypothesis that
the loss of Rab3D might potentially trigger the generation of a “secondary” secretory pathway
originating from endolysosomes in the inflamed exocrine glands that are affected by the
autoimmune disease, Sjögren’s Syndrome (SS). That change might in turn lead to changes in
exosome secretion from acinar cells since exosomes are present in endolysosomal membranes.
Both tear and LG exosome abundance, and exosomal marker proteins were analyzed in specimens
from different murine models including the Rab3DKO mouse and the SS disease model male Non-
Obese Diabetic (NOD) mouse and their respective controls, C57BL/6 and male BALB/c mice.
Previous study in the lab have shown that miR-375-3p is upregulated in Rab3DKO mouse tears.
Therefore, the target of miR-375-3p predicted by Ingenuity Pathway Analysis, the cancerous
inhibitor of protein phosphatase 2A (CIP2A)’s gene and protein expression were measured in
downstream cells. In this project, the protocol of tear exosome quantification in both murine
models was optimized, and the abundance of exosomal proteins TSG101, HSC70, LAMP2, CD9,
viii
CD81, and CD63 in mouse tear exosomes was analyzed. The protocols of measuring CIP2A’s
gene and protein level in Rab3DKO model were also developed and optimized.
1
Chapter One
1. Introduction
1.1. Sjögren’s Syndrome
Sjögren’s Syndrome (SS) is a chronic, autoimmune inflammatory disorder that affects nearly 1%
of the population (Vitali C. et al, 2002). The predominance of SS in women is approximately 20:1
female to male ratio (Chatzis L. et al, 2020), suggesting that sex might play a role in the disease
development. SS is mainly characterized by diminished lacrimal and salivary gland function due
to lymphocytic infiltration of exocrine glands, leading to severe dryness of the eyes and mouth
(Pilar BZ. et al, 2016). SS also causes systemic inflammation in other organs including vagina,
skin, larynx and trachea (Cornec D. et al, 2017; Ramos-Casals M. et al 2005). Other manifestations
including arthritis, nephritis, purpura, pneumonitis, and vasculitis are also observed among SS
patients (Baldini C. et al, 2014). The diagnosis of SS is difficult for several reasons: i) the most
seen symptoms, the dryness of the eyes and mouth can have many different causes; ii) the
manifestations of SS overlap with other diseases such as Rheumatoid Arthritis (RA) and Systemic
Lupus Erythematosus (SLE) and other dry eye diseases like Meibomian gland disease (MGD),
resulting in a delay in diagnosis or misdiagnosis (Pavlidis NA. et al, 1982); iii) currently there is
no non-invasive way to diagnose SS, and salivary gland biopsies are required. In all, it is
imperative to find an early and non-invasive test to diagnose SS.
1.2. Lacrimal Gland & Rab3D-enriched secretory pathway
The Lacrimal Gland (LG) is an exocrine gland (Obata H, 2006). The healthy LG continuously
secretes fluids to the ocular surface, lubricating and protecting it. These secretions are known as
2
tears, which consist of water, electrolytes, proteins, and nucleic acids. The LG mainly consists of
lacrimal gland acinar cells (LGAC), and they are the dominant cell type in the lacrimal gland, they
account for more than 80% of the total mass of the gland, and they are responsible for aqueous
tear secretion. These cells have unique apical and basolateral membranes linked via tight junctions
forming tubuloacinar structures. On the basolateral site, the membrane contains a lot of ion
channels, proteins for ion transportation, and receptors for neurotransmitters and growth factors
(Dartt DA, 2009; Edman M.C. et al., 2010). When the receptors on the basolateral membrane are
activated, intracellular signaling occurs, leading to the mature secretory vesicles (SV) moving to
the apical membrane at which the SV fuses with the membrane and get released to the luminal
area (Dartt DA, 2009).
Rab proteins are small Ras-like GTPases that play a key role in regulating exocytosis. They cycle
between the GTP-bound active state and GDP-bound inactive state, and this process can modulate
membrane association and dissociation (Magdalena D. et al., 2008; Sean W.D. & Vladimir I.G.
2001). Among all the subfamilies of Rab, Rab3, and Rab27 are reported commonly seen in
different secretory cell types including neurons, endocrine, exocrine, and immune cells (Tetsuro I.
et al, 2003), associated with secretory vesicles (SV). The Rab3 protein has four isoforms which
are Rab3A, 3B, 3C, and 3D. Rab3D has been reported as the most abundant Rab3 protein among
the Rab3 isoforms in LGAC, and it appears to be involved in the regulation of regulated exocytosis
via SV maturation (Oliver M.S. et al., 2002) and prevention of premature fusion (Dietmar R. et al.,
2002). In LGAC, most newly synthesized proteins meant for exocytosis are secreted through the
major pathway, which is regulated by Rab3D, from SV to the apical membrane. However, it is
hypothesized that a “secondary” secretory pathway originating from endolysosomes is triggered
in SS (Runzhong F. et al., 2021). Both pathways involve the maturation of endosomes forming
3
multivesicular bodies (MVB) or late endosomes (LE). Different from the major pathway, the
accumulation of endolysosomal-derived vesicles in SS LGAC may result in the direct fusion of
these vesicles and the apical membrane and the release (Runzhong F. et al., 2021).
1.3. Exosome biogenesis
Exosomes are a type of extracellular vesicle (EV), 30-150 nm in diameter known for delivering
bioactive cargoes such as nucleic acids, proteins, lipids, and other metabolites involved in
intracellular communication (Laura A.M. et al., 2014; Nina P.H. & Alicia L., 2018). The
biogenesis of exosomes begins within the endosomal pathway. The whole process first starts with
the inward budding at the plasma membrane of the organelle, leading to the formation of early
endosomes which contain interstitial and plasma membrane-bound materials (Clotilde T. et al.,
2002). Later, early endosomes mature into late endosomes (LE) or multivesicular bodies (MVB),
and at the same time, the endosomes membrane sprouts inward to form small intraluminal vesicles
(ILV) via the invagination. The ILV that accumulates in the MVB has two fates (Figure 1). One
is to be transported to the lysosomes resulting in the degradation of the contents of the vesicles.
The other is to fuse with the cytoplasmic membrane and then release the vesicles to the
extracellular space, which is known as exocytosis. And those small vesicles are called exosomes
(Nina P.H. & Alicia L., 2018; Suresh M. et al., 2010). More specifically, different trafficking
proteins can be associated with exosomes suggesting their cell of origin. The endosomal sorting
complexes required for transport (ESCRT) proteins such as ALIX, TSG101, and CD9 play an
important role in assisting the development of exosomes, and this pathway is also known as the
ESCRT-dependent pathway. In addition to the ESCRT-dependent pathway, in some cells, the
biogenesis of exosomes also requires ceramide. Once the MVB fuses with the plasma membrane,
exosomes are secreted. During this process, small GTPases from the Rab protein family including
4
Rab27a/b isoforms, Rab11, Rab7 together with Rab35 are required (Nina P.H. & Alicia L., 2018).
Besides, SNARE proteins, YKT6 and VAMP7 for instance, are also involved in mediating vesicle
fusion in the process of releasing exosomes (Nina P.H. & Alicia L., 2018).
Figure 1. Biogenesis of Exosomes.
Figure 1. Biogenesis of Exosomes. Early endosomes mature into multivesicular bodies (MVB)
or late endosomes (LE). Two destinies of MVB/LE, i. Degradation after the fusion with lysosomes;
ii. Fuse with plasma membranes and release exosomes. “Molecules shown to affect exosome
biogenesis and/or release” (Nina P.H. & Alicia L., 2018) is licensed under CC BY 4.0.
5
1.4. Exosomes in SS
Besides the dryness of eyes and mouth, studies show that the expression and activity of a tear
protein Cathepsin S (CTSS), a cysteine protease enriched in endolysosomes, are enhanced in tears
of an SS mouse model and of patients with SS (Hamm-Alvarez et al., 2014). It is also shown that
CTSS plays an important role in the maturation of MHC class II molecules and other inflammatory
pathways besides SS (Hamm-Alvarez et al., 2014; Shi et al., 1999). Notably, a study shows that
CTSS is involved in the regulation of MVB, the common progenitor of exosomes (Huang et al.,
2016). Therefore, the high levels of CTSS in SS may influence tear exosomes’ profile, for instance,
exosome abundance as well as the contents inside exosomes. In all, in light of previous studies,
one may suggest that the characteristics of exosomes isolated from SS tears may be a good place
where we can search for disease-specific biomarkers.
1.5. The male Non-Obese Diabetic (NOD) mouse – a murine model of SS
As stated previously, the diagnosis of SS is challenging, and currently, there aren’t any disease-
specific tear biomarkers that are clinically approved for diagnosis of SS prior to the hallmark
symptoms being observed. Therefore, a murine model that can share SS characteristics with human
SS patients is a precious asset for researchers to study the pathogenesis of SS as well as to develop
new therapies. The male NOD mouse is one of the most established murine models of SS. In
addition to developing SS disease manifestations, the male NOD mice also tend to develop other
autoimmune diseases such as autoimmune peripheral polyneuropathy, type 1 diabetes as well as
autoimmune thyroiditis (Many et al., 1996; Salomon et al., 2001). Notably, compared to the male
NOD mice, female NOD mice develop less profound LG disease at the age of 8 weeks, which may
be explained by sex steroids (Takahashi et al., 1997). And due to the fact that the risk of both male
6
and female NOD mice developing type 1 diabetes increases at a young age: male NOD <14 weeks
and female NOD <10 weeks are generally used for SS studies to avoid complicating the studies.
1.6. Aims and hypothesis
1.6.1. Aim 1: Exosomes as a biomarker for exploring the “secondary” pathway
As described above, previous studies from our lab have revealed that the expression and activity
of CTSS are enhanced in tears of an SS murine model and of SS patients (Hamm-Alvarez et al.,
2014). Moreover, these changes may be related to the dysfunction of an exocytosis regulator
Rab3D. Therefore, in this project, I wanted to elucidate the features of this potential “secondary”
pathway by exploring the presence of exosomes in tears, saliva, and serum from different murine
models including the Rab3DKO mice and the male NOD mice with SS-like dry eye. Ultimately,
we hoped that these studies would help us to have a better understanding of this potential pathway’s
role in SS.
1.6.2. Aim 2: Validation of predicted target of increased miR-375-3p in Rab3DKO tears
Parallel studies in the lab had identified that miR-375-3p was upregulated in Rab3DKO mice tears
and also in exosomes. Since corneas are exposed to tears, I wanted to evaluate the possible changes
in the gene and protein expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), a
major target of miR-375-3p predicted by Ingenuity Pathway Analysis (IPA), to probe whether this
increases in tear had a functional effect. Besides, I wanted to utilize miRNA mimics to see how
miR-375-3p has an effect on the Human corneal epithelial cell-transformed (HCE-T) cell line.
7
Chapter Two
2. Materials and Methods
2.1. Mice
Mouse strains used in this project include Rab3DKO, C57/BL6, Non-Obese Diabetic (NOD), and
BALB/c. Rab3DKO mice are generated and bred in-house from current breeding pairs. The
C57/BL6 strain is either bred in-house from existing breeding pairs or purchased from Jackson
Laboratory (Sacramento, CA). NOD and BALB/c are purchased from Jackson Laboratory
(Sacramento, CA). Rab3DKO and C57/BL6 mice were sacrificed for experimental purposes
between the age of 12 to 20 weeks. NOD and BALB/c mice were used at the age of 12 to 14 weeks.
The animal procedures listed in this thesis were performed strictly according to protocols approved
by Institutional Animal Care and Use Committee (IACUC) at University of Southern California
(USC) as well as the Guide for Care and Use of Laboratory Animals 8th edition (National Research
Council (US) Committee, 2013).
2.2. Tear collection
Mice anesthesia was performed via intraperitoneal injection which in accordance with the inhouse
anesthesia protocol: 80-100 mg of Ketamine and 5-10 mg of Xylazine per kilogram of body weight.
Once the mouse had ceased all movement, and no righting reflex was observed, an incision on
each side, approximately 1 cm below the ear was made. A Kimwipes
TM
tissue was cut into pieces
with the size close to the size of the incision, then placed into the incision site, on the top of the
LG. Then, 10 μL of dd.water was used to rinse the eyes of the mice. Next, 3 μL of Carbachol which
serially diluted into 50 μM was prepared and pipetted onto every placed tissue strip 4 times in total,
8
with an interval of 5 minutes. 32 mm, 2 μL glass capillaries were then used to collect mice tears
for 35 – 40 minutes. Tears were pooled in tubes from either 1 mouse or 3 mice depending on
different experiments (Figure 2), and mice were euthanized after the collection.
Figure 2. Tear Collection Procedure.
Figure 2. Tear collection procedure (Created with BioRender.com).
2.3. Saliva collection
Saliva collection was performed while collecting tears by using 40 mm 10 μL capillaries for 55 –
60 minutes. Saliva was collected from 1 mouse per sample group (Figure 3). After saliva collection,
mice were euthanized by cervical dislocation.
9
Figure 3. Saliva Collection Procedure
Figure 3. Saliva collection procedure (Created with BioRender.com).
2.4. Serum collection
After tear/saliva collection, while the mice were still anesthetized, the cardiac puncture was
conducted and whole blood was collected using a 1.0 mL syringe. All mice were euthanized via
cervical dislocation right after the procedure was finished. Then, the mice's whole blood was put
into 0.8 mL gold cap Separator tubes. And the blood sample was sat at room temperature for 20
minutes to clot. The blood sample was then centrifuged at 2,000 g for 15 minutes at 4 °C. After
the spin, serum was collected and put in new tubes. To remove cellular debris, the serum sample
was then spined for 20 minutes at 2,000 g, with the temperature being 4 °C, and the supernatant
10
was collected and transferred to a new tube. The supernatant was then spun at 12,000 g for 45
minutes at 4 °C to remove macrovesicles.
2.5. Exosome isolation
Before the isolation, PBS containing 25 mM Trehalose Dihydrate (PBS-Tre) was prepared and
filtered. After tear/saliva/serum collection, the sample volume was calculated and brought up to
500 μL with previously prepared PBS-Tre. The sample was then mixed thoroughly and spun to
remove cellular debris. After the supernatant was collected in a new tube, the sample was spun at
for 30 minutes at 4 °C, at 10,000 g to remove larger secreted vesicles such as microvesicles. 495
μL of the supernatant was collected in a new tube. PBS-Tre was centrifuged at 200 g for 5 minutes
at room temperature to remove bubbles. IZON qEVoriginal / 35 nm Gen 2 Column (iZON,
Christchurch, New Zealand) was used to isolate exosomes from tear/saliva/serum samples. Prior
A
11
to running the column, the column was placed in qEV rack at room temperature for enough amount
of time to be within the working temperature range of 18 – 24 °C (Figure 4).
Figure 4. Exosome Isolation
Figure 4. Exosome isolation. A) qEV rack for holding columns and collection tubes. B) Exosome
isolation detailed procedures (Created with BioRender.com).
The column was first washed with 17 mL centrifuged PBS-Tre. When the buffer fully entered the
filter, close the bottom cap, then 495 μL of the sample (tear/saliva/serum) was added to the top of
the filter carefully without introducing any air bubbles to enter the column. The bottom cap was
then opened. Once the sample fully entered the filter, 2 mL of PBS-Tre was added to the top of
the filter. At the same time, a 15 mL tube was placed at the bottom of the rack to collect the void
volume of 2.9 mL (first 6 fractions). Right after the void fractions were measured and collected,
1.6 mL (fractions 7-9) was then collected in a new 2 mL Eppendorf tube and placed on ice.
B
12
Table 1. iZON qEVoriginal 35 nm Size Exclusion Column Fraction collected.
Table 1. iZON qEVoriginal 35 nm Size Exclusion Column Fraction collected.
Volume to be collected Fractions to be collected
Void 2.9 mL 1-6
Exosome fraction 1.6 mL 7-9
The sample was then either processed for immediate Nanoparticle Tracking Analysis (NTA) or
concentrated down as stated below. Up to 500 μL of the sample was added into Amicon 500 μL
Centrifugal filters. The concentration step was finished in 8 minutes at 14,000 g at 4 °C. Repeat
until the remaining was concentrated in the filter. The filter was then flipped, and the sample was
recovered with 2 minutes of spinning with the settings of 1,000 x g (Figure 5). The concentrated
sample was then flash-frozen in liquid nitrogen and stored at -80 °C.
Figure 5. Amicon Ultra-10k Centrifugal Filters
Figure 5. Concentration using Amicon Ultra - 0.5 mL Ultracel – 10k Centrifugal filters
(https://www.emdmillipore.com)
13
2.6. ZetaView Nanoparticle Tracking Analysis
Exosome quantification and size measurement were done via ZetaView Nanoparticle Tracking
Analysis (ZVNTA). The instrument is capable of measuring the diameter of the particle, zeta
potential as well as particle concentration in the solution. Before every usage, the instrument was
calibrated with 100 nm standard beads (Applied Microspheres, The Netherlands), and optimized
focus. Each exosome sample was first diluted with PBS-Tre, in a 15 mL falcon tube. The sample
was then centrifuged at 200 g for 5 minutes at 4 °C to remove bubbles and was kept on ice until
running the analysis. 1 mL of the sample was injected into the instrument. When a relatively similar
number of particles can be detected at 11 locations within the machine, one can start the analysis.
Each sample was analyzed 3 times. Between each loading, HyClone HyPure Water for Injection
(WFI) Quality Water (Cytiva, USA) was used to remove the remaining sample inside the
instrument both manually through injection and automatically by the machine itself. Each analysis
was taken using the following settings: i) room temperature; ii) sensitivity was adjusted to 84; iii)
30 as the frame rate; iv) 65 as the shutter speed; v) a brightness of 20; vi) 500 pixels as the
maximum size; vii) 10 pixels as the minimum size.
2.7. BCA
The concentration of either LG lysates or tear protein was determined by using Pierce
TM
BCA
Protein Assay Kit (ThermoFisher Scientific
TM
). The BCA working solution was prepared by
mixing BCA reagents A, B, and C with a ratio of 25:24:1. Protein standards were first prepared by
diluting Bovine Serum Albumin (BSA; Sigma #A6003) with 20 mM Tris-HCl. The final
concentrations of BSA stock and BSA working stock were 10 mg/mL and 200 μL/mL, respectively.
Then, the protein standards were further diluted to 0.5, 1, 1.5, 2, and 2.5 mg/mL with dd. water.
14
Tissue samples were diluted to 1:100 and tear samples were diluted to 1:300 with dd. water as well.
20 μL of samples and protein standards were pipetted onto the ThermoFisher Scientific
TM
Pierce
TM
96-Well Plates in duplicates. 150 μL BCA working solution was then added to the plate using a
multichannel pipet. After loading all reagents, the 96-well plate was wrapped in alumina foil and
then incubated for 1 hour in a 37°C incubator. The absorbances of standards and samples were
measured at 570 nm.
2.8. Western blotting
2.8.1. Tissue
Mice were first euthanized in accordance with the inhouse mice procedure as described previously.
LGs were then collected and lysed in RIPA buffer containing protease/phosphatase inhibitor
cocktail (Cell Signaling Technology, Danvers, MA, USA) in 2 mL BeadBug™ prefilled tubes
(Sigma-Aldrich Corporation (St. Louis, MO, USA). The supernatants were collected. The protein
concentration of the lysates was measured using the BCA assay with the Micro BCA™ Protein
Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Lysate samples were left in a reducing
6X Laemmli SDS sample buffer (Thermo Fisher Scientific Rockford, IL, USA), containing β-
mercaptoethanol for 5 minutes at 98 °C. 10% Tris-Glycine Novex™ WedgeWell™ Mini Protein
Gels (Thermo Fisher Scientific Rockford, IL, USA), were used for gel running. 30 µg of protein
were loaded into each well and run with the setting of 120 V, at 4 °C cold room for 1 to 2 h.
Proteins were then transferred to nitrocellulose membranes, and the transfer sandwich was
prepared via iBlot 2 dry blotting system (ThermoFisher Scientific, Rockford, IL, USA). After gels
transfer, the Revert™ 700 Total Protein Stain Kits for Western Blot Normalization were used for
total protein staining as the loading control. After distaining, the transferred membranes were
blocked in the fluorescent blocking buffer (Rockland Immunochemicals Inc, Limerick, PA, USA)
15
for an hour. After the incubation, the membranes were shaken gently for approximately 12-16 h in
the primary antibody which was diluted in the same blocking buffer in 4 °C cold room. After
overnight incubation, the membranes were washed 3-5 times, with an interval of 5 minutes, with
Tris-buffered saline supplemented with 0.2% Tween (TBST). The membranes were gently shaken
in secondary antibodies for incubation at room temperature, for 1 h. After washing with TBST 3
to 6 times, with an interval of 5 minutes, the imaging step was processed with the use of the
Odyssey Licor imaging system (LI-COR Biotechnology, Lincoln, NE, USA). All primary
antibodies and secondary antibodies are listed in (Table 2, Table 3).
Table 2. Primary antibodies.
Table 2. Primary antibodies.
Primary Antibodies Host Reactivity Product No. Company
CIP2A Rabbit Mouse, Rat,
Human
PA588456 ThermoFisher
Scientific
LAMP2 Rabbit Mouse, Rat,
Human
PA1-655 ThermoFisher
Scientific
TSG101 Rabbit Mouse, Rat,
Human
ab125011 Abcam
ALIX Rabbit Mouse, Rat,
Human
PA5-52873 ThermoFisher
Scientific
CD63 Rabbit Mouse, Rat,
Human
ab217345 Abcam
CD81 Rabbit Mouse, Rat,
Human
MA5-32333 ThermoFisher
Scientific
CD9 Rabbit Mouse, Rat,
Human
MA5-31980 ThermoFisher
Scientific
Table 3. Secondary antibodies
Table 3. Secondary antibodies
Secondary antibodies Host Product No.
IRDye 680 LT Donkey
anti-Rabbit
926 - 68073 Li-Core
IRDye 680 RD Goat
anti-Rat
926 - 68029 Li-Core
16
2.8.2. Tears
After tear collection which discussed in section 2.2, each tear sample was brought up to 20 μL
using sterile PBS. 2 μL of diluted tear sample was then further diluted to 1:100 with sterile PBS.
The 1:100 diluted sample was used for BCA analysis. After determining the tear protein
concentration, the remaining steps were the same as described in section 2.8.1. Table 2 and Table
3 also listed out the antibodies which were used in tear Western Blotting.
2.9. Cell culture
An SV40-immortalized human corneal epithelial cell line, HCE-T cells, was obtained from the
RIKEN Cell Bank, Japan (Cat. # RCB2280). The cell culture medium was prepared in accordance
with the vendor’s instructions. For preparing the ready-to-use keratinocyte-SFM (KSFM), Bovine
Pituitary Extract (BPE), human recombinant EGF (rEGF), and antibiotic gentamycin (Gibco®,
Life Technologies, Grand Island, NY, USA) were used as supplements. When cells reached 80-
90% confluence, the cells were first digested by using 0.25% Trypsin-EDTA (Mediatech, Inc.,
Manassas, VA, USA). After the digestion, 10% of Heat-inactivated fetal bovine serum
(ThermoFisher Scientific, Rockford, IL, USA) which was prepared in sterile PBS was used to
quench trypsin digestion. All cells used were no more than passage 8.
2.10. Cell Transfection
Cells were cultured in 12-well plates with KSFM supplemented only without gentamycin which
can do harm to cell growth and affect transfection efficiency during the process. Cells at 60-80%
confluency were transfected with miRNA Mimic Negative Control #1 (Invitrogen
TM
, Carlsbad,
CA, USA), miR-375-3p (assay ID: MC10327), and miRNA mimic miR-1 Positive Control. The
17
preparation of all reagents followed the manufacturer's instructions (Figure 6). After overnight cell
seeding in the incubator, the cell medium was changed. Measurement time points were set at 48h
and 72h to test which one was more efficient. After these two time points, the efficiency of
transfection together with the silencing effects of these mimics was tested by measuring whether
there are changes in miRNA expression via qRT-PCR (hsa-miR-375-3p miRCURY LNA miRNA
PCR Assay GeneGlobe ID: YP00204362) as well as using miR-1 positive control and measuring
whether there are fold-changes in the expression of its target PTK9 (Gene Expression Assay ID:
Hs00911809_g1) at mRNA levels.
Figure 6. miRNA Mimic Transfection Protocol
Figure 6. miRNA mimic transfection protocol
(http://tools.thermofisher.com/content/sfs/manuals/OOBE_mirVana_miRNA_Mimics_man
.pdf).
18
2.11. Cornea isolation
Rab3DKO mice and C57/BL6 mice were euthanized via intraperitoneal injection as discussed in
the previous section. Mouse eyeballs were then dissected and placed on a sterile tissue culture Petri
dish with sterile PBS in it. With the petri dish under the microscope, forceps were used to grab the
optic nerve, and carefully cut the eyeball into halves by using a blade. The corneas were then
carefully cut along the sclera by using a micro-surgical scissor. Pool two corneas into one 2 mL
prefilled beads tube and proceed to total RNA isolation.
2.12. RNA isolation
2.12.1. Tissue
RNeasy Plus Universal Kit (Qiagen, Hilden, Germany) was used in the LG total RNA extraction.
The LG tissue was first placed in a 2 mL prefilled beads tube, and 900 μL of QIAzol Lysis buffer
was pipetted into the tube. Then, the tissue was disrupted and homogenized. After the tissue was
lysed, the homogenate was incubated at room temperature for 5 minutes. 100 μL of gDNA
Eliminator Solution was added into the homogenate and the tube was shaken vigorously for 15
seconds. Next, 180 μL of chloroform was added to the tube, and the previous step was repeated.
Incubate the sample at room temperature for 2-3 minutes. The sample was then centrifuged at
12,000 g at 4°C for 15 minutes. After the centrifugation was done, the upper aqueous phase was
transferred to a new tube, and 1 volume of 70% ethanol was added and vortexed. Up to 700 μL of
the sample was added to the spin column in a 2 mL collection tube. Centrifuge the tube at 8,000 g
for 15 seconds at room temperature, and then the flow through was discarded. The remaining
19
sample was added into the same tube, and the previous step was repeated. The purification and
collection steps are listed in Figure 7.
Figure 7. RNA Purification Procedure
Figure 7. RNA purification procedure.
20
2.12.2. Cell RNA
The HCE-T cells were used in this project to test whether miR-375-3p has an effect on the
expression of its predicted target CIP2A at the mRNA level, as well as to explore the efficiency of
miRNA mimic transfection. The miRNeasy Mini Kit was used. 700 μL of QIAzol Lysis Reagent
was added to each well. A cell scraper was used to harvest the cells from tissue culture flasks. The
cells were then transferred to the prefilled bead tube and homogenized. And the homogenate was
incubated at room temperature for 5 minutes. 140 μL of chloroform was added to the homogenate,
and the tube was shaken vigorously for 15 seconds. Then, incubate the homogenate at room
temperature for 2-3 minutes. The sample was then spun at the speed of 12,000 g at 4°C for 15
minutes. The aqueous phase on the top was pipetted to a new tube carefully without intervening
other phases and 1.5 volume of 100% ethanol was added to the tube and mixed thoroughly by
vortexing. And the following procedure was the same as in Figure 7.
2.13. RT-qPCR
Reagents used for reverse transcription and the following qPCR varied by samples and
experiments. For miRNA mimic functional studies, the LNA-enhanced SYBR Green miRNA kits
were used. All primers used are listed in Table 4.
Table 4. Qiagen primer list
Table 4. Qiagen primer list
Primer GeneGlobe ID
hsa-miR-375-3p miRCURY LNA miRNA PCR Assay YP00204362
Uni-Sp-6 miRCURY LNA miRNA PCR Assay YP00203954
cel-miR-39-3p miRCURY LNA miRNA PCR Assay YP00203952
21
SNORD48 (hsa) miRCURY LNA miRNA PCR Assay YP00203903
For gene expression of CIP2A at mRNA level studies, TaqMan
TM
Reverse Transcription reagents
and TaqMan
TM
primers were used. The primers used are listed in Table 5. The miRNA mimics
used are listed in Table 6.
Table 5. TaqManTM primer list
Table 5. TaqMan
TM
primer list
Primer Assay ID
Human CIP2A primer Hs00405413_m1
Human GAPDH primer Hs99999905_m1
Human PTK9 primer Hs00911809_g1
Mouse CIP2A primer Mm00553898_m1
Mouse miR-375-3p primer mmu481141_mir
Mouse GAPDH primer Mm99999915_g1
Table 6. miRNA mimic list
Table 6. miRNA mimic list
miRNA mimic Catalog No.
miRNA mimic Negative Control #1 4464058
miRNA mimic miR-1 Positive Control 4464062
hsa-mir-375-3p mimic 4464066
All samples and reagents for cDNA synthesis were thawed beforehand, spun down and stored on
ice before preparation. The Reverse Transcription protocols varied by different experiments. For
miRNA mimic functional studies, miRCURY LNA SYBR Green RT Kit was used. The reaction
setup was listed below in Table 7 and Table 8.
22
Table 7. miCURY LNA Reverse Transcription reaction setup per sample
Table 7. miCURY LNA Reverse Transcription reaction setup per sample
Reagent Volume used in each step
5X miRCURY SYBR® Green RT Reaction
Buffer
4 μL
10X miRCURY RT Enzyme Mix 2 μL
UniSp6 RNA spike-in 1 μL
Template RNA The volume of template RNA used in RT
depends on its concentration. The maximum
loading volume of RNA is 13 μL
RNase-free water The total volume of RNase-free water plus
template RNA is 13 μL.
Total 20 μL
Table 8. Reverse Transcription cycling setup
Table 8. Reverse Transcription cycling setup
Reaction step Time setting Temperature setting
1. Reverse Transcription
step
60 minutes 42°C
2. Inactivation of
reaction
5 minutes 95°C
3. Cool down & store Indefinitely 4°C
For the reverse transcription in CIP2A gene expression studies, TaqMan
TM
Reverse Transcription
Reagents and TaqMan
TM
Primer Assay were used. The RT reaction mixture was prepared as listed
in Table 9 and Table 10.
Table 9. TaqManTM Reverse Transcription reaction setup per sample
Table 9. TaqMan
TM
Reverse Transcription reaction setup per sample
Reagent Volume used in each step
10X TaqMan R.T. Buffer 5 μL
25 mM MgCl2 11 μL
MultiScribe Reverse Transcriptase 1.25 μL
dNTP Mixture 10 μL
RNase Inhibitor 1 μL
Random Hexamers 2.5 μL
Template RNA 4 μg of template RNA was used, the volume of
RNA was calculated based on concentration
with maximum RNA loading of 19.25 μL
Total 50 μL
T
23
able 10. Reverse Transcription cycling setup
Table 10. Reverse Transcription cycling setup
Step Time Temperature
Primer annealing 10 min 25°C
DNA polymerization 30 min 37°C
Inactivation of reverse
transcriptase
5 min 95°C
Cool down & store Indefinitely 4°C
Upon completion of RT, the concentrations of cDNA samples were measured. Then, all the
samples were diluted to 10 ng – 100 ng in accordance with the vendor’s instructions with nuclease-
free water. All samples were then added to MicroAmp
TM
Optical 96-Well Reaction Plate, and
qPCR was performed by using inhouse Real-Time PCR System. And finally, the Comparative CT
(ddCT) was chosen as type of experiment.
2.14. NanoDrop
TM
Spectrophotometer
NanoDrop
TM
Spectrophotometer was used to quantify the concentration of nucleic acids including
RNA and DNA as well as the purity of these biological samples. After the instrument was initiated,
Nanodrop was first washed with RNase-free water and the detection spot was then cleaned using
Kimwipe. When the instrument was ready for measurements, 2 μL of RNase-free water was loaded
for blanking. Then, 2 μL of sample was loaded to the detection spot and the RNA concentration
was measured, including the ratios of A260/A280 and A260/A230. After all measurements, the
instrument was cleaned.
24
Chapter Three
3. Results
3.1. The expression of CIP2A at mRNA level is higher in Rab3DKO female LG
Based on the previous finding that miR-375-3p is elevated in tears from Rab3DKO mice, we
thought this might reflect higher tissue levels which in turn, could be the expression of one of its
target genes, CIP2A. LGs were collected from both Rab3DKO and C57/BL6 mice, and each strain
could also be broken down into different groups based on two variations which were stimulation
(Carbachol treated for tear collection) and sex. After collection, one LG from one single mouse
was used for CIP2A RT-qPCR analysis. As shown in Figure 8A, female Rab3DKO had
significantly higher CIP2A expression compared to C57/BL6 females, in both stimulated (p <
0.0001) and unstimulated (p = 0.0007) groups, which indicates that the potential silencing effect
of miR-375-3p on its target CIP2A might have a sex difference. No significant difference was
observed in CIP2A gene expression in the male groups. Since there are three independent factors
including strain, gender, and stimulation, a three-way ANOVA was performed. As shown in Figure
8B, the influences of gender and strain were significant, with both p-values lower than 0.0001.
And no significant influence of stimulation on the expression was observed. This suggests that
miR-375-3p may not decrease CIP2A expression but that other factors such as sex hormones may
increase it.
25
Figure 8. The CIP2A expression at the mRNA level in LG from Rab3DKO and C57/BL6 mice.
Figure 8. The CIP2A expression at the mRNA level in LG from Rab3DKO and C57/BL6
mice. The RT-qPCR signal was normalized to the housekeeping gene GAPDH. Each group of
data was also normalized to C57/BL6 signal. A. The CIP2A expression in LG from unstimulated
male mice (Rab3DKO n = 12; C57/BL6 n = 11), unstimulated female mice (Rab3DKO n = 2;
C57/BL6 n = 3), stimulated male mice (Rab3DKO n = 9; C57/BL6 n = 12), and stimulated
female mice (Rab3DKO n = 3; C57/BL6 n = 3). The y-axis represents fold change B. The Three-
way ANOVA results. All error bars are SD.
26
3.2. The expression of CIP2A at mRNA level in mouse corneas
As corneas are exposed to tears that have elevated miR-375-3p in Rab3DKO strains, we
hypothesized that corneas from these mice might also show decreased CIP2A. Corneas were
isolated from Rab3DKO and C57/BL6 male mice, and each RNA sample was pooled from both
sides of the cornea from one mouse. As shown in Figure 9, no statistically significant difference
in CIP2A expression levels between Rab3DKO and C57/BL6 corneal sample was observed.
Figure 9. CIP2A expression at the mRNA level in the cornea from Rab3DKO and C57/BL6 mice.
Figure 9. CIP2A expression at the mRNA level in the cornea from Rab3DKO and C57/BL6
mice. The RT-qPCR signal was normalized to the housekeeping gene GAPDH. Each group of data
was also normalized to C57/BL6 signal. Each data point was pooled from both corneas from one
mouse. An unpaired t-test was performed, with a p = 0.1712 > 0.05. Each strain has an equal
number of mice, n = 6. The y-axis represents fold change. All error bars are SD.
3.3. CIP2A Western Blotting
In addition to the gene expression analysis, CIP2A western blotting on LG lysates from Rab3DKO
and C57/BL6 was also performed. Signals in both stimulated LG and unstimulated LG were
detected (Figure 10). The concentration of protein lysate was determined by BCA assay, and the
target protein signal was normalized to total protein staining. The results below indicate that there
is no significant difference in CIP2A protein expression level between male Rab3DKO and
C57 RAB3DKO
0.0
0.5
1.0
1.5
2.0
2^ddCt
Cornea CIP2A Expression Level
ns
27
C57/BL6 in both scenarios. At the time, we did not know of the altered CIP2A mRNA expression
in female mice and the sex difference, so this would be a good assay to repeat with female C57
and Rab3DKO mice in the future.
Figure 10. CIP2A Western Blotting of male Rab3DKO and C57/BL6 LG from 12-16 weeks mice.
Figure 10. CIP2A Western Blotting of male Rab3DKO and C57/BL6 LG from 12-16 weeks
mice. The estimated molecular weight of CIP2A is 90 kDa. Each data point represents one LG
collected from one mouse. A. 30 μg of unstimulated LG protein was loaded into the gel, the
dilutions of primary antibody and secondary antibody were 1:1000 and 1:2000, respectively. B. 18
μg of stimulated LG protein was loaded into the gel, the dilutions of primary antibody and
secondary antibody were 1:1000 and 1:2000, respectively. C. Rab3DKO CIP2A relative intensity
compared to C57/BL6. An unpaired t-test was performed, p = 0.3354 > 0.05, n = 3. D. Rab3DKO
CIP2A relative intensity compared to C57/BL6. An unpaired t-test was performed, p = 0.7753 >
0.05, n = 3. The y-axis represents relative intensity. All error bars are SD.
28
3.4. The CIP2A functional assay
3.4.1. Protein Tyrosine Kinase 9 (PTK9) gene expression
Micro RNA mimics are designed to mimic mature endogenous miRNA for functional analysis. To
develop the functional assay which uses miRNA mimics to transfect HCE-T cells and see whether
the miRNA mimic has an effect on its downstream targets, miR-1 positive control, and miRNA
negative control were first used. The PTK9 gene has been shown to be downregulated by miR-1.
The transfection procedure was as stated in section 2.10., 100 nM miRNA negative control and
the positive control were prepared and used in the cell transfection experiment. After 48 h of
incubation, total RNA isolation followed by RT-qPCR was performed. As shown in Figure 11A,
cells transfected with miR-1 positive control had significantly lower PTK9 expression, with a p <
0.0001.
Figure 11. miRNA mimic transfection validation experiments.
Figure 11. miRNA mimic transfection validation experiments. A. The RT-qPCR signal was
normalized to the housekeeping gene human GAPDH. An unpaired t-test was performed, the p <
0.0001 (n = 3). B. The RT-qPCR signal was normalized to the housekeeping gene human
SNORD48. An unpaired t-test was performed, the p = 0.0023 < 0.05 (n = 3). The y-axis represents
fold change. All error bars are SD.
29
3.4.2. miR-375-3p transfection efficiency
After the protocol was successfully developed, miR-375-3p mimics together with miRNA negative
control were transfected into HCE-T cells. The transfection efficiency was measured by the miR-
375-3p level in the cells, compared to the negative control (Figure 10B). The fold-change of miR-
375-3p was significantly higher in the miR-375-3p mimic-treated group, which indicates that the
transfection was successful.
3.4.3. The CIP2A expression after miR-375-3p mimic transfection
The cells were treated with 100 nM miR-375-3p mimic and miRNA negative control. After
overnight incubation, the cell medium was changed to KSFM with all the supplements. Since the
intervention of miRNA mimics on the expression of certain genes at the mRNA level usually takes
a longer period, the measurement time points were set at 48 h and 72 h (Figure 12). Total RNA
was isolated as stated in section 2.12.2., and the concentration was measured by NanoDrop. After
cDNA synthesis, equal amount of cDNA was prepared and then qPCR was performed. The
expression results as shown below, the difference in expression of CIP2A after 48 h and 72 h
treatment of miR-375-3p between treatment group and negative control was not significant,
suggesting at least in HCE-T cells, that the relationship between miR-375-3p and CIP2A is not as
hypothesized by IPA analysis.
30
Figure 12. The CIP2A expression after 48 h, 72 h incubation.
Figure 12. The CIP2A expression after 48 h, 72 h incubation. A. The RT-qPCR signal was
normalized to the housekeeping gene human GAPDH. An unpaired t-test was performed, the p =
0.6571 > 0.05 (n = 3). B. The RT-qPCR signal was normalized to the housekeeping gene human
GAPDH. An unpaired t-test was performed, the p = 0.3834 > 0.05 (n = 3). The y-axis represents
fold change. All error bars are SD.
3.5. ZetaView Nanoparticle Tracking Analysis (ZV-NTA)
The quantification of exosomes was done by using ZetaView Nanoparticle Tracking Analysis
(ZV-NTA). The instrument can measure the Zeta potential and particle size by capturing the
Brownian motion of nanoparticles. In this project, the exosome abundance in mouse tears from
Rab3DKO, C57/BL6, NOD, and BALB/C were measured. The exosome sample isolated from
Rab3DKO and C57/BL6, together with the serum exosome from male NOD mice were also
measured.
31
3.5.1. Tear exosomes in Rab3DKO and C57/BL6 mouse tears
As shown in Figure 13A, the exosome abundance in male Rab3DKO mouse tears was not
statistically significantly different from C57/BL6 (p > 0.05). However, there was an increased
trend in Rab3DKO tear exosomes compared to the control group. There was no significant
difference in the median size of exosomes between these strains (Figure 13B).
Figure 13. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from Rab3DKO and C57/BL6.
Figure 13. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from Rab3DKO
and C57/BL6. Each data point represents one group which consists of tear exosomes pooled from
5 mice. Exosomes were isolated as stated in section 2.5. A. The number of particles captured by
ZV-NTA was normalized per microliter of tears collected. An unpaired t-test was performed, p =
0.1742 > 0.05, (n = 2). B. The median particle size of captured particles. All error bars are SD.
32
3.5.2. Saliva exosomes in Rab3DKO and C57/BL6 mouse saliva
As shown in Figure 14A, the exosome abundance in male Rab3DKO mouse saliva was not
statistically significantly different from C57/BL6 (p > 0.05). No statistically significant difference
in the median size of exosomes between these strains was detected.
Figure 14. Saliva Exosome ZetaView Nanoparticle Tracking Analysis results, from Rab3DKO and C57/BL6.
Figure 14. Saliva Exosome ZetaView Nanoparticle Tracking Analysis results, from
Rab3DKO and C57/BL6. Each data point represents one mouse. Exosomes were isolated as
stated in section 2.6. A. The number of particles captured by ZV-NTA was normalized per
microliter of saliva collected. An unpaired t-test was performed, p = 0.0619 > 0.05, (n = 6). B. The
median particle size of captured particles. An unpaired t-test was performed, p = 0.5544 > 0.05, (n
= 6). All error bars are SD.
3.5.3. Tear Exosomes in NOD and BALB/C mouse tears
As mentioned previously, the male NOD mouse is an established SS disease model, and male
BALB/C is typically used as the control group. At the age of 12-14 weeks, NOD female mice
develop less serious LG manifestations compared to male NOD mice. Therefore, NOD female
mice were also used as an extra, non-diseased, and sex-based control group. In my thesis project,
16 weeks male NOD mice were also included because we wanted to see if older male NOD would
33
have a different exosome abundance compared to the younger ones. As shown in Figure 15A, the
exosome abundance in male NOD mouse tears was significantly less than in female NOD mice,
so as in the 16 weeks group. And there is no difference in exosome abundance between 16-week
male NOD and younger ones. As shown in Figure 15B, there are no differences in exosome
abundance between BALB/C, male NOD, and 16-week male NOD.
Figure 15. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from NOD and BALB/C.
Figure 15. Tear Exosome ZetaView Nanoparticle Tracking Analysis results, from NOD and
BALB/C. Each data point represents one group pooled from 3 mice. Exosomes were isolated as
stated in section 2.6. A. The number of particles captured by ZV-NTA was normalized to per
microliter of tears. A One-way ANOVA was performed, and multiple comparisons were also
performed, p (NOD F vs. NOD M) = 0.0021, p (NOD F vs. NOD M/16wk) = 0.0010, p (NOD M
vs. NOD M/16wk) = 0.6779, (n = 3). B. The number of particles captured by ZV-NTA was
normalized to per microliter of tears. A One-way ANOVA was performed, and multiple
comparisons were also performed, p (BALB/C vs. NOD M) = 0.3001, p (BALB/C vs. NOD
M/16wk) = 0.8813, p (NOD M vs. NOD M/16wk) = 0.5167, (n = 3). All error bars are SD.
34
3.5.4. Serum Exosomes in male NOD mice
The exosome isolation from serum samples was the same as stated in section 2.5. In this study, we
wanted to see if there was a difference in the exosome abundance between serum and tears. As
shown in Figure 16, there is no difference between each group. however, we did observe a
increased trend in NOD male 16-week serum exosomes compared to NOD male 16-week tear
exosomes.
Figure 16. Tear and serum Exosome ZetaView Nanoparticle Tracking Analysis results from male NOD.
Figure 16. Tear and serum Exosome ZetaView Nanoparticle Tracking Analysis results from
male NOD. The number of particles captured by ZV-NTA was normalized per microliter of
biofluid. A One-way ANOVA was performed, and multiple comparisons were also performed, p
(NOD M vs. NOD M/16wk) = 0.6305, p (NOD M vs. NOD M/16wk Ser) = 0.0.3915, p (NOD
M/16wk vs. NOD M/16wk Ser) = 0.1208, (n = 3). All error bars are SD.
35
3.6. Western Blotting of exosomal protein markers
3.6.1. Alix Tear Western Blotting
Western blotting analysis showed that Alix is enriched in tears, suggesting that it labels tear
exosomes. However, compared to both NOD female group and BALB/C group, the Alix protein
signal in the NOD male group was not significant difference. Shown in Figure 17.
Figure 17. Western Blotting analysis of Alix in tears of male and female NOD and BALB/C, aged 12-14 weeks.
Figure 17. Western Blotting analysis of Alix in tears of male and female NOD and BALB/C,
aged 12-14 weeks. The estimated molecular weight of Alix is 80 kDa. Each data point represents
one mouse. 30 μg of tear protein was loaded into each lane of the gel. The dilutions of primary
antibody and secondary antibody were 1:1000 and 1:2000, respectively. A. Alix signal in mouse
tear: male NOD mouse versus Balb/c control group. An unpaired t-test was performed, p = 0.3789 >
0.05, (n = 3). B. Alix signal in mouse tear: male NOD versus female NOD control group. An
unpaired t-test was performed, p = 0.2850 > 0.05, (n =3). All error bars are SD.
36
3.6.2. TSG101 Tear Western Blotting
Western blotting analysis showed that TSG was present in mouse tears, suggesting it labels tear
exosomes. However, the TSG signal in male NOD mouse tears was statistically significantly lower
than in female NOD mice. But there was no difference between BALB/C and male NOD. The
results are shown in Figure 18.
Figure 18. Western Blotting analysis of TSG101 in tears of male and female NOD and BALB/C, aged 12-14 weeks.
Figure 18. Western Blotting analysis of TSG101 in tears of male and female NOD and
BALB/C, aged 12-14 weeks. The estimated molecular weight of TSG101 is 52 kDa. Each data
point represents one mouse. 30 μg of tear protein was loaded into each lane of the gel. The dilutions
of primary antibody and secondary antibody were 1:1000 and 1:2000, respectively. A. TSG101
signal in mouse tear: male NOD versus NOD female control group. An unpaired t-test was
performed, p = 0.0226 < 0.05, (n = 3). B. TSG signal in mouse tear: male NOD versus BALB/C
control group. An unpaired t-test was performed, p = 0.4607 > 0.05, (n =3). All error bars are SD.
37
3.6.3. CD63 LG Western Blotting
Western blotting analysis showed that CD63, a member of the tetraspanin family enriched in the
membrane of exosomes, is expressed in male BALB/C mouse LG but not detected at comparable
levels in male NOD mice (Figure 19). Since the epitopes of CD63 as well as CD9 and CD81
require non-reducing conditions, none of the reducing regents were added during sample
preparation.
Figure 19. Western Blotting analysis of CD63 in LG from male NOD and BALB/C mice, aged 12-14 weeks.
Figure 19. Western Blotting analysis of CD63 in LG from male NOD and BALB/C mice,
aged 12-14 weeks. The estimated molecular weight of CD63 is 26-32 kDa. Each lane represents
one LG from one mouse. 50 μg of tear protein was loaded into each lane of the gel. The dilutions
of the primary antibody and secondary antibody were 1:1000 and 1:2000, respectively.
38
3.6.4. CD81 LG Western Blotting
Western blotting analysis showed that CD81, a member of the tetraspanin family enriched in the
membrane of exosomes, was not significantly differentially expressed between LG of male NOD
and BALB/c mice (Figure 20).
Figure 20. Western Blotting analysis of CD81 in LG of male NOD and Balb/c, aged 12-14 weeks.
Figure 20. Western Blotting analysis of CD81 in LG of male NOD and Balb/c, aged 12-14
weeks. The estimated molecular weight of CD63 is 25-28 kDa. Each lane represents one LG from
one mouse. 50 μg of tear protein was loaded into each lane of the gel. A. CD81 western blotting
signal in male NOD and Balb/c LG. The dilutions of primary antibody and secondary antibody
were 1:1000 and 1:2000, respectively. B. An unpaired t test was performed, p = 0.0646 > 0.05, (n
= 5). All error bars are SD.
39
3.6.5. CD9 LG Western Blotting
Western blotting analysis showed that CD9, a member of the tetraspanin family enriched in the
membrane of exosomes, was not detected by Western blotting in the lysates of either male NOD
LG or Balb/c mice LG (Figure 21).
Figure 21. Western Blotting analysis of CD9 in LG of male NOD and Balb/c, aged 12-14 weeks.
Figure 21. Western Blotting analysis of CD9 in LG of male NOD and Balb/c, aged 12-14
weeks. The estimated molecular weight of CD9 is 25kDa. Each lane represents one LG from one
mouse. 50 μg of tear protein was loaded into each lane of the gel. The dilutions of the primary
antibody and secondary antibody were 1:1000 and 1:2000, respectively.
40
3.6.6. LAMP2 LG Western Blotting
Western Blotting analysis showed that the total LAMP2 signal was not significantly different
between the LG of Balb/c and NOD (Figure 22A/B).
Figure 22. Western Blotting analysis of LAMP2 in LG of male NOD and Balb/c, aged 12-14 weeks.
Figure 22. Western Blotting analysis of LAMP2 in LG of male NOD and Balb/c, aged 12-14
weeks. The estimated molecular weight of CD9 is 110 kDa. Each lane represents one LG from
one mouse. 50 μg of tear protein was loaded into each lane of the gel. A. LAMP2 signal in the LG
of Balb/c and NOD mice. The dilutions of the primary antibody and secondary antibody were
1:1000 and 1:2000, respectively. B. An unpaired t test was performed, p = 0.2937 > 0.05, (n = 5).
All error bars are SD.
41
Chapter Four
4. Discussion
The aims and objectives of this thesis project were to begin exploring the role of trafficking of
exosomes from the potential “secondary” secretory pathway which may originate from
endolysosomes in exocrine glands including the LG and SG that are affected by SS. At the same
time, the gene and protein expression levels of CIP2A, a predicted target of potential tear
biomarker miR-375-3p, was tested by RT-qPCR, Western blotting as well as functional assays
utilizing miRNA mimics.
I will first summarize findings on CIP2A. Previous studies in the lab identified miR-375-3p as
upregulated in both male and female Rab3DKO tears, as well as in the exosome fraction. And
further IPA pathway analysis showed that CIP2A is one of the major targets of miR-375-3p such
that when the miRNA is increased, CIP2A gene expression should be decreased. I found that,
intriguingly, the mRNA levels of CIP2A were higher in Rab3DKO female mice LG with and
without topical stimulation. Since the increased tear miR-375-3p was found in both strains, this
may suggest a sex effect on CIP2A regulation in this strain that is independent of the miRNA. In
both corneal studies and miRNA functional studies, no marked effect of miR-375-3p was noted.
However, the corneal CIP2A levels were assessed only in male mice. Female Rab3DKO mice
might have more CIP2A transcripts in the lacrimal glands due to the presence of other bioactive
molecules such as miRNA, siRNA, and enzymes which regulate the expression of CIP2A, and at
the same time, are influenced by hormones. It will be of interest to repeat these studies in female
mice with a greater sample size, evaluating both CIP2A expression in LG and cornea. Therefore,
further experiments can: i) increase the sample size of female mice from both strains; ii) compare
42
the miR-375-3p level in tears between male and female mice from both strains; iii) re-do the
pathway analysis for miR-375-3p to find a more promising target.
The ZetaView Nanoparticle Tracking Analysis showed that the exosome concentration of
Rab3DKO tears was approximately three-fold higher than that in C57/BL6, although this change
was not statistically significant. The size of the particles was not different between these two strains.
The NTA also showed that no statistically significant difference was observed in the abundance of
exosomes which was isolated from saliva. These findings suggest that although the pathway
associated with mature secretory vesicle secretion in LG and SG is impaired, the change in the
total number of exosomes secreted may be minimal in Rab3DKO mice. However, the sample size
was low for the tear analysis since the samples need to be pooled from multiple mice. A future
experiment should increase the sample size to at least 5 groups per strain to see if the modest
increase becomes significant.
Interestingly, the NTA showed that the abundance of exosomes in male NOD tears was
significantly lower than that in female NOD mice, but not in male BALB/C mice. We previously
hypothesized that due to the inflamed exocrine glands which affected by SS, the “secondary”
secretory pathway originating from endolysosomes could be triggered and therefore an
upregulation of exosomes in tears might be observed. However, my results showed an opposite
trend. Nevertheless, it is still possible that with disease, that exosome composition is affected in
the male NOD mice. Surprisingly, the female NOD mice showed higher exosome content in tears
than either male NOD or BALB/c. This will be further pursued to see if there is a sex effect on
43
exosome composition within strains, and if it is unique to the NOD strain or present in C57/BL6
mice.
Tear Western blotting allowed me to evaluate the abundance of different exosomal proteins in
tears, which should be related to their abundance on exosomes from the strain. The exosomal
protein marker TSG101 which plays a role in endosomal sorting, was expressed more in tears of
NOD female mice. Notably, this result was aligned with the tear exosome NTA results which
showed that female NOD mice had more exosomes in tears. However, the other marker Alix, a
protein that regulates the secretion of exosomes showed no difference between strains. Lysosome-
associated membrane protein 2 (LAMP2) is a highly glycosylated membrane protein that is
thought to mediate the process of ILV loading which takes place on the sites of endosomal
membrane by capturing cargo during the formation of ILV (Ferreira et al., 2022). As a result, an
increase in LAMP2 expression can indicate that the formation of ILV is increased as well as the
exosome secretion. The LAMP2 western blot showed that there was no significant difference
between male NOD and BALB/c mice, however, the quantification of LAMP2 signal is tricky due
to glycosylation. Therefore, a de-glycosylation of protein lysates before blotting could aid in the
protein signal quantification in future studies.
CD63, CD81 and CD9 are the three most common tetraspanins that are expressed on the surface
of the exosome since they have a broad tissue distribution. Researchers often use the presence of
these three proteins to identify and capture exosomes. The Western blotting analyses of these three
tetraspanins in NOD and BALB/C showed that: i) CD9 was not able to be detected in the LG of
any of the strains; ii) there was no statistically significant difference in the expression of CD81
44
between male NOD and Balb/c; iii) CD63 can be detected in the LG of BALB/c mice, but not in
diseased male NOD mice. These interesting results might indicate that, in the SS disease model
male NOD, the composition of exosome surface markers is somehow changed. Further studies
such as detecting these three proteins in the LG at the mRNA level, using magnetic bead-based
isolation to pull down exosomes from tears biofluids, and a more comprehensive characterization
of additional exosomal marker abundance in tears and LG will be important.
45
Chapter Five
5. Conclusion
Exosomes were initially considered as cellular waste, but over the years, these nanosized
extracellular vesicles (EV) have drawn great attention because of their potential in therapies and
diagnosis (Nina P.H. & Alicia L., 2018). In addition to the exosome itself, the contents that
encapsulated inside its body are also precious assets that still have not been fully explored. Studies
show that, exosome protein cargo varies in different physiological and pathological conditions,
which gives them the potential as biomarkers in pathological conditions (Deng & Miller, 2019).
Notably, previous studies in the lab have found that the onset of SS may trigger the endolysosomal
secretory pathway via the dysregulation of an exocytosis regulator, Rab3D. Through exploring the
characteristics of extracellular vesicles in both disease model NOD and “major” secretory
pathway-deficit model Rab3DKO, we hope to identify non-invasive tear fluid biomarkers and have
a better understanding of the “secondary” pathway’s role in SS.
In this project, the NTA results of the abundance of tear/saliva/serum exosome together with
Western blotting analyses gave us a hint that a more specific exosome isolation protocol together
with more comprehensive analysis of the exosomal effector proteins across strains is needed to
better identify exosomal differences and to characterize those changes. For example, using
magnetic beads to isolate exosomes followed by immunostaining or flow cytometry to quantify a
more accurate amount or composition of EV in tears will be useful.
46
Although the miRNA mimic transfections did not validate the predicted trend by which the CIP2A
mRNA level was downregulated by miR-375-3p, the positive controls validated that the miRNA-
mimic protocol may be further used as a robust tool in elucidating the pathogenesis of SS in acinar
cells and ocular surface cells with other miRNAs (Shruti S.K. et al., 2023). We have recently
identified many miRNAs that differ in tears of male NOD mouse and in LG of male NOD mice,
and validation of their signaling effects can be accomplished using the miRNA mimic protocol.
In all, these data show that further studies on exosome characterizations and exploring the
endolysosomal secretory pathway are needed not only for searching biomarkers for diagnosis but
also for therapies.
47
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Sarah F. Hamm-Alvarez; The miRNA Landscape of Lacrimal Glands in a Murine Model of
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Abstract (if available)
Abstract
Sjo?gren’s Syndrome (SS) is a chronic, inflammatory disorder characterized by inflammation of the lacrimal gland (LG) and the salivary gland (SG) leading to dysfunction, with the most seen manifestations being dry eyes and dry mouth. Exosomes are nano-sized, cell-derived vesicles that play an important role in delivering a variety of biomolecules such as proteins, and lipids as well as nucleic acids to different cells in the body. Increased evidence shows that these bioactive cargoes in exosomes facilitate intercellular communication by transmitting phenotypic information to other cells under both physiological and pathological conditions. Rab3D is a GTPase that regulates exocytosis in exocrine epithelial cells in association with mature secretory vesicles. It is well-acknowledged that most proteins are secreted through the Rab3D-regulated secretory pathway in lacrimal gland acinar cells (LGAC). We then came up with a hypothesis that the loss of Rab3D might potentially trigger the generation of a “secondary” secretory pathway originating from endolysosomes in the inflamed exocrine glands that are affected by the autoimmune disease, Sjögren’s Syndrome (SS). That change might in turn lead to changes in exosome secretion from acinar cells since exosomes are present in endolysosomal membranes. Both tear and LG exosome abundance, and exosomal marker proteins were analyzed in specimens from different murine models including the Rab3DKO mouse and the SS disease model male Non-Obese Diabetic (NOD) mouse and their respective controls, C57BL/6 and male BALB/c mice. Previous study in the lab have shown that miR-375-3p is upregulated in Rab3DKO mouse tears. Therefore, the target of miR-375-3p predicted by Ingenuity Pathway Analysis, the cancerous inhibitor of protein phosphatase 2A (CIP2A)’s gene and protein expression were measured in downstream cells. In this project, the protocol of tear exosome quantification in both murine models was optimized, and the abundance of exosomal proteins TSG101, HSC70, LAMP2, CD9, CD81, and CD63 in mouse tear exosomes was analyzed. The protocols of measuring CIP2A’s gene and protein level in Rab3DKO model were also developed and optimized.
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Li, Xiaoyang
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Core Title
Secretion of exosomes through the potential endolysosomal pathway in Rab3DKO and Non-Obese Diabetic murine models
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School of Pharmacy
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Pharmaceutical Sciences
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2023-08
Publication Date
08/01/2023
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