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Defining the functional region of LINC00261 in lung adenocarcinoma
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Defining the functional region of LINC00261 in lung adenocarcinoma
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Content
Defining the Functional Region of
LINC00261 in Lung Adenocarcinoma
By: Gopika Nandagopal, B.S
A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree MASTER OF
SCIENCE (Biochemistry and Molecular Biology)
August 2019
2
Dedications
I would like to dedicate this thesis to my parents Veena Nandagopal and Nandagopal
Nair.
3
Acknowledgements
I would like to thank Dr. Crystal N. Marconett, Jonathan Castillo and all past and current members of
Marconett lab for their patience, support and mentorship throughout this project. I would also like
to thank Dr. Ite Offringa, Chunli Yan and the members of Offringa lab for their insights as well as for
the use of their equipment. I would also like to thank my thesis committee members, Dr.Crystal N.
Marconett, Dr. Ite Offringa and Dr. Michael Stallcup for their guidance.
This project would not have been possible without the funding provided by the USC Department
of Surgery, The Baxter Foundation, The Wright Foundation, and STOP cancer.
4
Table of Contents
Dedications ............................................................................................................................. 2
Acknowledgements ................................................................................................................. 3
ABSTRACT ............................................................................................................................... 6
Chapter 1: Introduction ........................................................................................................... 7
1.1: Characterizing Lung Adenocarcinoma ........................................................................................ 7
1.2 Long non-coding RNA ................................................................................................................ 10
1.3 The Role of lncRNA in Cancer .................................................................................................... 13
1.4 Preliminary Data ....................................................................................................................... 14
Chapter 2: Isolating the functional region of LINC00261 ........................................................ 20
2.1 Introduction .............................................................................................................................. 20
2.2 Materials and methods ............................................................................................................. 23
Cloning LINC00261 Truncations ......................................................................................................................... 23
Transient Transfection ........................................................................................................................................ 24
Generation of stable cell lines ............................................................................................................................ 25
Cell line maintenance ......................................................................................................................................... 26
Flow Cytometry .................................................................................................................................................. 26
RT-qPCR .............................................................................................................................................................. 27
2.3 Results ...................................................................................................................................... 27
Isolating Exon 4 of LINC00261 ............................................................................................................................ 28
Further truncating LINC00261 Exon 4 ................................................................................................................ 30
Isolating the conserved region of LINC00261 exon 4 ......................................................................................... 32
2.4 Discussion ................................................................................................................................. 34
Chapter 3: Understanding the role of LINC00261 in the DNA damage response ..................... 36
3.1 Introduction .............................................................................................................................. 36
3.2 Method ..................................................................................................................................... 37
Western Blots ..................................................................................................................................................... 37
3.3 Results ...................................................................................................................................... 39
DNA Damage Response Gene Westerns ............................................................................................................ 39
3.4 Discussion ................................................................................................................................. 40
Chapter 4: Summary ............................................................................................................. 41
Chapter 5: Future Directions .................................................................................................. 42
Supplementary Materials ...................................................................................................... 43
Plasmids .............................................................................................................................................................. 43
Primers used for cloning ..................................................................................................................................... 47
5
Primers used for qPCR ........................................................................................................................................ 48
References ............................................................................................................................ 50
6
ABSTRACT
Lung cancer is the leading cause of cancer related deaths in the United States. Prior research on
lung cancer primarily focused on protein coding oncogenes, however the role of long non-coding
RNA (lncRNA) in lung carcinogenesis is relatively underexplored. We have identified a lncRNA,
LINC00261 that is downregulated in multiple cancers, including lung adenocarcinoma (LUAD).
Recently published data has shown that LINC00261 acts as a tumor suppressor. This is supported
by the observation that ectopic over expression of LINC00261 in H522 LUAD cells inhibits cell
proliferation and migration through G2/M cell cycle arrest and activated expression of genes
associated with the DNA damage response pathway. The length of LINC00261 is 4.9kB
complicating the utilization of this RNA as an adjuvant therapeutic for LUAD. In order to package
this lncRNA into nanoparticles for delivery, the smallest functional unit of LINC00261, that that
evokes the tumor suppressive properties needs to be defined. Truncation of LINC00261 has
demonstrated that the isolated 1.3kb conserved region of exon 4 LINC00261 is sufficient to
induce G2/M cell arrest, implicating this region as the functional unit of LINC00261. Western blot
experiments have validated that downstream signaling targets of ATM become activated upon
ectopic expression of LINC00261.
7
Chapter 1: Introduction
1.1: Characterizing Lung Adenocarcinoma
Lung cancer is the most common cause of cancer related deaths in the United States, accounting for
~150,000 deaths per annum (Ridge et al., 2013), and 1.6 million deaths worldwide (Didkowska et al.,
2016). In fact, deaths from lung cancer surpass those attributed to breast, colorectal, and prostate
cancers combined (Figure 1). While the vast majority of lung cancers occur in tobacco smokers, ~10-
15% of cases are in those who have never smoked (Thun et al., 2008).
Figure 1: The epidemiology of lung cancer
In 2016 the total number of combined tracheal, bronchus and lung cancers in the world was ~1.71 million. This was
more than double that caused by stomach cancer, the next most common cause of cancer death (Institute for Health
Metrics and Evaluation, 2016).
8
Lung cancer is heterogenous in nature and is categorized based on histology. The two major
classes of lung cancer are small cell lung cancer (SCLC) and Non-small cell lung cancer (NSCLC).
Classifying lung cancer into different groups allows for more targeted therapies upon diagnosis,
as well as more accurate predictions on the outcomes of the disease. NSCLCs are the most
commonly occurring type of lung cancer and are also the focus of most molecular therapies used
today in the treatment of lung cancer. NSCLCs are further subcategorized into large cell
carcinoma, squamous cell carcinoma and adenocarcinoma (Figure 2). Lung adenocarcinoma
(LUAD) is the most common subtype, accounting for ~50% of all cases of lung cancer (Ridge et
al., 2013) (Travis et al., 2011). LUAD is also the most common type of lung cancer to affect never-
smokers, as well as younger patients (Dela Cruz et al., 2011).
Figure 2: Subtypes of lung cancer
Lung cancer is split into two broad categories – small cell and non-small cell lung cancer. Non-small cell lung cancers
are then further subcategorized into large cell carcinoma, adenocarcinoma, and squamous cell carcinoma.
9
Adenocarcinoma is the most prevalent type of lung cancer and is thought to originate from alveolar epithelial cells
(Jackson et al., 2014).
In recent years molecular target-based therapies have been increasingly popular and studied in
the treatment of cancer. These methods can be advantageous over conventional
chemotherapeutic strategies due to their specificity and lesser adverse effects in patients. Some
of the most common molecular alterations found in LUAD include changes in the EGFR, KRAS,
ALK, and MET genes (Villalobos et al., 2017) (Figure 3).
A B
Figure 3: Known mutations associated with lung adenocarcinoma
A. This table illustrates the incidence of the most frequently occurring mutations seen in adenocarcinoma and
squamous cell carcinoma, as well the types of mutations that have occurred (Villalobos et al., 2017). B. ~40% of lung
adenocarcinoma cases show no known mutations (Chan et al., 2015).
These molecular based therapies, as well as the screening patients for known mutations has
become increasingly more common practices in the treatment of lung cancer. One such example
10
of molecular target-based therapy in lung cancer treatment is Gefitnib, a drug used in patients
with mutated EGFR, which in 2015 became approved by the FDA as a first-line treatment for
NSCLC (Kazandjian et al., 2016).
Despite all this, the prognosis for lung cancer remains poor, with an overall 5-year survival rate
of ~16.8% (Ridge et al., 2013). Additionally, these known mutations do not account for all cases
of lung cancer, as the driver for ~20-40% of cases remains unknown (Kumarakulasinghe et al.,
2015). Furthermore, even in cases with a known mutation, this may not account all the symptoms
seen in the patient. Therefore, there are other molecular drivers causing lung cancer, that remain
unexplored. Much of the prior research has focused on proteins, but less than 2% of RNA
transcripts are translated into proteins (Fok et.al, 2017), with only a handful of functional RNAs
explored for their potential impact on cancer etiology. This leaves a huge class of molecules that
have not been characterized for their roles in this cancer. The discovery of these factors, and the
development of treatments to target them could be critical in improving the prognosis for lung
cancer.
1.2 Long non-coding RNA
Long non-coding RNAs (lncRNAs) are a new, relatively understudied class of regulatory molecule that
have been implicated to be key players in a variety of biological processes. lncRNAs are defined as
non-protein-coding RNAs that are greater that 200 nucleotides in length. lncRNAs undergo post-
transcriptional modifications, and mature products are capped and polyadenylated at the ends.
lncRNA genes also can contain introns that are spliced out post-transcriptionally.
11
LncRNA genes are classified according to their location in the genome. They are found on both
sense and antisense strands and can either be overlapping protein coding genes or be found in
intergenic regions of the genome (LincRNAs) (Fok et al., 2017) (Figure 4). In comparison to protein
coding mRNA, lncRNA is typically much lower in abundance, and is localized to the nucleus.
lncRNAs also have a tendency to be tissue specific and have much less conservation across
different species (Khorkova et al., 2015).
Due to their length, lncRNAs are able to fold into complex three-dimensional structures with
functional domains, that enable them to regulate cellular processes, including action as
transcriptional regulators, chromatin remodelers and post transcriptional modifiers, through a
variety of different mechanisms (Figure 4). They can act as guides, and recruit chromatin modifier
proteins to specific sites in the genome, as in the case of ANRIL which has been known to recruit the
polycomb repressive complex PRC2 (Kotake et al., 2011). lncRNAs can also function as scaffolds,
through interaction with RNA-binding proteins to form RNA-protein complexes, that can then go on
to recruit other factors to a target area of the genome. The HOTAIR gene is a prime example of this
kind of activity, as this lncRNA has two distinct domains, one with specificity for PRC2 and the other
with specificity for LSD1 of the LSD1/CoREST/REST complex, thus tethering these two separate
complexes together. In this way, HOTAIR is able to bring about coordinated modifications to target
chromatin (Tsai et al., 2010). Another way lncRNAs act as transcriptional regulators is through direct
binding with chromatin. This is the case with XIST, which coats one of the two X chromosomes in
females, thus blocking the binding of RNA Pol III and initiating the process of X chromosome
inactivation (Long et al., 2017). lncRNAs can also act
12
as decoys by competitively inhibiting other molecules. This can be seen in the case of certain
lncRNAs that contain the complimentary sequence for important micro RNAs (miRNAs), allowing
these lncRNAs to bind and siphon off target miRNAs as a means of repressing their function
(Banks et al., 2012).
B
A
I
II
III
IV
Figure 4: Organization and Function of LncRNA
A. LncRNAs are categorized based on genomic location. Intergenic lncRNAs are found in between protein coding genes
(A.I), bidirectional lncRNAs are found in close proximity to a protein coding genes but on the anti-sense strand, and
are regulated by the same promoter (A.II), Antisense lncRNAs overlap protein coding genes but on the opposite DNA
strand (A.III) , and sense-overlapping lncRNAs as the name suggests are located on the sense strand and overlap a
protein coding gene (Balas et al., 2018). B. lncRNAs can regulate transcription in many different ways, including
through direct binding to chromatin (B.I), through action as a competitive inhibitor (B.II), through recruitment of other
factors (B.III) and through action as a scaffold by binding other factors and tethering them to each other (B.IV) (Wang
et al., 2011)
13
1.3 The Role of lncRNA in Cancer
Long non-coding RNAs can be driving factors in cancer. Due to their important roles in chromatin
remodeling, as well as regulation of transcription, the deregulation of these molecules can lead
to cancerous outcomes, which has been summarized in Figure 5 below. LncRNAs have been
shown to have roles in cell cycle regulation, pluripotency, DNA damage response, immune
response, cell survival and mobility – the deregulation of which can lead to cancer phenotypes.
lncRNAs can act both as oncogenes, as in the case of MALAT1 and HOTAIR and as tumor
suppressors as in the case of DILC and TERRA (Balas et.al, 2018).
A B
14
Figure 5: Cancer associated LncRNAs
A. lncRNAs have been shown to be dysregulated in a variety of cancers throughout the body. Some lncRNAs such as
MALAT1 and HOTAIR are involved in many different types of cancer, whereas others like PNCR1 are specific to certain
types of cancer (Balas et al., 2018). B. These dysregulations can be both upregulations or downregulations, implicating
the role of lncRNAs as both oncogenes and tumor suppressors (Fang et.al, 2016).
One of the most well characterized lncRNAs in cancer, metastasis associated lung
adenocarcinoma transcript 1 (MALAT1) is a lncRNA that has been found to be upregulated in
numerous cancers, and was first identified as a potential prognostic marker for non-small cell
lung cancer, as it was shown to be associated with metastasis in these patients (Zhao et al., 2018).
In addition to being used as a diagnostic tool, MALAT1 is also a potential therapeutic target.
Research has shown that oligonucleotide-based inhibition strategies such as siRNAs and
antisense-RNAs have been able to successfully knock down the gene in preclinical models of
human cancers (Amodio et al., 2018). In vitro MALAT1 knock-out models on human lung cancer
cells, as well as mouse xenograft models with MALAT1 deficient cells both showed that the loss
of function of this gene led to a decrease in tumor migration and metastasis (Gutschner et.al,
2014). This supports the study of lncRNAs as a new branch of therapeutic targets for cancer.
1.4 Preliminary Data
Our lab has identified the lncRNA, LINC00261 as a functional tumor suppressor that plays a crucial
role in the onset of lung adenocarcinoma. Bioinformatic analysis revealed that LINC00261 is
significantly downregulated in LUAD cell lines compared to adjacent normal cells (Shahabi et al.,
15
2019). RNA-Seq data from The Cancer Genome Atlas database has shown that LINC00261 is
downregulated in LUAD as well as a variety of other cancers, including liver, prostate, and squamous
cell lung cancer. LUAD patients with low levels of LINC00261 had shorter overall survival than for
those with high expression of the gene (figure 6). This suggested to us the potential of LINC00261 as
a therapeutic target for LUAD, and an ideal candidate for further study.
A
B
C
Figure 6: Identifying the tumor suppressor capabilities of LINC00261
A. TCGA datasets illustrate that LINC00261 is significantly downregulated in a variety of different cancers including
LUAD. B. qRT-PCR experiments showed that LINC00261 was downregulated in LUAD cell lines when compared to AEC
cell lines, the presumed cell of origin for LUAD. C. The overall survival of patients with low LINC00261 expression
(indicated in black) was found to be significantly lower than in those patients with high LINC00261 expression
(indicated in red) (Shahabi et al., 2019).
16
LINC00261 is a long intergenic non-coding RNA (lincRNA) found on chromosome 20 of the human
genome. The mature transcript of LINC00261 contains 4 exons and has a total length of 4.9kb
(figure 7).
A
B
Figure 7: Organization and structure of LINC00261
A. The RefSeq prediction from NCBI of LINC00261 organization, taken from the UCSC genome browser B. The
proposed 3D structure of LINC00261 as predicted by Viennafold (Lorenz et al., 2011).
The functional role of LINC00261 in LUAD carcinogenesis was studied by ectopically introducing the
LINC00261 under the control of the cytomegalovirus constitutive promoter (CMV-LINC00261) into
the gene in H522 LUAD, and then comparing the phenotype of these cells to those where an empty
vector had been introduced (CMV-NEO). Proliferation assays revealed that cell lines with LINC00261
expression had a slower rate of proliferation compared to those without LINC00261.
17
Scratch assays using the same cell lines revealed that the introduction of LINC00261 lead to an
increase in cell migration (figure 8B). Cell cycle analysis through FACS sorting was done on H522
CMV-LINC00261 and H522 CMV-NEO cells in order to gain a better understanding of the
mechanism of action of LINC00261. Through these experiments it was demonstrated that cells
without LINC00261 expression were able to bypass the G2/M phase of the cell cycle, and that the
ectopic expression of LINC00261 allowed for the cells to arrest in the G2/M phase, all of which
indicates that the reintroduction of LINC00261 into cells that are deficient in it is able to reduce
their cancerous phenotype (Shahabi et al., 2019) (figure 8C).
A
B
C
Figure 8: Mechanistic studies of LINC00261
A. cells expressing LINC00261 had a significantly slower proliferation rate B. Migration rate than for CMV-
LINC00261 was lower than cells expressing CMV-NEO blank vector. C. CMV-LINC00261 was able to initiate
G2/M cell cycle arrest (Shahabi et al., 2019).
18
Although this data demonstrates the promise of LINC00261 reintroduction in LUAD patients that are
have lost expression in their tumors as a strategy for treating LUAD, the large 4.9kB length of this
lncRNA makes its packaging inside a nanoparticle not feasible. Isolating the functional region of
LINC00261 is critical in order for this work to have translational relevance in a clinical setting.
In order to better understand the mechanistic role LINC00261 plays in cell cycle regulation, IPA
pathway enrichment analysis had been performed on H522 CMV-LINC00261 cells compared to
CMV-NEO cells. This revealed significant differences in the expression of DNA damage response
related genes (both through upregulation and downregulation) upon ectopic expression of
LINC00261. Furthermore, preliminary western blot data had demonstrated that ectopic
LINC00261 expression leads to phosphorylation-mediated activation of ATM at Ser1981, which is
a critical event in the transmission of the DNA damage signal to downstream effector proteins of
the DNA damage response RNA immunoprecipitation assays were done in A549 LUAD cell lines
with endogenous LINC00261 expression. The pull down of ATM lead to the pull down of
LINC00261, implying that there is direct binding between these two factors.
The role of ATM in the cell, is to detect damaged DNA and initiate the DNA damage response
signaling cascade. The inability for ATM to be activated upon LINC00261 loss, would lead to cells
being able to bypass the G2/M cell cycle checkpoint as we had observed through FACS (Shahabi
et al., 2019).
19
A
B
C
Figure 9: Gene expression analysis of LINC00261 co-regulated genes
A) Illustrated above is a map of the DNA damage signaling cascade with genes differentially expressed in A549
shScrambled vs. A549-shLINC00261 cell lines. A549s are LUAD cells that do have endogenous expression of
LINC00261. A549-shLINC00261 have been treated with siRNA to known down LINC00261 expression, while A549-
shScramble have been treated with a control siRNA and have normal LINC00261 expression. Genes upregulated in
shLINC00261 samples are denoted in pink, while those that are downregulated are indicated in green. B) Western
blot images for phosphorylated ATM and CHK2 in H522 cell lines transfected with CMV-LINC00261 or CMV-NEO
controls. Hsp90 was used as a control. C) RNA immunoprecipitation using α-ATM in A549 cells to pull down
endogenous LINC00261. (Shahabi et.al, 2019)
20
Chapter 2: Isolating the functional region of LINC00261
2.1 Introduction
The use of RNAs as a therapeutic agent has become increasingly popular over recent years. One
promising method for delivering RNA to specific sites in the body, is through the use of lipid
vesicles (Adams et al., 2017). This method has shown promising results in vitro, as in the case of
the delivery of the lncRNA H19, through nanovesicles for the treatment of diabetic wounds (Tao
et al., 2018). Using the same concept, the delivery of LINC00261 to affected LUAD cells in the
body through such vesicles could be a viable new treatment, to be used as an adjunct therapy to
chemotherapeutics.
Lipid based non-coding RNA (ncRNA) delivery strategies are being researched for the treatment
of lung cancer, and there are currently 17 ongoing clinical trials involving the delivery of miRNA
in lung cancer (figure 10)(Tian et al., 2017), and 3 ongoing clinical trials for the delivery of lncRNA
to treat human cancer. One example is the use of a tumor suppressive miRNA, mir-133B to target
the pro-survival protein MCL-1 in lung cancer, thereby making these cells more susceptible to
apoptosis by chemotherapy (Crawford et al, 2009) (Wu et al., 2011).
21
Figure 10: ncRNA delivery in lung cancer treatment
ncRNA-delivery based therapies for lung cancer in clinical trial as of August 2016. (Tian et al, 2017)
Defining the region of LINC00261 that is responsible for the induction of the downstream anti-
proliferative effects, through G2/M cell cycle arrest is critical to translating preliminary findings
into clinically relevant therapeutics.
22
It has been demonstrated that lncRNAs have different functional domains that can have unique
functions. This can be seen for example, in the case of RepA, a lncRNA involved in X-chromosome
inactivation in mice. RepA is a 1.6kb lncRNA that has a sequence identical to the 5’ terminal
sequence of the human lncRNA XIST which brings about X-chromosome inactivation. When this
conserved region of XIST is deleted in humans, it was observed that X inactivation does not occur,
and that essential proteins for this process are not able to bind. This suggests that this smaller
region of XIST is able to bring about functional changes on its own in mice, and that it is also
integral for silencing in humans – essentially acting as a functional domain (Liu et al., 2017).
Similarly, deletion experiments of the NEAT1 lncRNA, a molecule that functions to assemble
paraspeckle nuclear bodies, found that the middle domain of this gene was sufficient for the
assembly of these structures, and that this was the site of protein binding (Yamazaki et al., 2018),
once again suggesting individual regions of lncRNAs are able to bring carry out specific functional
roles independently.
LINC00261 is 4.9kB long, and it is possible that it may have additional roles in cell regulation
outside of the effects we have seen on proliferation and cell cycle regulation (Shahabi et al.,
2019). If there is a region that is sufficient to correct the loss of G2/M seen in cells that do not
express this gene, then the isolation and targeted delivery of this fragment will be crucial for
designing a clinically relevant treatment approach for LUAD.
23
2.2 Materials and methods
Cloning LINC00261 Truncations
The full CMV-LINC00261 and CMV-LINC00261 Exon 4
bp882-4935
plasmids had been previously
generated. The smaller exon 1-3
bp1-881
, exon 4
bp839-2185
, exon 4
bp2219-3610
and exon 4
bp3585-4931
truncations were generated using PCR amplification. For smaller LINC00261 truncations, the PCR
reaction mix consisted of 5μL 5x Phusion HF buffer (NEB), 10mM dNTPs, 10μM forward primer (IDT),
10μM reverse primer (IDT), 250ng of CMV-LINC00261 plasmid served as the template DNA, 0.25μL
Phusion DNA polymerase (NEB), and ultrapure water to a total volume of 25μL. Primers used for PCR
amplification, as well as plasmid maps for CMV-LINC00261, CMV-LIN00261 Exon 4
bp882-4935
and
CMV-NEO have been included in the supplemental materials section.
Samples were then placed in the thermocycler for 1 cycle at 98 C for 30 seconds for initial
denaturation, then 30 cycles of 98 C for 10 seconds, 65 C for 30 seconds annealing, and 72 C for
30 seconds extension. Samples underwent a final extension at 72 C for 10 minutes to extend,
and then held at 4 C until retrieval.
The amplified PCR products were then run on an agarose gel, and the insert of interest was
isolated and purified through gel purification using the QIAGEN gel extraction kit (lot
#427110590, MD).
LINC00261 Exon 1-3
bp1-881
, LINC00261 Exon 4
bp839-2185
, and LINC00261 Exon 4
bp3585-4931
inserts
were digested with EcoRI-HF (NEB, catalog #R3101S, lot #0151708) and Kpn1-HF (NEB, catalog
24
#R3142S, lot#0071709). For LINC00261 Exon 4
bp2219-3610
, Hindi III (NEB, catalog #R3104S lot
#0061504) and EcoRI-HF were used instead for digestion. CMV-NEO vector was digested with the
same enzymes as the insert. Digestion mix consisted of 2μg of DNA, 5μl Cutsmart buffer (NEB)
and 2μl of enzyme. Digestions were left to incubate at 37 C for 4 hours.
Digested insert and CMV backbone were run on an agarose gel to verify successful digestion, and
then purified through gel purification using the QIAGEN gel extraction kit.
Purified products were then ligated. The ligation reaction mix consisted of 2μL T4 DNA ligase
buffer (NEB), 50ng cut vector DNA, an optimized amount of cut insert DNA calculated through
the NEB calculator, ultrapure water to 20μL, and 1μL of T4 DNA ligase (NEB). Samples were left
to incubate at 16 C overnight.
Transient Transfection
Prior to transfection, WT H522 cells were plated on 6-well tissue culture plates and allowed to
reach 70% confluency. Cell transfection mix for four wells consisted of 10,000ng of either CMV-
LINC00261 exon 4
bp882-4935
, CMV-NEO or CMV-LINC00261 insert DNA, 40μL of Lipofectamine 2000
(Invitrogen, lot #1699503, Carlsbad CA), and Opti-MEM Serum free media (Gibco) to a total volume
of 1ml. The mix was left to incubate at room temperature for 5 minutes. Cells were then aspirated of
existing media, and 250μL of reaction mix was added to each well. In this way, each sample DNA
insert had 4 transfection replicates. Cells were then left to incubate for 4 hours, before each well was
topped off with 3ml of RPMI-1640 1X media (CORNING, lot #06319004, Manassas VA). After 24 hours,
the cell media was replaced with new RPMI-1640. Cells were
25
passaged to achieve 30% confluency for FACS analysis if needed. Further experiments were done
48 hours after transfection.
Generation of stable cell lines
For the smaller truncations of LINC00261, stable polyclonal cell lines were generated in H522 cell
lines. Plasmids were first linearized, before transfection. All plasmids were linearized with AgeI-HF
(NEB, catalog #R3552S, lot #10025897), as this ensured no disruption of important plasmid elements.
Linearization reaction mix consisted of 20μg DNA, 20μl Cutsmart buffer, 3μl AgeI-HF enzyme, and
ultrapure water to a total volume of 200μl. Samples were left to incubate overnight
at 37 C and then purified using the QIAquick PCR purification kit.
Linearized plasmids were then transfected into WT H522 cells using the FuGENE HD reagent
(Promega, lot #0000336308, Madison WI). Transfections were done on cells in 6-well plates at
>80% confluency. Optimized amounts of for transfection at a ratio of 3:1 (reagent:DNA), were
calculated using the online FuGENE HD protocol database. The transfection mix consisted of
3.3μg of DNA diluted in Opti-MEM for a total volume of 155μl. To this, 9.9μl of FuGENE HD
reagent was added. The reaction was mixed by pipetting and allowed to incubate at room
temperature for 5 minutes. Existing media was aspirated from the cells and 150μl of complex
was added to the corresponding well. Cells were left to incubate at 37 C for 4 hours before each
well was topped up with 3ml of RPMI-1640 media. Existing media was aspirated off and replaced
with new RPMI after 24 hours.
26
48 hours after transfection, existing RPMI-1640 was aspirated off the cells and replaced with new
RPMI-1640 supplemented with G418 selection marker. The media was changed every 2 days, and
cells were passaged as needed until a stage where floating cells had ceased to show under the
microscope. After 2 weeks of maintenance, cell lines were verified using RT-qPCR.
Cell line maintenance
WT H522 cells were grown in RPMI media supplemented with 10% Fetal Bovine Serum (X&Y Cell
Culture, lot #7B0302, Kansas City MO) and 1% penicillin streptomycin (USC cell culture core
facility, lot #255). G418 was added to the media and used as a selection marker at a previously
optimized concentration of 166μg/mL for transfected cell lines.
Flow Cytometry
Flow cytometry was performed ~48 hours after transfection. Transfected cells were aspirated of
media and washed with 1ml DPBS (CORNING, lot #13018004, VA), and then harvested through
scraping with another 1ml DPBS. Cells were then pelleted by centrifugation at 3000RPM for 3
minutes and resuspended with 300ul of DNA staining mix. DNA staining mix consisted of
0.5mg/ml propidium iodide (sigma-aldrich), 0.1% sodium citrate and, 0.05% Triton X-100. The cell
resuspension was then passed through a 40um nylon cell strainer (VWR, lot #180621301, Radnor
PA) to prevent cell clumping. Fixed samples were placed on ice and flow cytometry was done
within an hour of fixation.
Samples were run on The BD SORP LSR II analysis cytometer with 355nm laser and a 675 long
pass filter. Cell cycles analysis was done using FlowJo v. 10.5 software.
27
RT-qPCR
Transfected cell lines were verified using qPCR. Cells were aspirated of existing media, washed with
1ml DPBS, then harvested through scraping with another 1ml DPBS. Cells were then pelleted by
centrifugation at 3000 rpm for 3 minutes, and DPBS was aspirated. If qPCR was to be done at
a later date, cells were frozen on dry ice and stored at -20 C.
RNA was isolated using the BioRad Aurum Total RNA Isolation mini kit (catalog #732-6803) and
followed the directed protocol.
cDNA was then prepared using the iScript cDNA synthesis kit (Bio-Rad). Reaction mix consisted of 5μl
iScript reaction mix, 1μl iScript reverse transcriptase, 1μg RNA sample, and nuclease free water to a
total volume of 20μl. Samples were then incubated in the thermocycler for 5 minutes
at 25 C, 30 minutes at 42 C, 5 minutes at 85 C and finally held at 10 C.
qPCR was used to test each transfected cell line for expression of their LINC00261 insert of
interest against H522 CMV-LINC00261 as a control. GAPDH was used as a housekeeping gene.
qPCR reaction mix consisted of 5μl cDNA (diluted 1:5 with water), 6μl water, 0.75μl reverse
primer, 0.75μl forward primer, and 12.5μl SYBR Green (bio-rad).
2.3 Results
The majority of LINC00261 is made up of the 4.1kb long exon 4 of this lncRNA. For this reason, it
was hypothesized that if there is a functional region on LINC00261 that is responsible for the
28
increase in population of cells in G2/M cell cycle arrest observed H522 CMV-LINC00261, then it
is likely that this region will be found within this exon.
Isolating Exon 4 of LINC00261
CMV-LINC00261 Exon 4
bp882-4935
, CMV-LINC00261 and CMV-NEO plasmids were transiently
transfected into WT H522 cells with 3 replicates of each insert (Figure 9A). Transfected cells were
verified with RT-qPCR, to ensure that inserts were being expressed at an appropriate level. Variations
in cell cycle between the 3 conditions were analyzed using FACS cell cycle analysis. It
was found that cells transfected with LINC00261 Exon 4
bp882-4935
and Full LINC00261 had a
significantly higher percentage of cells in G2 phase when compared to cells transfected with the
empty Neo vector (Figure 9B).
A
(bp882-4935)
B
29
(bp882-4935)
Figure 11: Ectopic expression of LINC00261 exon 4
bp882-4935
induces G2/M cell cycle arrest
A) Cell cycle curves derived from Propidium Iodide (PI) staining FACS analysis of CMV-LINC0026, CMV-LINC00261 Exon 4 and
CMV-NEO controls. The curves shown are representative of three independent experiments. The percentage population of
cells in G1 are highlighted in blue, those in S phase are highlighted in yellow, and those in the G2 stage are highlighted in
green. The Population was analyzed using FlowJo v10.5 N=3 (≥50,000 cells per sample). B)
Quantification of FACS analysis in (A), comparing CMV-LINC00261 and CMV-LINC00261 Exon 4 to the CMV-NEO
control. Significance was calculated using paired t-tests.
With these findings we decided to further narrow down the portion of LINC00261 acting as the
functional region of LINC00261 by truncating LINC00261 into smaller fragments and studying
whether the ectopic expression of these smaller fragments in H522 cell lines would be sufficient
to induce G2/M arrest.
To truncate Exon 4 of LINC00261, Exon 4 was sub-cloned into thirds. This truncation strategy was
decided on, as study of LINC00261 in the genome revealed that the bp2219-3610 middle region
of exon 4 was highly conserved in vertebrates.
30
Further truncating LINC00261 Exon 4
Figure 12 shows a schematic diagram of the Exon 4 truncations that were made. Smaller truncations
were made using through PCR amplification of sections of interest of the full LINC00261 plasmid and
cloned into the CMV-NEO backbone. Stable polyclonal H522 cell lines of CMV-LINC00261 Exon
4
bp882-4935
, CMV-LINC00261 Exon 4
bp839-2185
, CMV-LINC00261 Exon 4
bp2219-
3610
, CMV-LINC00261 Exon 4
bp3585-4931
, and CMV-LINC00261 Exon 1-3
bp1-881
were generated
and frozen down for future use.
(bp882-4935)
(bp1-881)
(bp839-2185)
(bp2219-3610)
(bp3585-4931)
A
B
31
Figure 12: Isolating the functional regions(s) of LINC00261
A) Schematic representation of truncated LINC00261 constructs. Indicated in red is a region of LINC00261 with high
conservation across vertebrates. B) Structural schematic of truncated LINC00261 constructs, depicting the
Vinennafold predicted structure of LINc00261 with regions corresponding to truncation constructed sequences
highlighted. CMV-LINC00261 Exon 4
bp839-2185
is highlighted in yellow, CMV-LINC00261 Exon 4
bp2219-
3610 in
green, CMV-LINC00261 Exon 4
bp3585-4931
in blue, and CMV-LINC00261 Exon 1-3
bp1-881
in red.
It was decided that stable cell lines would be the best method to continue the experiments.
Transient transfection had proved difficult to achieve successful transfection, while also maintaining
the low cell confluency need for FACs cell cycle analysis – as contact inhibition would initiate cell
cycle arrest in G0/G1 phase, and not allow for a true understanding of how the LINC00261
truncations were affecting the cells. Stable cell lines allowed for more control over the confluency
of the cells at time of harvest, ensured that the cell growth was not being inhibited by factors outside
of those being tested.
32
For FACs analysis of stable cell lines, it was decided that of the smaller truncations, exon 4
bp2219-3610
should be tested first, as this was the region of LINC00261 predicted to be responsible for inducing
the G2/M arrest. It was not possible to test all the truncations at once, as the time taken for FACs
analysis limited the number of samples that could be run in one session. Leaving cells on ice for longer
than an hour would have made them not viable for analysis.
Isolating the conserved region of LINC00261 exon 4
The generated stable H522 cell lines for CMV-LINC00261, CMV-LINC00261 Exon 4
bp882-4935
, and
CMV-LINC00261 Exon 4
bp2219-3610
were tested against CMV-NEO controls for variations in cell
cycle using the same FACS cell cycle analysis methods as seen in previous experiments (Figure
13A). It was found that cells transfected with LINC00261, whether it be the smaller fragments or
the full gene, had a significantly higher percentage of cells in G2 phase when compared to cells
transfected with the empty NEO vector. It was also observed that the reintroduction of
LINC00261 also led to a significant decrease of the population of cells in S phase (Figures 13B,
13C).
33
A
CMV-NEO
CMV-LINC00261
CMV-LINC00261
CMV-LINC00261
Exon 4
bp882-4935
Exon 4
bp2219-3610
B
(bp882-4935)
(bp2219-3610)
34
% population
(bp882-4935) (bp2219-3610)
Figure 13: Ectopic expression of LINC00261 exon 4
bp2219-3610
induces G2/M cell cycle arrest
A) Pictured above are cell cycle curves derived from Propidium Iodide (PI) staining FACS analysis of stable cell lines
transfected with CMV-LINC0026, CMV-LINC00261 Exon 4, CMV-LINC00261 exon 4
bp2219-3610
and CMV-NEO controls. The
curves shown are representative of three independent experiments. The percentage population of cells in G1 are highlighted
in blue, those in S phase are highlighted in yellow, and those in the G2 stage are highlighted in green. The Population was
analyzed using FlowJo v10.9 N=3 (≥50,000 cells per sample). B) Shows quantification of FACS analysis in (A). It is to be noted
that there are marked changes in the population of cells in S and G2 in all LINC00261
conditions, and not in the control. C) comparing CMV-LINC00261, CMV-LINC00261 Exon 4 and CMV-LINC00261 Exon
4
bp2219-3610
to the CMV-NEO control. Significance was calculated using paired t-tests.
2.4 Discussion
Through FACS mediated cell cycle analysis, we observed that the 4.1kb LINC00261 Exon 4
bp882-
4935
portion of LINC00261 was able to induce the same halt in G2/M that had been observed with
35
the full LINC00261, implicating the functional until of LINC00261 to be contained within this
region.
Upon further truncation, FACS analysis of H522 cells expressing the full LINC00261, just exon 4
bp882-
4935
, and the exon 4
bp2219-3610
piece all were shown to have an increase in G2 as compared to the
blank vector control. This suggested that the bp2219-3610 piece of LINC00261 exon 4 was indeed
sufficient to induce the G2/M arrest that had been observed with the larger truncations, allowing us
to narrow down the functional region of LINC00261 to this 1.3kB piece. It is known that G2/M is a
DNA damage repair checkpoint and based on the results we had seen, we hypothesized that cells
without LINC00261 expression are able to bypass this checkpoint, thus allowing for the accumulation
of mutations that give rise to cancerous phenotypes.
It was found that reintroduction of LINC00261 also lead to a significant decrease in cells in S
phase. This had not been observed in previous data, but the lack of replication in these cells upon
LINC00261 expression, as suggested by a decrease of cells in S phase would explain the slowed
rate of proliferation observed upon LINC00261 reintroduction. The observed change in S phase
could be attributed to many different factors. One possibility is mathematical error, which can
be determined by repeating these experiments to see if the change is reproduceable.
Additionally, it could be that truncated LINC00261 is causing cells to arrest at the G1 phase, and
not progress in the cell cycle past this point. This could be validated by western blot analysis of
truncated LINC00261 containing H522 cell lines for cyclins, to determine which phases of the cell
cycle these cells are able to enter.
36
Chapter 3: Understanding the role of LINC00261 in the DNA damage
response
3.1 Introduction
Preliminary data has demonstrated that LINC00261 binds ATM, and is able to transcriptionally
activate downstream effectors of ATM. However, it has not been verified if the downstream
signaling partners of ATM are similarly activated at the protein level, to bring about changes to
the DNA damage response pathway.
Other lncRNAs have been shown to play a role in DNA damage response through regulation of
the ATM/ATR pathway, and these examples give insights into the potential mechanism of
LINC00261. A study testing for the differential expression of lncRNAs in ATM
+/ +
mice compared
to ATM
-/-
mice revealed 100 lncRNAs became upregulated while a further 70 became
downregulated upon ATM activation (Su et al., 2018). One example of lncRNAs in the ATM
pathway, is DDSR1, which had been found to be upregulated upon the induction of DNA damage
to cells and was also found to become downregulated upon inhibition of ATM with KU55933
inhibitor (Su et al., 2018).
Further understanding how LINC00261 interacts with the ATM pathway will give us insight into
the best treatment approach for treating cases of LUAD where it is under expressed.
37
3.2 Method
Western Blots
cells were aspirated of media and washed with 1ml DPBS, and then harvested through scraping
with another 1ml DPBS. Cells were then pelleted by centrifugation at 3000g for 3 minutes and
resuspended with 100ul-300ul of RIPA buffer with Halt phosphatase and protease inhibitors
(thermo scientific) depending on the size of the cell pellet. Halt inhibitors were purchased at a
100X concentration and diluted to 1X in RIPA. The cell suspension was lysed using a 23g needle
and passed through 15 times. Lysed cells were then centrifuged at 10,000rpm for 20 minutes at
4 C. After centrifugation, the clear supernatant was collected and placed into a clean microtube,
while the pellet containing cellular debris was discarded. Protein lysate was stored in -20 C.
Protein lysates were quantified using the Bradford assay. All samples were standardized using a
1mg/ml stock concentration of Bovine Serum Albumin (BSA) (VWR, lot #18J2556269, Solon OH) and
30µg of lysate was added in each well. Samples were mixed with 5x GLB (Sodium Dodecyl Sulphate,
bromophenol blue, DDT and betamercaptoethanol) and then run on 10% stacked tris-polyacrylamide
gels for 3 hours at 15mA per gel. 10X Running Buffer consisted of 75g Glycine, 15g Tris and double
distilled water to a total volume of 1L. 1X Running buffer consisted of 800ml double distilled water,
80ml 10X running buffer, and 8ml 10% SDS. Gels were transferred at 20V
overnight at 4 C, onto PVDF membranes. Transfer buffer consisted of 11.6g Glycine, 5.8g Tris,
38
400ml Methanol, and double distilled water to a total volume of 2L. Prepared transfer buffer was
stored at 4 C.
After transfer, blots were stained with Ponceau S solution (Sigma Aldrich, catalog #P717-1L, lot
#SLBR3445V, St Louis MO) for 30 seconds to visualize actin bands and verify whether transfer had
been successful. Blots were then washed with TBST and cut to separate experimental protein
regions from control regions. Blots were placed into 10cm
2
petri dishes and blocked for 1 hour
in 8ml of 10% non-fat milk in TBST. Milk was then removed and replaced with 1:1000 primary
antibody diluted in 5% non-fat milk in TBST and left to shake gently overnight at 4 C. Antibody
specifications are listed in the supplementary figures section 6.4.
Blots were then washed 3 times with TBST for 15-minute intervals, before secondary antibody was
added at a dilution of 1:10,000 in 3% non-fat milk in TBST and left to shake gently at room
temperature for 1 hour. Blots were then washed first with 3% non-fat milk in TBST for 15 minutes,
then 1.5% non-fat milk in TBST for 15 minutes and finally in TBST for 15 minutes. Membranes
were visualized using Super Signal West Femto Maximum Sensitivity Substrate (thermo
scientific). Blots were visualized using the Bio-rad Chemi doc XRS machine on the
chemiluminescence high sensitivity setting. Blots were imaged between 3-300 seconds
depending band intensity.
39
3.3 Results
DNA Damage Response Gene Westerns
Western blots analysis of key DNA damage related proteins was done on stable cell lines of H522
CMV-LINC00261 and H522 CMV-NEO in order to validate the differential expression of these genes
that had been observed through earlier RNA-Seq experiments to see if they were carried on to the
protein level of these cell lines. Total protein and phosphorylated protein expression of key genes
was also tested in order to determine the role of LINC00261 in the phosphorylation mediated
activation of these genes. Genes tested were ATR, BRCA1, BRCA2 and Phospho-BRCA2. Analysis of
average fold difference between CMV-NEO and CMV-LCMV-IN00261 conditions revealed ATR and
BRCA1 show increased protein expression in CMV-LINC00261 compared to CMV-NEO. In the case of
BRCA2 it was found that while the levels of total BRCA2 were similar in H522 CMV-LINC00261 and
H522 CMV-NEO, the levels of phospho-BRCA2 were increased with the ectopic expression of
LINC00261 and higher than the levels seen in CMV-NEO (Figure 14).
40
Figure 14: LINC00261 plays a role in the DNA damage response pathway
Western blotting images for DDR pathway members in H522 cell lines expressing CMV-LINC00261 or CMV-NEO
controls. Loading controls were HSP90 or actin, as indicated. Representative blots of three independent experiments
are shown here. The average fold difference was calculated using the ImageJ software, however the background
correction resulted in underrepresentation of fold change in the quantification.
3.4 Discussion
Western blot results revealed increases in ATR, and BRAC1 levels upon ectopic expression of
LINC00261, suggesting that LINC00261 is activating these DNA damage pathway members. In the case
of BRCA2, there was an increase in the phosphorylated form of the protein, but no change in total
protein, implicating that LINC00261 has an important role in the phosphorylation mediated activation
of BRCA2. These findings further support previous findings that LINC00261 acts upstream of ATM,
and that its loss of function prevents ATM from identifying DNA damage
– as demonstrated by the decreased expression of downstream DDR members activated by ATM.
Western blots had also been done on H522 CMV-LINC00261 and CMV-NEO cell lines to test for
p53, in order to understand whether the effect of LINC00261 on the DNA damage response
pathway was a p53 dependent or independent mechanism. However, these blots did not yield
any signal, in either condition. This could be due to mutated p53 in the H522 cell line, carrying a
frameshift deletion (P191fs*56) (Leroy et al., 2014). Generally, frameshift deletion mutations are
deleterious, and lead to loss of function (Wang et al., 2017), which would explain why p53 could
not be detected through western blots. UV-irradiation of a control population of cells would allow
us to determine if p53 is inducible in H522 cells.
41
Chapter 4: Summary
Through this research, the region of LINC00261 required to rescue G2/M arrest in LUAD cells has been
narrowed down to a 1.3kb region. While this piece is still quite large, it is much more viable for
delivery when compared to the full 4.9kb LINC00261 and makes LINC00261 a viable candidate for
delivery to patients using a nanoparticle-based strategy. Additionally, new mechanistic information
was gathered on the roles of LINC00261 in cell cycle, as our experiments observed a decreased
population of cells in S upon LINC00261 reintroduction, suggesting that LINC00261 may also be
suppressing DNA replication in addition to its induction of G2/M cell cycle arrest. ATM is able to
initiate both G1 and G2/M cell cycle arrest, and as we know that LINC00261 binds to ATM, it is
possible that G1 arrest is being initiated through this mechanism.
The knowledge we have gained on the deregulation of the DNA damage repair pathway in the
absence of LINC00261 will have huge importance in the treatment of LUAD, in ways that had not
originally been considered. Many chemotherapeutic agents act by inducing DNA damage – if the
damage repair mechanism is compromised and cells are able to continue replicating with
damaged DNA, this would lower the efficacy of these treatments. Insights into a patient’s
LINC00261 expression levels may allow for clinicians to tailor a therapeutic approach for them
and predict how successful certain treatments may be for them.
42
By determining which piece of LINC00261 is functionally relevant, we will be able to target our
mechanistic studies to the specific signaling partners required to mediate LINC00261’s
downstream activity.
Chapter 5: Future Directions
While FACs analysis suggests that the bp2219-3610 middle region of LINC00261 exon 4 is
sufficient to rescue the G2/M phase of the cell cycle, it has not been confirmed that this piece is
also able to initiate the downstream signaling events that seen with the full LINC00261, such as
phosphorylation mediated activation of ATM and the subsequent increase in expression of its
downstream signaling proteins. To determine this, the H522 stable cell lines of the smaller
truncations that were generated through this project could be analyzed using western blots
treated for these DNA damage response genes.
It would also be important to know if ATM is able to bind this 1.3kB truncation of LINC00261.
This could be done by repeating the RNA immunoprecipitation ATM pull down assay on H522
CMV-LINC00261 Exon 4
bp2219-3610
cell line, like had previously been done with the full lncRNA.
Another aim is to better understand the kinetics of ATM-LINC00261 binding using the Dynamic
Biosciences DRX
2
. This was previously not possible due to the machine having limits on the size
of the RNA sample, but now with this smaller truncation, this interaction can be studied at a finer
level.
43
Additionally, it will be investigated whether this 1.3kb piece can be further narrowed down. This
will be done by using the viennafold program to predict the 3D folding of this truncated fragment,
and split it further based on structure.
Finally, it should be noted that due to time constraints we were not able to do FACs analysis on
the other smaller segments of LINC00261 that had been generated, and future experiments will
be done to test whether these pieces have any effect on the cell cycle.
Supplementary Materials
Plasmids
CMV-NEO Plasmid
CMV-LINC00261 plasmid
44
CMV-LINC00261 Exon 4
CMV-LINC00261 Exon 1-3
bp1-881
45
CMV-LINC00261 Exon 4
bp839-2185
46
CMV-LINC00261 Exon 4
CMV-LINC00261 Exon 4
bp2219-3610
bp3585-4931
47
Primers used for cloning
Primers for CMV-LINC00261 exon 1-3
bp1-881
Forward: TCGAGAATTCGCTAAAGTCAACAGTCGCTTG
Reverse: TCGGCGGTACCCAAGAAGAGTTATGCAGTCATTAGAC
Primers for CMV-LINC00261 Exon 4
bp839-2185
Forward: GGGCGGCGAATTCTCTCTTTTCAAACAAAAGTCTAAT
Reverse: TAGCGGTACCGAAATATTGATCCTGCAAATGA
Primers for CMV-LINC00261 Exon 4
bp2219-3610
Forward: TAGCGAATTCACGTCTCACTTTTTCCTTGACA
Reverse: TAGCAAGCTTCCGATCACAGCTTGCAAG
Primers for CMV-LINC00261 Exon 4
bp3585-4931
Forward: TAGCGAATTCTTTAAATGGAAGGGTTCATTG
Reverse: TAGCGGTACCGCAAAGAAATACAGAACAATTTAT
All primers were ordered from IDT
48
Primers used for qPCR
Primers for CMV-LINC00261 and CMV-LIN00261 Exon 1-3
bp1-881
Forward: GGATAAAGACCAGCTCAACCA
Reverse: CTCCAAGACAAAGAAGAGTAGG
Primers for CMV-LINC00261 Exon 4
bp839-2185
Forward: TGGGTGCTGTGTGTGAATAC
Reverse: TGGCTGTTGGTCTATTGGTTC
Primers for CMV-LINC00261 Exon 4
bp2219-3610
Forward: GCACACAGGGCTTCTATAACT
Reverse: AGAATTGCCCGGGATGAAATA
Primers for CMV-LINC00261 Exon 4
bp3585-4931
Forward: ATGAAGCCATTGTCCAGTAGAG
Reverse: ACACACGCACACACATACA
Primers for CMV-LINC00261 Exon 4
49
Forward: GCACACAGGGCTTCTATAACT
Reverse: AGAATTGCCCGGGATGAAATA
All primers were ordered from IDT
Western blot antibodies
beta-actin (Cell Signaling #4970)
BRCA2 (Origene #TA313520)
phospho-BRCA2 (Invitrogen #PA537499)
HSP90 (GeneTex GTX109753)
ATR (cell signaling #2790S)
All antibodies were diluted 1:1000 in 5% milk in TBST
50
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Abstract (if available)
Abstract
Lung cancer is the leading cause of cancer related deaths in the United States. Prior research on lung cancer primarily focused on protein coding oncogenes, however the role of long non-coding RNA (lncRNA) in lung carcinogenesis is relatively underexplored. We have identified a lncRNA, LINC00261 that is downregulated in multiple cancers, including lung adenocarcinoma (LUAD). Recently published data has shown that LINC00261 acts as a tumor suppressor. This is supported by the observation that ectopic over expression of LINC00261 in H522 LUAD cells inhibits cell proliferation and migration through G2/M cell cycle arrest and activated expression of genes associated with the DNA damage response pathway. The length of LINC00261 is 4.9kB complicating the utilization of this RNA as an adjuvant therapeutic for LUAD. In order to package this lncRNA into nanoparticles for delivery, the smallest functional unit of LINC00261, that that evokes the tumor suppressive properties needs to be defined. Truncation of LINC00261 has demonstrated that the isolated 1.3kb conserved region of exon 4 LINC00261 is sufficient to induce G2/M cell arrest, implicating this region as the functional unit of LINC00261. Western blot experiments have validated that downstream signaling targets of ATM become activated upon ectopic expression of LINC00261.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Nandagopal, Gopika
(author)
Core Title
Defining the functional region of LINC00261 in lung adenocarcinoma
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Publication Date
07/26/2021
Defense Date
06/21/2019
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer,DNA damage response,LINC00261,lncRNA,lung adenocarcinoma,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Marconett, Crystal (
committee chair
), Offringa, Ite (
committee member
), Stallcup, Michael (
committee member
)
Creator Email
gnandago@usc.edu,Gopika.Nandagopal@uvm.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-199526
Unique identifier
UC11662952
Identifier
etd-Nandagopal-7675.pdf (filename),usctheses-c89-199526 (legacy record id)
Legacy Identifier
etd-Nandagopal-7675.pdf
Dmrecord
199526
Document Type
Thesis
Format
application/pdf (imt)
Rights
Nandagopal, Gopika
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
DNA damage response
LINC00261
lncRNA
lung adenocarcinoma