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University of Southern California Dissertations and Theses
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Generation of an epigenetic toggle switch to test LINC00261 function on lung adenocarcinoma cellular response to the chemotherapeutics oxaliplatin and carboplatin
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Generation of an epigenetic toggle switch to test LINC00261 function on lung adenocarcinoma cellular response to the chemotherapeutics oxaliplatin and carboplatin
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Content
Generation of an epigenetic toggle switch to test
LINC00261 function on lung adenocarcinoma
cellular response to the chemotherapeutics
oxaliplatin and carboplatin
by
Lin Miao, B.S.
A Thesis Presented to the
FACULTY OF THE USC [KECK SCHOOL OF MEDICINE]
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)
August 2021
Copyright [2021] Lin Miao
ii
Dedication
For my parents Kongyou Miao and Yongbo Zhou.
A million of thanks for their selfless support and deepest love.
iii
Acknowledgements:
I would like to thank all my committee members for their valuable advice and kind guidance
during my whole project. I would like to thank Dr. Marconett first for her altruistic support and
patient mentorship. And I would also like to thank Dr. Offringa and Dr. Zhou for their support
and suggestions about my project.
I would like to express my sincere thanks to all members of Marconett and Offringa labs for
their kindly help and support: Jonathan Castillo, Chunli Yan, Evelyn Tran, Tianchun Xue,
Yinchong Wang, Minxiao Yang, Evan Mihalakakos, Lars St. Pierre, Daniel Mullen, Chris Li
and Ziben Zhou. I would like to give my special thanks to Jonathan Castillo for his great help
and kind advice for me as a senior lab member, and to preeminent Chunli Yan for her skilled
technical support in all of my experiments.
iv
Contents
Dedication .................................................................................................................................. ii
Acknowledgements .................................................................................................................. iii
List of Tables .............................................................................................................................. v
List of Figures ........................................................................................................................... vi
Abstract .................................................................................................................................... vii
CHAPTER 1: Introduction ........................................................................................................ 1
1.1 Lung cancer .................................................................................................................. 1
1.2 Long non-coding RNA ................................................................................................ 2
1.3 CRISPR and dox-inducible system .............................................................................. 4
1.4 DNA damage response ................................................................................................. 8
1.5 Chemotherapy and DDR ............................................................................................ 10
1.6 Preliminary data ......................................................................................................... 11
CHAPTER 2: Methods and Materials ..................................................................................... 15
2.1 Plasmid cloning, purification and transformation ...................................................... 15
2.2 Cell culture ................................................................................................................. 24
2.3 Total RNA extraction ................................................................................................. 25
2.4 Quantitative real-time PCR (qPCR)........................................................................... 26
2.5 Mammalian cell transfection...................................................................................... 26
2.6 Viability assay ............................................................................................................ 28
2.7 Genomic DNA extraction .......................................................................................... 29
2.8 Non-linear regression model and statistical analysis ................................................. 30
CHAPTER 3: Results .............................................................................................................. 33
3.1 Design of doxycycline-inducible CRISPR-mediated knock down system................ 33
3.2 Transient transfection of dox-inducible dCas9-KRAB plasmid decreases LINC00261
expression after dox treatment ......................................................................................... 34
3.3 Generation of A549 cell lines stably expressing dCas9-KRAB plasmids ................. 36
3.4 LINC00261 confers resistance to the cisplatin derivatives oxaliplatin and carboplatin
in H522 LUAD cell culture.............................................................................................. 38
CHAPTER 4: Discussion ......................................................................................................... 42
References ................................................................................................................................ 45
v
List of Tables
Table 1. lncRNAs in lung cancer, and their association with lung cancer tumorigenesis,
metastasis and drug resistance. .................................................................................................. 4
Table 2. List of primers used for PCR, fragment clone and sequencing ................................. 31
Table 3. LD50 of drugs for both CMV-LINC00261 and CMV-NEO cell lines. ...................... 39
vi
List of Figures
Figure 1. Leading sites of new cancer cases and deaths by sex, United States, 2021 ............... 1
Figure 2. The mechanisms of CRISPR immunity in bacteria. ................................................... 6
Figure 3. DNA response network. ............................................................................................. 9
Figure 4. LINC00261 has a functional role in DNA damage response pathways and genome
stability. .................................................................................................................................... 13
Figure 5. PB-TRE-dCas9-VPR plasmid sequence and enzyme cutting sites. ......................... 16
Figure 6. The pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro plasmid sequence and
cloning region. ......................................................................................................................... 17
Figure 7. The ligated PB-gRNA(emp)-TRE-dCas9-VPR plasmid sequence. ......................... 19
Figure 8. The cloning preparation for Gibson assembly.......................................................... 22
Figure 9. The Gibson assembly mechanism. ........................................................................... 24
Figure 10. Schematic of the doxycycline-inducible CIRSPR-mediated knock down system. 34
Figure 12. PCR validation of dCas-KRAB plasmid stable transfection colonies.................... 37
Figure 13. LINC00261 confers resistance to cisplatin derivatives, oxaliplatin and carboplatin
in H522 LUAD cell lines. ........................................................................................................ 40
vii
Abstract
Many tumors lack known oncogenic driver mutations, which underscores the importance of
discovering novel biomarkers for LUAD diagnosis and treatment. One of the classes of
biomolecules that may resolve this lack of understanding into the molecular origins of lung
cancer is long intergenic non-coding RNA (lincRNA). Our lab has identified LINC00261 as a
tumor suppressor in lung adenocarcinoma (LUAD), the major subtype of lung cancer and
discovered that this lncRNA plays a functional role in the response to DNA damage. We have
also observed that the ablation of LINC00261in lung cancer cells results in the formation of
macronucleated cells that have higher-order DNA content. Based on these preliminary results,
my thesis hypothesis was that LINC00261 alters the sensitivity of lung cancer cells to
chemotherapeutics through affecting the DNA damage response (DDR). To test the role of
LINC00261 the DNA damage response, I created an inducible CRISPR-mediated system that
can utilize doxycycline as an epigenetic toggle switch to modulate endogenous LINC00261
expression. With this system, our lab will be able to further characterize the DNA content of
the previously observed macronucleated cells, and test the ability of DDR pathway members
to localize to sites of DNA damage in the presence or absence of LINC00261. Also, since many
chemotherapeutic drugs work by targeting cancer cells and inducing DNA damage, to test if
LINC00261 influences chemotherapy, I treated H522 cells into which LINC00261 has been
reintroduced and NEO control with different chemotherapeutics drugs and found that
LINC00261 confers resistance to the cisplatin derivatives oxaliplatin and carboplatin in H522
LUAD. These findings may provide a mechanistic basis for the function of LINC00261 in the
DDR pathway and genome stability.
1
CHAPTER 1: Introduction
1.1 Lung cancer
Lung cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-
related death in the United States as well as worldwide. Among those diagnosed with lung
cancer in 2021, the incidence of lung cancer is up to 12% in male and 13% in female of all
cancer diagnoses, and the mortality of lung cancer is ~22% in male and female, the largest
proportion of cancer-related death cases in both sexes (1). Therefore, lung cancer plays an
outsized role in overall cancer burden.
Figure 1. Leading sites of new cancer cases and deaths by sex, United States, 2021
(Siegel RL et al., 2021)
2
Lung cancer can be subclassified into two categories: small cell lung cancer (SCLC) and non-
small cell lung cancer (NSCLC). SCLC accounts for around 15% of total lung cancer cases,
and NSCLC, for the remaining ~ 85%. In NSCLC, lung adenocarcinoma (LUAD) is the
largest histologic subtype, which comprises around 50% of total NSCLC cases (2). A number
of driver mutations have been identified in a large proportion of NSCLC cases, such as KRAS
and EGFR, which are major oncogenes (~45%) that lead to LUAD. Many targeted therapies
toward these mutant genes for NSCLC have been used clinically, including inhibitors
directed at EGFR (Erlotinib, Carey KD et al, 2006), ALK (Crizotinib, Ou SH et al, 2010),
ROS1 (Entrectinib, Pfizer, 2021), BRAF (Dabrafenib, Novartis Pharmaceuticals Corporation
et al, 2021), MET (Trametinib, Novartis Pharmaceuticals Corporation et al, 2021) and RET
(Pralsteinib, Blueprint Medicines Corporation, 2020) (3-8).
1.2 Long non-coding RNA
Long non-coding RNAs (lncRNAs) are defined as RNAs longer than 200 nucleotides. Most
also have a 5’-cap, poly-A tail, and undergo splicing (9). Thousands of lncRNAs have been
identified and predicted, but very few have been functionally annotated, and little is known
about the expression patterns in different cell types (10). So far, cancer-associated lncRNAs
have shown functions across a wide spectrum of processes, including influencing genomic
alterations, cancer diagnosis and monitoring, cancer prognostic, and predicting therapeutic
3
responsiveness (11). For instance, elevated levels of lncRNA HOTAIR is highly predictive of
progression to metastasis and overall survival in the early stage of surgically-resected breast
cancer (12). Elevated HOTAIR levels are also associated with cancer progression in 26 human
tumor types (13). In addition, the lncRNA- AA174084 was identified as a biomarker capable
of differentiating between gastric cancer and benign disorders of the gastric epithelium (14).
Overall, the cataloging of lncRNA with functional relevance in the carcinogenic processes has
grown exponentially over the past few years, owing to new computational methods for
transcriptome assembly, enhanced lncRNA annotation, and a growing awareness within
Glaucoma Hemifield Test (GHT) field of the significance of these functional RNAs (15, 16).
In lung cancer cases, lncRNA expression may impacted by chemical compounds and local
tumor microenvironments. In addition, epigenetic modification in tumor progression may
influence lncRNAs. The dysregulation of lncRNAs could result in dysregulated gene signaling
network at the transcriptional, post-transcriptional and post-translational level, and thus, lead
to various malignant behaviors and treatment responses of lung cancer (17). The lncRNAs
known to be associated with lung cancer tumorigenesis, metastasis and drug resistance are
shown in Table 1. In this study, we focused on a novel long non-coding RNA called LINC00261,
which is a tumor suppressor that our lab has previously identified, and has a role in DNA
damage response (DDR).
lncRNAs Expression Functions and treatment responses
MALAT1 Upregulation Increased proliferation and invasion of lung cancer
4
Cisplatin resistance
HOTAIR Upregulation Increased proliferation and invasion of lung cancer
Cisplatin, gefitinib and crizotinib chemoresistance and
radiation resistance
H19 Upregulation Increased NSCLC cell proliferation
PVT1 Upregulation Increased proliferation of lung cancer
Radiation resistance
ANRIL Upregulation Increased proliferation and invasion of lung cancer
LINC00473 Upregulation Increased growth of LKB1-inactivated NSCLC cells
LINC00963 Upregulation Promotes metastasis of lung cancer
DLX6-AS1 Upregulation Promotes proliferation
SOX2OT Upregulation Promotes cell proliferation
UCA1 Upregulation EGFR-TKIs resistance
ZXF1 Upregulation Increased migration and invasion of lung cancer
CAR10 Upregulation Increased proliferation
BANCR Downregulation Increased proliferation and migration
Radiation sensitivity
AK126698 Downregulation Inhibits cell proliferation and migration; induces apoptosis
Cisplatin sensitivity
PICART1 Downregulation Suppresses proliferation and induces apoptosis
PANDAR Downregulation Induces apoptosis
MIR22HG Downregulation Suppresses proliferation and invasion of lung cancer
MEG3 Downregulation Cisplatin sensitivity
GAS5 Downregulation EGFR-TKIs sensitivity
SPRY4-IT1 Downregulation Inhibits proliferation and metastasis
Table 1. lncRNAs in lung cancer, and their association with lung cancer tumorigenesis,
metastasis and drug resistance. (modified from Jiang L et al., 2019)
1.3 CRISPR and dox-inducible system
5
Our lab previously used small hairpin RNA (shRNA) to knockdown LINC00261 expression.
However, the LINC00261 knock down cell line (A549-shLINC000261 cell line) has some
drawbacks. The A549-shLINC00261 cell line lost repression over ~15 passages in culture, and
the stable clones that did not lose expression died off after becoming hyper-aneuploid (Shahabi
et al., 2019 supplemental figure). In addition, we were only able to attain shRNA knock down
of ~85% of total LINC00261 expression, which still left some endogenous expression of
LINC00261. Finally, this method cannot modulate the endogenous LINC00261 expression to
be turned on and off according to experimental conditions. To address these disadvantages of
using an shRNA system, we decided to design an inducible CRISPR-mediated system for
further characterization of LINC00261 function in genomic stability.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful
technology for editing genomes, which was adapted from the natural defense mechanisms of
bacteria and archaea. These organisms use CRISPR-derived RNA (crRNA) coupled with Cas
proteins to save snippets of viral genomes from past infections and to activate a defense system
upon re-exposure, thus forming a primitive unicellular immune response (18). The CRISPR
system has been repurposed to take advantage of these processes to add, remove or alter genetic
sequences at highly site-specific locations in the genome (19).
6
Figure 2. The mechanisms of CRISPR immunity in bacteria. CRISPR loci contain clusters
of repeats (black diamonds) and spacers (colored boxes), which are flanked by a “leader”
sequence and Cas genes. The CRISPR precursor transcript is processed by Cas
endoribonucleases to generate small crRNAs, and the crRNA spacer is complementary with
the target sequences, which specify the nucleolytic cleavage (red cross). (Rodolphe et al., 2014)
CRISPR-associated protein 9 (Cas9) is a subtype of RNA-guided DNA nucleases, and forms a
complex with a guide RNA (gRNA) that provides the 18-22nt complementary sequences that
the cleave specific strands and create DNA double-strand breaks, and it is guided by two RNA
molecules: crRNA and trans-activating crRNA (tracrRNA). The tracrRNA is complementary
to the repeat sequence. Its transcription and the primary CRISPR transcript contribute base
7
pairing and the formation of dsRNA at the repeat sequence, which subsequently produce
crRNAs (20). The Cas9 utilizes both the crRNA and the tracrRNA to function and cleave the
DNA by different endonuclease domains to cut two strands of the DNA (21).
In this study, we utilized the endonuclease-deficient Cas9 (dCas9) fused to the Krüppel
associated box (KRAB) epigenetic repression domain. The dCas9-KRAB system is a powerful
tool for transcriptional repression of genes within their endogenous genomic loci (22).
Epigenetic repression of the transcriptional locus is preferable for lncRNA gene silencing as
simply cutting part of a lncRNA out may not block the ability of the RNA to function. The
dCas9 protein directs the transcriptional repression complex to target sites indicated by the
guide RNA, where it can still bind to the DNA target sites but cannot cleave the genomic DNA
sequences. The KRAB domain is a category of transcriptional repression domains in zinc finger
protein-based transcription factors, which can repress gene expression by decreasing H3
acetylation and increasing H3 lysine 9 methylation (23). As one of the most effective CRISPRi
methods, the dCas9-KRAB system can repress transcription of the target gene up to 99% in
human cells (22). By combining with the tetracycline-responsive promoter element, we can
further control the inducible gene knockdown by doxycycline, a synthetic tetracycline (TET)
analog. In this study, we took advantage of this TET-inducible dCas9-KRAB system as well as
the rTTA domain that is needed for doxycycline activation to study the effects of LINC00261
loss in lung adenocarcinoma cell lines.
8
1.4 DNA damage response
Genomic instability is a characteristic of most cancer cells, because cancer frequently results
from damage to multiple genes controlling cell division and tumor suppressors (24). It is known
that genomic integrity is closely monitored by several surveillance mechanisms, including
DNA damage checkpoints, the DNA repair machinery and mitotic checkpoints (25). A defect
in the regulation of any of these mechanisms often results in genomic instability, which
predisposes the cell to malignant transformation (24). Therefore, lack of appropriate DNA
repair or response to DNA damage will result in a higher propensity for genetic mutations,
which often leads to various human diseases, including cancer (26). Thus, it is important to
study the relationship between drivers of DNA damage response or DNA repair to understand
LUAD etiology.
DDR is a phosphorylation-driven signaling event initiated by multiple types of damage to the
DNA. ATM (ataxia-telangiectasia mutated) and ATR (ATM- and Rad3-Related) are two
important proteins that are DDR transducers that act as a central node of the signaling cascade,
activating downstream targets, such as p53, checkpoint kinase (CHK) 1&2, BRCA1/2, and the
G2/M cell cycle arrest apparatus (27). Damage to the DNA that results in double-strand break
formation attracts the MRN complex, which is composed of MRE11, RAD50, and NBS1
proteins (28). The histone variant H2AX is then phosphorylated to become γH2AX ,which
functions as a localization signal for DNA double-strand breaks, to which ATM associates and
subsequently activates a phosphorylation cascade to slow down the cell cycle for repair to occur.
9
In single-strand breaks, RPA, which is a single-strand DNA (ssDNA) binding protein, acts as
a sensor working together with a 9-1-1 complex (composed of Rad9, Hus1, and Rad1 protein)
to activate the ATR pathway (29). Then, downstream DDR mediators such as 53BP1, MDC1,
and BRCA1, are activated via phosphorylation. TP53 (p53) plays an important role in gauging
the overall damage to the cell, and exerting appropriate responses, including cell cycle arrest,
DNA repair, and if the damage is extreme, apoptosis (30).
Figure 3. DNA response network. (Mohammad et al., 2018) The DDR sensors first enters to
detect DNA damages and recruit downstream transducer molecules when damages occur. The
double strand break sensors involved in the ATM pathway are MRN complex, and the histone
variant H2AX, which get phosphorylated producing γH2AX and in turn functions as a signal
10
of DNA double strand breaks. In single strand DNA damages, RPA and 9-1-1 complex act as
sensors and activate ATR pathway. Then the sensors transduction of damage signal to DDR
mediators, where P53 plays a key role to guide cells exerting a response according to severity
of DNA damage.
1.5 Chemotherapy and DDR
Platinum-based chemotherapeutic drugs are still largely applied to the treatment of multiple
cancers based on their ability to induce DNA damage in proliferation cells, therefore inhibiting
tumor growth (31). However, these treatments produce many side effects as a consequence of
DNA damage to normal cells that divide rapidly, such as cells in the bone marrow, digestive
tract and hair follicles (32).
The types of chemotherapy drugs can be divided into alkylating agents, platinums, replication
disrupting agents and radiomimetics based on the type of DNA damage they induce. The drugs
used in this study are cisplatin, oxaliplatin, carboplatin and taxol. Cisplatin, oxaliplatin and
carboplatin are commonly used drugs for cancer treatment in platinum category, which often
induce bulky DNA damage intra-strand or inter-strand (31, 33). These platinum drugs undergo
aquation, which is a key step in the drug forming a complex with the target DNA, and lead to
the formation of a positively charged molecule that then crosslinks to DNA, forming the
DNA/platinum adducts. By covalently linking to DNA, drugs are able to disrupt the nuclear
11
metabolism, inducing DNA damage as well as inhibiting DNA replication and repair (34). In
contrast, taxol (paclitaxel) is unique among chemotherapeutic agents as it has a specific binding
site on the microtubule polymer. It is an anti-tumor drug that stops the uncontrolled cell
divisions of cancer by forming extremely stable and nonfunctional microtubules. The
microtubules are the means of chromosome motion during mitosis, so that the mitosis is halted
when these microtubules fail to form a normal mitotic apparatus (35).
1.6 Preliminary data
Our lab has previously identified the LINC00261 as a tumor suppressor (Shahabi et al., 2019).
To determine the functional role of LINC00261 in LUAD carcinogenesis, we reintroduced
LINC00261 expression in H522 cells, which is a LUAD cell line that lacks endogenous
LINC00261 expression. This was done using an ectopic vector containing the CMV promoter
driving expression of the full-length LINC00261 transcript. Bulk RNA sequencing of target
genes whose expression was altered allowed us to perform Ingenuity Pathways Analysis (IPA),
which showed that the major pathways altered due to the reintroduction of LINC00261 were
G2-M DNA damage checkpoint signaling, GADD45, and RAN signaling. LINC00261 also
altered expression of genes associated with G2-M cell-cycle arrest and the DDR (Figure 4A).
LINC00261 reintroduction was also able to induce phosphorylation of ATM, CHK2, BRCA etc,
which are downstream targets of ATM. This indicates that LINC00261 is sufficient to activate
the DNA damage response pathway. Also, we determined that LINC00261 acts at or above the
12
level of ATM in the phosphorylation cascade, as the ATM inhibitor Ku-55933 ablates
LINC00261-mediated increased phosphorylation of DDR targets (Figure 4B).
It was also previously observed that reduction of LINC00261 using the short hairpin RNAs
(A549-shLINC00261) in the A549 cell line resulted in an increase in the proportion of cells
in >G2 phase, which suggested LINC00261 is involved in chromosomal stability and
aneuploidy (Figure 4C-D). Consistent with this, ~ 10%-15% of A549 cells where LINC00261
levels were reduced formed macronucleated structures, however the mechanism by which this
occurred is unknown (Figure 4E) (36).
13
Figure 4. LINC00261 has a functional role in DNA damage response pathways and
genome stability. A) IPA pathway enrichment of differentially expressed genes. Purple,
14
correlated, and anticorrelated genes with LINC00261 expression in TCGA LUAD, as
computed by TANRIC. Dark blue, differentially expressed genes between H522 CMV-NEO
and CMV-LINC00261. B) Western blotting of H522 CMV-NEO and H522 CMV-LINC00261
upon exposure to multiple doses of ATM inhibitor Ku-55933. C) Flow cytometric analysis of
DNA content of LUAD cells using propidium iodide. The population was analyzed using
FlowJo v10.1. N ¼ 3 (10,000 cells per sample). D) Quantification of the flow analysis in C),
E) Brightfield image of A549-shLINC00261 stable cell line and A549-shScrambled control.
Magnification = 40X. Arrows indicate multinucleated cells (~10-15% of the total population).
(Shahabi et al., 2019)
15
CHAPTER 2: Methods and Materials
2.1 Plasmid cloning, purification and transformation
The plasmid containing the TRE promoter and the Cas9m4 fused with VP64 domain was first
purchased from Addgene (Addgene # 63800) (Figure 5) (37). The plasmid backbone was first
digested with SpeI and PspXI (New England Biolabs, Ipswitch, MA # R0133S and R0656S)
in a 50 l digestion reaction system that contained: 1 l SpeI enzyme (New England Biolabs,
Ipswitch, MA # R0133S), 1 l PspXI (New England Biolabs, Ipswitch, MA # R0656S) enzyme,
2 g plasmid, 1X Cutsmart buffer and water to 50 l in 37℃ overnight. The digested plasmid
was verified on agarose gel and followed by gel extraction (Qiagen # 28304) to get the purified
backbone fragment. For 1 volume of reaction mix, 3 volumes of QG were added. The mixture
was incubated in 50℃ for 10 minutes, and vortex every 2 minutes to make sure there is no gel
residue left. Add 1 gel volume of isopropanol to the sample and mix. Then, the mixture was
transferred to a QIAquick column (Qiagen) and centrifuged for 1 minute. The column was
washed with 750 l buffer PE and centrifuged 2 minutes to remove residual reagent. The
column was then saturated with 50 l elution buffer for 3 minutes and centrifuged for 1 minute
for plasmid backbone elution.
16
Figure 5. PB-TRE-dCas9-VPR plasmid sequence and enzyme cutting sites. The enzyme
cutting sties for ligation, the plasmid has been digested with PspXI and SpeI.
The plasmid that contains the U6 promoter and gRNA region was first purchased from Addgene
(Addgene # 71236) (Figure 6) (38). In order to get the U6 promoter and gRNA fragment, and
17
PCR was done in 50 l reaction system and the purchased plasmid serves as template. The
single stranded DNA oligonucleotide primers (Table 2, primer 1-2) were ordered from
Integrated DNA Technologies (IDT). PCR purification was done next to remove the residual
enzyme.
Figure 6. The pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro plasmid sequence and
cloning region. The schematic of clone region, U6 promoter and the gRNA scaffold region.
18
After isolating the plasmid digestion and PCR products, ligation was performed. The 20 l
ligation reaction system contained: 2 l T4 ligation buffer (10X), 50 ng plasmid backbone, 13
ng insert fragment, which was the PCR product. 1 l T4 DNA ligase (New England Biolabs,
Ipswitch, MA # M0202S) and distilled water to a total volume of 20 l. They were mixed by
pipetting up and down several times and incubated overnight at 16℃. The ligation reaction
mix was directly subjected to transformation reactions. In order to amplify plasmids longer
than 10kb, NEB 10-beta Competent E.coli (New England Biolabs, Ipswitch, MA # C3019I)
was used as the host bacteria. Transformation was performed using 5 l of the ligation reaction
mix added into 50 l of host bacteria solution that the factory premade and incubated on ice
for 30 minutes, and then underwent 42℃ heat shock for exact 30 seconds. 950 l pre-warmed
SOC media (New England Biolabs, Ipswitch, MA) was added to the mixture. Then, the tube
was moved to the incubator under vigorous rotation for 1 hour to encourage optimal growth.
50-100 l of the bacteria suspension was then spread onto LB agar plates with ampicillin (VWR,
Radnor, PA # 97061-442 )for colony formation overnight at 37℃ using sterile technique. The
ligated plasmid map was shown in Figure 7.
19
Figure 7. The ligated PB-gRNA(emp)-TRE-dCas9-VPR plasmid sequence. The backbone
of PB-TRE-dCas9-VPR plasmid ligated with the U6 promoter and the gRNA scaffold region.
For the purpose of adding any guide RNA by using the BsmBI restriction cutting sites between
U6 promoter and gRNA scaffold region, extra BsmBI restriction sites were destroyed by using
GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher # A14604). In order to
destroy the extra BsmBI restriction sites but keep the amino acids the same so that the functional
region will not be affected, three point mutations were introduced. The positions of the point
mutations are located at position 12545 (C-G); 4610 (G-C); and 6683 (C-G). The single
20
stranded DNA oligonucleotide primers (Table 2, primers 3-8) were ordered from IDT. The
primer mix was generated first for the following methylation and mutagenesis reaction. Primer
mix 1 contained 16 l PCR water, 2 l Primer 1 reverse (100 M) and 2 l Primer 3 forward
(100 M). Primer mix 2 contained 16 l PCR water, 2 l Primer 1 forward (100 M) and 2 l
Primer 2 reverse (100 M). Primer mix 3 contained 16 l PCR water, 2 l Primer 2 forward
(100 M) and 2 l Primer 3 reverse (100 M). The methylation and mutagenesis reaction
system are consisting of: 6 l AccuPrimeTM Pfx Reaction buffer (10X) (Thermo Fisher #
12344024), 6 l Enhancer (10X), 1.2 l ligated plasmid DNA (20 ng/ul), 1.2 l DNA methylase
(4U/ l), 2.4 l SAM (25X),0.5 l AccuPrime TM Pfx (2.5U/ l) (Thermo Fisher # 12344024)
and PCR water to 57 l. Mix the reaction mixture by pipetting it up and down, and aliquot 19
l each into three PCR tubes labeled 1, 2, and 3. Then, add 1 l of Primer mix 1 into tube 1, 1
l of Primer mix2 into tube 2, and 1 l of Primer mix 3 into tube 3. Then, perform the
methylation reaction and PCR using the parameters as follows: 37℃ for 20 minmutes, 94℃
for 2 minutes, 18 cycles for the steps including 94℃ for 20 seconds, 57℃ for 30 seconds, 68℃
for 30 seconds/kb, and finally 68℃ for 5 minutes, and 4℃ for hold. After the reaction 5 l for
each tube was used to analyze on agarose gel, and 2 l from each tube was used for
recombination reaction, 4 l PCR water was added, and 10 l GeneArt 2X enzyme mix was
added. The mixture was incubated at room temperature for 15 minutes, and the reaction was
stopped by adding 1 l 0.5M EDTA. 2 l of the mixture was used directly to the transformation.
As DH5αTM-T1R E.Coli competent cells were provided in the kit, it was used as the host
bacteria.
21
For the purpose of generating the plasmid that can knock down gene expression by dox-
inducible manner, the previously generated dCas9-VPR with gRNA expression directing to
LINC00261 promoter was used as the backbone. The plasmid was digested with PmeI and NheI
(New England Biolabs, Ipswitch, MA, # R0560S and R3131S) restriction enzyme, and the
digested fragments were loaded on agarose gel followed by gel extraction (Figure 8). The
Cas9m4 and the VPR region were replaced by the dCas9 fused with KRAB gene to repress
LINC00261 expression in the presence of dox. The dCas9-KRAB fragment was obtained by
PCR, using the previously purchased plasmid from Addgene (Addgene # 71236) (Figure 2) (38)
as the template. The single stranded DNA oligonucleotide primers (Table 2, primers 9-10) were
ordered from IDT. The cloning products were loaded on agarose gel for validation and followed
by PCR purification.
22
Figure 8. The cloning preparation for Gibson assembly. A. The cloning of dCas9-KRAB
region from pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro plasmid. B. The digestion of PB-
gRNA(emp)-dCas9-VPR plasmid backbone by PmeI and NheI enzymes.
23
In order to merge the fragments obtained from different source of plasmids together, Gibson
Assembly was used in this case. Gibson assembly is an efficient and quick molecular cloning
method to join multiple DNA fragments together based on sequence identity. It requires that
the DNA fragments contain 20-40 base pair overlap with adjacent DNA fragments and no
restriction digestion requirement of DNA fragments after PCR (39). Thus, the primers were
designed to have overhang that overlap to adjacent DNA fragments (Figure 9). The Gibson
assembly reaction contained: 10 l NEBuilder HiFi DNA Assembly Master Mix (New England
Biolabs # E5520S), 80 ng backbone, 87.62 ng insert DNA fragment, and water to 20 l. The
mixture was incubated at 50℃ for 15 minutes. 2 l of the mixture was directly used for
transformation, and NEB 10-beta Competent E.coli (New England Biolabs Ipswitch, MA #
C3019I) was used as the host bacteria.
24
Figure 9. The Gibson assembly mechanism. After the insertion fragment and the digested
plasmid backbone were ready, they were merged together by Gibson Assembly Master Mix
(New England Biolabs, Ipswitch, MA).
2.2 Cell culture
The H522-CMV-LINC000261 and H522 -CMV-NEO cell lines were both previously generated
in the lab. The A549 cell line was obtained from the laboratory of E. Haura or the ATCC and
fingerprinted to verify their identity prior to experimentation at the University of Arizona
25
(Tucson, AZ). The cell lines were cultured in RPMI-1640 (Corning) media with 10% fetal
bovine serum (FBS) and 1% penicillin streptomycin (P/S). Geneticin (G418) (VWR, Radnor,
PA # 97064-358) with a concentration of 166 g/ml was used to apply long-term selection
pressure for H522-CMV-LINC00261 and H522-CMV-NEO cell lines. All cell lines were
verified as H522 or A549 cell line by genotype authentication before, and the expression of
LINC00261 were also verified by qPCR.
2.3 Total RNA extraction
Culture media was aspirate when the cells reach around 90% confluency, and immediately
washed by DPBS. RNA was isolated by using the Aurum
TM
Total RNA Mini Kit (Bio-rad,
Hercules, CA # 7326820) according to the manufactures protocol. Cells were first treated with
350 l lysis solution, and pipet up and down to lysis thoroughly. Then, add 70% ethanol and
pipet up and down to denature any protein fraction. The mixture was transferred to the RNA
binding column and washed with 600 l low stringency wash solution successively with
highest spinning speed for 30-60 seconds each. Dilute 5 l reconstituted DNase I with 75 l
DNase dilution solution, and add 80 l diluted DNase I. Then, incubate 25min at room
temperature, and spin down with highest spinning speed. Add 600 l high stringency wash
solution and 600 l low stringency in order, and centrifuge at highest spinning speed. To elute
the total RNA, add 40 l elution solution onto membrane stack, and let it saturate the column
for 1 minute. Centrifuge to get the eluted total RNA. cDNA was prepared using the iScript
26
cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. The
reaction system was composed of 4 l 5x iScript RT advanced reaction mix, 1 l iScript
Reverse Transcriptase, 1 g total RNA, and variable nuclease-free water to 20 l. The mixture
was incubate 5 minutes at 25℃ for priming, 20 minutes at 46℃ for reverse transcription, and
1 minute at 95℃ for RT inactivation by the MJ Mini Thermocycler (Bio-Rad). cDNA libraries
were diluted 1:5 by adding 80 l nuclease-free water prior to qPCR set up.
2.4 Quantitative real-time PCR (qPCR)
For each well of qPCR run, 6.25 l SYBR-green, 0.375 l 3 M forward/reverse primer of
GAPDH or LINC00261 (Table 2, primer 16-17 or 18-19), and 5 l cDNA was used for a total
volume of 12 l system. For each PCR cycle, the temperature setting was: 30 seconds at 95 C
for DNA unwinding, 30 seconds at 57 C for primers annealing, 30 seconds at 72 C for Taq
extension. A melting curve was performed after each run to ensure proper amplification of
primers.
2.5 Mammalian cell transfection
The final TRE-dCas9-KRAB;U6-LINC00261[gRNA];EF1a-rTTA plasmid was first linearized
by restriction digestion with EcoRV-HF (New England Biolabs, Ipswitch, MA # R3195S) and
27
column purified to enhance integration for stable transfection. The transfected cells were plated
in antibiotic and penicillin/streptomycin free culture media one day before transfection. Stable
transfection was done by using FugeneHD (Promega, Madison, WI # E2311) and its protocol.
For the 6-well plate, 200,000 cells were seeded into each well, and 3.3 g of plasmid were
treated into each well. The plasmid was first diluted in the Opti-Mem (Gibco, Gaitherburg, MD)
media to the concentration of 0.02 g/ l, and then mixed with FugeneHD reagent for the ratio
of 1 g:3 l of FugeneHD. The mixture was incubated at room temperature for 15 minutes,
and distributed to each well. The selection media (regular culture media with 250 g/ml
hygromycin) was added 48 hours after transfection. The selection media was refreshed every
48-72 hours. When isolated colonies (more than 100 cells) with similar morphology to the wild
type of cell line were found in the plate, colonies were picked using a sterile pipette tip and
transferred into a new 96-well plate for monoclonal expansion. Cells were then transferred into
a 48-well plate, 24-well plate, 12-well plate, 6-well plate, and subsequently to a 10 cm dish to
expand the monoclonal cell line.
For the transient transfection, the plasmids do not need to be linearized by enzymatic digestion.
The protocol is similar to the stable transfection with the cell seeding steps, and transfection
steps. However, the cells do not need to treat with selection drugs. In contrast, they were treated
with doxycycline hyclate (Sigma-Aldrich, Burlington, VT # D9891-1G) 24 hours after
transfection and were subsequently collected 48 hours after doxycycline treatment.
Doxycycline was refreshed every 24 hours. The doxycycline treatment plates were put into the
incubator and wrapped in foil to avoid light, because doxycycline is a light sensitive drug.
28
2.6 Viability assay
In order to test the different drugs effect in H522 cells with or without LINC00261 expression,
drug resistant viability assay was performed. 7500 cells of the H522-CMV-NEO and H522-
CMV-LINC00261 cell lines were seeded in 96-well plates for each well one day before drug
treatment. After 24 hours, the cells were treated with Cisplatin (VWR, Radnor, PA # 10187-
512), Oxaliplatin (Tocris Bioscience, Minneapolis, MN # 61825-94-3), Carboplatin (Tocris
Bioscience, Minneapolis, MN # 41575-94-4) and Taxol (Tocris Bioscience, Minneapolis, MN
# 33069-62-4) platin respectively. The drugs cisplatin, oxaliplatin and carboplatin were diluted
in saline vehicle (sodium chloride from AMRESCO, Radnor, PA # 0983C336, dissolved in
sterile water to 0.9% concentration), and taxol was dissolved in DMSO (Dimethyl sulfoxide
from Sigma-Aldrich, Burlington, VT # 67-68-5). The treated concentration of each cell line
was 0.098, 0.196, 0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 M for cisplatin and oxaliplatin.
For carboplatin, the treated concentration of each cell line was 0.196, 0.391, 0.781, 1.562, 3.125,
6.25, 12.5, 25, 50, 100 M. For Taxol the treated concentration of each cell line was 0.098
0.196, 0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 nM. All drug treatments were done using
three biological replicates representative of independent plasmid integrations as well as three
technical replicates within each biological repeat. The drugs were refreshed every 24 hours and
treated for three days. After drug treatment, the cells were treated with regular culture media
mixed with WST-8 solution (Abcam, Cambridge, MA # ab228554), the ratio of culture media
29
and WST-8 solution was 10:1. WST-8 reagent was incubated with cells in the 37℃ incubator
to avoid light exposure for 4 hours. Then, absorbance was measured at 460 nm. Three technical
replicates were performed on three different stably transfected cell lines per assay.
2.7 Genomic DNA extraction
Culture media was aspirate when the cells reach around 90% confluency, and immediately
washed by DPBS. DNA was isolated by using the DNeasy Blood & Tissue Kit (Qiagen # 69504)
according to the manufactures protocol. Cells were first trypsined and centrifuged to a pellet.
Cells were resuspended in 200 l PBS. Then, 20 l proteinase K and an additional 200 l of
buffer AL were added before being mixed thoroughly by vortexing. Mixtures were then
incubated at 56 C for 10 minutes. A 200 l 100% ethanol was seubsequently added to the
sample, and again mixed thoroughly by vortexing. The mixture was pipetted into DNeasy Mini
spin column and placed in a 2 ml collection tube, then centrifuged at 8000 rpm for 1 min. The
flow-through was discarded, and a new collection tube used to collect the eluate in 500 l of
buffer AW1. Then, centrifuge for 3 min at 14000 rpm, and discard flow-through and collection
tube. Place the column in a new collection tube, and 500 l buffer AW2 was added followed
by another centrifuge for 3 min at 14000 rpm. Discarded flow-through and collection tube.
Finally, the DNeasy Mini spin column was placed in a clean 1.5 ml microcentrifuge tube, and
200 l buffer AE was used to elute the genomic DNA.
30
2.8 Non-linear regression model and statistical analysis
For data smoothing, the degree of fit of different non-linear regression model were compared.
The regression model of b-spline was used for the drug treatment resistance kill curve, which
uses basis functions for the entire spline (40). R studio (R 4.0.5 2021-03-31) was used for
plotting and regression model calculation. Packages used were “tidyverse” (1.3.1), caret (6.0-
88), splines (0.4.3) and ggplot2 (3.3.3). For testing the statistical significance of regression
model between tests and controls, the code is shown below:
fit <- lm(viability ~ bs(conc, df=5) + linc, data=data)
summary(fit)
anova(fit)
The data was imported through csv files, and the data frame contained three columns:
concentration of drugs, cell viability and LINC. The LINC column is to separate the data of
the two cell lines. In this case, the H522-CMV-NEO cell line is annotated with 0, and the
H522-CMV-LINC00261 cell line is annotated with 1, so that the statistical significance
testing can be performed between the two groups. P values are denoted as follows: *, 0.05;
**, 0.01; ***, 0.001.
31
Table 2. List of primers used for PCR, fragment clone and sequencing
Number Primer Name Primer Sequence
1 U6 and gRNA cloning
forward
TATACTAGTAATCGACACGGGTTAATTAA
2 U6 and gRNA cloning
reverse
TTACCTCGAGTATAACAGCAGAGATCCAGTT
3 Site directed mutagenesis
primer 1 forward
GTTCGACTCCGGGGAAACGGCCGAAGCCAC
G
4 Site directed mutagenesis
primer 1 reverse
CGTGGCTTCGGCCGTTTCCCCGGAGTCGAA
C
5 Site directed mutagenesis
primer 2 forward
GGATCCCGATGAAGAAACGAGCCAGGCTGT
C
6 Site directed mutagenesis
primer 2 reverse
GACAGCCTGGCTCGTTTCTTCATCGGGATCC
7 Site directed mutagenesis
primer 3 forward
AAAGTTCGACAGCGTGTCCGACCTGATGCA
G
8 Site directed mutagenesis
primer 3 reverse
CTGCATCAGGTCGGACACGCTGTCGAACTTT
9 dCas9-KRAB region
cloning forward
AACTTTCCGTACCACTTCCTACCCTCGTAAA
GGTCTAGAGTCTAGAGCCACCATGGACTACA
AAGACC
10 dCas9-KRAB region
cloning reverse
GTATTGTCTCCTTCCGTGTTTCAGTTAGCCTC
CCCCGTTTAAACTTTGCGTTTCTTTTTCGGA
ACTGA
11 Plasmid verification
sequencing – EF1a
ATAAGTGCAGTAGTCGCCGTGA
12 Plasmid verification
sequencing – HSV TK
polyA
CGCCCGGTTTTATTCTGTC
13 Plasmid verification
sequencing – Rta AD
CCAACACCAACCGGTCCAGTAC
14 Plasmid verification
sequencing – TET-on
GACCTTGACATGCTCCCCGGG
15 Plasmid verification
sequencing – TRE
CCGTACCACTTCCTACCCTCG
16 GAPDH qPCR forward GGTGAAGGTCGGAGTCAACG
17 GAPDH qPCR reverse GTTGAGGTCAATGAAGGGGTC
32
18 LINC00261 qPCR
forward
GGATAAAGACCAGCTCAACCA
19 LINC00261 qPCR
reverse
CTCCAAGACAAAGAAGAGTAGG
20 A549 colony validation
LINC00261 forward
GCCATTCTAGATCCGAATTCCA
21 A549 colony validation
LINC00261 reverse
AAGTCTGCATACGTTCTCTATCAC
22 A549 colony validation
scrambled forward
ACTCCAATTGGCGAAGTTT
23 A549 colony validation
scrambled reverse
AACTCTTCATACGTTCTCTATCAC
24 A549 colony validation
dcas9-1 forward
CTGACTTGCGGTTGATCTATCT
25 A549 colony validation
dcas9-1 reverse
AGCTGTGCGATGAGGTTT
26 A549 colony validation
dcas9-2 forward
AATTCAAAGTTCTGGGCAATACC
27 A549 colony validation
dcas9-2 reverse
AGAAAGAGTCATCCACCTTAGC
33
CHAPTER 3: Results
3.1 Design of doxycycline-inducible CRISPR-mediated knock down system
The Marconett lab has previously generated a LINC00261 knock down A549 cell line using a
short hairpin RNA (shRNA). However, this method has some drawbacks. First, shRNA is a
post-transcriptional regulator, which works after LINC00261 transcription. So, some of
LINC00261 could have already functioned in cells. Also, the shRNA knockdown was only
around 75% efficient, so that there is still 25% of the normal level of LINC00261 in the cells.
Furthermore, the A549-shLINC00261 cell line lost repression over 15-20 passages, or died off
after becoming hyperaneuploid (36).
Thus, to improve the knock down model, we designed a doxycycline-inducible CRISPR-
mediated knock down system. We used the two guide RNAs that our lab designed before. One
is the guide RNA targeting the promoter region of LINC00261, another is a scrambled guide
RNA that targets nothing in the cell, which will be the control. The LINC00261 guide RNA
was located 200 nucleotides upstream the transcription start site, and the scrambled guide RNA
sequence was from the GFP gene, which was confirmed in the University of California Santa
Cruz (UCSC) genome browsere by BLAT (BLAST-like alignment tool) to make sure that the
scramble sequence not existed in the host cell. Both these two guide RNAs was previously
designed by the lab. The system also contains a mutant Cas9 protein that lacks endonuclease
activity fused with the KRAB domain to repress LINC00261 expression at the transcriptional
34
level. Since KRAB would repress gene expression by decreasing histone H3-acetylation and
increasing H3 lysine 9 methylation at the cell level, KRAB mediates a reversible and long-
range transcriptional repression through heterochromatin spreading (38). Then, to make the
system inducible, a promoter carrying tetracycline response elements, also called a TRE
promoter, was added to control the dCas9-KRAB expression. The system also contains the
modified reverse tetracycline-controlled transactivator (rtTA) protein that can bind tightly to
the TRE promoter in the presence of doxycycline followed by a consecutive promoter (Figure
10). Therefore, when adding doxycycline, the TRE promoter will be activated and dCas9-
KRAB will be expressed, and the guide RNA will lead dCas9-KRAB to the LINC00261 locus
and repress its expression at a transcriptional level.
Figure 10. Schematic of the doxycycline-inducible CIRSPR-mediated knock down system.
(Created with BioRender.com)
3.2 Transient transfection of dox-inducible dCas9-KRAB plasmid decreases
LI N C 0 0 2 61 expression after dox treatment
The sequence of the TRE-dCas9-KRAB;U6-LINC00261[gRNA];EF1a-rTTA and TRE-dCas9-
35
KRAB;U6-Scramble[gRNA];EF1a-rTTA plasmids were both validated first through digestion
and size determination via agarose gel electrophoresis, then subsequently by sequencing. To
optimize the doxycycline concentration for maximum knock down efficiency, transient
transfection of both TRE-dCas9-KRAB;U6-LINC00261[gRNA];EF1a-rTTA and TRE-dCas9-
KRAB;U6-Scramble[gRNA];EF1a-rTTA plasmids in A549s were performed in a 12-well plate,
and 1ug of plasmid was used for each well. Based on the doxycycline-A549 kill curve, the
doxycycline concentration gradient was set to 0 ng/ml 20 ng/ml 500 ng/ml 1000 ng/ml 1500
ng/ml 2000 ng/ml 2500 ng/ml 3000 ng/ml 3500 ng/ml 4000 ng/ml 4500 ng/ml 5000 ng/ml.
Doxycycline was given to the transient transfected cells after 24 hours, and the cells were
harvested 24 hours after doxycycline induction, and subsequent qPCR was done to test the
LINC00261 expression. The result indicated that 3500 ng/ml doxycycline was the most
effective dose to reduce LINC00261 expression (Figure 11).
Figure 11. LINC00261 repression based on doxycycline concentration in A549 cells
0.00
0.50
1.00
1.50
2.00
2.50
0 20 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000
LINC00261 Fold Change
Doxycycline Concentration ng/ml
TRE-dCas9-
KRAB;U6-
Scramble[gRNA];
EF1a-rTTA
TRE-dCas9-
KRAB;U6-
LINC00261[gRN
A];EF1a-rTTA
36
transiently transfected with CRISPR-mediated epigenetic toggle switch. qPCR was
performed to determine LINC00261 expression in A549 using a dose response of multiple
doxycycline concentrations from 0ng/ml to 5000 ng/ml. LINC00261 expression was decreased
at a doxycycline concentration of 3500 ng/ml.
3.3 Generation of A549 cell lines stably expressing dCas9-KRAB plasmids
In order to generate stable cell lines for subsequent functional testing, a stable transfection was
performed in A549 cells. One colony of TRE-dCas9-KRAB;U6-LINC00261[gRNA];EF1a-
rTTA plasmid stable transfection and three colonies of TRE-dCas9-KRAB;U6-
Scramble[gRNA];EF1a-rTTA plasmid stable transfection were obtained. To validate the
colonies, genomic DNA was extracted from the stable transfection colonies. PCR was
performed to amplify a part of the dCas9 of all the colonies. Then, to validate the unique gRNA
targeting sequence, I designed the primers for both LINC00261 gRNA and scrambled gRNA
target seq to amplify the LINC00261 gRNA or scrambled gRNA and a part of the TRE promoter
(Figure 12). All the expected PCR products were around 200 bp, and for different colonies
PCR product bands were of the expected size. This suggests that stable cell lines for both
A549::TRE-dCas9-KRAB;U6-LINC00261[gRNA];EF1a-rTTA and A549::TRE-dCas9-
KRAB;U6-Scramble[gRNA];EF1a-rTTA were generated. However, to further verify the stable
colonies, the stable colonies will be treated with doxycycline to see whether the LINC00261
expression can be successfully knock down.
37
Figure 12. PCR validation of dCas-KRAB plasmid stable transfection colonies. 1: 100bp
ladder, 2: LINC00261 gRNA and part of TRE promoter of TRE-dCas9-KRAB;U6-
LINC00261[gRNA];EF1a-rTTA colony (253bp), 3: part of dCas9 of TRE-dCas9-KRAB;U6-
LINC00261[gRNA];EF1a-rTTA colony (201bp), 4: scrambled gRNA and part of TRE
promoter of TRE-dCas9-KRAB;U6-Scramble[gRNA];EF1a-rTTA colony 1 (209bp), 5: part of
dCas9 of TRE-dCas9-KRAB;U6-Scramble[gRNA];EF1a-rTTA colony 1 (241bp), 6:
scrambled gRNA and part of TRE promoter of TRE-dCas9-KRAB;U6-
Scramble[gRNA];EF1a-rTTA colony 2 (209bp), 7: part of dCas9 of TRE-dCas9-KRAB;U6-
Scramble[gRNA];EF1a-rTTA colony 2 (241bp), 8: scrambled gRNA and a part of TRE
promoter of TRE-dCas9-KRAB;U6-Scramble[gRNA];EF1a-rTTA colony 1 (209bp), 9: part of
dCas9 of TRE-dCas9-KRAB;U6-Scramble[gRNA];EF1a-rTTA colony 1 (241bp), 10: TRE-
dCas9-KRAB;U6-LINC00261[gRNA];EF1a-rTTA plasmid, 11: TRE-dCas9-KRAB;U6-
Scramble[gRNA];EF1a-rTTA plasmid, 12: 1kb ladder
38
3.4 L I N C 0 0 2 61 confers resistance to the cisplatin derivatives oxaliplatin and
carboplatin in H522 LUAD cell culture
We previously determined that LINC00261 was sufficient to activate the DNA damage
response phosphorylation cascade in LUAD cell culture, and that LINC00261 levels are
inversely correlated to tumor mutational burden (TMB) across the Cancer Genome Atlas
Program (TCGA) primary LUAD patient cohort. Also, it is widely accepted that current
platinum-based chemotherapeutics drugs used to treat LUAD clinically work by inducing DNA
damage through bulky adduct formation (41). Thus, we set out to determine if ectopic
LINC00261 expression can alter LUAD cell line resistance to chemotherapeutics drugs. We
decided to test oxaliplatin and carboplatin, as their utilization for LUAD treatment is part of
the current standard of care. We also included cisplatin, which is the parental compound and
has also been showed to have differential dose activity based on LINC00261 expression (data
not yet published). We also included Taxol, as it is used clinically in combination with
platinum-based therapeutics and has a completely separate mode of activity, namely the
disruption of microtubule polymerization and subsequent mitotic chromosomal separation (35).
Therefore, as we are still working out the experimental details of the epigenetic toggle switch
described in Chapter 2, I performed a dose response curve for cisplatin, oxaliplatin, carboplatin,
and taxol in H522 LUAD cells stably transfected with either CMV-LINC00261 or CMV-NEO
empty vector control to determine the half lethal dose (LD50) for each drug under each gene
39
expression condition. Cells were treated for 3 days to mimic acute exposure, and cell viability
was performed using the viability assay. The resulting dose response curves allowed us to
determine the half lethal dose (LD50) and their standard deviation (SD) for the drugs (Table
3).
Drugs CMV- L I N C 0 0 2 6 1 SD CMV-NEO SD
Cisplatin 2.40 ( M) 1.22 1.54 ( M) 0.63
Oxaliplatin 3.27 ( M) 2.94 2.60 ( M) 2.27
Carboplatin 26.02 ( M) 1.58 22.56 ( M) 5.96
Taxol 5.70 (nM) 1.06 4.67 (nM) 1.74
Table 3. LD50 of drugs for both CMV- L I N C 00261 and CMV-NEO cell lines.
To determine if the differential LD50 and dose response curves significantly differed based on
LINC00261 expression, the dose curve was fit through a non-linear regression model. The dose
curves of cisplatin, oxaliplatin, and carboplatin were significantly different between the H522-
CMV-LINC00261 and H522-CMV-NEO control cells (Figure 13 A-C). These three drugs are
all platinum-containing compounds used intravenously in the chemotherapy treatment of lung,
colorectal, ovarian, breast, head and neck, bladder, and testicular cancers (37). Since these
platinum drugs cross-link to DNA, forming the DNA/platinum adducts, induce DNA damage
as well as inhibit DNA replication and repair (38), we conclude that LINC00261 confers
resistance to platinum-based chemotherapeutics drugs that induce DNA damage or interfere
40
DNA repair. By contrast, taxol treatment of H522-CMV-LINC00261 and H522-CMV-NEO
cells showed no significant difference in drug sensitivity based on LINC00261 expression
(Figure 13 D). Taxol acts through a completely different mechanism to inhibit cellular
proliferation. Rather than targeting the DNA, it blocks cancer cell proliferation by inhibiting
microtubule formation, which is critical for separation of chromatids during mitosis. Therefore,
the ability of LINC00261 to confer resistance to chemotherapeutic agents is specific to
mechanisms affecting DNA repair, and not a generalized function of inhibiting cellular
proliferation.
Figure 13. LINC00261 confers resistance to cisplatin derivatives, oxaliplatin and
carboplatin in H522 LUAD cell lines. H522-CMV-LINC00261 cells and H522-CMV-NEO
41
cells were treated with a range of doses for the indicated chemotherapeutic. Green lines are the
H522-CMV-LINC00261 cells and the black lines are the H522-CMV-NEO controls. A)
Cisplatin treatment B) Oxaliplatin treatment C) Carboplatin treatment D) Taxol treatment.
P<0.05 (*), P<0.01(**), P<0.001(***).
42
CHAPTER 4: Discussion
Lung cancer is one of the most prevalent and lethal cancers in the United States and worldwide,
and lung adenocarcinoma (LUAD) is the major histologic subtype of lung cancer. In order to
further understand LUAD development, we interrogated the long non-coding RNA
LINC00261, which is a tumor suppressor in multiple cancer types including LUAD, and its
functional role in response to DNA damaging chemotherapeutics.
To better understand the role of LINC00261 in the regulation of the DNA damage response,
our lab has tried multiple different methods to ablate LINC00261 expression in cells. Originally,
we transfected a short hairpin RNA targeting LINC00261 exons to generate stable knock-down
A549-shLINC00261 cells. However, the A549-shLINC00261 cell line lost repression over
many passages, and the cells that did not lose expression also died off after becoming
hyperaneuploid (36). We then utilized the CRISPR-Cas9 system that is not inducible. However,
we were unable to generate stable cell lines using this construct. One potential reason for this
could be that the knock down efficiency was so high that it could affected cell viability. To
overcome this issue, I created a doxycycline-inducible CRISPR-mediated epigenetic repression
system to knock down LINC00261 endogenous expression. The system takes advantage of the
mutant Cas9 protein that lacks endonuclease activity fused with the KRAB domain to knock
down LINC00261 expression at the transcriptional level. Also, the dCas9-KRAB is activated
by the TRE promoter, which is a tetracycline response element, and the TET-on domain will
43
provide a modified rTTA that binds to the TRE promoter in the presence of doxycycline.
Another advantage of this system is that all functional domains are in one plasmid so that there
will be no need for co-transfection. In this case, the dCas9-KRAB can be activated by adding
dox, and inactivated by removing dox, and we are thus able to control the expression level of
LINC00261 by applying different concentration of doxycycline.
By using this system and the stable A549 LINC00261 knock down cell lines, we can now
effectively characterize the effect loss of LINC00261 in vitro and in vivo. Since we have
previously demonstrated that LINC00261 is sufficient to activate the DNA damage response
(Shahabi et al., 2019), future experiments in the lab will test whether LINC00261 is necessary
to activate DDR signaling and recruitment to sites of DNA damage in vitro. In addition, since
DDR genes play key roles in maintaining human genomic stability, loss of DDR function,
conversely, is an important determinant of cancer risk, progression, and therapeutic response
(30). Based on our previous data that shows shRNA knock down A549 cells tend to have a
higher DNA content and become macronucleated, further validation will be required to test if
LINC00261 is necessary to maintain genomic stability.
The dysregulation of DNA repair is a major contributing factor in carcinogenesis, and can be
exploited for cancer therapy. Consistent with the previous finding that LINC00261 has a role
in activation of the DNA damage response, the H522-CMV-LINC00261 cells are more resistant
to chemotherapeutics drugs that induce DNA damage, including Cisplatin, Oxaliplatin and
Carboplatin. In contrast, ectopic LINC00261 did not confer any significant difference in
44
response to taxol treatment, which may be due to the different mechanism taxol utilizes to
prevent cell division to impede cancer development (35). As the experiments in Chapter 3 were
done using ectopic LINC00261 reintroduction, follow-up work should include experiments that
test whether reintroduction of endogenous LINC00261 expression affects the resistance to
platinum-based chemotherapy drugs. Furthermore, in vivo testing of chemotherapeutic
resistance in mice is also necessary to understand the translational implications of this work,
and how these results can be leveraged to improve patient care.
45
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Abstract (if available)
Abstract
Many tumors lack known oncogenic driver mutations, which underscores the importance of discovering novel biomarkers for LUAD diagnosis and treatment. One of the classes of biomolecules that may resolve this lack of understanding into the molecular origins of lung cancer is long intergenic non-coding RNA (lincRNA). Our lab has identified LINC00261 as a tumor suppressor in lung adenocarcinoma (LUAD), the major subtype of lung cancer, and discovered that this lncRNA plays a functional role in the response to DNA damage. We have also observed that the ablation of LINC00261in lung cancer cells results in the formation of macronucleated cells that have higher-order DNA content. Based on these preliminary results, my thesis hypothesis was that LINC00261 alters the sensitivity of lung cancer cells to chemotherapeutics by affecting the DNA damage response (DDR). To test the role of LINC00261 in the DNA damage response, I created an inducible CRISPR-mediated system that can utilize doxycycline as an epigenetic toggle switch to modulate endogenous LINC00261 expression. With this system, our lab will be able to further characterize the DNA content of the previously observed macronucleated cells and test the ability of DDR pathway members to localize to sites of DNA damage in the presence or absence of LINC00261. Also, since many chemotherapeutic drugs work by targeting cancer cells and inducing DNA damage, to test if LINC00261 influences chemotherapy, I treated H522 cells into which LINC00261 has been reintroduced and NEO control with different chemotherapeutics drugs and found that LINC00261 confers resistance to the cisplatin derivatives oxaliplatin and carboplatin in H522 LUAD. These findings may provide a mechanistic basis for the function of LINC00261 in the DDR pathway and genome stability.
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Asset Metadata
Creator
Miao, Lin
(author)
Core Title
Generation of an epigenetic toggle switch to test LINC00261 function on lung adenocarcinoma cellular response to the chemotherapeutics oxaliplatin and carboplatin
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Degree Conferral Date
2021-08
Publication Date
08/02/2021
Defense Date
06/04/2021
Publisher
University of Southern California
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Tag
chemotherapeutics,CRISPR,DNA damage response,long non-coding RNA,lung adenocarcinoma,OAI-PMH Harvest
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English
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Marconett, Crystal Nicole (
committee chair
), Offringa, Ite A. (
committee member
), Zhou, Beiyun (
committee member
)
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linmiao@usc.edu,linmiao2020@gmail.com
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Tags
chemotherapeutics
CRISPR
DNA damage response
long non-coding RNA
lung adenocarcinoma