Cloning and Expression of Human DLC1 in SCC-9 OSCC
Cell line
submitted in partial fulfilment for the award of the degree of
Bachelor of Technology
in
Genetic Engineering
By
DEEPIKA N (RA1911037010034)
Department of Genetic Engineering
School of Bioengineering
SRM Institute of Science and Technology
Kattankulathur, Tamil Nadu
India 603 203.
Month Year- May 2023
DISSERTATION
submitted in partial fulfilment for the award of the degree of
Bachelor of Technology
in
Genetic Engineering
by
DEEPIKA N (RA1911037010034)
Under the guidance of
Dr Sitaram Harihar
(Assistant Professor, Department of Genetic Engineering)
Department of Genetic Engineering
School of Bioengineering
SRM Institute of Science and Technology
Kattankulathur, Tamil Nadu
India 603 203.
Month Year- May 2023
ACKNOWLEDGMENTS
We appreciate the opportunity we got to work on this project under this esteemed
institution. We would like to thank Dr SITARAM HARIHAR for giving us the
excellent opportunity to study under his brilliant supervision and we would have not
succeeded without his constant encouragement. We would like to show courtesy to Dr
M Ramya, HOD of the Department of Genetic Engineering for supporting the
execution of this research. We extend our gratitude to our PhD student Mrs
Krishnaveni for advising us throughout the duration of the project and Ms
S.Sathammai, our M Tech student for assisting with arranging instruments and
materials for our project.
DECLARATION
I hereby declare that the project report entitled “Cloning and Expression of
Human DLC1 in SCC-9 OSCC Cell line” is a record of original work carried out by
us under the supervision of Dr Sitaram Harihar, Department of Genetic Engineering,
SRM Institute of Science and Technology, Kattankulathur, 603 203. This project has
not been submitted earlier in part or full for the award of any degree, diploma,
associate ship, or fellowship.
Kattankulathur
(Signature)
Date
Name
CERTIFICATE
Certified that the dissertation entitled “Cloning and Expression of Human
DLC1 in SCC-9 OSCC Cell line” submitted by “Deepika. N (RA1911037010034)"
is a record of project work done for the partial fulfilment of the award of the degree of
Bachelor of Technology in Genetic Engineering under the guidance of Dr Sitaram
Harihar, Department of Genetic Engineering, SRM Institute of Science and
Technology, Kattankulathur 603 203. This project has not formed the basis for the
award of any degree, diploma, associateship, or fellowship.
GUIDE
(Name and designation of the guide with
seal)
For the purpose of viva voce
Examiner 1
HEAD OF THE DEPARTMENT
Examiner 2
CONTENTS
Particulars
Page
List of Tables
List of Figures
List of abbreviations
Abstract
10
1.Introduction
1.1 Oral Squamous Cell Carcinoma
11
1.2 Deleted in Liver Cancer 1: A metastasis Suppressor gene
12
2.Objectives
13
3.Review of Literature
3.1 DLC1 : A tumour suppressor gene belonging to RhoGAP family 14
3.2 DLC1- A switch bridging the GAP
15
3.3 The role of DLC1 as a metastasis suppressor
16
3.4 Molecular Cloning of DLC1
17
3.5 Study of DLC1 in cell lines
17
3.6 Study of localization of DLC1gene
19
4.Materials and Methods
4.1 Materials Used
4.1.1 Cell lines
20
4.1.2 Plasmid details
20
4.1.3 Enzyme details
21
4.2 Methods :
4.2.1 Plasmid Isolation using Alkaline Lysis Method
4.2.2 Plasmid Concentration and purity Determination
21
using NanoDrop™
24
4.2.3 Digestion and extraction of DLC1 insert
24
4.2.4 Ligation using T4 DNA Ligase
24
4.2.5 Transformation of pEGFP-N2-DLC1 into DH5 strain
25
4.2.6 Transfection into SCC9 cell lines and determining its
Localization
26
5.Results
5.1 Results for Objective 1
5.1.1 Streaking and Isolation of Plasmid
27
5.1.2 Restriction Digest of Plasmid to confirm presence of insert
30
5.3 Results for Objective 3
5.3.1 Preparation of Competent DH5a cells
34
6.Discussion
35
7.Conclusion
36
8.References
37
LIST OF TABLES
Table No.
Title
Page No.
Table 1
Table illustrating the concentration and volume of reagents
used to prepare Solution I
22
Table 2
Table illustrating the concentration and volume of reagents
22
used to prepare Alkaline Lysis solution II.
Table 3
Table illustrating the concentration and volume of reagents
23
used to prepare Alkaline Lysis solution III.
Table 4
Table showing the proportion of digested vector and insert to
25
be used in a standard ligation reaction of 20μL using T4
DNA Ligase.
Table 5
Concentration of DNA and absorbance ratios of the four
28
isolated plasmids as determined by NanoDrop™.
Table 6
Volumes of reagents added to perform the EcoRI digest of all
30
the samples.
Table 7
Volumes of reagents added to perform the double digest of
pcDNA3.1-T7-DLC1.
32
LIST OF FIGURES
Figure
No.
Title
Page No.
Fig.1
Figure showing regulation of Rho-GTPase through signalling and
14
transition between inactive Rho-GDP in an ‘OFF’ state to active Rho-GTP
state turning the signal to an ‘ON’ state leading to a cascade of signals between
cells
Fig.2
Image of SCC-9 cells under 10X microscope after 7-day passage
20
Fig 3
Plasmid map of pCDNA3 T7 DLC1 from depositing lab adapted from
Addgene. Plasmid number #24266 is listed on the Addgene website.
21
Fig 4
Colonies of plasmid pCDNA3.1-T7-DLC1 grown on LB Agar plate with 100
μg/ml of Ampicillin.
27
Fig 5
Agarose Gel image of Isolated Plasmid run on 1%TBE Agarose Gel at 100V
for 30 minutes
29
Fig 6
Agarose Gel image of EcoR1 digest of the DLC1 plasmids run on 0.8% TBE
Agarose Gel at 100V for 40 minutes.
31
Fig 7
Undigested and double Digest of the plasmid run on 1.5% Agarose Gel at
100V for 50 minutes.
33
Fig 8
DH5α cells streaked on LB agar plate and incubated overnight.
34
LIST OF ABBREVIATIONS
1.
2.
3.
4.
DLC1
OSCC
Rho-GTP
Rho GAP
proteins
5. MAPK/ERK
Deleted-in-Liver Cancer 1
Oral Squamous Cell Carcinoma
Ras Homolog GTP
Ras Homolog GTPase-activating
6. CAF
7. NSCLC
8. HT29
9. SR
10. SEOC
11. SDS
12. NaOH
13. EDTA
14. M
15. mL
16. μL
17. g
18. μg
19. LB
Mitogen-activated protein kinase/
extracellular-regulated kinase
Cancer-Associated Fibroblasts
Non-small cell lung cancer
Human Colorectal Adenocarcinoma
Serine Rich
Serous epithelial ovarian cancer
Sodium dodecyl Sulphate
Sodium Hydroxide
Ethylenediaminetetraacetic acid
Molar
Millilitre
Microlitre
Grams
Micrograms
Luria Bertanii
ABSTRACT
The ability to propagate a gene from any species through isolation and insertion into a
vector has allowed us to study its mechanism and function using Molecular Cloning.
DLC1 a protein that activates Rho GTPases is frequently deactivated in tumours and
is actively involved in inhibiting metastasis by controlling GTPase activity that
focuses on cytoskeletal control and adhesion of cells. This project aims to clone the
gene and check for its cellular localization in oral carcinoma cells. The cDNA
encoding for DLC1 was isolated from a plasmid with a pCDNA3 backbone, and the
insert containing DLC1 was digested using highly specific restriction endonucleases
and further ligated into the mammalian expression vector pEGFP-N2 establishing
pEGFP-N2-DLC1 construct. The recombinant plasmids expressing the characteristics
of the insert will be selected and further transfected into the Oral Squamous Cell
Carcinoma cell line SCC-9 where the expression and localization of this gene will be
studied.
Key words: Metastasis Suppressor Genes, DLC1, Oral Squamous Cell Carcinoma,
localization, Molecular Cloning.
Page | 10
1. Introduction
1.1 Oral Squamous Cell Carcinoma
Oral squamous cell carcinoma is ranked sixth place among common malignancies
worldwide. It poses a problem mainly in India ranking first among the top three types
of cancer found in the country. Of the reported cases, around 90% are caused by
smoking, drinking alcohol and tobacco use in various forms the most common being
chewing raw tobacco leaves. Oral cancer originates from stratified epithelium made
up of squamous cells in the oral cavity, but the most common site of occurrence is the
tongue. (Xun Chen, Mutation profiles of oral squamous cell carcinoma cells, 2021)
Malnutrition, immune system suppression and poor oral hygiene give rise to risks of
persistent irritation and infection of human papillomavirus (HPV). The signs
indicating OSCC vary from ulcerated bleeding lesions to detached, mobile teeth and
bleeding, pain, or numbness of regions in the mouth. (eña-Oyarzu´nD, 2020) With the
help of various cancer tissues, we can create cancer cell lines crucial resources for
research. The OSCC cell lines SCC-4, SCC-9 and SCC-25 have been utilized
extensively to study tumours and the several signal pathways that are involved in the
initiation and advancement of oral cancer. (Xun Chen, 2021) In addition, there is the
progression of cancer-associated fibroblasts from normal fibroblasts that can infiltrate
and invade the immune system, further promoting OSCC to spread by metastasis.
This occurs when modification of oral keratinocytes to accustom themselves to any
environmental stimulus like stress by altering their shape or number. Even further
exposure to these catalysts will promote transformation into malignant cancer cells
and these cells will start to exhibit metastatic potential. OSCC cells predominantly
proliferate to the cervical lymph nodes on the same side of the face utilizing the
lymphatic vessels. (Suresh, 2019) Through these external conditions, cancer has
developed capacities of tissue invasion and metastasis, largely linked to death in
OSCC. Determining the basis of these mechanisms is crucial in evaluating the
progression of cancer to create more effective, successful therapies, as the death of
most patients can be attributed to metastasis or recurrence of the tumour. (Yang,
2013) . Metastasis of the tumour is a significant factor in mortality due to cancer due
to its poor prognosis of the disease. Invasion of tumour cells to cells within proximity
of the tumour, transportation through the circulatory system by extravasation, and
Page | 11
fixation to secondary organ promoting colonisation at the secondary site. (Khan,
2021) OSCC is frequently located on the tongue, and this connection may be
attributed to the presence of highly extensive blood vessel supply to large surfaces in
the mobile region of the tongue. (Lima, 2022) To understand the steps leading up to
the metastasis of a tumour, the mechanism of both metastasis suppressor and
metastasis-promoting genes must be understood. These genes influence many steps of
the metastatic cascade and inhibit metastasis while not affecting the growth of the
primary tumour. DLC1 has been identified as a metastasis suppressor gene, that
subdues metastasis through Rho-GTPase activation. In patient tumour samples the
metastasis suppressor’s expression is decreased in comparison to tumour suppressors
(Yan, 2013). The production of pro-inflammatory cytokines by OSCC cells
encourages the progression of CAF’s from normal fibroblasts that evade the immune
system by infiltration and immunosuppressive characteristics. A state of inflammation
for lengthy durations of the oral cavity will generate a diverse array of responses to
stress such as autophagy that allows them to survive in this environment. (Peña,
2020)
1.2 Deleted in Liver Cancer 1: A metastasis suppressor Gene
Deleted in Liver cancer, DLC1 is a gene mapped to chromosome 8p22-p21.3, a
region in the short arm of chromosome 8 that is frequently deleted in hepatocellular
carcinoma, prostate, lung, colorectal, and breast cancers are just a few of the tumours
where DLC1 acts as a tumour suppressor (Basak, 2015). DLC1 belongs to the family
made up of three proteins, and behaves as Rho-GTPase-activating proteins that are
crucial to controlling the cytoskeletal structure, proliferation and attachment of cells.
This gene is often deleted in many cancers by deletion, mutation, or DNA
methylation. Studies have shown heterozygous deletion of DLC1 in around 50% of
liver and breast, 70% of colon, and 50% of lung cancer cases. Subsequent expression
of DLC1 in cancer cells inhibits cell growth by the formation of focal adhesions and
altering the actin cytoskeleton, resulting in a rounded morphology and making it
easier for tumour cells to detach and metastasize (Liao, 2007). Many cancers with
decreased DLC1 expression have poor outcomes. DLC1 has been disrupted in several
cancers and its downregulation enhances migration and invasion of the tumour cells.
(Hui-Ta Wu, 2018) In the absence of the Rho-GAP family of proteins like DLC1, the
recurrence of the cell cycle results in more proliferation of cells that when combined
Page | 12
with changes in cytoskeletal morphology and failed focal adhesion formation
enhances the chance of metastasis. (Tai Young Kim, 2007)
2. Objectives
Objective 1: To digest and extract DLC1 from the pcDNA3.1-T7-DLC1 vector.
Objective 2: To ligate the DLC1 insert with pEGFP-N2 vector to establish
pEGFPN2-DLC1.
Objective 3: To transfect the pEGFP-N2-DLC1 vector in the SCC-9 OSCC cell
line and check for its localization
Page | 13
3. Review of Literature
3.1 DLC1: A Tumour suppressor gene belonging to the RhoGAP
family
Deleted in Liver Cancer 1 is a gene mapped to the 8p22-p21.3 region of the
chromosome and is observed to be frequently deleted in hepatocellular carcinomas.
This gene has been proposed as a tumour suppressor as it is downregulated and
decelerates tumorigenesis by regulating the transformation of the active GTP-bound
state of the protein, and its inactive GDP state. (Popescu, 2014) The Rho group of
proteins typically govern the interaction between the external environment of the cell
and internal pathways and signalling between cells and foreign substances. This
allows the binding of Rho proteins and promotes the initiation of an assortment of
signals that regulate many structural and biological functions across the cell.
(Mosaddeghzadeh N, 2021)
Fig.1: Figure showing regulation of Rho-GTPase through signalling and transition
between inactive Rho-GDP in an ‘OFF’ state to active Rho-GTP state turning the
signal to an ‘ON’ state leading to a cascade of signals between cells. A study of these
mechanisms could help design remedies in disorders like tumour invasion and
metastasis adapted from (Mosaddeghzadeh N, 2021)
DLC1 belongs to a family of three proteins DLC1, DLC2, and DLC3. It has been
found that reintroducing the expression of DLC1 in cancer cells decreases tumour
development by inhibiting the RhoA pathway and making the cells undergo apoptosis
Page | 14
and senescence. This prevents them from growing, migrating and turning invasive
which are hallmark signs of metastasis of a tumour. (Yang Zhang, 2019) When the
expression of the three genes belonging to the DLC group, the downregulation of
DLC1 was to a greater extent compared to DLC2 and DLC3. The study of point
mutations in these genes is very scarce, but their downregulation has been attributed
to the deletion of this gene in a variety of cancers, primarily liver and DNA
methylation of the promoter region also causes diminished expression of this gene
resulting in cancer. (Wang, et al., 2020)
3.2 DLC1- A switch bridging the GAP :
The presence of atypical Rho-GAP domain activity has been detected in several
human cancers. Upon deletion of genes like DLC1, the expression of Rho proteins is
altered leading to loss of tumour suppression ability. Excessive expression of RhoA is
linked typically to the development of tumours and has been detected in the stages of
cellular modifications ultimately leading to migration and metastasis of the cells.
(M.J. Lukey, 2016) While there may be other pathways and signals to activate Rho
GTPases, it is frequently observed when there is a deletion of the GAP. DLC1
expression in cancer cell lines has prevented the proliferation of tumours and arrests
migration by impeding anchorage-independent growth. However, it cannot be
concluded that DLC1 loss caused the formation of the tumour in the first place as it
just slows down the spread of cancer cells. (Lahoz, 2008)
Three conserved domains have been found in the DLC-1:
1. SAM domain: Sterile α Motif Domain
2. START domain: Steroidogenic acute regulatory regulated lipid transfer
domains
3. Rho-GAP domain
The SAM domain is thought to play a role in interactions between proteins. This Nterminal domain has a Serine Rich region that interferes with the normal roles of
protein interactions that regulate DLC1 function regularly (Joshi, 2021). This SR
region has been shown to interact with tensin homologs, talin and focal adhesion
kinases. (Li G., 2011) The C- terminal of DLC-1 has a START domain that ceases
actin fibre and focal adhesion formation. Along with modifications in the actin
cytoskeleton and maintaining focal adhesion of tumour cells, binding of DLC-1
Page | 15
protein leads to hydrolysis of PIP2 that generates DAG and IP3. This leads to a buildup of IP3 and PKC releasing calcium ions in the intracellular matrix allowing it to
cross the cell membrane. The final step activates the ERK1/2 signalling pathway
causing a cascade of signals downstream, one of these is found to suppress
metastasis. (Yi-Chun Liao, 2008)
3.3 The role of DLC1 as a metastasis suppressor:
As previously mentioned, DLC1 inhibits metastasis by controlling GTPase activity.
The gene has been extensively studied as a tumour suppressor gene, although it
demonstrates decreased expression only in certain cancer models and cell lines that
end up leading to metastasis. (Douglas R. Hurst, 2011) Rho proteins belong to the
Ras superfamily and control cell proliferation and actin cytoskeleton formation.
These proteins alter cell structure, mobility and migration in the presence of external
factors. The ability of cancer to proliferate at the new site of metastasis can be
significantly altered by molecular interactions between the destination organ and
migratory cancer cells. (Goodison, et al., 2005) DLC1 is reduced in cells and tissues
of lung adenocarcinomas. When DLC1 was expressed or upregulated in cells it
suppressed migration, proliferation and increased invasion capability of the cells
while its inactivation advanced the metastatic potential of cancer cells. This was
achieved by repressing the MAPK/ERK signalling pathway. (Niu Niu, 2021) In
prevalent malignancies including those of the lung, colon, pancreatic, and breast, loss
of DLC1 occurs almost as often as deletion of p53. The overexpression of DLC1 is
indeed able to inhibit the growth of carcinomas. A majority of studies conducted on
this gene have solely depended on its overexpression in prominent cancers. But in a
mouse model using RNAi the expression of DLC1 was knocked out and the cell’s
ability to form tumours in the mouse resulted in many positive nodules. Upon
analysis, these tumours had a high index of proliferation and mimicked the traits of
human hepatocellular carcinomas, indicating that the loss of DLC1 can effectively
advance the progression of liver cancer. (Xue, 2008)
Page | 16
3.4 Molecular Cloning of DLC1 :
Molecular cloning of DLC1 can give us an insight into its expression and functioning
in various cell and tumour environments. Transfection of cells that express the DLC1
gene will allow us to draw a basic understanding of how these genes are involved in
regulating the cell cycle and apoptosis. Western Blot Analysis revealed that lower
levels of DLC1 protein are observed in non-transfected Ovarian cancer cells. (Shi,
2012) The role of DLC1 in SEOC is known to have a rapid progression and higher
potential to metastasize. Total RNA was isolated from twenty-two SEOC samples
and RNA quantity and purity were checked using NanoDrop® ND-1000
spectrophotometer (Thermo Scientific, MA, USA) to assess the purity of RNA with
an OD260/280 ratio ranging between 1.8-2.1. and the miRNA molecules were
transfected into SEOC cells using Lipofectamine 2000 and assays such as migration
assays, and transwell formation assays were conducted. (Ibrahim, 2015)
3.5 Study of DLC1 in cell lines :
A-549 cells were transfected with pcDNA3.1-DLC1 where mRNA levels and
expression in DLC1
were evaluated. After seeding A549 cells transfected with
DLC1, cell proliferation was calculated using optical density at 450nm. The study
concluded that a higher expression of DLC1 suggested a higher chance of survival, by
halting proliferation and reducing apoptosis in human lung cancer cell line A549.
(Sun, 2019) By using a monoclonal antibody to assess DLC1 protein levels in
NSCLC cell lines, it was found that DLC1 was lost in almost 67% of the cell lines.
The selection of two NSCLC cell lines that are lacking DLC1 and restoring its
expression decreased Rho-GTP activity, implying that a loss of DLC-1 will increase
Rho GTPase activity. Analysis of Colony Formation Assay and a Wound Healing
Assay along with an assay on anchorage independence it can be concluded that DLC1 uses GAP-dependent and independent pathways to arrest the invasion and migration
of NSCLC. (Kevin D. Healy, 2007) The most common route to studying DLC1
function was to create a pcDNA3.1 vector containing DLC1 and transfect it into
various cancer cells. When HT29 colon cancer cells were transfected to establish
pCDNA3.1-DLC1-HT29 cells an MTT assay revealed that cell proliferation was
decreased in these cell lines. Trans well migration assays revealed that lesser HT29
cells that were transfected with DLC1 migrated showing that DLC1 reduced in vitro
Page | 17
invasion of cells from this assay. (Wu, 2009) A study of the suppressive effects of the
DLC1
gene
in gallbladder
cancer
transfected
with
pcDNA-DLC1
using
LipofectAmine 2000. The cells were selected after transfection using G418
resistance. Cell proliferation and migration assays conducted concluded that the
restoration of DLC1 expression inhibited the growth of gallbladder cancer and
induced apoptosis in these cells with a major portion of cells transfected with DLC1
having a higher rate of apoptosis due to activation of the intrinsic and extrinsic
apoptotic signalling pathways. (Qin, 2014) A lot of studies have been conducted on
DLC1 and its role as a tumour suppressor gene in cancer, lung, breast, and colorectal
cancer whose expression leads to halting of tumour cell progression and invasion. In a
study of pancreatic cancers, it was found that a number of patients at stages 3-4 of
pancreatic cancer exhibited lower expression levels of DLC1 than patients at stages 1
and 2, which could be due to the increased proliferation and metastasis of the tumour
during the onset of later stages of cancer. The entire sequence of DLC1 was cloned
into a lentiviral vector PCDH-puro (Addgene Inc). Cells were transfected using
Lipofectamine 2000 and selected using antibiotic resistance against puromycin. The
transfected SW1990 cells of pancreatic cancer exhibited low expression levels of
DLC1. Transfection of the vector containing DLC1 into SW1990 pancreatic cancer
cells showed increased levels of protein and mRNA in the cells. Using flow
cytometry analysis, the effect of DLC1 on the cell cycle was studied and it was
concluded that there was slight moderation in the cell cycles. Lastly, the transfected
cell’s ability to invade pancreatic cancer cells was evaluated using a tumour invasion
assay kit that showed transfection with DLC1 had reduced tumour invasion by around
47% leading the data to indicate that DLC1 can reduce the invasive capability of
tumours in pancreatic cancer. (Chen, 2019) A study to establish and analyse the
relationship of DLC1 at chromosome 8p and its invasion ability in Human
Hepatocellular Carcinoma where DLC1 expression was lower in normal samples and
on the other hand were higher in metastatic cell lines. This further justifies its role as
a metastasis suppressor gene. (Song LJ, 2005)
Page | 18
3.6 Study of localization of DLC1 gene:
The re-expression of DLC1 in numerous cancer cell lines has proved to suppress
tumour formation. DLC1 in various cell cultures typically have localization to the
cytoplasm, but in certain cells, they have been identified in focal adhesions, and are
often present alongside actin-stress fibres. (Marian E. Durkin, 2007) DLC1 was found
to show diminished RhoA activity utilising both Rho-GAP dependent and
independent mechanisms. But the SAM domain of DLC1 was not found to be limited
to focal adhesions and also contributed to a significant alteration in the breakdown of
the actin cytoskeleton. (Dandan Zhong, 2009) A study of protein-lipid interactions
revealed that DLC1 activity is mediated through SAM and START domain and its
localization is governed by expression of DLC1. Localization of focal adhesions was
reduced when a lower level of DLC1 was expressed and DLC1 was constrained to the
periphery of detached metastatic cells. This further implies these sites would be
exhibiting inactivation of Rho proteins. (Patrik Erlmann, 2009) But the localization of
DLC and its ability to inhibit metastasis of OSCC is yet to be determined. But in
OSCC, loss of DLC1 has been regarded as a prognostic factor and its expression may
be reduced in these cells. It was compared by immunohistochemistry in normal oral
mucosa providing more evidence that lack of DLC1 expression shows a poor outcome
in Oral Cancer. (Satyendra Chandra Tripathi, 2012)
Page | 19
4. Materials and Methods:
4.1 Materials Used:
4.1.1 Cell Lines:
SCC9 is an Oral Cancer Cell line harvested from the stratified squamous epithelium
of oral mucosa. OSCC is indicative of about 90% of head and neck cancers. These
cells were cultured and maintained by our M tech student Ms S.Sathammai at SRM
Institute of Science and Technology, Kattankulathur
10
x 7-day passage performed by
Fig.2: Image of SCC-9 cells under 10X microscope after
our Mtech student Ms S Sathammai
4.1.2 Plasmid Details:
pCDNA3 T7 DLC1 was a gift from Roger Davis (Addgene plasmid # 24266
http://n2t.net/addgene:24266 ; RRID:Addgene_24266) received from Addgene. The
plasmid is supplied in an agar stab and has a pcDNA3 backbone (Invitrogen). It
shows antibiotic resistance to 100μg/ml of Ampicillin.
Page | 20
Fig.3:
Plasmid map of pCDNA3 T7 DLC1 from depositing lab adapted from
Addgene. Plasmid number #24266 is listed on the Addgene website.
4.1.3 Enzymes Details:
1. EcoRI with a 20U/μL and 400 units were purchased from GeNei™ Laboratories
through the SouthernIndia Scientific Corporation.
2. High Fidelity HindIII with a rCutSmart Buffer to reduce incubation time and star
activity was obtained from New England Biolabs, Inc. (NEB)
4.2 Methods:
4.2.1 Plasmid Isolation using Alkaline Lysis Method:
The Addgene plasmid was streaked onto LB Agar plates containing a final
concentration of 100μg/ml of Ampicillin. The plasmid was isolated from prominent
colonies using Alkaline lysis solution I, II and III. The protocol was adapted from The
International Genetically Engineered Machine (iGEM) Foundation, 2015 and the
volumes were adjusted to prepare 30mL of each solution.
Stock solutions:
1. 1M of Glucose prepared by dissolving 1.8g in 10ml of water. And filter
sterilized using a 0.2μm syringe filter.
2. 10N Sodium Hydroxide was prepared by dissolving 4g in 10ml sterilised
water.
Page | 21
3. 1% SDS was prepared by dissolving 0.1g of SDS in 10 ml sterile water.
4. 8.83g of Potassium Acetate was dissolved in 18 mL of water to bring a
final concentration of 5M Potassium acetate.
4.2.1.a Alkaline lysis solution I: Resuspension Solution
Reagent and its concentration
Volume
1M Glucose
1.5 mL
1M Tris HCl
0.75 mL
0.5M EDTA
0.3mL
Sterile Water
27.45mL
(Nuclease-free or de-ionised)
Total Volume
30mL
Table 1: Table illustrating the concentration and volume of reagents used to prepare
Solution I
The Resuspension solutionwas sterilised using an autoclave and stored at 4°C until
use.
4.2.1.b Alkaline lysis solution II: Lysis Solution
Reagent
and
its
Volume
concentration
0.2N NaOH
600μL
1% SDS
3mL
Sterile Water
26.4mL
(Nuclease-free or de-ionised)
Total Volume
30mL
Page | 22
Table 2: Table illustrating the concentration and volume of reagents used to prepare
Alkaline Lysis solution II.
4.2.1.c Alkaline lysis solution III: Neutralization Solution
Reagent and its concentration
Volume
5M Potassium Acetate
18mL
Glacial Acetic Acid
3.45mL
De-ionized or Nuclease-free water
8.55mL
Total Volume
30mL
Table 3: Table illustrating the concentration and volume of reagents used to prepare
Alkaline Lysis solution III.
Neutralization Solution was stored at 4°C and transported to a container with ice to
maintain the temperature just before use. Incubate on ice for 15 minutes after adding
Solution III. Taken around 4mL of overnight inoculum of plasmid in LB broth.
Centrifuged the culture to obtain a pellet of cells. Repeated the process twice after
discarding the supernatant each time or until the pellet is dry. The dissolved pellet
obtained was resuspended in around 150μL of resuspension solution and vortexed.
Added 200μL freshly prepared lysis solution and mix contents by gently agitating the
tube 4-6 times, then repeat the same with 300μL neutralizing solution on ice. After
centrifugation at 10000rpm for 5 minutes, the supernatant was transferred and added
an equal volume of ice-cold isopropanol to precipitate the plasmid DNA. The tube
was centrifuged at 14000rpm until a pellet of plasmid DNA was seen, upon which
supernatant is discarded and 70% ethanol is added to the pellet and centrifuged to
wash the DNA. This pellet is finally air-dried until the ethanol evaporates and is
dissolved in 30uL Nuclease-free water to be stored at -20C refrigerator.
Page | 23
4.2.2. Plasmid Concentration and Purity Determination using
NanoDrop™ :
The amount of DNA in the isolated plasmid and its purity can be checked using
spectrophotometry or more commonly using a NanoDrop™ Spectrophotometer. This
instrument measures the absorbance of the sample at 260nm and it is used to
determine the approximate concentration of DNA in the sample and its purity
(García-Alegría, 2020). This can be used to select the concentration and volume of
plasmid DNA we want to use in further applications and experiments.
4.2.3 Digestion and Extraction of DLC1 Insert:
Restriction enzymes are now used to cleave DNA into fragments of predetermined
size and at certain predicted ends. This can further enable us to introduce targets for
modifications like deletions in our gene like DLC1 that can be used to study its
expression. (Di Felice, 2019)
the plasmid vector pCDNA3 T7 DLC1 contained
HindIII at the 5’ cloning site and EcoRI at the 3’ cloning site. So a double digestion
reaction with both enzymes should release the insert containing DLC1. The enzymes
are incubated for a period of 1-3 hours and EcoRI is heat inactivated at 65C for 20
minutes whereas HindIII is heat inactivated at 80C for 20 minutes. The insert size is
confirmed using Agarose Gel Electrophoresis and can be further extracted and
purified from the agarose gel band with the gene of interest using Aura™ pure
Agarose Gel Extraction kit. Using a spin column for fast extraction and purification
of DNA fragments ranging from 100 base pairs to 10kb size from agarose gels run the
TBE or TAE buffers. After purification, the insert is ready to be used for further
applications like ligation, sequencing, and Colony PCR.
4.2.4 Ligation using T4 DNA Ligase:
Using a destination vector that has been digested with the same restriction enzymes,
in this case, we used pEGFP-N2 we can ligate our insert to create a vector expressing
our gene of interest. This DNA Ligase has been isolated from a T4 bacteriophage and
it forms phosphodiester bonds between 3’ hydroxyl and 5’ phosphate. First, we have
to try different ratios of insert to vector as too low concentration would inhibit
Page | 24
annealing and too high concentration can result in abnormally long ligated products.
DNA ligase essentially repairs the ‘cuts’ caused during digestion with restriction
enzymes. (Kroemer, n.d.) a 3:1 ratio of insert to vector should be efficient to enable
the formation of more recombinants. The calculation is done using the following
formula:
𝑛𝑔 𝑜𝑓 𝑖𝑛𝑠𝑒𝑟𝑡 =
𝑛𝑔 𝑜𝑓 𝑑𝑖𝑔𝑒𝑠𝑡𝑒𝑑 𝑣𝑒𝑐𝑡𝑜𝑟∗𝑠𝑖𝑧𝑒 𝑜𝑓 𝑖𝑛𝑠𝑒𝑟𝑡 𝑖𝑛 𝑘𝑏
𝑉𝑒𝑐𝑡𝑜𝑟 𝑠𝑖𝑧𝑒 𝑖𝑛 𝑘𝑏
* 3:1
The ligation reaction was set up using 4 microliters or 1 microgram of the digested
plasmid DNA, which can be calculated after determining the concentration of insert
in the weight of agarose gel extracted and incubated for 1 hour at 37C. The ligated
product can now be used for transformation. For 4μL of the digested fragment 1 unit
of T4 DNA Ligase (GeNei™) was used in the following reaction:
Component
Volume
DNA fragment eluted from the 4μL
gel
10X Ligation Buffer
2μL
Digested pEGFP -N2 vector
2μL
T4 DNA Ligase
1μL
Nuclease-Free
or
De-ionized 11μL
water
Total
20 μL
Table 4: Table showing the proportion of digested vector and insert to be used in a
standard ligation reaction of 20 μL using T4 DNA Ligase.
4.2.5 Transformation of pEGFP-N2-DLC1 into DH5α strain:
Now that we have our vector ligated with the gene of interest, it must be transformed
into a growth strain of bacteria. DH5α is a versatile strain that can establish the
stability of expressed DNA products. Most DNA cloning studies end with
transformation into bacteria that have been altered chemically or physically to allow
Page | 25
the uptake of DNA. (Kostylev, 2015 ) Bacterial cells are made competent by using
ice-cold Calcium chloride that has divalent cations and is most effective even when
compared with electroporation. The divalent cations have been proven to increase
transformation efficiency as they create salient pores in the cell wall (Asif, 2017).
This is frequently followed by heat shock, a rapid change in temperatures from hot to
cold and circling back to hot allowing cells to take up the DNA. Optimal
transformation is found to occur at 42◦C as higher temperatures may destroy the cell
itself, reducing transformation. The return of cells to a lower temperature increases
transformation efficiency significantly so the heat followed by the cold shock step
must be optimised depending on the nature of the cells used for transformation.
(Mahipal Singh*, 2010) The positive clones expressing our gene of interest, DLC1 in
a pEGFP-N2 vector should grow only on a plate containing the antibiotic kanamycin,
which confirms the transformation.
4.2.6 Transfection into SCC9 cell lines and determining its
localization:
The expression of DNA methylation has been studied in OSCC cell lines using single
RNA interference and its effect on cell replication. OSCC cells showing siRNAs were
transfected using Lipofectamine™2000 transfection reagent. (Lei Zhang, 2009) by
following the manufacturer’s protocols and incubating for 24 hours. In OSCC cells,
the expression and localization of genes have been studied using immunofluorescence
assays, Western Blots and in-vitro studies like the wound-healing assays previously
mentioned. Cell invasion assays should be showing reduced activity and can be
evaluated using a fluorescent microplate reader. (Hamakawa, 2011)
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5. Results
5.1. Results for Objective 1:
5.1.1 Streaking and Isolation of Plasmid:
The plasmid received from Addgene was streaked from the agar stab onto an LB
Agar plate with a final concentration of 100μg/ml of Ampicillin using a quadrant
streak for single, isolated colonies. Since the plasmid has an antibiotic resistance to
ampicillin it will grow only in ampicillin plates. From this plate, four medium size
colonies were inoculated and plasmid was isolated from them and stored at -20.
Fig.4: Colonies of plasmid pCDNA3.1-T7-DLC1 grown on LB Agar plate with 100
μg/ml of Ampicillin.
Next, the isolated plasmid was checked for concentration and purity using
NanoDrop™. The apparatus was calibrated using a blank of nuclease-free water and
1μL of the sample was loaded in each well named A02, B02, C02, D02 and E02.
Page | 27
Well
Sample
DNA
concentration Absorbance
Absorbance
measured at 260nm (in at 260/280
at 260/230
ng/μL)
A02
Blank: Nuclease Free 67.70
1.094
0.5939
1.628
1.747
1.180
1.212
1.1891
2.092
1.544
1.672
water
B02
DLC1 isolated from the 3522
first colony
C02
DLC1 isolated from the 3575
second colony
D02
DLC1 isolated from the 3183
third colony
E02
DLC1 isolated from the 3537
fourth colony
Table 5: Concentration of DNA and absorbance ratios of the four isolated plasmids as
determined by NanoDrop™.
All four plasmids were run on a 1% TBE Agarose Gel at 100V for 30 minutes to
visualize the bands and see which corresponded to around 5000bp size of the vector
backbone.
Page | 28
L
1
2
3
4
10037bp
8000bp
6000bp
5000bp
4000bp
3000bp
2500bp
2000bp
1500bp
5000bp
1000bp
800bp
600bp
400bp
Fig.5: Agarose Gel image of Isolated Plasmid run on 1%TBE Agarose Gel at 100V
for 30 minutes
Lane
Sample
Size
L
Ladder (HiMedia)
1kb
1
Isolated Plasmid 1
Around 3000bp
2
Isolated Plasmid 2
Around 6000bp
3
Isolated Plasmid 3
Around 6000bp
4
Isolated Plasmid 4
Around 5000bp
From the above gel image, it is clear that only the plasmid in fourth lane
corresponded to the correct backbone size of 5000bp. Next step was to check for
presence of insert using restriction digestion with both ECOR1 and HindIII enzymes.
Page | 29
5.1.2 Restriction Digestion of Plasmid to confirm presence of insert:
A diagnostic digest to see if the plasmid is of the correct size was done using the
ECORI enzyme. A 20 microlitre reaction was setup using the following components
in the order of: water, 10X Reaction Buffer supplied with Enzyme, Nuclease free
BSA, Template DNA and lastly the enzyme.
Component of Reaction Mixture
Volume
Water
15.8μl
10X Reaction Buffer
1μl
Nuclease- free BSA
0.2μl
Template DNA
2μl
Enzyme
1μl
Total Reaction
20 μl
Table 6 : Volumes of reagents added to perform the EcoRI digest of all the samples.
Heat Inactivation:
1. EcoRI was heat inactivated at 65◦C for 20 minutes.
The digested plasmids were run on 0.8% Agarose Gel to confirm single digest.
Lane
Sample
Size
L
DNA Ladder
1kb
1
EcoRI digest of plasmid 1
4500bp
2
EcoRI digest of plasmid 2
4000bp
3
EcoRI digest of plasmid 3
4500bp
4
EcoRI digest of plasmid 4
5000bp
Page | 30
L
10037bp
8000bp
6000bp
5000bp
4000bp
3000bp
2500bp
2000bp
1500bp
1
2
3
4
5000bp
1000bp
800bp
600bp
400bp
200bp
Fig.6: Agarose Gel image of EcoR1 digest of the DLC1 plasmids run on 0.8% TBE
Agarose Gel at 100V for 40 minutes.
From the above figure, we can clearly see that the DLC1 plasmids in lanes 1,2 and 3
are not positive for the insert as cleavage with EcoRI should give rise to only one
band. Hence proceeded to make a double digest reaction for plasmid isolated that is
seen in fourth lane as it may contain insert. The sample was re-inoculated into 5mL
fresh LB Broth containing 100μg\ml concentration of ampicillin. Then plasmid was
isolated. And restriction digest was performed first using EcoRI and after heat
inactivation, HindIII buffer and enzyme was added to the same tube to make a 20
microlitre double digest. The digest was then checked 0.8% Agarose gel. Bands were
not clearly visible and so around 5ug of plasmid was used to make a reaction to check
size.Since DNA concentration was determined to be 825.1 ng/uL , 5ug of sample is
around 6ul. So the new reaction mix for restriction digestion is tabulated as:
Page | 31
Component of Reaction Mixture
Volume
Water
7.8μl
10X Reaction Buffer
1μl
Nuclease- free BSA
0.2μl
Template DNA
6μl
Enzyme
2μl
Total Reaction
20 μl
Table 7 : Volumes of reagents added to perform the double digest of pcDNA3.1-T7DLC1.
The digest was first set up for HindIII and then after heat inactivation at 80◦C,1 μl of
EcoRI buffer along with 2 μl of EcoRI was added and the whole reaction was
incubated at 37C for almost three hours. Lastly, EcoRI was heat inactivated at 65◦C
for 20 minutes. To check the presence of the insert and confirm its size, controls were
established: Undigested plasmid, the plasmid with only EcoRI digest, the plasmid
with only HindIII digest and finally the double digest was loaded in the final well of
the gel. Bands were visualised on a 1.5% agarose gel and insert size was determined.
Lane
Sample
Size
L
1kb DNA Ladder
1kb
1
Undigested Plasmid
5000bp
2
EcoRI & HindIII digest to Around 750bp
visualize insert
Page | 32
Fig.7: Undigested and double Digest of the plasmid run on 1.5% Agarose Gel at
100V for 50 minutes.
The presence of two bands in the undigested plasmid compared to the single band at
5000bp in the double digest indicates the double digest of the plasmid was successful.
But the insert was observed as a very faint band around 750bp, 400 bases higher than
its actual size. So, it was verified that the isolated plasmid did not have any positive
colonies of the vector expressing our insert with DLC1.
Page | 33
5.3 Results for Objective 3:
5.3.1 Preparation of competent DH5α cells:
Fig.8: DH5α cells streaked on LB agar plate and incubated overnight.
These cells were grown to an OD of 0.2-0.5 in fresh LB Broth and all steps were
carried out at 4◦C. The pellet was settled using centrifugation at 3000rpm for ten
minutes at 4◦C cooling centrifuge. Then 0.1M CalCl2 was added gradually and
repeated centrifugation. Supernatant was discarded and cells were put back into a
suspension of around 800μl of CaCl2 and vortexed gently before a final resuspension
in 100μl of 0.1M Calcium chloride. 17% glycerol was added to the aliquots of
competent cells and they were stored at -80◦C to be used for the transformation step.
Page | 34
6. Discussion
DLC1 has been identified as a prominent tumour suppressor and metastasis inhibitor
studied in various cancer cell lines. Previous work has been conducted to create
plasmids expressing DLC1 and studying its effect in cancer cell lines mainly colon
cancer and A549 cell lines (Song LJ, 2005) (Chen, 2019). Its effect has also been
studied in OSCC cells (Satyendra Chandra Tripathi, 2012) but its localization has not
yet been studied. Reduced DLC1 expression patterns are consistent with metastasis of
cancers as DLC1 was found to control hallmark signs of metastasis when reintroduced into certain cancer cells. (Wu, 2009) Our study aimed to clone DLC1 from
the plasmid and clone it into the destination vector that would be further transfected
to determine localization in SCC9 cells. The method has been followed in various
other genes as well to carry out histopathological studies and clinical analysis of
disseminated tumours. (Barbara A. Yoshida, 2000) This information can potentially
be used to screen patients effectively using markers mapped to certain chromosomal
locations and maybe even the determination of sub-cellular localization. This could
help in early intervention and control of metastasis, which has been the leading cause
of cancer-related deaths around the world. Such recombinant vectors showing
localization in certain carcinomas can be used to develop therapeutic mediators to
prevent rapid progression and deterioration of quality of life in patients with
metastasized tumours.
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7. Conclusion
This research project focused on expression of an anti-metastatic gene in a
mammalian vector by constructing a clone and using the recombinants to study
localization of the gene in cancer cell lines. This was attempted by traditional cloning
methods to create a plasmid expressing DLC1. Releasing insert from a commercially
available plasmid which was attempted to clone in the pEGFP-N2 vector that shows
expression in a strain of E.Coli cells, DH5α. Difficulties were encountered in
obtaining a large concentration of the insert, and hence proceeding with further
downstream purification and processes was not possible. After screening for positive
clones with insert through Colony PCR, they could have been transfected into the oral
Cancer cell line that was selected as a model to verify its localization. Further effects
on how the localization changes with respect to the tumour environment could have
been carried out. If localization of metastasis suppressors can be investigated using
already available recombinants carrying their genes, the same genes can be
comparatively studied under various adverse conditions like novel mutations on the
chromosome, different stressors in the cellular environment and can check the affinity
of drugs that target suppression of these cancers.
Page | 36
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