SHORT TERM PROJECT LAB REPORT

Supervised by: - Dr. Anindya Dasgupta

HOD of Biochemistry Department
Calcutta National Medical College
Kolkata – 700014
West Bengal, India

Presented by: - Debabrata Samanta

2nd year student, Biotechnology dept.

Haldia institute of technology

Haldia, West Bengal

Title:- Detection of a particular SNPs in VEGF 2
gene in renal cell cancer

Submitted By- DEBABRATA SAMANTA

SUPERVISED BY- DR. ANINDYA DASGUPTA
(HOD OF BIOCHEMISTRY DEPERTMENT)
 Abstract

Background: - The aim of the study is the significance of VEGF-2 (vascular endothelial
growth factor) gene and detection of +936 C/T polymorphism in VEGF-2 gene of renal
cell carcinoma (RCC) patients.

Methods: - A total of 20 patients with diagnosed RCC were enrolled in the study of
VEGF-2 gene expression. It was done by taking particular set of primer for VEGF-2
(+936 C/T) gene and determined by PCR based restriction fragment length
polymorphism (RFLP) method.

Result: - We have got genotypic distribution as well as Allelotypic distribution of RCC

patients in this study. 45% of the population had more dangerous CC genotype with
only 25% bearing the safer TT genotype. And 60% of alleles belong to wild safe allele
while the mutant T allele was only 40%.

Conclusion: - The C allele in +936 C/T SNP increases the synthesis of VEGF-2 gene.
Early use of inhibitor for VEGF-2 gene may stops the angiogenesis. Therefore, this
study revealed that VEGF +936 C/T polymorphism might be risk factor for RCC
patients.

Keywords: - VEGF 2 gene polymorphism, renal cell carcinoma, restriction fragment

length polymorphism method, Hind III.

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 Table of Content
Page No.

 ABSTRACT ---------------------------------------------------------- 01

 INTRODUCTION ------------------------------------------------- 03-07

 AIM AND OBJECTIVE---------------------------------------------- 08

 METHODOLOGY---------------------------------------------------- 09-15

 CALCULATION------------------------------------------------------- 16

 RESULT AND ALALYSIS------------------------------------------- 17-18

 DISCUSSION----------------------------------------------------------- 18

 BIBLIOGRAPHY------------------------------------------------------ 19-20

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 Introduction

Background of Project:-

Renal cell carcinoma (RCC) is also called hypernephroma, renal adenocarcinoma, or

renal or kidney cancer. It’s the most common kind of kidney cancer found in adults. Renal
cell carcinoma (RCC) is the most common malignancy of the kidney, accounting for
approximately 80%-85% of all renal tumors. [1]

The kidneys are organs in your body that help get rid of waste while also regulating fluid
balance. There are tiny tubes in the kidneys called tubules. These help filter the blood,
aid in excreting waste, and help make urine. RCC occurs when cancer cells start growing
uncontrollably in the lining of the tubules of the kidney. RCC is a fast-growing cancer and
often spreads to the lungs and surrounding organs. [2]

Renal Cell Carcinoma (RCC) has the highest mortality rate of the genitourinary cancers
and the incidence of RCC has risen steadily. In recent decades, the incidence of RCC
has been steadily rising by 2–4% each year and RCC is now the 7th leading cancer type
in men in the US. In 2010, it is projected that in the US there will be approximately 58,000
new cases of, and 13,000 deaths from, kidney cancer, the vast majority being RCC. [3]

In this project I have collected the data of human mortality as a cause of renal cell cancer.
In this project I have shown all SNPs of Vascular Endothelial Growth Factor (VEGF) gene
associated with RCC. From here we can know how VEGF gene is effected during RCC
(renal cell cancer). Also VEGF plays role in other cancer like breast, bladder etc. But here
we are focusing on +936 C/T polymorphism of VEGF 2 gene. From this study we can say
how much dangerous this polymorphism is for mankind.

***

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Importance of renal cell cancer as a cause of human mortality
RCC is most common types of cancer in worldwide. It comes on 8 th rank according to
data of National Cancer Institute of The U.S.

Estimated
New Estimated
Common Types of Cases Deaths
Rank Cancer 2018 2018
Breast Cancer
1. 266,120 40,920
(Female)
Lung and Bronchus
2. 234,030 154,050
Cancer
3. Prostate Cancer 164,690 29,430
4. Colorectal Cancer 140,250 50,630
Melanoma of the
5. 91,270 9,320
Skin
6. Bladder Cancer 81,190 17,240
Non-Hodgkin
7. 74,680 19,910
Lymphoma
Kidney and Renal
8. 65,340 14,970
Pelvis Cancer
9. Uterine Cancer 63,230 11,350
10. Leukemia 60,300 24,370

Kidney and renal pelvis cancer represents 3.8% of all new cancer
cases in the U.S. In 2018, it is estimated that there will be 65,340
new cases of kidney and renal pelvis cancer and an estimated
14,970 people will die of this disease. [4]

3.8%

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Kidney cancer is more common in men than women and among African Americans and
American Indian and Alaska Native populations. The number of new cases of kidney and
renal pelvis cancer was 15.9 per 100,000 men and women per year based on 2011-2015
cases. Kidney and renal pelvis cancer is most frequently diagnosed among people aged
65-74. Median age at diagnosis is 64 for RCC patients. The percent of kidney and renal
pelvis cancer deaths is highest among people aged 65-74. Median age at death for RCC
is 71. [4]

This is the rate of incidence of male and female who are affected by RCC. It is the data
of American Cancer Society (2019). Kidney cancer is more common in men than women
and among African Americans and American Indian and Alaska Native populations. [5]

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This is the data of death rate of male and female of US. This data is published in American
Cancer Society at 2019. [5]

***

Role of SNP in different gene responsible for RCC

The best characterized oncogenic gene in human RCC is the tumor suppressor gene von
Hippel-Lindau (VHL). VHL along with elongin B, elongin C and cullin 2 form an E3
ubiquitin-ligase complex, and are considered to serve an important role in RCC. [6]
Mutations in VHL can result in the over accumulation of HIF, and its target genes such as
VEGF (vascular endothelial growth factor), PDGF (platelet derived growth factor) and
EGRF (epidermal growth factor receptor), thus resulting in carcinogenesis. [7] There are
many researches focusing on the pathogenesis of RCC, but it remains unclear by now.
In recent years, the studies suggested that the genes of APOE, VHL and MTHFR were
involved in the onset of RCC. [8]
***

Importance of VEGF-2 gene Mutation in renal cell cancer

Angiogenesis plays an important role in growth, progression, and metastasis of tumors.
The most important regulator of angiogenesis is vascular endothelial growth factor
(VEGF). [9] Angiogenesis is the formation of new blood vessels from endothelial
precursors and it is one of the fundamental processes in carcinogenesis. [10,11] VEGF
also known as critical angiogenesis factor, could promote tumor development and
progression both in vitro and in vivo experiment. [12]

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The VEGF rs699947 polymorphism is obviously associated with an increased risk of
bladder cancer carcinoma, particularly in Asian population and renal cell. [13] And VEGF
polymorphisms may be associated with the progression and prognosis of RCC. Several
SNPs, such as VEGF -2578C/A, −1156G/A, +1612G/A, +936C/T, and -634G/C, have
been reported to be associated with cancer susceptibility, tumor growth and metastases
in RCC patients. [14-16] Among all the SNPs of the VEGF gene addressed, −2578C/A,
+936C/T, +1612G/A, −634G/C, +460T/C, +405 G/C, −1154G/A are the most common.
The process of carcinogenesis might be accelerated when the VEGF gene expression
was influenced by some functional gene variations [17]
This study suggests that −2578C/A and +460T/C polymorphisms of VEGF modulate the
risk of developing RCC in Chinese population. [18]
VEGF -2578C/A, +936C/T, +405 G/C polymorphisms are associated with an elevated
susceptibility to RCC, indicating that these three polymorphisms might be risk factors for
RCC, especially in Asian populations. Moreover, VEGF -1154G/A and -634C/G
polymorphisms are found significantly associated with poor prognosis of RCC and might
become a predicted biomarker in the future. [17]
Among several VEGF 2 mutation, +936 C/T polymorphism has been studied extensively
where it is found that +936 C/T SNP (rs- 3025039) potentiates the renal cell carcinoma
patients for worse prognosis and aggressive metastasis. This SNP can be identified by
restriction digestion of its PCR product (207 bp). If C allele is replaced by T allele, then
the restriction enzyme Hind III cuts it into 2 fragments (122 bp, 85 bp). And we have done
this for 20 population.
But the linkage between this SNP and worse prognosis of renal cell carcinoma has been
found to be much more inconsistent from place to place with significant variation between
different ethnic population groups.

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Aim:-

To detect the +936 C/T Single Nucleotide Polymorphism (SNP) of VEGF 2

gene in renal cell cancer.

Objective:-

I. To amplify the VEGF gene by polymerase chain reaction (PCR).

II. To find out +936 C/T SNP in PCR product of VEGF by using Restriction

Enzyme Hind III.

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 Methodology

 Collection of blood sample

Material required:-
Syringe needle, EDTA
Methodology:-
3 ml EDTA venous blood collected from a patient

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  Isolation and purification of DNA from human blood.
Principle:-
DNA was extracted from the cell using cell lysis buffer and nucleic lysis buffer. The
isolated DNA was purified from protein using, Tris saturated phenol and from lipid using
chloroform Isoamyl alcohol.
Material requirement:-
 Incubator
 Spectrophotometer
 Centrifugation refrigerant machine
 Hot air oven
 Hot water bath chamber
Reagent Required:-
 1 ml lysis buffer (10 mM EDTA, 50 mM Nacl, pH 7.5)
 Cell lysis buffer(5 mM Mgcl, 1% Triston X-100, 10 mM Tris Hcl, 320 mM
Saccharose, pH 7.5)
 Nucleic lysis buffer(400 micro liter, 10 mM Tris-Hcl, 10 mM EDTA, 50 mM Nacl,
pH 7.5)
 Tris Saturated phenol and chloroform
Procedure:-
 We take the blood sample. It suspended in 1 ml of erythrocyte lysis solution.
 We centrifuged the sample at 10000 rpm for 10 minutes.
 Then we add nucleic lysis buffer containing proteinase K (0.1 mg/ml).
 Then place it into incubator for 3 hrs. at 55˚ ºC.
 The solution is then added with Tris saturated phenol of pH=7.4 of amount 1ml.
 Then we again centrifuged it at 10000 rpm for 10 minutes.
 The supernatant is extracted carefully which should be devoid of proteins.
 Then we add 1 ml of chloroform: iso-amyl alcohol (24:1) to the solution.
 Solution is again centrifuged at 10000 rpm for 10 minutes.
 The supernatant will contain the DNA which is then precipitate with Sodium acetate
and chilled ethanol.
 At last extracted DNA is then washed with 70% ethanol, dried and stored in TEA
(Tris EDTA acetate) buffer and hence preserve for long duration.

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  Amplification of VEGF-2 (vascular endothelium growth
factor) gene using PCR (polymerase chain reaction).
Principle:-
Polymerase chain reaction (PCR) is a method widely used in molecular biology to make
many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA
sequence is exponentially amplified to generate thousands to millions of more copies of
that particular DNA segment. PCR employs two main reagents - primers and a DNA
polymerase. Almost all PCR applications employ a heat-stable DNA polymerase, such
as Taqpolymerase, an enzyme originally isolated from the thermophilic bacterium
Thermus Aquaticus.
PCR cycles include three different steps: denaturation of the template DNA into single
strands at about 90°C, a moderate temperature (around 50° to 60°C) to anneal the
primers to the target sequence, and an extension step in which DNA polymerase
elongates a second strand of DNA complementary to each strand of the target.
A machine called a thermo cycler is employed during the process of PCR to cycle
between several temperatures needed for the reactions to occur.
The first temperature in PCR is about 90°C. At this high temperature, the template DNA
is melted or denatured. The hydrogen bonds between the nitrogenous bases are broken
and the double-stranded helix becomes two single strands of DNA. The denaturation
stage of PCR takes about 1–2 minutes.
The thermo cycler then lowers the temperature to about 50° to 60°C, which allows the
short, oligonucleotide primers to anneal to their complementary sequences on the single-
stranded DNA molecules. The annealing stage of PCR lasts about 30 seconds.
Finally, the thermo cycler machine raises the temperature slightly to near 70°C, which is
the elongation step of PCR. In this step, DNA polymerase binds to the 3´ ends of each
primer and, using the single-stranded DNA as a template, incorporates new nucleotides
from a 5´ to 3´ direction. The length of this stage depends upon the length of the target
sequence.
Following elongation, the thermo cycler raises the temperature to 90°C, which starts the
denaturation cycle over again. Normally, PCR cycles 30–35 times through a set of
temperatures.

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Vascular endothelial growth factor (VEGF), originally known as vascular permeability
factor (VPF), is a signal protein produced by cells that stimulates the formation of blood
vessels. VEGF's normal function is to create new blood vessels during embryonic
development and new blood vessels after injury. When VEGF is overexpressed, it can
contribute to disease like cancer.

Material requirement:-
 Incubator
 Thermal cycler for PCR
 Centrifugation refrigerant machine
 Gel electrophoresis kit
 Hot water bath chamber
 Gel doc system

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Reagent Required:-
 PCR master mix
 All dNTP
 Taq polymerase
 Mgcl2 -1.5 mM
 Tris buffer (pH=8.5)
 Primer
 Forward primer(2 µl)
 Backward primer(2 µl)
 Template DNA(1 µl)
 Agarose gel (2%)
 TEA (Tris EDTA acetate) buffer

Procedure:-
 At first we take out all reagents from refrigerator.
 After that we make Master Mix by adding all dNTPs, Taq polymerase, Mgcl2, and
Tris buffer inside laminar air flow.
 Then we mixed two types of primer, template DNA and type 1 water with master
mix.
 We placed micro tube inside PCR thermo cycler machine.
 In this machine mainly three steps alters for 35 times to get the millions of gene
i.e. Denaturation (at 94°C), Annealing (at 50-70°C) and extension (at 72°C). It
takes 1 hr. 40 minutes to complete entire process.
 After this all process completed, we take out the sample from PCR machine.
 The sample is then pass through agarose gel (2%) to get the result. It takes 40-50
minutes to run the sample.
 We observe the agarose gel sample in gel doc system.

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  To observe restriction digestion of VEGF-2 in PCR
product by Hind III enzyme to detect the +936 C/T SNP
(Single Nucleotide Polymorphism).
Principle:-
RFLP (Restriction Fragment Length Polymorphism) is an enzymatic procedure for
separation and identification of desired fragments of DNA. Using restriction endonuclease
enzymes fragments of DNA is obtained and the desired fragment is detected by using
restriction probes. Southern hybridization using restriction endonuclease enzymes for
isolation of desired length of DNA fragments is an example of RFLP.
Here are some Application of RFLP test:
I. Genome mapping: helps in analysis of unique pattern in genome for organism
identification and differentiation. It also helps in determining recombination rate in
the loci between restriction sites.
II. Genetic disease analysis: After identification of gene for particular genetic or
hereditary disease, that gene can be analyzed among other family members.
III. To detect mutated gene.
IV. DNA finger printing (forensic test): It is the basis of DNA finger printing for paternity
test, criminal identification etc.
Hind III is a type II site-specific deoxyribonucleic restriction enzyme isolated
from influenza that cleaves the DNA palindromic sequence AAGCTT in the presence of
the cofactor Mg2+ via hydrolysis.
Hind III restrictions process results in formation of overhanging palindromic sticky ends.
The cleavage of this sequence between the AA's
results in 5' overhangs on the DNA called sticky
ends:
5'-A | A G C T T-3'
3'-T T C G A | A-5'

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Material requirement:-
 Incubator
 Gel electrophoresis kit
 Hot water bath chamber
 Gel doc system

Reagent Required:-
 Tango buffer - 3µl of pH=7.6
 Sample(PCR product) - 15µl
 Restriction endonuclease(Hind III) - 2µl
 Distilled Water - 15µl
 Agarose gel (3%)
 TEA buffer
 DNA ladder of 100 base pair

Procedure:-
 We, at first take the sample of 12 µl in micro tube.
 Then, we take 3 µl of Tango buffer i.e. prepared before and add to micro tube.
 We add 2 µl of restriction enzyme (Hind III) to the micro tube.
 After this, we mix 15 µl of distilled water to make the volume upto 35 µl.
 The micro tube is then allowed to incubate for an hour at 37°C.
 After incubation, gel electrophoresis is performed against DNA ladder track dye.
 After migration across the gel, the sample is placed under the gel doc system to
observe fragment of DNA.

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 Calculation

Concentration of DNA extracted from sample and its purity

DNA concentration = 50 µg/ml × OD at 260 nm × Dilution Factor

= 50 × 0.015 × 150

= 112.5 µg/ml

𝑂𝐷 𝑎𝑡 260 𝑛𝑚
Purity of DNA extracted =
𝑂𝐷 𝑎𝑡 280 𝑛𝑚

0.015
= = 2.14
0.007

Since, the ratio value is 2.14 which is greater than 1.5. Therefore, the extracted DNA is
pure and free of contamination.

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  Result and analysis:-
Figure 1 shows the patterns of distribution of different genotypes in agarose gel
electrophoresis.
Figure 1: Restriction digestion pattern of the PCR product of VEGF gene.

Lane 1: 100 bp ladder, Lane 2, 4, 5 and 7 show heterozygotes (CT) with 3 bands at 207
bp
and 122 bp and 85 bp. Lane 3 shows the uncut homozygote (CC) at 207 bp. Lane 6
shows the cut homozygote (TT) with two bands at 122 and 85 bp regions (Lane numbers
are counted from left to right).
Data obtained from 20 cases of renal cell carcinoma were tabulated in form of genotypes
and Allelotypic in Table 1 and Table 2 respectively.
Table 1: Genotypic distribution of renal cell cancer patients in the present study (n = 20)
CC (uncut) homozygote CT (partially cut) TT (completely cut)
heterozygote homozygote
9 (45%) 6 (30%) 5 (25%)

Data showed that 45 % of the total cases studied had the more dangerous CC genotype
which has been reported in several studies as non-linked genotype with renal cell
cancers. On the other hand, 25% of the study population contained the mutant genotype
TT that was reported to be protective against renal cell cancers. To study the distribution

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of the C and T alleles in the study group, number of homozygote genotypes were
multiplied by two followed by addition of the total number of heterozygotes. Data for allelic
distribution are shown in Table 2.
Table 2: Allelotypic distribution of renal cell cancer patients in the present study.
C (wild) allele T (mutant) allele
24 (60%) 16 (40%)

It is evident from the Table 2 that 60% of the alleles belong to wild safe allele while the
mutant T allele was only 40%.

 Discussion:-
The present study showed that 45% of the population had the more dangerous CC
genotype with only 25% bearing the safer TT variety among the renal cell cancer patients.
Our data is a little bit lower than other observations earlier which suggested an association
between the +936 C/T SNP in the VEGF gene and renal cell cancer. Shen et al (2015),
Lu et al (2015) and Xian et al reported 62%, 63% and 26% of prevalence of TT allele
among the renal cell cancer patients(14,18,19). The “C” allele in the +936 C/T SNP
increases the synthesis of VEGF that in turn potentiates the chance for metastasis
through newly formed blood vessels.
Hence, several drugs like inhibitors of VEGF are being used as inhibiting agents for risk
of metastasis where angiogenesis is supposed to play a major critical role. In view of this,
early use of VEGF inhibitors may invoke an effective method of treatment against
prognostically worse varieties of renal cell cancers.

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