ITGA2 C807T Polymorphism And Risk Of Colorectal Adenoma And Colorectal Cancer

Backgroud and aimsColorectal cancer(CRC) is the most common neoplasm of the gastrointestinal tract in humans and the third cause of cancer death worldwide. With the improvement of people’s living standard and the change of dietary structure, the incidence of CRC is ascending in China and ranking from the third to the fifth. Since 1978, Morson put forward that CRC occurs according to the pattern of adenoma progressing to adenocarcinoma. A great deal of research indicated that normal mucosa can progress to colorectal adenoma(CRA), eventually develping to advanced CRC. Colorectal carcinogenesis is a complicated multistep progress involving the interaction of many genetic and environmental factors.Environmental factors mainly include poor dietary habit(an irrational food structure:high fat, high protein, high quantity of heat, low cellulose, etc.),tobacco smoking,carcinogenic factors and so on.Nevertheless, Not all the persons who are exposed to the above environmental factors will develop CRC. In a word, the occurrence of lagre intestine tumor is associated with certain genetic susceptibility. Genetic polymorphisms have been recently considered as the main genetic elements involved in the development of colorectal cancer. Therefore,it is particularly important to study the relative genes.A single nucleotide polymorphism (SNP) is a source variance in a genome. A SNP is a single base mutation in DNA. SNPs are the simplest form and most common source of genetic polymorphism in the human genome (90% of all human DNA polymorphisms). In the human genome, an average of about one out of every 1000 base pairs is a SNP, the degree of which within a population is not less than 1%. Moreover, SNPs have been recently considered to be play a major role in the susceptibility and resistance to diseases, the diversity of clinical manifestations and the difference of different individual responses to drugs.Only the non-synonymous SNPs (nsSNPs), also called as missense variants are particularly important as they result in to changes in the translated amino acid residue sequence. It is likely that nsSNPs play a major role in the functional diversity of coded proteins in human populations and have been linked with many diseases. nsSNPs may affect the protein function by reducing protein solubility or by destabilizing protein structure and they may affect gene regulation by altering transcription and translation. Our previous epidemiological studies provided the evidence that genetic polymorphisms were closely associated with the risk of large intestine tumor.Integrins are members of a family of cell-surface heterodimeric proteins that cell-extracellular matrix and cell-cell interactions. The 18 α-subunits (α1 ～α11、 αD、 αE、αL、αM、αV、αX、αⅡb) and 8 β-subunits(β1～β8) form together at least 25 different integrins, each Pair being specific for a unique set of ligands. Integrins such as α2β1, αⅡbβ3 and αvβ3 are known as key factors for tumorgenesis and progression. The α2 integrin subunit exclusively with the β1 integrin subunit forms the heterodimer α2β1, which almost expressed on all the epithelial cells. Integrin α2β1, is also considered as platelet glycoprotein Ⅰα-Ⅱα and its level of expression in tumor cells is bound up with motility, invasion and differentiation. Besides, several studies have shown that integrin α2β1 expression is closely associated with large intestinal epithelium differentiation and proliferation, invasion and metastasis of colorectal cancer cell.The integrin, a2 gene (ITGA2) is located on chromosome 5q23-31. A silent change in the coding region at nucleotide 807 (TTT/TTC at codon Phe253) has been identified. The C807T single nucleotide Polymorphism (NCBI SNP ID:rs1126643) of the ITGA2 gene affected the integrin α2β1 density. The genotype 807 TT was related to the highest receptor density and the genotype 807 cc with a lowest density. Recent studies showed that ITGA2 C807T gene polymorphism are closely related to all kinds of diseases, including breast cancer, ovarian cancer, prostate cancer, gastric cancer, ischemic stroke, retinal vein occlusion, acute coronary syndrome. However, as far as we know, In China there has been no study that evaluated the association between the polymorphism and the risk of colorectal adenoma and cancer. And the prior studies suggested that the integrin α2β1 density and its functional change were involved with the develoPment and metastasis of colorectal cancer. Hence, In order to make clear that the correlation between ITGA2 C807T gene polymorphism with the risk of CRA and CRC, we conducted a hospital-based case-control study in a Jiangxi han population.Methods1.Subjects:This hospital-based case-control study consisted of 232 individuals including 89 with histologically confirmed CRC,48 with histologically confirmed CRA and 95 normal individuals as controls.The CRC patients were without synchronous and/or metachronous secondary malignancy and didn’t accept chemotherapy and radiotherapy. The control group comprised 95 healthy people without tumor or history of genetic disease who received physical examination in our hospital during the same time period. All subjects were of unrelated Han nationality from Pingxiang in Jiangxi province or its surrounding regions.2.The collection of 3 groups peripheral venous blood samples and 232 individual information:The study was approved by the Ethics Committee of the Affiliated Pingxiang Hospital of Southern Medical University and informed consent was obtained from all the participating subjects. Two milliliter of peripheral blood was collected and reserved in the minus eighty degree laboratory freezer. Information on the family history of colorectal cancer, residence (urban or rural), age, gender, body weight, smoking, drinking alcohol and drinking tea status was collected by questionnaire.Individuals who formerly or currently drunk more than one time per day for at least 3 months were defined as drinkers. Individuals who formerly or currently smoked ≥10 cigarettes per day for at least 2 years were defined as smokers. Individuals who formerly or currently drunk tea more than twice per day for at least 3 months were defined as tea drinkers. More than one in the relatives and/or more than two secondary relative risk of colorectal cancer was defined as a positive family history of colorectal cancer. Depth of tumor invasion and local lymph node status metastasis were classified in accordance with the TNM classification criteria of International Union Against Cancer. Differentiation was graded in line with World Health organization classification.3.Leukocyte DNA was extracted by DNA extraction kit in strict accordance with the instructions.4.The polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP) was used to identify the ITGA2 C807T genotypes. ITGA2 was amplified by PCR. The reaction was performed using 0.15 mmol/L dNTP,1.75 mmol/L MgCl2,2 μL 10 × PCR buffer,0.25 μmol/L each primer(forward 5’-GTGTTTAACTTGAACACATAT-3’,reverse5’-ACCTTGCATATTGAATTGCTT-3’ ),150 ng genomic DNA and 1 unit Taq polymerase,brought to a 20μL total reaction volume with sterilised double-distilled water. Amplification proceeded in a thermal cycler (Bio-Rad) under the following conditions:95℃ for 5 minutes;and 94℃ for 30 seconds,55℃ for 30 seconds,72℃ for 1 minutes and 72℃ for 5 minutes for 35 cycles. The 115 bp PCR amplification products were digested with restriction enzyme Taq I at 65 ℃ for 12 h, and then separated on a 3% bromide-stained agarose gel stained with GoldenviewTM.Eventually, electrophoresis products were analyzed by ultraviolet analyzer and in the meantime visualised by digital camera,which meant the results would be analysised with the semi-quantitative assessment method.5.1n addition, a 10% masked samples were randomly selected and retested, and the reproducibility was 100%.6.Statistical analysis:Statistical analyses were performed using SPSS 16.0 software. All statistical tests were two-tailed and a P-value of less than 0.05 was defined as a statistical significance. Quantitative variables departing from the normal distribution were analyzed by Mann-Whitney rank sum test. Pearson’s x2 test was used to compare the difference in the distribution of classified variables and genotype frequencies between cases and controls. The Hardy-Weinberg equilibrium of the ITGA2 genotypes was estimated for cases and controls by a goodness-offit x2 test. Odds ratio (OR) and 95% confidence interval(CI) were calculated to evaluate the association between genetic polymorphisms and the risk of CRA and CRC. The adjusted OR was calculated by unconditional logistic regression method, with adjustment for age, gender, tobacco smoking, alcohol drinking, tea drinking, hypertension, diabetes and family history of colorectal cancer.7.Evaluation of ITGA2 gene polymorphism and genetic susceptibility to CRA and CRC. 1)Demographic information of the total of subjects.2)ITGA2 genotype distribution in cases and controls and risk assessment.3)Sratification analysis of ITGA2 polymorphism and the risk of CRA and CRC.4)Analysis of polymorphism and the clinicopathological characteristics of CRC.Results1.The wild-type homozygotes (CC) produced two bands at 92 and 23 bp, while the variant homozygotes (TT) produced one band at 115 bp, and the heterozygous (CT) produced three bands at 115,92 and 23 bp.2.The gender in cases and controls was not statistically significant.CRA group and control group had no statistical differences in age(within five years), Relatively, the age of CRC patients was older.Therefore,age and gender in all the subjects were matched successfully. Statistical analysis by diabetes history, alcohol drinking, tea drinking and marital status found that the correlation was not statistically noteworthy in both cases and controls.Whereas, Statistical analysis by hypertension family, family history of CRC, educational background, occupation and residence indicated that a significant association was observed in both cases and controls. In comparison with controls, both the medians of Body Mass Index and height and the mean of body mass were lower in cases. Statistical analysis according to smoking status shown an statistical differences between CRA group and normal group.3.Genotype and allele frequencies of ITGA2 C807T were significantly different between patients and controls (P<0.05), but no difference was detected in genotype or allele frequencies between CRA and controls. Compared with individuals with the wild genotype CC, subjects with the variant genotypes (CT+TT) had a significantly higher risk of CRA and CRC.4.Stratified analysis showed that the variant genotypes were not associated with the increased risk of CRA. In stratified analyses, the elevated CRC risk was especially evident in older individuals females, smokers, drinkers, highly-educated background, mental workers and urban subjects. However, when stratified by gender, the elevated risk of CRA associated with the variant genotypes was not noteworthy.5.When stratified by clinicopathological features such as lesion distribution, pathology subtypes, tumuor size, differentiation degree, depth of invasion, lymph node metastasis and Duke’s stage in the patients with CRC, no associations were observed in the polymorphism distributions.Conclusions1. Individuals with T allele(CT and TT) may have significantly evaluated risk to develop CRA and CRC.2.There might be a significant association of ITGA2 C807T polymorphism with increased risk of CRC among individuals of older individuals females, smokers, drinkers, highly-educated background, mental workers and urban subjects,which means those above individuals may have noteworthy evaluated risk to develop CRC.3.There is no significant association between ITGA2 C807T polymorphism with the clinicopathological features of CRC patients.