The Single Nucleotide Polymorphisms And Expression Of HPOT1 And HTERT On Infection Of H.pylori And The Microsatellite Instability In Gastric Carcinoma

Gastric cancer(GC) is one of the most common malignant tumors in china, which is a multi-gene based disease caused by the interaction between enviromental and genetic factors. A large amount of epidemiological evidence has accumulated indicating a significant relationship between Helicobacterpylori (H.pylori,Hp) infection and gastric adenocarcinoma development. In 1994, the World Health Organization/International Agency for Research on Cancer concluded that Hp is a definite carcinogen based on the epidemiological findings.Single nucleotide polymorphisms(SNPs) is nucleotide sequent polymorphism caused by single-base mutation of genome that has been generally thought to be the third era mark of heredity following microsatellite. Vast investigation of the SNPs data has shown that it can provide us with new approaches to understand multi-gene involved diseases and to evaluate the inter-individual and inter-groups difference in tumor risks and treatment response.Through the common study of SNPs in seven genes of telomere (TERT,TERC, TERF1,TERF2,TINF2,TERF2IP,POT1 andTNKS), it can be presumed that these variants will be of significant meaning in study of gene-associated diseases as cancer, aplastic anemia,autoimmune diseases,and degenerative disorders,as well as chromosomal abnormalities and/or abnormal telomeres. Human protection of telomeres 1(hPOT1), a member of the telomeric family, is a telomeric single-strand DNA binding protein that widely exist in eucaryotic cell as a house-keeping gene. It plays an important role in regulating telomeric length, attendancing telomeric function and protecting chromosome stability. Telomeric disfunction is a common occurrence in genom instability, which may trigger the development of gastric cancer. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of human telomerase, which regulates the telomer length by taking part in telomerase activity. An average of over 85 percent of human malignant tumors has telomerase expression, which can induce the infinite generation of cells and participate in tumorigenesis. However, in normal adult cells and tissues, there is no telomerase activity or telomerase expression. Therefore, it draws increasingly attention at its role in tumorigenesis.In current study we investigated the SNPs of hPOT1IVS13-98G/T and hTERT Ex2-659A/G, the expression of hPOT1,hTERT protein and microsatellite instability, with the hope of understanding the correlation between GC risk and SNPs of hPOT1,hTERT.MATERIALS AND METHODSOne hundred and sixty-eight cancer and corresponding normal tissues were obtained from surgically resected gastric carcinoma in three hospitals of west Gansu,China. A 5cm section was cut from each tissue and stained with hematoxylin/ eosin in order to ascertain whether the cancer cells in tissues were predominant or not. Normal mucosa was collected from local residents who come for regular health examining, exclused chronic gastritis, intestinal metaplasia, atypical hyperplasia, ulceration and canceration under gastroscopy. These samples were frozen immediately and stored at -70℃until analyzed.Age and sex of two groups has been matched and ethical approval has been informed consent. All carcinomas were staged according to UICC criteria and typed according to WHO 2000 criteria of histopathological classification. Genomic DNA was isolated by standard proteinase-K digestion and phenol-chloroform extraction protocols. SNPs were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP),microsatellite instability were detected by PCR-single strand conformation polymorphism (PCR-SSCP). The expression of hPOT1 and hTERT protein was detected by immunohistochemistry method. H.pylori infection was detected by PCR of urea enzyme gene of H.pylori and Warthin- Starry.RESULTSThe frequencies of TT,GT and GG genotypes of hPOT1IVS13-98 G/T were 36.90%,41.67% and 21.43 % in patients with gastric cancer 24.36%, 51.92% and 23.72% in controls,respectively. The OR for any GT and GG genotype were 0.439 (95 %CI : 0.251-0.767,P=0.004) and 0.514(95%CI: 0.264-0.999,P=0.050) when compared with TT genotype, respectively. There were no significantly difference in genotype distribution and allele frequency of hPOT1IVS13-98 G/T polymorphism (χ2=3.5853,P= 0.0583). The frequencies of GG, AG and AA genotypes of hTERTEx2-659A/G were 41.88%, 47.5% and 10.63% in patients with gastric cancer and 28.29%, 56.58% and 15.13% in controls,respectively. The OR for any AG and AA genotype were 0.519 (95%CI:0.310-0.870, P=0.013), 0.550(95%CI: 0.255-1.188, P=0.128) when compared with GG genotype,respectively. There were significant difference in genotype distribution and allele frequency of hTERTEx2-659A/G polymorphism (χ2=5.3734, P=0.0204).The positive expression ratio of hPOT1 in neoplasms, gastric atypical hyperplasia, intestinal metaplasia, normal mucosa were respectively 91.33%, 80.00%, 55.00% and 28.00%, and hTERT positive expression rate were 76.67%, 55.00%, 25.00%, and 0.00% , respectttively.All the three parameters significantly increased in atypical hyperplasia, neoplasms and intestinal metaplasia compared to normal mucosa( P < 0.05). Expression of hPOT1 protein increased significantly inⅢ/Ⅳstaging gastric cancer with deep invading and bad differentiation (P <0.01), Expression of hTERT protein increased significantly in poorly differentiated group ( P <0.01). There were significant relationships among the two parameters in neoplasms and dysplasia,respectively(rGC=0.212,P =0.009;rDYS=0.504,P =0.023).The ratio of H.pylori infection in gastric cancer, atypical hyperplasia, intestinal metaplasia and normal mucosa were 54.00%,70.00%,55.00% and 20.00,% respectively. Among three genotypes of hPOT1IVS13-98G/T, the ratio of H.pylori infection were 51.52% in GG, 45.16% in GT and 65.45% in TT, with no difference among the three genotype (χ2= 4.937,P=0.085).It strongly implied that hPOT1IVS13-98 G/T polymorphism was not correlated with H.pylori infection in gastric cancer. Of the three genotype of hTERT Ex2- 659A/G, the ratio of H.pylori infection were 66.67% in AA, 52.86% in AG and 50.88% in GG, there were no significant difference among three genotype (χ2=1.215,P=0.545).This results suggested that hTERTEx2-659A/G polymorphism was not correlated with H.pylori infection in gastric cancer. The positive rate of hPOT1 protein in patients with H.pylori infection (96.30%) was significantly higher than that of patients with H.pylori infection negative (85.51%,P < 0.05).The positive ratio of hTERT protein of patients with H.pylori infection positive (85.19%) was significantly higher than that of patients with H.pylori infection negative (71.01%, P < 0.05) .Seventy-five cases of gastric carcinoma were studied for MSI by using two microsatellite markers. The positive ratio of MSI was detected in 29 of 75(38.67%) in two microsatellite markers, among which 9(12.00%) was detected at BAT26 locus and 20 (26.67%) was detected at D5S346 locus. MSI is not to related to sex, age,invading to serosa and histological type (P >0.05). However,the frequence of MSI in groups without lymph node metastasis and I and II staging is significantly higher than that of groups with lymph node metastasis and staging III and IV(P<0.01). MSI was not correlated with hPOT1 IVS13-98G/T and TERTEx2-659A/G polymorphism in gastric cancer; MSI was not correlated with H.pylori infection in gastric cancer.CONCUSIONOur results indicate:①IVS13-98 G/TSNP of hPOT1 gene and Ex2-659A/GSNP of hTERT gene are probably associated with reduced risk for gastric carcinoma of west Gansu,China.②Expressions of hPOT1 and hTERT are up-regulated at an early stage of the gastric carcinonogenesis and they may play an important role in activity of telomerase. It may be useful to detect expression of hPOT1 and hTERT for predicting the biological behevor of gastric carcinoma.③H.pylori infection is not associated with IVS13-98 G/T SNP of hPOT1 and Ex2-659A/G SNP of hTERT, but associated with expression of hPOT1 and hTERT. There were no significant relationships between the IVS13-98G/TSNP and expression of hPOT1 in gastric carcinoma. There were no significant relationships between in gastric carcinoma.④MSI pathway is not related to hPOT1 IVS13-98 G/T, hTERTEx2- 659A/G polymorphism and H.pylori infection in gastric cancer.