| As one of the most common digestive malignant tumors, the incidence and mortality of gastric cancer(GC) have kept high in China during the past few decades. GC has become a public health issues, bringing a serious burden for individual, society and country in the long run. Numerous studies showed that GC was a complex desease with many risk factors,such as infection, dietary, environmental and genetic factors. Increasing evidence demonstrated Helicobacter pylori(H. pylori) infection was one of the most important factors in GC. But familial GC only accounts for 1% to 3% of all GC in China. The sporadic GC accounts for more than 90% and shows weaker genetic susceptibility,which is probably related to gene single nucleotide polymorphisms(SNPs).Many related researches, both in China and abroad, confirmed the SNPs of inflammatory pathway gene are closely related to inflammatory response before the development of GC. Micro RNA(mi RNA) can regulate inflammatory gene expression by targeting the 3’ untranslated region(3’-UTR) of inflammatory cytokines gene. The SNPsin the 3’-UTR of inflammatory gene maylocated in mi RNA target sites, called mi R-SNPs, which would strengthen or weaken mi RNA-mediated regulation. The above factors could cause immune response and have been shown to connected with GC progression.Objectives This case-control study aimed to examine whether four mi R-SNPs in three IL23/Th17 genes(rs3748067 of IL17 A,rs10889677 of IL23 R rs887796, rs1468488 of IL17RA) were associated with GC risk, and then explored their gene-environmental interactions and their roles in the GC progression. According to the above results, we chose the mi R-SNPs for following functional analysis, which had the strongest correlation with GC.Under the above results, we would explore the mechanisms of mi RNA-mediated regulation of IL23/Th17 pathway genes and progression of GC, which will provide deep insights into GC progression, biomarkers for prevention and early diagnosis of GC.Methods Potential mi R-SNPs of IL23/ Th17 pathway genes were searched the db SNP database by using bioinformatics methods, and selected according to corresponding standards. Then mi RNA target prediction programs were used to find those mi RNAs which may bind to related mi R-SNPs. Finally, the binding free energy for both alleles were assessed by RNAhybrid. This case-control study included 500 GC patients from the First Affiliated Hospital of Zhengzhou University and 500 healthy controls from community. Patients were matched by controls with sex and age(± 5 years). Polymerase chain reaction–restriction fragment length polymorphism(PCR-RFLP) were used for genotyping of the four mi R-SNPs. Goodness-of-fit chi-squared test was used for testing Hardy-Weinberg Equilibrium(HWE). The connection of selected mi R-SNPs and GC risk was performed by unconditional logistic regression. Haplotype analyses was evaluated by an online tool: SHEsis. Gene-environment and gene-gene interactions were accomplished by MDR 2.0 software. The above statistical analysis was tested by SPSS 21.0 software. Mi R-10a-3p over expression vector, mi R-10a-3p inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells respectively, then mi R-10a-3p and IL17 A relative expression were detected by q RT-PCR. IL17 Aprotein expression were detected by using Western-blot. Relative luciferase activity was detected with double luciferase report genes, to determine whether IL17 A is atarget gene of mi R-10a-3p.Results(1) The individuals with CT genotype(ORadjusted=0.59; 95%CI: 0.44-0.79) and TC+TT carriers(ORadjusted=0.58; 95%CI: 0.43-0.77) of IL17 A rs3748067, compared with CC genotype, were associated with decreased GC risk. IL23 R rs10889677 CC genotype(ORadjusted =2.22; 95%CI: 1.27-3.87) carriers had an increased GC risk.(2) Stratified analyses results showed, among those people, who were men (ORadjusted =0.55; 95%CI: 0.40-0.75), or with age > 50 years(ORadjusted =0.57; 95%CI: 0.41-0.78), or who ever smoked(ORadjusted=0.43; 95%CI: 0.29-0.63) or drank(ORadjusted=0.50; 95%CI: 0.31-0.79) or without family history of GC(ORadjusted =0.58; 95%CI: 0.43-0.79), IL17 A rs3748067 had an obviously decreased GC risk. Meanwhile, among those people, with a smoking history(ORadjusted =1.40; 95%CI: 1.05-1.85) or without family history of GC(ORadjusted =1.33; 95%CI: 1.06-1.66), IL23 R rs10889677 had a significantly higher risk of GC.(3) Analysis of gene- environment interactions showed GC risk was 2.25 fold in those who ever smoked, with GC family history, and IL17 A rs3748067 TC+TT genotype.(4) q RT-PCR results showed, mi R-21 over expression vector, mi R-21 inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells respectively. Relative expression of mi R-21 and PTEN was significant difference with their respective control group by t test(P<0.05). Additionally, mi R-10a-3p over expression vector, mi R-10a-3p inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells respectively. Relative expression of mi R-10a-3p and IL17 A was significant difference with their respective control group by t test(P<0.05).(5) Western-blot results showed, mi R-21 over expression vector, mi R-21 inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells respectively. Relative expression of PTEN protein was significant difference with their respective control group by t test(P<0.05). Additionally, mi R-10a-3p over expression vector, mi R-10a-3p inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells respectively. Relative expression of IL17 A protein was significant difference with their respective control group in SGC-7901 cell line by t test(P<0.05). However, relative expression of IL17 A protein was not statistically significant in BGC-823 cell line(P>0.05).(6) Luciferase report gene assay results showed, relative luciferase activityof IL17A3’-UTR-luciferase reporter vector and mi R-10a-3p mimic transient cotransfection group was significant difference with its control group by t test(P<0.05). The relative luciferase activitydifference between IL17A3’-UTR-luciferase reporter vector and mi R-10a-3p inhibitor transient cotransfection group and its control group, was also statistically significant(P<0.05).Conclusion(1) IL17 A rs3748067 CT and TC+CC genotypes could decrease the risk of GC, while IL23 R rs10889677 CC genotype could increase the GC risk.The gene-environment interactions between smokers, drinkers, GC family history and rs3748067 might improve probability of the development of GC.(2) After mi R-10a-3p over expression vector, mi R-10a-3p inhibitor and their respective control group transfected to SGC-7901 and BGC-823 cells, IL17 A m RNA and protein expression could be regulated by mi R-10a-3p in SGC-7901 cells, but not in BGC-823 cells.(3) Luciferase report gene assay verified that mi R-10a-3p could regulate IL17 A expression by binding IL17A-3’-UTR, suggesting IL17 A was a target gene of mi R-10a-3p. |
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