Bioinformatic Prediction Of SNPs Within MiRNA Binding Sites Of Inflammatory Genes Associated With Gastric Cancer And A Preliminary Study Of MiR-328

ObjectiveInflammation is an important factor leading to gastric cancer. miRNA binding toinflammatory genes acts as positive or negative regulation role in the occurrence anddevelopment of gastric cancer. However, studies on single nucleotide polymorphisms(SNPs) of inflammatory genes and miRNAs were not inadequate andcomprehessive.the design of this tudy is to retrieve SNPs within miRNAs bindingsites of inflammatory genes associated with gastric cancer and the related miRNAscomprehensively and get the effect of SNPs on miRNA binding site, in order toprovide theoretical data for subsequent control-study of gastric cancer, and study ofmiRNAs’ fuctions and the target of SNPs; and to investigate whether theinflammatory gene IL10RB associated with gastric cancer is the target of miR-328.MethodInflammatory genes related to gastric cancer were selected from theexisting domestic and foreign literature database. And a complete list of SNPs in3-untranslated region (3’UTR) of these genes in HCB was identified from dbSNP.Bioinformatic methods and miRNA target prediction databases were used to predictwhether there are SNPs of these genes within miRNA binding sites.Based on the prediction of miRNAs and SNPs from the first step and previousstudy on the relationship between IL10RB and gastric cancer, over expressionvector of miR-328was generated by using molecular cloning technique, thencombined with miR-328inhibitor and control, tranfected into gastric cancer cellsSGC7901. Total RNA of cells was extracted and reverse transcription PCR was used.The expression of miR-328and IL10RB of the four groups were examed byreal time PCR technique. SPSS21.0software and t test were used to analysis therelative expression of miR-328and IL10RB.Results(1)72inflammatory genes were found to be in relation to gastric cancer through database retrieval. Among these genes, there are99SNPs in3’UTR regions of47genes. After using bioinformatic methods and miRNA target prediction databases, themiRNA binding sites were found in47SNPs of25inflammatory genes. And therewere95putative miRNAs.(2) Over expression vector of miR-328was constructed successfully by usingrestriction enzyme digestion and sequence analysis.(3) Real time PCR results showed that over expression vector of miR-328andinhibitors were transfected into cells successfully. Compared with control group,relative expression of miR-328in pGenesil-1-miR-328group is significantly higher(P<0.001), the relative expression of which are respectively3.17±0.31and0.26±0.05Compared with miR-328inhibitor control group, Relative expression of miR-328inmiR-328inhibitor group is significantly higher (P=0.005), the relative expression ofwhich are respectively0.12±0.04and1.05±0.29. However, the relative expression ofIL10RB in pGenesil-1-miR-328group and pGenesil-1control group are respectively0.52±0.02and0.78±0.21, the difference of which is not significant (P=0.097).While,the relative expression of IL10RB in miR-328inhibitor group and miR-328inhibitorcontrol group are respectively1.02±0.02and0.83±0.12, and the difference is nosignificant(P=0.054).ConclusionBioinformatic methods could identify a set of SNPs within miRNA binding sitesof the inflammatory genes associated with gastric cancer and the correspondingmiRNAs. miRNA binding site was affected by SNP in the3’UTR of target gene.IL10RB gene may not be the target gene of miR-328.