Effects Of Gene Sequence Polymorphisms In Functional Study Of Susceptible Genes And On Resistance Of FMDV To RNAi

With the development of human genome project, gene sequence polymorphism increasingly shows comprehensive promise in pathogenesis of complex disease, individual difference in drug susceptibility, existence and resistance of virus, and other aspects in biomedicine. In this study, the effect of a single nucleotide polymorphism within 5'UTR of TNF-αgene on the expression of AS susceptible gene TNF-α, the effect of an insertion/deletion polymorphism site within 3'UTR of IGF2R gene on the expression of type 2 diabetes susceptible gene IGF2R and the effect of a variation site within FMDV RNA genome on the resistance of FMDV for siRNA targeting were explored, separately. The verification of functional effects of gene sequence polymorphism/variation from different control layer will contribute to the elucidation for those issues, such as the mechanism underlying the susceptibility to specific complex disease, resistance of virus for antiviral gene therapeutics, and so on.PartⅠ: Effects of a single nucleotide polymorphism within gene 5'UTR region on the expression of human TNF-αTumor necrosis factorα(TNFα) plays a prominent role in inflammation and is a proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious disease. Recent association studies have found that the TNFα-857T allele was associated with several disorders. Here we demonstrate, with reporter genes under the control of the two allelic TNFαpromoters, that the minor allele -857T is a much stronger transcriptional activator than the major allele -857C in RAW264.7 cell line in response to lipopolysaccharide (LPS) stimulation. However, the result was not consistent in HeLa cell line. Furthermore, for the quantitative analysis of TNFαsynthesis between the -857C/C genotype from healthy subjects and the -857C/T genotype from AS patients, the quantitative reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed, separately. There was no significant difference between the two groups at the level of mRNA and protein. These results show that this polymorphism may have a direct effect on TNFαregulation in a tissue-specific manner, and aside from the polymorphism at -857 in the TNFαpromoter, there may be other factors affecting the expression of TNFα. PartⅡ: Effects of an insertion/deletion polymorphism within gene 3'UTR region on the expression of human insulin-like growth factor 2 receptorThe biological mechanism of a recent discovered association of type 2 diabetes with the ACAA-insertion/deletion polymorphism at the 3'UTR of the IGF2R gene has remained unclear. A very recently emerging novel polymorphic control layer by microRNAs (miRNAs) makes it possible to elucidate this issue. In this study, a prediction from web tools microInspector and miRanda demonstrated that DNA sequence polymorphism (DSPs) ACAA-insertion/deletion in IGF2R 3'UTR is located within hsa-miR-657 and hsa-miR-453 binding sites. And luciferase reporter assay revealed that hsa-miR-657 acts directly at the 3'-UTR of the IGF2R. Furthermore, ACAA-deletion exerted a further repression compared with ACAA-insertion, indicating that hsa-miR-657 regulates IGF2R gene expression in a polymorphic control manner. Importantly, we also demonstrated that hsa-miR-657 can translationally regulate the IGF2R expression levels in HepG2 cells. Thus, our findings testify the possibility that the ACAA-insertion/deletion polymorphism may result in the change of IGF2R expression levels at least in part by hsa-miR-657-mediated regulation, contributing to the elucidation for the pathogenesis of type 2 diabetes and raise the possibility that miRNAs or in combination with functional DNA sequence polymorphism may be valuable in the treatment of human type 2 diabetes.PartⅢ: Effects of a variation within VP1 gene on the resistance of FMDV to RNAiFoot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a severe, clinically acute, vesicular disease of cloven-hoofed animals. RNA interference (RNAi) is a mechanism for silencing gene expression posttranscriptionally that is being exploited as a rapid antiviral strategy. To identify efficacious small interfering RNAs (siRNAs) to inhibit the replication of FMDV, candidate siRNAs corresponding to FMDV VP1 gene were designed and synthesized in vitro using T7 RNA polymerase. In reporter assays, five siRNAs showed significant sequence-specific silencing effects on the expression of VP1-EGFP fusion protein from plasmid pVP1-EGFP-N1, which was cotransfected with siRNA into 293T cells. Furthermore, using RT-qPCR, viral titration and viability assay, we identified VP1-siRNA517, VP1-siRNA113 and VP1-siRNA519 that transiently acted as potent inhibitors of FMDV replication when BHK-21 cells were infected with FMDV. In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV, and that FMDV may escape the antiviral activity of siRNA by a specific single nucleotide variation in targeted sequence. Second generation siRNAs that recognize the variatioin may be recommended to prevent the emergence of resistant FMDV.In conclusion, our results demonstrate that -857SNP within TNF-α5'UTR promoter region could change the promoter transcription activity, contributing to the susceptibility to AS, that ACAA-insertion/deletion polymorphism could stabilize the interaction between hsa-miR-657 and IGF2R, contributing to the susceptibility to type 2 diabetes, and that the 410nt variation within VP1 gene region could decrease the inhibition of a specific siRNA targeting FMDV, indicating the need for a new generation of siRNAs corresponding to virus genome variation. Therefore, the verified effects of three different type of gene sequence polymorphism/variation on related gene expression show novel molecular models for pathogenesis of complex disease and virus escape from antiviral therapeutics.