miR-134靶基因筛选与鉴定在卵巢癌耐药中的应用研究

目的 应用Hybrid-PCR 技术，筛选 miR-134在人卵巢癌紫杉醇耐药SKOV3-TR30细胞中潜在靶基因，为卵巢癌耐药的研究提供理论依据。方法 2011年9月至2013年9月在中国医科大学附属盛京医院通过RNA抽提，Hybrid-PCR 引物设计，Hybrid-PCR及测序，BioEdit和DNAclub分析所有测序结果并运用BLAST分析确定候选靶mRNA的确切信息。最后通过候选靶基因的3’UTR荧光素酶报告基因质粒及miRNA共转染HEK293细胞进行双荧光素酶报告基因检测。结果 Hybrid-PCR方法筛选出SKOV3-TR30中miR-134的潜在靶基因有：C16orf72、PNAS-105、spermidine synthase、VIM2、F-box protein 2、GAPDH、PRPF6和RPL41，成功构建以上8个靶基因3’-UTR荧光素酶报告基因质粒；通过荧光素酶报告基因检测显示miR-134对8个候选靶基因3’-UTR区均具有直接结合作用。结论 在SKOV3-TR3O 细胞中，运用Hybrid-PCR方法共筛选得到8个miR-134候选靶基因；miR-134可能通过调控这些靶基因在卵巢癌耐药形成中发挥作用。

Study of target gene screening of miR-134 in ovarian cancer chemoresistance.

Abstract：Objective Applying Hybrid-PCR method to screen target genes of miR-134 in ovarian cancer chemoresistant cells and to provide a theoretical basis onthe formation of ovarian cancer chemoresistance.Methods RNA extraction, primer design, Hybrid- PCR was carried out to screen putative targets of miR-134 in SKOV3-TR30 cells,Dual Luciferase Reporter Assay System was used to evaluate the binding of miR-134 with 3’UTR of candicate targets predicated by Hybrid-PCR.Results By applying Hybrid -PCR we find in SKOV3-TR30 cells target mRNA of miR-134：C16orf72, PNAS-105,spermidine synthase, VIM2,F-box protein 2,GAPDH,PRPF6 and RPL41. We successfully constructed 3'-UTR plasmids of the 8 putative targets of miR-134 in SKOV3-TR30 cells.By co-transfecting mRNA 3'UTR PMIR-REPORT and Psilencer-miR-134 to HEK293 cell respectively, we demonstrated that miR-134 directly binds to 3'UTR of these 5 mRNAs.Conclusion Hybrid-PCR is an effective and rapid approach for screening putative miRNA targets and could be applied to find target mRNA of miRNA in SKOV3-TR30 cells. C16orf72, PNAS-105,spermidine synthase, VIM2,F-box protein 2,GAPDH,PRPF6 and RPL41 could be target mRNA of miR-134 in SKOV3-TR30 cell and miR-134 could take part in chemoresistance formation of SKOV3-TR30 cells via these targets.