miR-134靶基因筛选与鉴定在卵巢癌耐药中的应用研究  

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

作　　者：双婷[1] 王敏[1] 常爽[1] 周莹莹[1] 闫效宇[1] 史聪[1] 胡婷[1] 

机构地区：[1]中国医科大学附属盛京医院妇产科,辽宁沈阳110004

出　　处：《中国实用妇科与产科杂志》2014年第1期51-54,共4页Chinese Journal of Practical Gynecology and Obstetrics

基　　金：国家自然科学基金(30973189);辽宁省自然科学基金(2013021040)

摘　　要：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。Objective Applying Hybrid-PCR method to screen target genes of miR-134 in ovarian cancer chemoresistant cells and to provide a theoretical basis on the 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 Hy- brid-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 plas- mids 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 mR- NAs. Conclusion Hybrid-PCR is an effective and rapid approach for screening putative miRNA targets and could be ap- plied 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.

关 键 词：卵巢肿瘤 耐药 miR-134 靶基因筛选 

分 类 号：R737.3[医药卫生—肿瘤]