中国实用妇科与产科杂志

中国实用妇科与产科杂志

• 研究与探索 • 上一篇    

miR-134靶基因筛选与鉴定在卵巢癌耐药中的应用研究

双婷,王敏，常爽,周莹莹,闫效宇,史聪,胡婷   

  1. 作者单位： 中国医科大学附属盛京医院妇产科, 辽宁 沈阳110004
  • 出版日期:2014-01-02 发布日期:2014-01-02
  • 通讯作者: 王敏
  • 基金资助:

    国家自然科学基金（30973189）;辽宁省自然科学基金（2013021040）

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

SHUANG Ting, WANG Min,CHANG Shuang, ZHOU Ying-ying, YAN Xiao-yu, SHI Cong, HU Ting.   

  1. Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University, Shenyang  110004, China
  • Online:2014-01-02 Published:2014-01-02

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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。

关键词: 卵巢肿瘤, 耐药, miR-134, 靶基因筛选

Abstract:

Abstract：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 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.

Key words: ovarian carcinoma, chemoresistance, miR-134, target gene screening

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