Has-miR-29a与盆腔器官脱垂相关靶基因的预测与验证  

Prediction and validation the target gene of Has-miR-29a associated with pelvic organ prolapse

作　　者：李周兰 洪新如 何春妮 宋岩峰 LI Zhoulan;HONG Xinru;HE Chunni;SONG Yanfeng(Department of Obstetrics and Gynecology,Medical University of Fujian, FuZhou Fujian 350025,China)

机构地区：[1]福建医科大学福总临床医学院(第九○○医院),350025 [2]福建省龙岩市第一医院 [3]联勤保障部队第九○○医院

出　　处：《中国妇产科临床杂志》2019年第2期124-127,共4页Chinese Journal of Clinical Obstetrics and Gynecology

基　　金：科技部国家支撑计划课题(2014BAI05B02);福建省自然科学基金(2014J01428)

摘　　要：目的预测和验证Has-miR-29a与盆腔器官脱垂(POP)相关的靶基因。方法应用miRBase、PicTar以及TargetScan三大数据库进行生物学信息分析,预测Has-miR-29a全部作用靶点,结合前期实验结果,筛选出与POP可能相关的靶基因为COL1A1、COL3A1;构建靶基因的3'UTR的表达载体,双荧光素酶报告基因检测系统验证miR-29a对靶基因的靶向作用;将miR-29a对照组、miR-29a过表达组和miR-29a抑制组分别共转染离体培养的盆底组织成纤维细胞,免疫印迹法检测靶蛋白Ⅰ、Ⅲ胶原蛋白的表达变化。结果在线预测结果提示,COL3A1 mRNA-3'UTR和COL1A1 mRNA-3'UTR均有与miRNA-29a完全互补的配对序列,确定二者均为miR-29a的靶基因。miR-29a的过表达和抑制均能够相应的引起Ⅰ、Ⅲ型胶原蛋白的含量变化。结论COL1A1和COL3A1是miR-29a直接作用的靶基因,且主要结合位置在COL1A1和COL3A1的3'UTR区域,在转录后水平抑制COL1A1和COL3A1基因,即miR-29a可能靶向调控Ⅰ、Ⅲ型胶原蛋白的表达,参与POP的发生、发展过程。Objective To predict and verify the target gene of Has-miRNA-29 a associated with pelvic floor organ prolapsed(POP).Methods Biological information was analyzed by miRBase,PicTar and TargetScan to predict all the targets of Has-miR-29 a.Based on previous experimental results,the target genes related to miR-29 a and POP were selected as COL1 A1,COL3 A1.The expression vector of target gene 3’UTR was constructed,and the dual-luciferase reporter gene detection system was used to verify the targeting effect of miR-29 a.miR-29 a control,miR-29 a mimics and miR-29 a inhibitor were co-transfected into cultured pelvic floor tissue f ibroblasts respectively.The expression of target proteins collgagen Ⅰ and Ⅲ was detected by Western blot.Results The results of online prediction indicated that both COL3 A1 mRNA-3’UTR and COL1 A1 mRNA-3’UTR had pairing sequences complementary to miR-29 a,and both of them were identified as target genes of miR-29 a.The overexpression and inhibition of miR-29 a can result in the changes of type Ⅰ and type Ⅲ collagen.Conclusion The COL1 A1 and COL3 A1 were the direct target genes of miR-29 a.The binding site is mainly in the 3’UTR region of COL1 A1 and COL3 A1,and miR-29 a directly inhibits the expression of COL1 A1 and COL3 A1 genes at post-transcriptional level.miR-29 a may target to regulate the expression level of type Ⅰ and Ⅲ collagen,and may participate in the occurrence and development of POP.

关 键 词：Has-miR-29a 盆腔器官脱垂 胶原蛋白 

分 类 号：R711.2[医药卫生—妇产科学]