miR-99a对宫颈癌Hela细胞增殖影响机制研究

Effect and related mechanism of miR-99a on the proviferation of cervical carcinoma hela cell

目的 miR-99a在宫颈癌等多种肿瘤组织中异常表达,且与肿瘤细胞的多种生物学行为密切相关。本研究旨在探讨miR-99a沉默HOXA1表达抑制宫颈癌Hela细胞增殖的影响及相关机制。方法通过瞬时转染将miR-99a mimics导入宫颈癌Hela细胞,采用MTT法和平皿克隆形成实验观察miR-99a增加对Hela细胞增殖的影响;应用Targetscan 6.2软件预测miR-99a与HOXA1基因3’UTR的靶向性作用,荧光素酶报告基因分析miR-99a对HOXA1基因表达的影响,采用qRT-PCR和蛋白质印迹法检测miR-99a对HOXA1表达的作用;构建HOXA1干扰载体,转染Hela细胞,筛选后利用MTT法和平皿克隆实验观察HOXA1表达降低后对Hela细胞增殖的影响。结果 miR-99a被导入宫颈癌Hela细胞后,细胞的生长速度减慢,miR-99amimics组Hela细胞克隆数为156±30,与miR-ctr组(386±49)比较,克隆数减少,F=26.093,P=0.001。采用Targetscan6.2(http://www.targetscan.org/)软件分析miR-99a与HOXA1基因的作用位点发现,miR-99a与HOXA1基因3’UTR的1 485~1 497位核苷酸位点存在结合,miR-99a表达后,抑制Hela细胞中HOXA1的表达。干扰HOXA1表达后,Hela细胞生长速度减慢,平皿克隆形成能力降低,siHOXA1克隆数为165±35,si-control克隆数为390±46,P<0.05。结论miR-99a与HOXA1基因3’UTR 1 485~1 497位核苷酸位点结合沉默其表达,抑制宫颈癌Hela细胞的增殖。

OBJECTIVE To investigate the mechanisms on miR-99a affecting the proliferation of cervical cancer He la cells and to provide experimental basis for elucidating the molecular mechanisms of cervical cancer. METHODS miR 99a mimics was introduced into the Hela cells by transient transfection,and the proliferation of Hela cells was observed by MTT and cloning forming assay. The HOXA1 gene 3＇UTR targeting regulated by miR-99a was predicted by Target-scan 6.2 software. After miR 99a mimics was introduced into the Hela cells,the expression of HOXA1 gene was analyzed by the lueiferase reporter gene,qRT-PCR and Western-blot. The interference vector of HOXA1 was constructed and trans fected into the Hela cells. The proliferation of Hela cells affected by the HOXA1 expression was observed by cell growth curve and cloning experiment. RESULTS Firstly, after miR-99a mimics were introduced into the Hela cells, miR-99a slowed down the growth and decreased the proliferation ability in Hela cells （ the clones number of miR-99a mimics vs miR-control：156±30 vs 386±49,P〈0. 05）. Secondly, miR-99a combined with the 1 485--1 497 nucleotide site of HOXA1 gene 3＇UTR. Moreover,the expression of HOXA1 gene was inhibited by miR 99a in Hela cells. After the expres- sion of HOXA1 gene was interfered,the growth of Hela ceils was slowed down and the proliferation was decreased（the clones number of si-HOXA1 vs si-control：160±35 vs 390±46,P〈0.05）. CONCLUSION MiR-99a combined with the 1 485-1 497 nucleotide sites of HOXA1 gene ＇ UTR targeting can silence its expression to inhibit the proliferation of cervical cancer Hela cells.