长链非编码RNA染色体失活特异转录本靶向调控微小RNA-106a-5p影响肾癌细胞增殖和凋亡的研究

Study on the regulation of renal cancer cell proliferation and apoptosis function by long-chain noncoding RNA XIST targeting microRNA-106a-5p

目的探讨长链非编码RNA染色体失活特异转录本(lncRNA XIST)靶向调控微小RNA(miRNA,miR)-106a-5p对肾癌细胞增殖和凋亡的影响及其机制。方法通过实时定量反转录聚合酶链反应(RT-qPCR)检测人肾癌ACHN、Caki-1、Caki-2和786-O细胞和正常肾脏上皮细胞HK-2中XIST的表达水平。将体外培养的Caki-2细胞分为Empty组(转染空白载体)和XIST组(转染XIST过表达载体),采用RT-qPCR检测各组细胞中XIST和miR-106a-5p的表达水平,噻唑蓝(MTT)法和流式细胞仪检测各实验组细胞增殖和凋亡。采用双荧光素酶报告基因实验检测XIST和miR-106a-5p的靶向结合关系。将miR-106a-5p激动剂与XIST过表达质粒共转染后观察上调miR-106a-5p表达对XIST过表达的Caki-2细胞增殖和凋亡的影响。多组间比较使用单因素方差分析,组间多重比较采用学生纽曼·基尔斯(Student-Newman-Keuls,SNK)-q,两组间比较采用独立样本t检验。结果与HK2细胞比较,ACHN、Caki-1、Caki-2和786-O细胞中XIST的表达水平明显降低(相对表达量分别为0.516±0.031、0.605±0.021、0.241±0.024、0.355±0.042,t=11.666、11.321、20.321、12.992,P均<0.05)。与Empty组比较,过表达XIST组细胞中XIST的表达水平升高(相对表达量为175.829±1.021,t=20.471,P<0.05)、细胞增殖能力降低(为对照组0.759倍,t=6.388,P<0.05)、细胞凋亡增加(为对照组1.304倍,t=11.371,P<0.05),而细胞中miR-106a-5p的表达水平明显降低(相对表达量为0.415±0.035,t=13.717,P<0.05)。双荧光素酶报告基因实验证实miR-106a-5p可与XIST靶向结合。转染miR-106a-5p激动剂成功上调miR-106a-5p表达后,过表达XIST对Caki-2细胞增殖的抑制作用及促凋亡作用明显逆转。结论过表达XIST可通过靶向调控miR-106a-5p抑制肾癌Caki-2细胞增殖并促进其凋亡。

Objective To investigate the effect of long-chain noncoding RNA(lncRNA)XIST targeting microRNA(miRNA,miR)-106a-5p on the proliferation and apoptosis of renal cancer cell line and its mechanism.Methods The expression of XIST in ACHN,Caki-1,Caki-2,786-O cells and HK2 cells was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).Caki-2 cells cultured in vitro were divided into Empty group(transfected with empty vector),and XIST group(transfected with pCDNA3.1 vector containing full-length XIST sequence).The expression levels of XIST and miR-106a-5p were detected by RT-qPCR,and cell proliferation,cell apoptosis were detected by methyl thiazolyl tetrazolium(MTT)method and flow cytometry.The target binding relationship between XIST and miR-106a-5p was detected by double luciferase reporter gene assay.After co-transfection of miR-106a-5p mimics and XIST vector,the effect of up-regulation of miR-106a-5p expression on the proliferation and apoptosis of Caki-2 cells which were XIST over expression was observed.Results Compared with HK2 cells,the expression level of XIST in ACHN,Caki-1,Caki-2,786-O cells decreased significantly(relative expression level was 0.516±0.031,0.605±0.021,0.241±0.024,0.355±0.042,t=11.666,11.321,20.321,12.992,P<0.05).Compared with the Empty group,the expression level of XIST(relative expression level was 175.829±1.021,t=20.471,P<0.05),cell apoptosis rate(1.304 times vs.control group,t=11.371,P<0.05)in XIST group were significantly higher,while the expression level of miR-106a-5p(relative expression level was 0.415±0.035,t=13.717,P<0.05)and cell viability(0.759 times vs.control group)were significantly lower(t=6.388,P<0.05).The double luciferase reporter gene experiment confirmed that miR-106a-5p could target with XIST.After miR-106a-5p mimics was successfully transfected to up-regulate the expression of miR-106a-5p,the inhibition of proliferation and promotion of apoptosis of Caki-2 cells by high expression of XIST were reversed.Conclusion Over expression of XIST could inhibit the proliferation and promote the apoptosis of Caki-2 cells by targeting miR-106a-5p.