miR-125b靶基因鉴定及对卵巢癌细胞增殖和凋亡能力影响实验研究

Identification of miR-125b target gene and its effect on proliferation and apoptosis of ovarian cancer cells

目的既往研究结果显示,HER2基因在卵巢癌组织中存在着过表达,且在卵巢癌细胞系中,沉默HER2基因后,能显著降低卵巢癌细胞的增殖、侵袭能力,诱导细胞凋亡。本研究拟探讨靶向HER2的miR-125b对卵巢癌SKOV-3细胞增殖及凋亡的影响。方法分别采用miRbase、miRanda和targetscan 3个数据库进行miR-125的靶基因预测,对获得的靶基因分别进行GO注释描述、GO富集分析和Pathway分析;pre-miR-125b转染卵巢癌细胞系,通过荧光定量PCR验证;同时构建含HER2的3′UTR野生型和突变型荧光素酶载体,分别和pre-miR-125b及control miR共转染SKOV-3细胞,应用荧光素酶报告系统检测HER2是否是miR-125b的靶基因;蛋白质印迹法检测转染后HER2的表达水平变化;采用MTT法检测SKOV-3细胞的增殖率变化,AnnexinⅤ方法检测SKOV-3细胞凋亡率的变化。结果生物信息学方法分析显示,miR-125b与HER2的3′UTR区域存在可能的结合位点。pre-miR-125b转染组miR-125b的表达(5.48±1.169)较对照组(1.35±0.391)明显升高,差异有统计学意义,t=5.811,P=0.018;荧光素酶报告基因实验结果显示,pre-miR-125b能明显降低Wt-HER2的荧光素酶活性,与其他各组相比,差异有统计学意义,P<0.01;HER2是miR-125b的预测靶位点。蛋白质印迹结果显示,转染pre-miR-125b组HER2的表达水平(0.292±0.059)较对照组(0.799±0.101)明显下降,差异有统计学意义,t=7.485,P=0.002;转染48h后,对照组和实验组的细胞存活率分别为0.35±0.013和0.21±0.025,差异有统计学意义,t=8.63,P=0.001;转染72h后,对照组和实验组的细胞存活率分别为0.58±0.020和0.32±0.028,差异有统计学意义,t=13.062,P<0.001。说明上调miR-125b后可抑制卵巢癌细胞SKOV-3的增殖能力。在转染后,实验组凋亡率[(11.47±1.16)%]较对照组[(4.73±1.14)%]明显增加,差异有统计学意义,t=7.168,P=0.002。结论 miR-125b能通过下调HER2基因的表达,抑制细胞增殖,诱导细胞凋亡,参与卵巢癌的生物学功能。

OBJECTIVE Previous studies showed that overexpression of HER2 gene existed in ovarian cancer tis- sues and silencing HER2 expression can significantly reduce ovarian cancer cell proliferation,invasion and induced cell ap- optosis in vitro. The aim of this study is to investigate the effect of microRNA-125b targeting HER2 on the proliferation and apoptosis of ovarian cancer SKOV-3 cells. METHODS miRbase,miRanda and targetscan were used to predict target genes of miR-125b. The intersection targets of the three results were analyzed separately by GO annotations describing, GO enrichment analysis and pathway analysis. Pre-miR-125b was transfected into ovarian cancer cell lines and real-time quantitative PCR was used to identify the effect. Construct HER2 containing 3＇UTR of wild type and mutant luciferase vectors and transfected into SKOV-3 cells with pre-miR-125b and control miR,respectively. Luciferase reporter assay was used to determine whether HER2 was the direct target of miR-125b. The change of expression level of HER2 was detected by Western method. MTT method was used to detect SKOV-3 cell proliferation rate and Annexin V method for detection of SKOV-3 cell apoptosis rate. RESULTS The bioinformatics analysis showed that there were possible binding sites be- tween 3＇ UTR region miR-125b and HER2; the expression of pre-miR-125b in miR-125b transfection group （5. 48 ± 1. 169） was significantly higher than that of the control group （1.35±0. 391）, the difference was statistically significant （t= 5.811, P = 0.018） ;the luciferase reporter assay results showed that pre-miR-125b could significantly decrease the lu- ciferase activity of Wt-HER2 compared with other groups, the difference was statistically significant （P〈0.01）. HER2 was the predicted target sites of miR-125b. Western results showed that the expression levels of HER2 in transfected miR-125b group （0. 292 ± 0. 059） were decreased significantly compared with the control group （0. 799 ±0. 101）, the difference was statistically significant（t= 7. 485,P=0. 002）. The rate of cell proliferation in miR-125b transfection group （0.21±0. 025） ,（0.32±0. 028） was slower than that of the control group （0.35±0. 013） ,（O. 58±0. 020） after the up- regulation of the expression of miR-125b in 48 and 72 h, respectively, and the difference was statistically significant （t= 8.63,P=0. 001;t=13. 062,P%0. 001）. The rate of apoptosis in miR-125b transfection group [-（11.47±1.16）M] was significantly increased compared with the control group [-（4.73±1.14）%], and the difference was statistically significant （t=7. 168,P=0. 002）. CONCLUSION miR-125b can effecitively decrease the expression of HER2,participate in the bio- logical function of ovarian cancer by inhibiting cell proliferation and inducing apoptosis.