miR-320e靶向调控FHIT对前列腺癌细胞增殖、凋亡的影响

Effects of miR320e targeting FHIT on the proliferation and apoptosis of the prostate cancer cells in vitro

目的研究微小RNA-320e(miR-320e)在前列腺癌细胞增殖和凋亡中的作用及其分子机制。方法利用实时荧光定量PCR(qPCR)检测前列腺癌细胞DU145和前列腺正常上皮细胞RWPE-1中miR-320e表达。在DU145细胞中转染anti-miR-320e或pcDNA3.1-FHIT,评估其对DU145细胞增殖和凋亡的影响。TargetScan预测及双荧光素酶报告基因实验分析miR-320e和脆性组氨酸三联体(FHIT)之间的靶向关系。噻唑蓝(MTT)法和流式细胞术分别检测细胞增殖与凋亡,蛋白质印迹法(Western blot)检测细胞周期蛋白D1(CyclinD1)、P21、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、FHIT蛋白表达。将anti-miR-320e与si-FHIT共转染,观察抑制FHIT表达对抑制miR-320e诱导的DU145细胞增殖和凋亡的影响。结果与前列腺正常上皮细胞RWPE-1相比,前列腺癌细胞DU145内miR-320e表达量明显上调(P<0.05)。抑制miR-320e表达或过表达FHIT明显降低24h、48h、72h时的DU145细胞活性、Cyclin D1、Bcl-2蛋白表达量(P<0.05),显著提高细胞凋亡率、P21、Bax蛋白水平(P<0.05)。miR-320e靶向调控FHIT表达。抑制FHIT能逆转抑制miR-320e对细胞DU145增殖、Cyclin D1、Bcl-2蛋白表达的抑制作用,并逆转其对细胞凋亡、P21、Bax蛋白表达的促进作用。结论miR-320e通过靶向调控FHIT影响前列腺癌细胞增殖、凋亡。

Objective To investigate the effects of microRNA320e(miR320e)on the proliferation and apoptosis of the prostate cancer cells and its molecular mechanism.Methods The expression levels of miR320e in the prostate cancer cell line DU145 and the prostate normal epithelial cell RWPE1 were detected by real-time fluorescence quantification(qPCR).Anti-miR320e or pcDNA3.1 FHIT were transfected into DU145 cells to evaluate their effects on the proliferation and apoptosis of DU145 cells.TargetScan prediction and dual luciferase reporter assays were used to analyze the targeting relationship between miR320e and FHIT.The cell proliferation and apoptosis were detected by MTT assay and flow cytometry,respectively.The protein expression levels of CyclinD1,P21,Bcl2,Bax and FHIT detected by Western Blot.The effects of inhibiting FHIT on the proliferation and apoptosis of DU145 cells induced by inhibiting miR320e were observed by cotransfection of anti-miR320e and siFHIT.Results The expression levels of miR320e in prostate cancer cells DU145 were significantly upregulated,as compared with those in prostate normal cells RWPE1(P<0.05).The inhibition of miR320e expression or overexpression of FHIT significantly decreased the DU145 cell activity at 24h,48h,72h after treatment,and decreased the protein expression levels of Cyclin D1 and Bcl2(P<0.05),moreover the inhibition of miR320e expression or overexpression of FHIT significantly increased the apoptosis rate,and the protein levels of P21 and Bax(P<0.05).The miR320e targeting regulated the FHIT expression.To inhibit FHIT could reverse the inhibitory effects of miR320e on the proliferation of cell DU145 and the protein expressions of Cyclin D1 and Bcl2,and could reverse the promotion effects on the cell apoptosis,and the protein expressions of P21 and Bax.Conclusion The miR320e affects the proliferation and apoptosis of prostate cancer cells by targeting regulating FHIT.