微小RNA-20a对膀胱癌T24细胞增殖和凋亡的影响

Effects of microRNA-20a on the proliferation and apoptosis of bladder cancer T24 cell

目的探讨微小RNA-20a(miR-20a)在膀胱癌中的表达情况及其对人膀胱癌细胞增殖和凋亡的影响。方法收集2015年11月至2016年10月本院收治的65对膀胱癌组织和癌旁组织标本,采用实时荧光定量PCR(QPCR)检测癌组织和癌旁组织的miR-20a水平。采用Lipofectamine^(TM)2000脂质体法将miR-20a模拟物(mimics组)和抑制剂(inhibitor组)转染人膀胱癌T24细胞,以不行任何转染的T24细胞为对照组,采用QPCR法检测各组转染48 h后的miR-20a水平,采用CCK-8法和AnnexinⅤ-FITC/PI双染流式细胞术检测各组的增殖和凋亡情况。采用生物信息学软件Targetscan预测miR-20a的靶基因并利用cytoscape 3.5.1软件及其插件Clu GO对靶基因进行GO功能注释。结果膀胱癌组织的miR-20a水平为3.181±1.669,高于对应癌旁组织的1.198±0.756(P<0.05)。对照组、mimics组和inhibitor组转染48 h后的miR-20a水平分别为1.024±0.468、2.756±0.941和0.412±0.075,与对照组相比,mimics组的miR-20a水平升高而inhibitor组的miR-20a水平降低,差异有统计学意义(P<0.05)。与对照组相比,mimics组转染24、48、72 h后的吸光值和增殖倍数均升高,而inhibitor组的吸光值和增殖倍数均降低,差异有统计学意义(P<0.05)。对照组、mimics组和inhibitor组的转染48 h凋亡率分别为(12.16±1.67)%、(7.22±0.97)%和(36.13±4.15)%,与对照组相比,mimics组的凋亡率降低而inhibitor组的凋亡率升高,差异有统计学意义(P<0.05)。MiR-20a的预测靶基因共筛选出147个,GO功能主要富集于细胞周期、磷脂酰肌醇Akt信号通路和细胞代谢过程。结论 MiR-20a在膀胱癌组织中表达上调并可以促进癌细胞的增殖,发挥类似促癌基因的作用,有望成为膀胱癌的新型生物治疗靶点。

Objective To investigate the expression of microRNA-20a （miR-20a） in bladder cancer tissues and its effect on the proliferation and apoptosis of human bladder cancer cells. Methods From November 2015 to October 2016, 65 pairs of bladder cancer tissues and para-cancerous tissues were collected in our hospital Real-time fluorescence quantitative PCR （QPCR） was used to detect the level of miR-20a in cancer tissues and para-cancerous tissues. LipofectamineTM 2000 liposome was used to transfect miR-20a mimics （mimics group） and inhibitor （inhibitor group） into human bladder cancer T24 cells, and T24 cells without transfection were used as control group. The level of miR-20a at 48 h after transfection was detected by QPCR. The proliferation and apoptosis of each group were detected by CCK-8 and Annexin V-FITC/PI double staining via flow cytometry. The target genes of miR-20a were predicted by bioinformatics software Targetscan, and function annotation of the target genes was carried out for Gene Oncology （GO） by cyto- scape3.5.1 and its plug-in CluGO. Results The expression of miR-20a in bladder cancer tissues was 3.181± 1. 669, which was high- er than 1. 198±0. 756 of the corresponding para-cancerous tissues （P〈0. 05）. The miR-20a levels were 1. 024±0. 468, 2. 756±0. 941 and 0. 412±0. 075 at 48 h after transfection in the control group, mimics group and inhibitor group. Compared with the control group, the level of miR-20a increased in the mimics group and decreased in inhibitor group, and the difference was statistically significant （P〈 0. 05）. Compared with the control group, the absorbance and proliferative ratio of mimics group increased at 24, 48 and 72 h after transfection, while the absorbance and proliferative ratio of inhibitor group decreased （P〈0. 05）. The apoptotic rates were （12. 16_± 1.67）%, （7. 22_±0. 97）% and （36. 13_±4. 15）% at 48 h after transfection in control group, mimics group and inhibitor group, re- spectively. Compared with the control group, the apoptotic rates decreased in mimics group but increased in the inhibitor group, and the difference was statistically significant （P〈0. 05）. One hundred and forty-seven target genes were screened for miR-20a. The func- tion of GO was mainly enriched in cell cycle, phosphatidyl inositol Akt signaling pathway and cell metabolic process. Conclusion MiR-20a is upregulated in bladder cancer tissue, and it can promote the proliferation of cancer cells and plays a similar role as onco- gene. It is expected to become a new target for biological treatment of bladder cancer.