miR-124通过靶向调控PKM2基因的表达抑制前列腺癌PC3细胞的增殖

miR-124 suppresses the proliferation of human prostate cancer PC3 cells by targeting PKM2

目的:探讨miR-124抑制前列腺癌PC3细胞增殖活性的机制。方法:利用荧光素酶报告基因验证miR-124能否靶向作用于丙酮酸激酶M2型(PKM2)3'端非编码区(UTR)。将miR-124 mimic转染至PC3细胞后,采用实时荧光定量PCR和Western印迹检测前列腺癌PC3细胞PKM2 mRNA和蛋白的表达水平,MTT法检测miR-124 mimic与PKM2 siRNA对PC3细胞生长活性的影响。结果:与人前列腺上皮细胞RWPE-1比较,PC3细胞中PKM2 mRNA和蛋白水平表达分别上调(5.12±0.35)倍和(4.05±0.20)倍(P<0.05)。荧光素酶报告基因结果证实,PKM2是miR-124调控的靶基因,miR-124可特异性地结合于PKM2 mRNA的3'-UTR。转染miR-124 mimic 24 h后,PKM2蛋白水平下调至阴性对照组(0.16±0.04)倍(P<0.05),但对PKM2 mRNA表达无显著影响(P>0.05)。MTT结果显示,转染miR-124 mimic和PKM2 siRNA都能显著抑制PC3细胞的增殖,但miR-124 mimic对PC3细胞生长活性的抑制能力较PKM2 siRNA强。转染miR-124 mimic和PKM2 siRNA 24 h和48 h后,PC3细胞的增殖率分别为(66.20±5.10)%和(82.10±6.35)%(P<0.05)、(49.34±2.37)%和(70.10±5.80)%(P<0.05)。结论:miR-124可通过靶向调控PKM2基因的表达而抑制前列腺癌PC3细胞的增殖。

Objective ： To explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells. Methods： Luciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR （qRT-PCR） and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay. Results ： The expressions of PKM2 mRNA and protein were upregulated （5.12 ±0.35） times and （4.05 ±0.20） times respectively in the PC3 cells as compared with those in the RWPE-1 cells （P 〈0.05）. Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated （0.16 ±0.04） times （P 〈0.05）, while the PKM2 mRNA level was not changed significantly （P 〉 0.05 ）, as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were （66.20 ±5.10）% vs （82.10 ±6.35）% at 24 hours （P 〈 0.05） and （49.34 ±2.37）% vs （70.10±5.80）% at48 hours （P〈0.05）. Conclusion： miR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.