MicroRNA570通过Wnt/β-catenin通路抑制人结肠癌HT-29细胞的增殖及调控机制

MicroRNA570 inhibits human colon cancer HT-29 cell proliferation via Wnt/β-catenin pathway

目的:探讨microRNA570(miR-570)对人结肠癌细胞株HT-29生物学特性的影响,并阐明其对Wnt/β-catenin通路的影响机制.方法:将miR-570 mimics转染人结肠癌细胞株HT-29后,采用cell counting(CCK-8)法检测细胞增殖变化,应用实时定量PCR技术检测转染后HT-29细胞中miR-570的表达,Western blot技术及实时定量PCR技术检测各组中胞核及胞浆内β-catenin的变化,及对Wnt/β-catenin通路下游基因c-myc、cyclinD1和survivin表达的影响.结果:HT-29细胞转染miR-570 mimics后,HT-29细胞增殖能力下降,72 h细胞增殖抑制率最为明显,约为53.23%±1.22%(P<0.05).实时定量PCR结果显示miR-570表达上调20倍(20.01±0.32)(P<0.05).Western blot技术及实时定量PCR技术检测显示,β-catenin、c-myc、cyclinD1和survivin蛋白表达和mRNA表达均下降(P<0.05),而空白对照组与阴性对照差异无统计学意义.Western blot结果进一步证实胞核内β-catenin的蛋白表达量下降明显,而胞浆内,β-catenin的蛋白表达量无明显变化.结论:miR-570能明显抑制人结肠癌细胞株HT-29的增殖,并下调Wnt相关基因的表达,其可能作为抑癌基因参与Wnt/β-catenin信号通路的调节.

AIM： To investigate the effect of microRNA570 （miR-570） on the proliferation and biological properties of HT-29 cells and the possible mech- anisms involved. METHODS： An miR-570 mimic was transfected into human colon cancer HT-29 cells. Cell prolif- eration was studied by cell counting kit-8 assay （CCK-8）. Total rniRNA was extracted and the relative expression of miR-570 was quantified by real-time quantitative PCR. Western blot and real-time quantitative PCR were performed to analyze the expression of β-catenin in the cy- toplasm and nucleus. The expression of c-myc,cyclinD1 and survivin was also detected by real- time quantitative PCR and Western blot. RESULTS： After transfection with the miR-570 mimic, the proliferation ability of HT-29 cells decreased. The inhibitory effect was most signif- icant at 72 h （53.23% ± 1.22%） （P 〈 0.05）. The ex- pression of miR-570 was up-regulated more than 20 times （20.01 ± 0.32） （P 〈 0.05）. The mRNA and protein expression of β-catenin, c-myc, cyclinD1 and survivin was decreased in the miR-570 mimic group, compared with blank and negative control groups. Western blot results further confirmed that the protein expression of β-catenin in the nucleus decreased significantly, although its expression in the cytoplasm did not change significantly. CONCLUSION： MiR-570 significantly inhibits the proliferation of HT-29 cells possibly by regulation of the Wnt/β-catenin signal pathway.