MicroRNA-9通过下调TGFBR2表达抑制肝星状细胞增殖和胶原合成

Effect of MicroRNA-9 on cell proliferation and collagen synthesis in hepatic stellate cells by down-regulation of TGFBR2

目的探讨MicroRNA-9（miR-9）对肝星状细胞增殖和胶原合成的影响并研究其相关机制。方法设计合成miR-9特异性模拟物（miR-9mimics），将其转入肝星状细胞中，培养48h后收集细胞，采用实时定量PCR技术（qRT—PCR）验证miR-9mimics转染细胞中miR-9的变化水平、Westernblot检测I型胶原、Ⅲ型胶原蛋白表达、噻唑蓝（MTY）法检测细胞增殖情况；通过qRT—PCR和Westernblot检测转染miR-9mimics后其潜在靶点转化生长因子-01受体2（TGFBR2）韵表达水平。结果与对照组相比，转染miR-9mimics后，检测肝星状细胞中miR-9表达量明显升高（P〈0．05）。过表达miR-9后I型胶原、Ⅲ型胶原蛋白分别降低约（44±2）％和（50±3）％（P〈0．05），细胞增殖活性降低约（48±4）％（P〈0．01），TGFBR2的表达水平降低（P〈0．05）。结论过表达miR-9可抑制肝星状细胞胶原合成和细胞增殖，此机制可能是通过下调TGFBR2的表达来实现。

Objective To investigate the effect of MicroRNA-9 （miR-9） on cell proliferation and collagen synthesis in hepatic stellate cells （ HSCs）, and to explore the potential mechanism. Methods HSC-T6 cells were cultured and transfected with miR-9 mimics with lipofeetamine 2000. After incubation 48 h, the cells were collected and total proteins and RNAs were extracted. The expression of miR-9 was detected by quantitative real time polymerase chain reaction （ qRT-PCR）. The protein expression of type I collagen and type III collagen were measured by Western blot. The methyl thiazolyl tetrazolium （MTT） method was used to asses the proliferation of HSC-T6 cells. The expression of transforming growth factor-β1 receptor 2 （TGFBR2） was detected by qRT-PCR and Western blot. Results Compared to the control group, miR- 9 expression in HSCs was increased in the miR-9 mimics group （P 〈0. 05）, type I and type Ill collagen protein expression was reduced by （44±2） % and （ 50±3） % （ P 〈 0. 01 ）, respectively. The proliferation activity of HSCs was decreased by （48±4） % （P 〈 0. 05 ）. The expression of TGFBR2 was inhibited in the miR-9 mimics group. Conclusions Upregulation of miR-9 plays a role on suppressing cell proliferation and collagen synthesis in HSCs. This process might be mediated by downregulation of TGFBR2.