miR-155通过下调BMP9/Smad信号通路抑制间充质干细胞C3H10T1/2成骨分化  

miR-155 Suppresses the Osteogenic Differentiation of Mesenchymal Stem Cells Line C3H10T1/2 via Down-Regulation of BMP9/Smad Signal Pathway

作　　者：刘红霞[1] 施琼[1] 安利钦 周一青[1] 张汝益[1] 严树涓[1] 翁亚光[1] 

机构地区：[1]重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国细胞生物学学报》2017年第4期410-418,共9页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:NSFC81672103、NSFC31200971);教育部高等学校博士点基金(批准号:20115503110009);重庆市渝中区科委科技项目(批准号:20130136)资助的课题~~

摘　　要：该文研究了在诱导小鼠间充质干细胞C3H10T1/2细胞成骨分化过程中miR-155的作用及其是否是通过调控BMP9/Smad(bonemorphogenetic protein 9/drosophila mothers against de-capentaplegic)信号通路发挥作用。在诱导C3H10T1/2细胞成骨分化过程中,采用实时定量PCR(Real-time quantitative PCR,q RT-PCR)检测miR-155水平的变化。转染miR-155模拟剂(miR-155)至C3H10T1/2细胞后,miR-155水平显著增高(P<0.001),而转染其抑制剂(anti-miR-155)至C3H10T1/2细胞后,miR-155水平显著降低(P<0.001)。转染后成骨诱导培养基诱导成骨7 d,碱性磷酸酶(alkaline phosphatase,ALP)活性及染色结果显示,miR-155能显著降低C3H10T1/2细胞成骨分化过程中的ALP活性(P<0.01)、减弱ALP染色,而anti-miR-155则能逆转其作用。成骨诱导14 d茜素红S染色结果显示,miR-155组钙盐结节较对照组少,下调miR-155的水平后,钙盐沉积结节增多。q RT-RCR检测结果显示,miR-155显著降低BMP9 m RNA水平(P<0.001),且miR-155组成骨基因Runx2和ALP表达均显著低于对照NC组(P<0.05、P<0.01)。Western blot检测BMP9、Runx2和p-Smad1/5/8蛋白质水平,结果显示,miR-155组蛋白质水平均显著降低(P<0.01、P<0.05、P<0.001)。该研究结果提示,miR-155对C3H10T1/2细胞成骨分化的抑制作用可能是通过抑制BMP9/Smad信号通路发挥作用的。The aim of this study was to investigate the regulatory effect of miR-155 on the osteogenic differentiation of mesenchymal stem cells line C3H 10T 1/2 and its mechanisms. This study tested the miR- 155 level during the process of osteogenic differentiation of C3H10T1/2 cells by Real-time quantitative PCR （qRT-PCR）. Transfected with miR-155 mimic （miR-155） into C3H10T1/2 cells, the miR-155 level was significantly increased （P〈0.001）, however, when transfected with miR-155 inhibitor （anti-miR-155） into C3H10T1/2 cells, the miR-155 level was significantly decreased （P〈0.001）. After transfecting, induced osteogenic differentiation with osteoblast inducing conditional medium for 7 d, the results of Alkaline phosphatase （ALP） activity and ALP staining shown that miR-155 markedly decreased ALP activity （P〈0.01） and attenuated ALP staining in the process of osteogenesis of C3H10T1/2 ceils, however anti-miR-155 could reverse this effect. Induced osteogenic differentiation for 14 d, Alizarin red S staining shown that compared with negative control （NC） group, miR-155 notably reduced the deposition of calcium salt, and inhibition of miR-155 increased the deposition of calcium salt of C3H 10T1/2 cells. It was also found that miR-155 significantly reduced the mRNA level ofBMP9 （P〈0.001）, and the mRNA levels of osteogenesis genes Runx2 and ALP decreased obviously （P〈0.05, P〈0.01）. miR-155 decreased the protein levels of BMP9, Runx2 and p-Smadl/5/8 significantly （P〈0.01, P〈0.05, P〈0.001）. We concluded that the inhibitory effect of miR- 155 on the osteogenic differentiation of C3H 10T 1/2 cells might through downregulated BMP9/Smad signal pathway.

关 键 词：MIR-155 C3H10T1/2细胞 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]