miR-93-5p靶向调控Smad5表达抑制小鼠C3H10T1/2细胞成骨分化的研究  

miR-93-5P SUPPRESSES OSTEOGENIC DIFFERENTIATION OF MOUSE C3H10T1/2 CELLS BY TARGETING Smad5

作　　者：徐练[1] 李晓龙[1] 刘印[1] 孔清泉[1] 龙丹[2] 李胜富[2] 

机构地区：[1]四川大学华西医院骨科,成都610041 [2]四川大学华西医院卫生部移植工程与移植免疫重点实验室,成都610041

出　　处：《中国修复重建外科杂志》2015年第10期1288-1294,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81171731;30700837);AOspine中国研究基金资助项目[AOSCN(R)2014-16]~~

摘　　要：目的探讨mi R-93-5p是否通过靶向调控其预测靶基因Smad5的表达,从而抑制小鼠MSCs C3H10T1/2细胞的成骨分化。方法构建Smad5 3’-UTR-荧光素酶报告载体(pmi R-RB-REPORTTM),通过双荧光素酶报告基因检测,观察mi R-93-5p对Smad5 3’-UTR荧光素酶活性的影响,鉴定Smad5是否为mi R-93-5p的靶基因。将mi R-93-5p mimics(M组)与mi R-93-5p inhibitor(In组)及其对应的阴性对照组mi R-93-5p mimics阴性对照(MC组)与mi R-93-5p inhibitor阴性对照(In C组)分别转染至C3H10T1/2细胞中,并进行成骨诱导培养,48 h后分别采用实时荧光定量PCR(real-time fluorescent quantitative PCR,q RT-PCR)及Western blot检测各组细胞Smad5在m RNA及蛋白水平的相对表达量;14 d后通过茜素红染色检测各组细胞外钙盐的沉积情况,了解mi R-93-5p对C3H10T1/2细胞成骨分化的调控效应。结果双荧光素酶报告基因检测结果显示,mi R-93-5p能与Smad5 m RNA3’-UTR特异性结合,抑制其荧光素酶活性(P<0.05)。q RT-PCR检测示,M组及In组Smad5的m RNA相对表达量与对应阴性对照组MC组及In C组比较差异均无统计学意义(P>0.05);Western blot检测示,M组及In组Smad5蛋白相对表达量与对应阴性对照组比较差异均有统计学意义(P<0.05),其中M组较MC组表达量下调,而In组则较In C组表达量上调。茜素红染色示,M组钙盐沉积较MC组明显减少,而In组则较In C组钙盐沉积明显增多。结论Smad5是mi R-93-5p的靶基因,mi R-93-5p可通过靶向调控Smad5的表达,抑制小鼠C3H10T1/2细胞成骨分化。Objective To investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells （C3H10T1/2） by targeting Smad5, a predicted target in silicon.Methods Smad5 3’-UTR-luciferase vector （pmiR-RB-REPORTTM） was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3’-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics （group M）, miR-93-5p inhibitor （group In）, miR-93-5p mimics negative control （group MC）, and miR-93-5p inhibitor negative control （group InC） were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR （qRT-PCR） and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. ResultsDual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3’-UTR specificity, and inhibited its luciferase activity （P〈0.05）. After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay （P〉0.05）; however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC （P〈0.05）. At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. Conclusion Smad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by dire

关 键 词：C3H10T1/2细胞 miR-93-5p SMAD5 MSCs 成骨分化 小鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]