Experimental Cell Research. 362(2):293–301, JAN 2018
DOI: 10.1016/j.yexcr.2017.11.030
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PMID: 29197556
Issn Print: 0014-4827
Publication Date: 2018/01/01
miR-195 inhibited abnormal activation of osteoblast differentiation in MC3T3-E1 cells via targeting RAF-1
Chen Chao;Feng Li;Zhiping Tan;Weizhi Zhang;Yifeng Yang;Cheng Luo;
+ Author Information
aDiabetes Center, Institute of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha 410011, PR ChinabDepartment of Cardiovascular Surgery, The Second Xiangya Hospital of Central South University, 139 Renmin Road, Changsha 410011, PR China
Abstract
Recent reports have demonstrated that RAF-1L613V (a mutant of RAF-1) mutant mice show bone deformities similar to Noonan syndrome. It has been suggested that RAF-1L613V might abnormally activate osteoblast differentiation of MC3T3-E1 cells.To demonstrate that RAF-1 is associated with bone deformity and that RAF-1L613V dependent bone deformity could be inhibited by microRNA-195 (miR-195), we first investigated the amplifying influence of wild-type RAF-1 (WT) or RAF-1L613V (L613V) on the viability and differentiation of MC3T3-E1 cells induced by bone morphogenetic protein-2 (BMP-2) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Subsequently, we investigated the blocking effect and its mechanism of miR-195 for abnormal activation of osteoblast differentiation of MC3T3-E1 cells via targeting RAF-1.RAF-1, especially RAF-1L613V, abnormally activates osteoblast differentiation of MC3T3-E1 cells induced by BMP-2. Meanwhile, miR-195 could inhibit the cell viability and differentiation of MC3T3-E1 cells. Transfection of miR-195 largely suppressed the L613V-induced viability and osteoblast differentiation of MC3T3-E1 cells and attenuated the accelerative effect of L613V on runt-related transcription factor-2 (Runx2), Osterix (OSX), alkaline phosphatase (ALP), osteocalcin (OCN), and distal-less homeobox 5 (DLX5) osteogenic gene expressions. In addition, miR-195 decreased the expression of RAF-1 mRNA and protein by directly targeting the 3′-untranslated regions (3′-UTR) of RAF-1 mRNA in MC3T3-E1 cells.Our findings indicated that miR-195 inhibited WT and L613V RAF-1 induced hyperactive osteoblast differentiation in MC3T3-E1 cells by targeting RAF-1. miR-195 might be a novel therapeutic agent for the treatment of L613V-induced bone deformity in Noonan syndrome.
