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| Overexpression of miR-155 Inhibits the Osteogenic Differentiation of Mesenchymal Stem Cells C3H10T1/2 Induced by BMP9 |
| LIU Hong-xia, SHI Qiong, ZHOU Yi-qing, AN Li-qin, YAN Shu-juan, ZHANG Ru-yi, WENG Ya-guang |
| Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine, Chongqing Medicine University, Chongqing 400016, China |
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Abstract Purpose:To investigate the effect of overexpressed miR-155 on the osteogenic differentiation of mesenchymal stem cells C3H10T1/2 cells induced by BMP9, and the related mechanisms contained in this process. Methods:① Used adenovirus BMP9 (BMP9) to induced the osteogenic differentiation of C3H10T1/2 cells, tested the expression of miR-155 by quantitative PCR (qPCR), detected the expression of Runx2 and ALP use RT-PCR. ② Cotransfected BMP9 and miR-155 into C3H10T1/2 cells, measured the expression of miR-155 by qPCR, ALP activity and ALP staining evaluated the early osteogenesis ability of C3H10T1/2 cells. ③ Cotransfected BMP9 and miR-155 into C3H10T1/2 cells, used Alizarin red S staining to estimate the osteogenesis ability in the later stage of differentiation in 14d. ④ Treated C3H10T1/2 cells with BMP9 and miR-155, tested osteogenesis-related genes by qPCR, include Runx2, OSX, COL1A1, ALP, OCN and OPN. ⑤ Treated C3H10T1/2 cells with BMP9 and miR-155, detected the expression of p-Smad1/5/8, OCN and OPN on protein levels used Western blot. ⑥ qPCR and Western blot examined the expression of HIF1α and VEGF on mRNA and protein levels respectively. ⑦ Confirmed the target gene of miR-155 by luciferase reporter assay. Results:During the process of osteogenic differentiation of C3H10T1/2 cells induced by BMP9, overexpressed miR-155 decreased ALP activity, ALP staining and Alizarin red S staining. Repressed the expression of bone-related genes, such as Runx2, OSX, COL1A1, ALP, OCN and OPN, also. On the protein levels, overexpressed miR-155 inhibited the expression of p-Smad1/5/8, OCN and OPN, the expression of HIF1α and VEGF were decreased on mRNA and protein levels, too. Through luciferase reporter assay, confirmed that HIF1α is one of the target genes of miR-155, miR-155 decreased the expression of HIF1α on protein level, not on mRNA level, yet. Conclusion:Overexpressed miR-155 could inhibit the osteogenic differentiation induced by BMP9 in mesenchymal stem cells C3H10T1/2 cells, it may through Smad/BMP signaling pathway and its target gene HIF1α to play the inhibitory effect in this process.
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Received: 08 November 2016
Published: 25 May 2017
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