过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化

Overexpression of miR-155 Inhibits the Osteogenic Differentiation of Mesenchymal Stem Cells C3H10T1/2 Induced by BMP9

目的:研究过表达miR-155对BMP9诱导间充质干细胞C3H10T1/2成骨分化的影响。方法:(1)用重组腺病毒Ad-BMP9(BMP9)诱导C3H10T1/2细胞成骨分化,定量PCR(qPCR)检测miR-155的表达,RT-PCR检测Runx2和ALP的表达。(2)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测miR-155的表达,ALP活性和染色检测早期成骨能力。(3)miR-155和BMP9共同处理C3H10T1/2细胞,诱导分化14d茜素红S染色检测晚期成骨能力。(4)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN的表达。(5)miR-155和BMP9共同处理C3H10T1/2细胞,Western blot检测p-Smad1/5/8、OCN和OPN蛋白水平的表达。(6)qPCR和Western blot分别检测HIF1α和VEGF的mRNA表达水平和蛋白质表达水平。(7)应用荧光素酶报告基因对miR-155的靶基因进行筛选和验证。结果:在BMP9诱导C3H10T1/2细胞成骨分化过程中,过表达miR-155降低ALP活性及染色;减少钙盐沉积;成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN表达降低;抑制p-Smad1/5/8、OCN和OPN蛋白水平的表达;HIF1α和VEGF的mRNA和蛋白表达水平减少。在对靶基因的检测中,过表达miR-155可以抑制HIF1α蛋白水平的表达,但对其mRNA水平无明显影响。结论:miR-155过表达减弱BMP9诱导间充质干细胞C3H10T1/2成骨分化,可能是通过抑制Smad/BMP信号通路发挥作用,也有可能是通过抑制靶基因HIF1α的表达来发挥作用。

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 TreatedC3H10T1/2 cells with BMP9 and miR- 155, tested osteogenesis-related genes by qPCR, include Runx2, OSX, COLlAl, ALP, OCN and OPN. ⑤ TreatedC3H10T1/2 cells with BMP9 and miR-155, detected the expression of p-Smadl/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, COLIA1, ALP, OCN and OPN, also. On the protein levels, overexpressed miR-155 inhibited the expression of p-Smadl/5/8, OCN and OPN, the expression of HIFα 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.