LncRNA-CWF19L1调控miR-132-3p抑制人骨髓间充质干细胞成骨分化的作用研究

LncRNA-CWF19L1 inhibits osteoblast differentiation of human mesenchymal stem cells by regulating MiR-132-3p

目的探讨lncRNA-CWF19L1通过调控miR-132-3p抑制人骨髓间充质干细胞成骨分化的作用机制。方法通过双荧光素酶试验验证lncRNA-CWF19L1与miR-132-3p以及miR-132-3p与Smad2的相互关系;通过茜素红(ARS)染色和碱性磷酸酶(ALP)染色计量钙结节数量变化;通过qRT-PCR和Western Blot检测成骨基因(ALP、BMP2和Runx2)的表达量变化。结果过表达lncRNA-CWF19L1抑制骨髓间充质干细胞成骨分化。利用生物信息学预测方法发现lncRNA-CWF19L1可直接靶控miR-132-3p,而Smad2作为miR-132-3p的下游靶基因。过表达lncRNA-CWF19L1致miR-132-3p表达上调;而上调miR-132-3p致Smad2表达下调,进而抑制人骨髓间充质干细胞成骨分化。结论lncRNA-CWF19L1可通过调控miR-132-3p来抑制Smad2的表达,进而抑制人骨髓间充质干细胞成骨分化。

Objective To investigate the mechanism of lncRNA-CWF19 L1 in inhibiting osteoblast differentiation of human bone marrow mesenchymal stem cells by regulating miR-132-3 p. Methods Dual luciferase reporter assay was conducted to investigate the relationships between lncRNA-CWF19 L1 and miR-132-3 p, and between miR-132-3 p and smad2.qRT-PCR. Western blot analyses were conducted to detect the expressions of osteoblast molecular markers(ALP, BMP2 and Runx2). The staining and activity assays of Alizarin red staining(ARS) and alkaline phosphatase(ALP) revealed the capacity of osteogenesis differentiation in hBMSCs.Results Knockdown of lncRNA-CWF19 L1 increased the activities of ALP and BMP2, and promoted osteoblast differentiation in hBMSCs. By utilizing bioinformatics prediction approaches, lncRNA-CWF19 L1 was identified to be bound directly to miR-132-3 p, while Smad2 was also identified as a direct target of miR-132-3 p. Furthermore, shRNA against lncRNA-CWF19 L1 demonstrated a significant decrease in miR-132-3 p expression, which further upregulated Smad2, and led to increased expressions of BMP2 and ALP.Conclusion A novel mechanism in osteoblast differentiation of human mesenchymal stem cells by which lncRNA-CWF19 L1 inhibits osteoblast differentiation is via sponging miR-132-3 p, so as to down-regulates Smad2 hBMSCs.