Human hair follicle mesenchymal stem cells (hHF-MSCs) are capable of differentiating into smooth muscle cells (SMCs) in response to transforming growth factor-β (TGF-β), and thus can be used for cardiovascular tissue engineering and regenerative medicine. However, the precise molecular mechanisms underlying SMC conversion of hHF-MSCs are still undefined. MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression post-transcriptionally by binding to the complementary sequences of targeted mRNAs. Accumulating evidence indicates that miRNAs are associated with SMC differentiation in vitro andin vivo. In this study, we revealed that miR-18b was significantly downregulated during TGF-β1-induced hHF-MSCs differentiation into SMC using miRNA array profiling and quantitative RT- PCR (qRT-PCR). Over-expression of miR-18b in hHF-MSCs led to remarkable downregulation of SMC-specific markers such as SMA and calponin proteins. On the contrary, inhibition of endogenous miR-18b by its antisense oligonucleotide antagomir-18b reversed the changes of SMA and calponin proteins. We also showed that SMAD2, a key transcription regulator in TGF-β signaling which was involved in SMC differentiation, is regulated by miR-18b. miR-18b could suppress the expression of SMAD2 protein by targeting the 3'UTR of SMAD2 gene without affecting its mRNA level in hHF-MSCs. Moreover, knockdown of SMAD2 by RNA interference could block the effect of inhibition of miR-18b on SMC differentiation, indicating that SMAD2 contributed to miR-18b mediated regulation of TGF-β-induced SMC differentiation. In conclusion, this study demonstrated that miR-18b regulated the TGF-β1-induced differentiation of hHF-MSCs into SMCs by targeting SMAD2 gene, and provided novel insights into the regulatory mechanisms of TGF-β-induced SMC differentiation.

译文

人毛囊间充质干细胞 (hhf-mscs) 能够响应转化生长因子-β (TGF-β) 分化为平滑肌细胞 (SMCs),因此可用于心血管组织工程和再生医学。然而,hHF-MSCs的SMC转化的确切分子机制仍未确定。Microrna (mirna) 是小的非编码rna,通过与靶向mrna的互补序列结合来调节转录后的基因表达。越来越多的证据表明,mirna在体外和体内与SMC分化有关。在这项研究中,我们揭示了使用miRNA阵列谱和定量rt-pcr (qRT-PCR) 在tgf-β1诱导的hHF-MSCs分化为SMC期间miR-18b显着下调。hHF-MSCs中miR-18b的过表达导致SMC特异性标记 (例如SMA和钙蛋白) 的显着下调。相反,其反义寡核苷酸对内源性miR-18b的抑制antagomir-18b逆转了SMA和钙蛋白的变化。我们还表明,SMAD2是参与SMC分化的TGF-β 信号传导中的关键转录调节剂,受miR-18b调节。miR-18b可以通过靶向SMAD2基因的3'UTR来抑制SMAD2蛋白的表达,而不影响其在hHF-MSCs中的mRNA水平。此外,通过RNA干扰敲除SMAD2可以阻断miR-18b对SMC分化的抑制作用,表明SMAD2有助于miR-18b介导的TGF-β 诱导的SMC分化的调节。总之,本研究表明,miR-18b通过靶向SMAD2基因调控了tgf-β1诱导的hHF-MSCs向SMC的分化,并为TGF-β 诱导的SMC分化的调控机制提供了新的见解。

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