MicroRNA-195-5p调节Bmpr1α表达对骨髓间充质干细胞成脂分化的影响

MicroRNA-195-5p affects adipocyte differentiation in bone mesenchymal stem cells by regulating Bmpr1α gene expression

目的探讨miR-195-5p在补肾方含药血清干预大鼠原代骨髓间充质干细胞(bone mesenchymal stem cell,BM SC)中的表达及其对成脂分化的影响。方法体外贴壁法原代培养的大鼠BM SC细胞经流式细胞术、CD44和CD34免疫荧光染色鉴定后,与椎间盘软骨细胞和成骨细胞一起采用荧光定量RT-PCR方法检测细胞中rno-miR-195-5p的表达情况;采用荧光素酶报告基因实验验证骨形态发生蛋白受体-1α(Bmpr1α)是否为rno-miR-195-5p的靶基因,采用Western印迹法和油红O染色检测rno-miR-195-5p对Bmpr1α表达和BMSC成脂分化的影响。结果流式细胞术与免疫荧光分析显示所培养细胞具备BMSC特征。rno-miR-195-5p在补肾方含药血清干预的原代BM SC中表达明显升高(P<0.01),且在分化完成的软骨细胞和成骨细胞中高表达。双荧光素酶报告实验与Western印迹法证实Bmpr1α是rno-miR-195-5p的靶基因。转染rno-miR-195-5p mimics上调BM SC中rno-miR-195-5p可抑制Bmpr1α的表达(P<0.05),降低成脂分化能力;而转染rno-miR-195-5p inhibitor可显著增加BM SC中Bmpr1α的表达(P<0.01),增强成脂分化能力。结论补肾方含药血清干预BMSC上调rno-miR-195-5p,通过降低其靶基因Bmpr1α的表达而降低BMSC的成脂分化,改善骨质疏松症状。

Objective To investigate the effect of rno-miR-195-5p on modulating adipocyte differentiation related to sensitivity for Bushen decoction-medicated serum of bone marrow mesenchymal stem cells （BMSCs） and its mechanism.Methods BMSCs were isolated and cultured in vitro by adherent culture method.The flow cytometric analysis and immunofluorescent staining were used to identify primarily cultured BMSCs in vitro. Quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR） was performed for verifying the expression of rno-miR-195-5p in primary BMSCs, intervertebral disc chondrocytes and osteoblasts.Bioinformatics was used for predicting target genes for rno-miR-195-5p.Dual-luciferase reporter assay system and Western blot were performed for verifying the target gene and detecting the expression of Bmpr1α.Oil red O staining was used to assess the differentiation of BMSCs to adipocytes by transfection with miRNA mimics or inhibitors.Results The adherent culture method was practicable to obtain a large number of primary rat BMSCs with typical BMSCs characteristics identified by flow cytometry and immunofluorescent staining.After comparing rno-miR-195-5p expression in Bushen decoction-containing serum culture with that from normal saline control culture,the results of RT-PCR showed that rno-miR-195-5p was markedly up-regulated （ P 〈0.01） in BMSCs cultured with Bushen decoction-containing serum.Dual luciferase report assay and Western blotting confirmed that rno-miR-195-5p can affect 3′-UTR sequence in Bmpr1α gene.Bioinformatics result showed that Bmpr1α was the direct target gene for rno-miR-195-5p.Over-expression of rno-miR-195-5p in BMSCs significantly suppressed Bmpr1α expression （ P 〈0.05）, and significantly inhibited adipogenic differentiation.We also observed that inhibition of rno-miR-195-5p in BMSCs significantly up-regulated Bmpr1α （ P 〈0.01）, and enhanced adipogenic differentiation.Conclusion The expression of rno-miR-195-5p in rat BMSCs is up-regulated after Bushen decoction-containing serum intervention.rno-miR-195-5p can suppress adipogenic differentiation via regulating Bmpr1α, which may relieve the symptom of osteoporosis.