Objective: To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation. Methods: BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 μl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured. Results: On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups. Conclusions: miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.
目的： 研究高脂与正常环境下大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）成骨分化过程中4种微RNA对蓬乱蛋白2的靶向调节作用及其对BMSC成骨分化的影响。 方法： 分别用普通（对照组）和高脂（高脂组）成骨诱导液培养BMSC，第3、5、7、14、21天实时荧光定量PCR（quantitative real-time PCR，qPCR）分析4种微RNA（micro RNA, miR）（miR-21-5p、miR-29c-3p、miR-138-5p及miR-351-5p）、蓬乱蛋白2、碱性磷酸酶（alkaline phosphatase，ALP）、Runt相关基因2（Runt-related transcription gene 2，Runx2）mRNA相对表达量，蛋白质印迹法分析蛋白表达；BMSC分别转染微RNA模拟物、抑制物和相应阴性对照（分别为模拟物组、抑制物组和相应阴性对照组），高脂成骨诱导后检测第1、3、5、7天ALP活性；第7天行ALP染色，蛋白质印迹法分析蓬乱蛋白2、ALP、Runx2的蛋白表达，qPCR分析蓬乱蛋白2 mRNA表达。构建蓬乱蛋白2野生型和突变型双荧光酶报告质粒，每种质粒分别与4种微RNA模拟物或相应阴性对照共转染293T细胞，检测荧光素酶活性。 结果： 成骨诱导各时间点高脂组BMSC的miR-21-5p、miR-29c-3p、miR-138-5p及miR-351-5p相对表达量均较对照组高，第21天分别高20%、60%、340%、4 420%（P<0.05）。转染miR-21-5p、miR-29c-3p模拟物后BMSC蓬乱蛋白2、ALP、Runx2蛋白表达均降低；其中miR-29c-3p模拟物组蓬乱蛋白2比相应阴性对照组下降35%（P<0.05），miR-29c-3p抑制物组蓬乱蛋白2比相应阴性对照组上调269%（P<0.05）。共转染野生型质粒与miR-29c-3p模拟物后，293T细胞荧光素酶活性受到抑制，较相应阴性对照组下降60%（P<0.05）。 结论： 高脂环境下BMSC成骨分化受到抑制，miR-21-5p、miR-29c-3p、miR-138-5p及miR-351-5p可能参与BMSC成骨分化调控；miR-29c-3p通过靶向调控蓬乱蛋白2表达而发挥抑制BMSC成骨分化的作用。.
Keywords: Bone marrow; Dishevelled 2; Mesenchymal stem cells; MicroRNAs.