中国骨质疏松杂志

LncRNA MEG3调控miR-133a-3p/TGF-β1/Smads轴促进骨髓间充质干细胞成骨分化的作用机制

LncRNA MEG3 regulates miR-133a-3p/TGF-β1/Smads axis to promote osteogenic differentiation by bone marrow mesenchymal stem cells

DOI：10.3969/j.issn.1006-7108.2023.06.009

中文关键词: LncRNA MEG3 miR-133a-3p TGF-β1 骨髓间充质干细胞成骨分化

英文关键词:LncRNA MEG3 miR-133a-3p TGF-β1 bone marrow mesenchymal stem cells osteogenic differentiation

基金项目:深圳市科技计划项目（JCYJ20180302144355408，JCYJ20190808100818959）

作者	单位
林晓生1，2* 杜根发2 张震1 王宏波1 庄加川1 肖庆华1 韩林静1 朱建宗1	1.深圳市中西医结合医院，广东深圳 518104 2.广州中医药大学，广东广州 510405

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中文摘要:

目的探究LncRNA MEG3调控miR-133a-3p/TGF-β1/Smads信号轴对人骨髓间充质干细胞（human bone marrow mesenchymal stem cell，hBMSC）成骨分化中的影响。方法通过构建慢病毒载体及慢病毒包装，将含有目的基因的慢病毒感染细胞。将hBMSC分为Control组、NC组、MEG3、sh-MEG3、MEG3+miR-133a-3p sponge及MEG3+miR-133a-3p组，成骨诱导后，采用碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红染色检测ALP活性及钙盐沉积，qRT-PCR检测各组细胞中MEG3、miR-133a-3p、TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN mRNA表达水平，Western blot检测TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN蛋白表达量。结果与NC组比较，MEG3组的ALP活性及茜素红钙盐沉积明显减弱，MEG3 mRNA上调，miR-133a-3p mRNA表达显著升高，TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN mRNA和蛋白表达水平显著降低，差异均具有统计学意义(P＜0.05)；sh-MEG3组的ALP活性及茜素红钙盐沉积明显增强，MEG3 mRNA下调，miR-133a-3p mRNA表达显著降低，TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN mRNA和蛋白表达水平显著升高(均P＜0.05)。与MEG3组比较，MEG3+miR-133a-3p sponge组ALP活性及茜素红钙盐沉积明显增强，TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN mRNA和蛋白表达水平显著升高(P均＜0.05)；MEG3+miR-133a-3p组ALP活性及茜素红钙盐沉积明显减弱，TGF-β1、TGF-βR1、smad2、smad3、smad4、Runx2及OPN mRNA和蛋白表达水平显著降低(P均＜0.05)。结论 LncRNA MEG3可能通过调控miR-133a-3p/TGF-β1/Smads信号轴促进hBMSC成骨分化。

英文摘要:

Objective To explore the effect of LncRNA MEG3 on osteogenic differentiation by human bone marrow mesenchymal stem cells (hBMSC) through regulating miR-133a-3p/TGF-β1/ Smads signal axis. Methods Lentivirus vector and lentivirus package were constructed to infect cells with lentivirus containing target genes. HBMSC were divided into Control group, NC group, MEG3, sh-MEG3, MEG3+miR-133a-3p sponge group, and MEG3+miR-133a-3p group. After osteogenic induction, alkaline phosphatase (ALP) staining and Alizarin red staining were used to detect ALP activity and calcium deposition. Real-time fluorescence quantitative (qRT-PCR) analysis was used to detect the expression levels of MEG3, miR-133a-3p, TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN mRNA in each group. Western blotting analysis was used to detect the protein expressions of TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN. Results Compared with those in the NC group, the ALP activity and Alizarin red calcium salt deposition reduced significantly in the MEG3 group. MEG3 mRNA was up-regulated, the expression of miR-133a-3p mRNA increased significantly, and the mRNA and protein expression levels of TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN decreased significantly in the MEG3 group (P<0.05). In the sh-MEG3 group, ALP activity and Alizarin red calcium salt deposition increased significantly, MEG3 mRNA was down-regulated, the expression of miR-133a-3p mRNA decreased significantly, and the mRNA and protein expression levels of TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN increased significantly (P<0.05). Compared with those in the MEG3 group, ALP activity and Alizarin red calcium salt deposition increased significantly in the MEG3+miR-133a-3p sponge group, and the mRNA and protein expression levels of TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN increased significantly (P<0.05). In the MEG3+miR-133a-3p group, ALP activity and Alizarin red calcium salt deposition decreased significantly, and the mRNA and protein expression levels of TGF-β1, TGF-βR1, smad2, smad3, smad4, Runx2, and OPN decreased significantly (P<0.05). Conclusion LncRNA MEG3 may promote the osteogenic differentiation of hBMSC by regulating miR-133a-3p/TGF-β1/Smads signal axis.

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