LncRNA MEG8靶向miR-203调控人牙周膜干细胞成骨分化的机制研究  

LncRNA MEG8 regulate osteogenic differentiation of human periodontal ligament stem cells by targeting miR-203

作　　者：袁清敏 付娟[2] 王美玲 赵西博 冯保静[1] YUAN Qing-min;FU Juan;WANG Mei-ling;ZHAO Xi-bo;FENG Bao-jing(The Six People’s Hospital of Anyang City,Anyang 455000,China;Henan Nursing Vocational College,Anyang 455000,China;Department of Oral Medicine,Qingdao Chengyang People’s Hospital,Qingdao 266109,China)

机构地区：[1]安阳市第六人民医院,安阳455000 [2]河南护理职业学院,安阳455000 [3]青岛市城阳区人民医院口腔科,青岛266109

出　　处：《口腔颌面修复学杂志》2024年第6期401-408,共8页Chinese Journal of Prosthodontics

摘　　要：目的:体外研究长链非编码RNA(long non-coding RNA,lnc RNA)MEG8调控人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)成骨分化能力并探究其调控机制。方法:体外分离、纯化培养hPDLSCs,q PCR检测hPDLSCs成骨诱导前后lnc RNA MEG8和成骨相关基因Runx2、Osx、Ocn、Opn的表达改变;通过MEG8过表达及sh RNA慢病毒转染hPDLSCs并鉴定转染效率;通过Western blot检测RUNX2、OSX、OCN、OPN的表达变化,茜素红染色法检测hPDLSCs成骨诱导后钙化结节的生成情况;借助生物信息学方法分析lnc RNA MEG8的候选靶基因,并通过双荧光素酶报告实验进一步验证潜在靶基因;通过共转染miR-203 mimics和lnc RNA MEG8过表达慢病毒,分析共转染后hPDLSCs成骨分化能力的变化。结果:Runx2、Osx、Ocn、Opn成骨基因及lnc RNA MEG8的表达在hPDLSCs成骨诱导后显著上调;敲降lnc RNA MEG8可抑制hPDLSCs成骨分化并下调RUNX2、OSX、OCN、OPN蛋白的表达,而在hPDLSCs中过表达lnc RNA MEG8,则结果相反;通过相关性分析发现miR-203与lnc RNA MEG8呈负相关;进一步通过生物信息学分析lnc RNA MEG8的潜在靶基因含有miR-203,双荧光素酶报告实验验证miR-203为MEG8的靶基因;共转染实验结果发现miR-203 mimics可逆转lnc RNA MEG8对hPDLSCs成骨分化的促进作用。结论:Lnc RNA MEG8可靶向调控miR-203促进hPDLSCs成骨分化能力,提示lnc RNA MEG8可作为牙周炎的潜在干预和治疗靶点。Objective:To investigate the effect and mechanism of long non-coding RNA(lncRNA)MEG8 on the osteogenic differentiation of hPDLSCs.Methods:hPDLSCs were isolated,purified and cultured in vitro.The expression of Osx,Runx2,Ocn,Opn and MEG8 was detected by qPCR.MEG8 overexpression and shRNA lentivirus were transfected in hPDLSCs and the transfection efficiency were identified.The RUNX2,OSX,OCN and OPN protein expression in hPDLSCs was detected by Western blot.The osteogenic differentiation of hPDLSCs was estimated by ARS(Alizarin red staining assays)demonstrated by the formation of calcified nodules.Bioinformatics methods were used to analyze candidate target genes of lncRNA MEG8,and potential target genes were further validated through dual luciferase reporter assays.The changes in osteogenic differentiation ability of hPDLSCs were analyzed after cotransfection with miR-203 mimics and lncRNA MEG8 overexpressing lentivirus.Results:The expression of Runx2,Osx,Ocn,Opn osteogenic genes and lncRNA MEG8 was significantly upregulated after osteoinduction in hPDLSCs.Knockdown of lncRNA MEG8 inhibited osteogenic differentiation of hPDLSCs and downregulated the expression of RUNX2,OSX,OCN,and OPN proteins,while overexpressing lncRNA MEG8 in hPDLSCs led to the opposite result.Through correlation analysis,it was found that miR-203 was negatively correlated with lncRNA MEG8.Further bioinformatics analysis revealed that the potential target gene of lncRNA MEG8 contained miR-203,and dual luciferase reporter experiments confirmed that miR-203 was the target gene of MEG8.The co-transfection experiment results showed that miR-203 mimics reversed the promoting effect of lncRNA MEG8 on the osteogenic differentiation of hPDLSCs.Conclusion:Our results demonstrate that lncRNA MEG8 absorbs miR-203 to promote osteogenic differentiation of hPDLSCs and shed new insights and targets for periodontitis therapeutics.

关 键 词：lncRNA MEG8 miR-203 人牙周膜干细胞(hPDLSCs) 成骨分化 

分 类 号：R780.2[医药卫生—口腔医学]