LncRNA MALAT1通过miR-34c/SATB2轴对脂肪间充质干细胞成骨分化的促进作用

Promotion effect of LncRNA MALAT1 on osteogenic differentiation of adipose-derived mesenchymal stem cells through miR-34c/SATB2 axis

目的:探讨长非编码RNA肺癌转移相关转录本1(LncRNA MALAT1)通过微小RNA(miR)-34c/特异AT序列结合蛋白2(SATB2)轴对脂肪来源的间充质干细胞(ADSCs)成骨分化的影响,并阐明其作用机制。方法:人ADSCs转染Lenti-NC、Lenti-MALAT1、sh-NC、sh-MALAT1、miR-NC和miR-34c,RT-PCR法检测ADSCs中LncRNA MALAT1、miR-34c和SATB2 mRNA表达水平;miRcode和TargetScan 7.1网站预测并通过荧光素酶报告基因实验验证miR-34c与LncRNA MALAT1、miR-34c与SATB2之间的靶向结合作用;Western blotting法检测ADSCs中成骨标志物Runt相关转录因子2(Runx2)、骨桥蛋白(OPN)和骨钙蛋白(OCN)表达水平;碱性磷酸酶(ALP)染色和茜素红S(ARS)染色检测ADSCs中ALP和ARS水平及钙盐沉积。结果:与第0天比较,ADSCs成骨诱导第3、7、14和21天后,细胞中LncRNA MALAT1、SATB2、Runx2、OPN和OCN蛋白表达水平明显升高(P<0.05),miR-34c表达水平明显降低(P<0.05)。与Lenti-NC组和sh-NC组比较,Lenti-MALAT1组ADSCs中LncRNA MALAT1表达水平,Runx2、OPN和OCN蛋白表达水平,ALP和ARS水平明显升高(P<0.01),sh-MALAT1组上述指标明显降低(P<0.01)。与miR-NC+Lenti-NC组比较,miR-34c+Lenti-NC组ADSCs中Runx2、OPN和OCN蛋白表达水平明显降低(P<0.01),ALP和ARS水平明显降低(P<0.01),miR-34c+Lenti-MALAT1组ADSCs中上述各项指标比较差异无统计学意义(P>0.05)。与Lenti-NC+miR-NC组比较,Lenti-SATB2+miR-NC组ADSCs中Runx2、OPN和OCN蛋白表达水平及ALP和ARS水平明显升高(P<0.01),Lenti-SATB2+miR-34c组上述各项指标差异无统计学意义(P>0.05)。结论:LncRNA MALAT1通过miRNA-34c/SATB2轴促进ADSCs的成骨分化。

Objective:To explore the effect of LncRNA MALAT1 on the osteogenic differentiation of adiposederived mesenchymal stem cells(ADSCs)through the microRNA-34c/SATB2 axis,and to clarify its mechanism.Methods:The human ADSCs were transfected with Lenti-NC,Lenti-MALAT1,sh-NC,sh-MALAT1,miR-NC and miR-34c.RT-PCR method was used to detect the expression levels of LncRNA MALAT1,miR-34c and SATB2 mRNA in the ADSCs.MiRcode and TargetScan 7.1 website were used to predicte and the targeted binding effect between miR-34c and LncRNA MALAT1,miR-34c and SATB2 were verified through luciferase reporter gene experiment;Western blotting method was used to detect the expression levels of osteogenic markers Runx2,OPN and OCN proteins in the ADSCs;ALP staining and Alizarin red S(ARS)staining were used to detect the levels of ALP and ARS and calcium salt deposition in the ADSCs.Results:Compared with day 0,the expression levels of LncRNA MALAT1,SATB2,Runx2,OPN,and OCN proteins in the cells were significantly increased on the 3rd,7th,14th,and 21st days after osteogenic induction of ADSCs(P<0.05);the expression levels were significantly reduced(P<0.05).Compared with Lenti-NC group or sh-NC group,the expression level of LncRNA MALAT1,the expression levels of Runx2,OPN and OCN proteins,and the levels of ALP and ARS in the ADSCs in Lenti-MALAT1 group were significantly increased(P<0.01),and the indexes mentioned obove in sh-MALAT1 group were significantly decreased(P<0.01).Compared with miR-NC+Lenti-NC group,the expression levels of Runx2,OPN and OCN proteins in the ADSCs in miR-34c+Lenti-NC group were significantly reduced(P<0.01),and the levels of ALP and ARS were decreased(P<0.01),but the indexes mentioned obove in miR-34c+Lenti-MALAT1 group had no statistically significant differences(P>0.05).Compared with Lenti-NC+miR-NC group,the expression levels of Runx2,OPN and OCN proteins in the ADSCs in Lenti-SATB2+miR-NC group were significantly increased(P<0.01),and the levels of ALP and ARS were significantly increased(P<0.01);but the indexes mentioned above in Lenti-SATB2+miR-34c group had no statistically significant differences(P>0.05).Conclusion:LncRNA MALAT1 promotes the osteogenic differentiation of ADSCs through the miRNA-34c/SATB2 axis.