LncRNA TERC调控H3K27me3、DKK1在地塞米松干预间充质干细胞成骨细胞分化中的相关研究

LncRNA TERC modulation of H3K27me3 and DKK1 in osteoblast differentiation of Dexamethasone-intervened mesenchymal stem cells

目的 研究地塞米松干预骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化后,过表达LncRNA TERC对组蛋白H3K27三甲基化(H3K27me3)及DKK1表达的影响,从而进一步验证其对成骨分化的作用。方法 (1)地塞米松干预BMSCs成骨分化,实验分为对照组(单纯成骨诱导组)及干预组(地塞米松干预成骨组),第14天使用茜素红染色检测的成骨分化能力,RT-PCR检测第3、7、14、21 d各组细胞中BMP-2、SMAD-1、Dlx5、Dlx3等成骨相关基因的表达。(2)地塞米松干预BMSCs成骨分化,构建TERC过表达载体,分别设置:空白对照组(地塞米松干预成骨组)、空白载体组、TERC过表达组。RT-PCR检测第3、7、14、21 d各组细胞中BMP-2、SMAD-1、Dlx5、Dlx3等成骨相关基因的表达,Western blot实验检测各组细胞中H3K27me3、DKK1的蛋白水平。结果 (1)地塞米松干预BMSCs成骨分化后,茜素红染色显示其成骨能力下降(P均<0.05),且RT-PCR显示,随着地塞米松干预时间的延长,BMP-2、SMAD-1、Dlx5、Dlx3等成骨相关基因均下调(P均<0.05)。(2)TERC过表达转染实验中,TERC过表达组细胞中BMP-2、SMAD-1、Dlx5、Dlx3等成骨相关基因与其他两组比较均上调(P均<0.05),其他两组以上成骨基因表达水平比较,差异无统计学意义(P均>0.05);TERC过表达组细胞中H3K27me3的蛋白表达水平较其他两组显著上升(P均<0.05),DKK1的蛋白水平较其他两组显著下降(P均<0.05)。其他两组中以上基因蛋白表达水平相近,差异无统计学意义(P>0.05)。结论 过表达LncRNA TERC能够显著调高地塞米松干预BMSCs向成骨细胞分化能力,并与H3K27me3及DKK1在地塞米松干预BMSCs向成骨细胞分化中存在有相关性。

Objective To study the effect of overexpression of LncRNA TERC on the expression of histone H3K27 trimethylation(H3K27me3)and DKK1 after dexamethasone intervention in osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)to further validate its role in osteogenic differentiation.Methods①Dexamethasone intervention in osteogenic differentiation of BMSCs.The experiment was divided into the control group(osteogenic induction alone)and the intervention group(Dexamethasone).On the 14th day,using alizarin red staining to detect the osteogenic differentiation ability,RT-PCR detection was performed on the 3rd,7th,14th,and 21st day of each group of cells in the expression of osteogenic-related genes,such as BMP-2,SMAD-1,Dlx5 and Dlx3.②Dexamethasone intervened in the osteogenic differentiation of BMSCs;a TERC overexpression vector was constructed;and the following groups were set up:a blank control group(osteogenic group with Dexamethasone intervention);a blank vector group;and a TERC overexpression group.RT-PCR was performed to detect the expression of osteogenic genes,such as BMP-2,SMAD-1,Dlx5 and Dlx3,in the cells of each group on day 3,7,14 and 21,and Western blot was performed to detect the protein levels of H3K27me3 and DKK1 in the cells of each group.A Western blot assay was performed to detect the protein levels of H3K27me3 and DKK1 in each group of cells.Results①After Dexamethasone intervened in the osteogenic differentiation of BMSCs,alizarin red staining showed a decrease in osteogenic capacity(all P<0.05),and RT-PCR showed that osteogenesis-related genes,such as BMP-2,SMAD-1,Dlx5 and Dlx3,were down-regulated with the prolongation of intervention time(all P<0.05).②In the TERC overexpression transfection experiment,osteogenesisrelated genes such as BMP-2,SMAD-1,Dlx5,Dlx3,etc.were up-regulated in the cells of the TERC overexpression group compared with those of the other two groups(P<0.05),and there was no difference in the expression levels of the osteogenic genes in the other two groups(P>0.05);the protein expression level of H3K27me3 in the cells of the TERC overexpression group was significantly increased compared with the other two groups(P both<0.05),and the protein level of DKK1 was significantly decreased compared with the other two groups(P both<0.05).The protein expression levels of the above genes in the other two groups were similar,with no significant difference(P>0.05).Conclusion Overexpression of LncRNA TERC significantly increased the ability of dexamethasone to interfere with BMSCs differentiation into osteoblasts,and was correlated with H3K27me3 and DKK1 in the intervention of dexamethasone in BMSCs differentiation into osteoblasts.