骨髓炎状态下成骨诱导大鼠骨髓间充质干细胞转录组的测序分析

Transcriptome sequencing analysis of osteogenic rat bone marrow mesenchymal stem cells induced by osteomyelitis

背景:近来发现骨髓炎环境会显著抑制骨髓间充质干细胞的成骨分化能力,进而对骨形成和骨代谢造成影响,但其具体的作用机制尚不十分清楚。目的:通过对葡萄球菌A蛋白作用下诱导成骨分化的大鼠骨髓间充质干细胞进行转录组分析,寻找潜在差异表达长链非编码RNA(lncRNA)及相关调控网络。方法:将大鼠骨髓间充质干细胞分为实验组和对照组,前者在葡萄球菌A蛋白(1μg/mL)模拟的骨髓炎环境下成骨诱导分化7,14 d,后者正常成骨诱导分化7,14 d;通过茜素红染色以及碱性磷酸酶活性检测观察两组细胞成骨诱导分化情况;收集两组细胞进行转录组测序,进行GO、KEGG生物信息学分析;采用实时荧光定量PCR进一步验证差异表达lncRNA。结果与结论:①成骨诱导分化7,14 d,对照组茜素红着色面积均高于实验组,对照组碱性磷酸酶活性明显高于实验组(P<0.01);②转录组测序结果显示328个lncRNAs表达差异,其中显著上调的184个,显著下调的144个;③基因本体富集分析发现差异表达基因功能主要涉及生物过程、细胞组分以及分子功能,KEGG富集分析发现主要参与的信号转导途径有肿瘤坏死因子信号通路、MAPK信号通路、肠道炎症疾病通路等,与骨形成、骨代谢相关;④选取与成骨分化相关的差异表达明显的6个lncRNAs,qPCR验证后发现ENSRNOT00000092811、TCONS_00023877、XR_001836916.1与高通量测序中的表达趋势一致;⑤结果表明,多个lncRNA参与骨髓炎状态的大鼠骨髓间充质干细胞成骨分化能力调节,且可能通过信号转导途径肿瘤坏死因子信号通路、MAPK信号通路等发挥作用。

BACKGROUND:Recently,osteomyelitis environment has been found to significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells,thereby affecting bone formation and bone metabolism,but the specific mechanism remains unclear.OBJECTIVE:Transcriptome analysis was conducted on rat bone marrow mesenchymal stem cells with osteogenic differentiation induced by staphylococcal protein A to search for potential differentially expressed lncRNAs and related regulatory networks.METHODS:Bone marrow mesenchymal stem cells of rats were divided into experimental group and control group.The former group was induced osteogenic differentiation for 7 and 14 days in staphylococcal protein A(1μg/mL)(to simulate osteomyelitis environment),while the control group was induced osteogenic differentiation for 7 and 14 days without any treatment.The content detection of alkaline phosphatase and alizarin red staining were used to observe the osteogenic induction of the cells.Two groups of cells were collected for transcriptome sequencing,gene ontology and KEGG bioinformatics analysis.Differential expression levels of lncRNAs were further verified by real-time fluorescence quantitative PCR.RESULTS AND CONCLUSION:(1)After 7 and 14 days of osteogenic differentiation,the staining area of alizarin red in the control group was larger than that in the experimental group,and the alkaline phosphatase activity in the control group was significantly higher than that in the experimental group(P<0.01).(2)Transcriptome sequencing results found 328 lncRNAs with significant differential expression level,with 184 significantly up-regulated and 144 significantly down-regulated.(3)Gene enrichment analysis showed that the function of differentially expressed genes mainly involved biological process,cell classification and molecular function.KEGG enrichment analysis found that the main signal pathways included tumor necrosis factor,MAPK and intestinal inflammatory disease signaling pathways,which were related to bone formation and bone metabolism.(4)Six differentially expressed lncRNAs related to osteogenesis differentiation were selected,and the expression trends of ENSRNOT00000092811,TCONS_00023877,and XR_001836916.1 were consistent with highthroughput sequencing after qPCR validation.(5)The results showed that multiple lncRNAs were involved in the regulation of osteogenic differentiation ability of rat bone marrow mesenchymal stem cells in osteomyelitis,and they may play a role through signal pathway,such as tumor necrosis factor and MAPK signaling pathways.