The Effect And Mechanism Of Long Noncoding RNA Lnc-ob1 To Promote BMSCs Osteogenic Differentiation

Objectives: To study the biological effect and molecular mechanism of lnc-ob1 to promote BMSCs osteogenic differentiation.Methods: 1.We established mouse BMSCs osteogenic differentiation model,and detected the expression level of lnc-ob1 during BMSCs osteogenic differentiation by QPCR.2.After transfected with lnc-ob1 expression vector to overexpressed lnc-ob1 or transfected with lnc-ob1 ASO to knockdown lnc-ob1,mouse BMSCs were induced osteogenic differentiation.The expression of osteogenic marker genes Alp and Bglap were detected by QPCR.The extracellular matrix mineralization was observed by alizarin red staining.The alkaline phosphatase activity was detected by alkaline phosphatase detection kit.3.We performed RNA-seq to screen differential expression genes in osteoblasts which transfected with lnc-ob1 overexpression vector or lnc-ob1 ASO.4.GO analysis was performed to obtain functional cluster of differential expression genes.KEGG analysis was used to cluster the signaling pathways.5.The key factor and downstream genes in the signaling pathway were detected by QPCR and Western blotting.6.We performed RNA pull-down and mass spectrometry analysis to identify lnc-ob1 binding protein.RNA pull-down and RIP assays were performed to further confirmed the interaction of lnc-ob1 and binding protein.7.The effect of lnc-ob1 on the expression of binding protein was analyzed by QPCR.The effect of lnc-ob1 on the level of binding protein was analyzed by Western blotting.Ch IP assay was performed to detect the H3K27me3 enrichment in gene promoter.Results: 1.Compared to undifferentiated BMSCs(0 week),the level of lnc-ob1 was upregulated about 2.3 times in 1 week,4.8 times in 2 week and 8 times in 3 week after induced BMSCS osteogenic differentiation.2.Two weeks after BMSCs which overexpressed lnc-ob1 were induced osteogenic differentiation,the expression of osteogenic marker genes Alp and Bglap were upregulated,mineralized nodules staining was enhanced and Alp activity was improved.3.Two weeks after BMSCs which knockdown lnc-ob1 were induced osteogenic differentiation,the expression of osteogenic marker genes Alp and Bglap were down-regulated,mineralized nodules staining was reduced and Alp activity was declined.4.According to RNA-seq data,there are 161 differentially expressed genes in osteoblasts overexpressed lnc-ob1,the expression of 98 genes were upregulated and 63 genes were down-regulated.There are 374 differentially expressed genes in osteoblasts knockdown lnc-ob1,the expression of 228 genes were upregulated and 146 genes were down-regulated.Integrated the two part,there are 70 differentially expressed genes.5.GO analysis suggested that the differentially expressed genes showed a significant enrichment in some function terms,such as osteogenic differentiation(P<0.05).KEGG analysis suggested that the osteogenic differentiation related genes were mainly enriched in Sp7 signaling pathway.6.The expression level of Sp7 was increased in osteoblasts when lnc-ob1 was overexpressed,and the expression levels of Bsp and Fgf3,the downstream genes of Sp7,were also upregulated.While the expression level of Sp7 was reduced in osteoblasts when lnc-ob1 was knockdown,and the expression levels of Bsp and Fgf3 were also down-regulated.7.The RNA pull-down and mass spectrometry analysis identified lnc-ob1 binding protein Suz12 in osteoblasts,subsequent RNA pull-down and RIP assays proved specific binding of lnc-ob1 to Suz12 protein.8.QPCR data showed that the expression level of Suz12 have no significant difference when overexpressed or knockdown lnc-ob1.Western blotting data suggested that the level of Suz12 protein have no significant difference when overexpressed or knockdown lnc-ob1.9.Ch IP analyses showed that lnc-ob1 overexpression significantly reduce H3K27me3 enrichment in Sp7 promoter,while lnc-ob1 knockdown obviously enhances H3K27me3 enrichment of Sp7 promoter in osteoblasts.Conclusion:1.The expression of lnc-ob1 was gradually up-regulated during BMSCs osteogenic differentiation.2.The overexpression of lnc-ob1 in BMSCs promote osteogenic differentiation.3.The knockdown of lnc-ob1 in BMSCs inhibit osteogenic differentiation.4.lnc-ob1 mainly regulates the expression levels of osteogenic differentiation related genes in osteoblasts.5.lnc-ob1 regulates Sp7 signaling pathway.6.lnc-ob1 promote the expression levels of Sp7 and its downstream genes.7.lnc-ob1 can binding to Suz12.8.lnc-ob1 have no effect on the expression levels of Suz12.9.lnc-ob1 can significantly reduce H3K27me3 enrichment in Sp7 promoter,consequently promote the expression of Sp7.