lncTIM3 promotes osteogenic differentiation of bone marrow mesenchymal stem cells via miR-214/Smad4 axis to relieve postmenopausal osteoporosis

Abstract

Abstract Background Promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the main therapeutic goal for postmenopausal osteoporosis (PMOP). Recently, several long non-coding RNAs (lncRNAs) have been reported to be involved in PMOP; however, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIM3) remains to be investigated. Methods The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIM3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay. Results The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and OCN were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIM3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIM3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIM3 exhibited the reverse results. Subsequently, lncTIM3 was confirmed to be located in the cytoplasm of BMSC, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIM3 and Smad4 were elucidated in vitro. Conclusion lncTIM3 attenuated PMOP via miR-214/Smad4. Possibly, these findings provided lncTIM3 as a therapeutic molecule for PMOP.