Screening and validation of autophagy-related mRNAs and lncRNAs in bone nonunion based on transcriptome sequencing...

This study aims to screen for differentially expressed mRNAs (DEmRNAs) and lncRNAs (DELncRNAs) related to autophagy in bone nonunion through transcriptome sequencing, and explore their molecular mechanisms at the cellular level. In this study, 5 pairs of bone nonunion and bone healing tissue samples were collected for RNA sequencing to obtain DEmRNAs and DElncRNAs. The protein-protein interaction (PPI) network of autophagy-related differential genes was constructed. In addition, an lncRNAs-mRNAs interaction network was constructed. qRT-PCR was used to verify key autophagy-related genes. shBNIP3 was transfected into Wistar rat bone marrow mesenchymal stem cells (BMSCs), and 14 days after osteoinduction, cell migration and osteoblast differentiation were detected by transwell, alizarin red staining, and ALP staining. A total of 1108 DEmRNAs and 46 DElncRNAs were identified, and further screening resulted in 15 autophagy-related DEmRNAs. GO and KEGG analysis showed that autophagy-related DEmRNAs are mainly involved in autophagy-related pathways. In addition, the lncRNAs network included 356 relationship pairs, and 5 central autophagy-related mRNAs (BNIP3, DDIT3, SIRT2, PINK1, and BAG3) were identified. QRT-PCR verified that the expressions of BNIP3, SIRT2, PINK1, and BAG3 were up-regulated, while DDIT3 was down-regulated in patients with bone nonunion compared with patients with normal bone union. Besides, sh2BNIP3 inhibited BMSCs cell migration and osteogenic differentiation compared with Mock and BNIP3-NC groups in vitro. In this study, central autophagy-related DEmRNA may play an important role in bone nonunion, especially BNIP3, which will help us to further understand the pathogenesis of bone nonunion.