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2022, Vol. 26 ›› Issue (11): 1705-1713High-throughput sequencing-based osteoporotic circRNA-miRNA-mRNA network construction and Has_ circ_0076906 functional verificationWang Hailong1, Li Long1, Wang Jianyuan1, Yan Bin1, Xier Aili1, Chen Hongtao2, Yilihamu · Tuoheti1
Abstract: BACKGROUND: Studies have shown that circular RNA (circRNA) is an endogenous non-coding RNA widely present in a variety of tissues, which can act as a "sponge" of microRNA (miRNA) to regulate mammalian gene expression. However, the expression characteristics and molecular mechanism of circRNA in osteoporosis and its potential circRNA-miRNA-mRNA regulatory network have not been reported yet. OBJECTIVE: Based on the high-throughput and high-throughput sequencing data of osteoporosis-related non-coding RNAs, to construct an interaction network and functional enrichment analysis, screen the possible mechanisms of competitive endogenous RNA (ceRNA), and verify the function and mechanism of circRNAs involved in the development of osteoporosis. METHODS: The circRNA-miRNA-mRNA network was constructed using the high-throughput sequencing data of liver cancer non-coding RNA in the GEO database. Subsequently, GO and KEGG analyses were performed on the co-expressed target genes predicted by miRNA. Through human mesenchymal stem cells-induced osteoblast differentiation, qRT-PCR and western blot were used to detect the expression of circ_0076906 and its targeted miR-29b and osteoglycoprotein (OGN) at mRNA and protein levels. Alkaline phosphatase activity and alizarin red staining were used to detect bone formation, and luciferase reporter assay used to detect gene interactions. RESULTS AND CONCLUSION: The experiment finally screened 5 co-expressed circRNAs (such as circ_0076906, circ_0004276 and circ_0003060, etc.), 10 co-expressed miRNAs (such as miR-183-5p, miR-29b and hsa-miR-15b-3p, etc.), and 241 co-expressed mRNAs (such as OGN, SMARCC1 and LRP6, etc.) from the GEO database, to successfully construct a circRNA-miRNA-mRNA network related to osteoporosis. GO analysis results revealed 78 biological processes, mainly including osteoblast proliferation, apoptosis and migration. KEGG analysis results indicated that FoxO signaling pathway and PI3K-AKT signaling pathway were closely related to the occurrence and development of osteoporosis. The verification results of circ_0076906, miR-29b and OGN, which have significant differences and target relationships, showed that circ_0076906, miR-29b and OGN were differentially expressed in the bone tissue and serum of patients with porosity and the control group. circ_0076906 could induce osteogenic differentiation, and its silencing could inhibit the expression of osteogenic genes (OCN and RUNX2) in human mesenchymal stem cells. circ_0076906 could act as miR-29b regulated OGN expression sponge. The above-mentioned data show that the osteoporosis circRNA-miRNA-mRNA network is successfully established in this study, and circ_0076906 and its targeted miR-29b and OGN screened out may play an important role in the progression of osteoporosis. The study further confirms that circ_0076906 in hMSCs promotes OGN expression by competitively inhibiting miR-29b, alleviating osteoporosis and promoting osteogenic differentiation.The study protocol was approved by the Ethics Committee of the Seventh Affiliated Hospital of Xinjiang Medical University (approval No.XY20190501-1). Key words:bone, osteoporosis, high-throughput sequencing, biological analysis, circ_0076906, miR-29b, osteoglycoprotein, targeting |