Biological activity and expression profile of long non-coding RNAs in jaw bone marrow-derived mesenchymal stem cells under hypoxic condition

Background In recent years, jaw bone marrow-derived mesenchymal stem cells (JBMMSCs) have drawn attentions for their good biological characteristics. As a common microenvironment, low oxygen plays a major role in enhancing MSCs resistance and is worthy of further exploration. However, the effect of lncRNAs on the regulation of the biological performance of JBMMSCs is not yet reported.

Objective To investigate the effect of hypoxia on the biological activity of JBMMSCs and the differentially expressed lncRNAs and mRNAs after hypoxic treatment, so as to provide theoretical references for further exploring the mechanism of lncRNAs in regulating the biological properties of JBMMSCs.

Methods JBMMSCs were isolated and cultured, and their mesenchymal stem cell characteristics were identified. JBMMSCs were stimulated with 50 µmol/L cobalt chloride (CoCl₂) to establish a hypoxic cell model of JBMMSCs, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence and Western blot to verify the cell model. The effects of hypoxia on the proliferation, migration and other biological activities of JBMMSCs were observed by CCK-8, cell scratch test and Ki67 immunofluorescence staining. High-throughput sequencing was performed in JBMMSCs under hypoxic and normoxic conditions, and |log2FC|≥1 and P＜0.05 was applied for screening criteria, so as to screen differentially expressed lncRNAs and mRNAs and forecast its biological function and mechanism of action.

Results JBMMSCs extracted from the mouse jaw grew in a long fusiform and whirlpool shape, which was identified as mesenchymal stem cells. After treatment with 50 µmol/L CoCl₂ for 72 hours, the expression of HIF-1α, a marker of hypoxia, was significantly higher than that of the control group (P=0.0023), suggesting that the hypoxic cell model was successfully established. CCK-8 and Ki67 immunofluorescence staining results showed that the proliferation ability of JBMMSCs was significantly increased under hypoxia (P=0.0057). Wound healing assay showed that hypoxia significantly enhanced the migration ability of JBMMSCs (P=0.0040). Using high-throughput sequencing technology, we identified 12 differentially expressed lncRNAs under hypoxia, of which 4 were up-regulated and 8 were down-regulated. Bioinformatics analysis was used to predict the downstream miRNAs and target genes of lncRNA-4632427E13Rik with the most significant difference, and an interaction network of lncRNA-miRNA-Mrna was preliminarily formed. Through GO and KEGG analysis, 14 signaling pathways were significantly enriched after hypoxia treatment, and many of them might be related to the regulation of stem cell proliferation, differentiation and plasticity maintenance.

Conclusion Hypoxia can promote the proliferation, migration and other activities of JBMMSCs, and lnc-4632427E13Rik may play a role in the regulation of biological properties of JBMMSCs under hypoxia.