The effects of bleomycin and rapamycin on cellular senescence and differentiation of rabbit annulus fibrosus stem cells (AFSCs) were investigated using a cell culture model. The results showed that bleomycin induced cellular senescence in AFSCs as evidenced by senescence-associated secretory phenotype. The morphology of AFSCs was changed from cobblestone-like cells to pancake-like cells. The senescence-associated β-galactosidase activity, the protein expression of P16 and P21, and inflammatory-related marker gene levels IL-1β, IL-6, and TNF-α were increased in bleomycin-treated AFSCs in a dose-dependent manner. Rapamycin treatment decreased the gene expression of MMP-3, MMP-13, IL-1β, IL-6, TNF-α, and protein levels of P16 and P21 in bleomycin-treated AFSCs. Furthermore, neither bleomycin nor rapamycin changed the ribosomal S6 protein level in AFSCs. However, the phosphorylation of the ribosomal S6 protein was increased in bleomycin-treated AFSCs and decreased in rapamycin-treated AFSCs. AFSCs differentiated into adipocytes, osteocytes, and chondrocytes when they were cultured with respective differentiation media. Rapamycin inhibited multi-differentiation potential of AFSCs in a concentration-dependent manner. Our findings demonstrated that mammalian target of rapamycin (mTOR) signaling affects cellular senescence, catabolic and inflammatory responses, and multi-differentiation potential, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.

Osteosarcoma is the most common type of malignant tumor arising from bone in children and adolescents. Accumulating evidences have shown the aberrant expression of numerous miRNAs is associated with the development and metastasis of osteosarcoma. The present study was conducted to investigate miR-27a expression in osteosarcoma tissues and cells. In the present study, quantitative RT-qPCR was used to measure the expression levels of miRNA and mRNA in osteosarcoma tissues and cells. Transwell assays were used to detect the effects of miR-27a on the invasive and migratory potential of cells. Luciferase reporter and western blot analysis were conducted to confirm cyclin G1 (CCNG1) as the target gene of miR-27a. The results showed that miR-27a was significantly upregulated in human osteosarcoma tissues and cell lines. The western blot analysis revealed that the overexpression of miR-27a suppressed CCNG1 protein expression. Luciferase reporter assays confirmed that CCNG1 is a direct target of miR-27a in osteosarcoma cells. The results suggest that miR-27a downregulates CCNG1 expression in osteosarcoma and acts as an oncogene directly targeting CCNG1. Thus, the miR-27a/CCNGI axis is a potential therapeutic target for human osteosarcoma.

The present study investigated the key genes, which cause switch from adipogenic to osteogenic differentiation of human mesenchymal stem cells (hMSCs). The transcriptomic profile of hMSCs samples were collected from Array Express database. Differential expression network was constructed by calculating the Pearson's correlation coefficient and ranked according to their topological features. The top 5% genes with degree ≥2 were selected as ego genes. Following the KEGG pathway enrichment analysis and the relevant miRNAs prediction, the miRNA-mRNA-pathway networks were constructed by combining the miRNA-mRNA pairs and mRNA-pathway pairs together. In total, we obtained 84, 119, 94 and 97 ego-genes in B, BI, BT and BTI groups, and DLGAP5, DLGAP5, NUSAP1 and NDC80 were the ego-genes with the highest z-score of each group, respectively. Beginning from each ego-gene, we identified 2 significant ego-modules with gene size ≥4 in group BI, and the ego-genes were PBK and NCOA3, respectively. Through KEGG pathway analysis, we found that most of the pathways enriched by ego-genes were associated with gene replication and repair, and cell proliferation. According to the miRNA prediction results, we found that some of the predicted miRNAs have been validated to be the regulatory miRNAs of these corresponding mRNAs. Finally we constructed a miRNA-mRNA-pathway network by integrating the miRNA-mRNA and mRNA-pathway pairs together. The constructed network gives us a more comprehensive understanding of the mechanism of osteogenic differentiation of hMSCs.