| Mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, due to their capabilities of self-renewal and differentiating into multiple cell lineages. Under defined conditions, they can differentiate into multiple cell lineages including osteoblasts, chondrocytes, and adipocytes, suggesting their potential use in cell-based regenerative therapy. Despite the extensive potential in cell therapy and wide interest in clinical applications of MSCs, the molecular mechanisms regarding the regulation of their fates remains to be fully elucidated. MiRNAs are a class of small endogenous (-22 nucleotides) and single-stranded noncoding RNAs. By transcriptional or post-transcriptional regulation of gene expression, they may play crucial roles in various cellular and biological processes. Recently, emerging evidence has shown that miRNAs are closely involved in controlling cellular proliferation, differentiation and apoptosis. MiR-144-3p, a well-characterized tumor suppressor, has been reported to regulate cell proliferation in multiple cancer or tumor cells. However, to our knowledge, no report is available on the involvement of miR-144-3p in proliferation and differentiation of MSCs. Thus, the present study aims to identify the functions of miR-144-3p in proliferation, differentiation and apoptosis of MSCs by gain and loss of function, dual luciferase reporter assays, qRT-PCR, western blotting, real-time cell analysis and flow cytometry. The results we have found are listed as follows: 1. Effects of miR-144-3p on the osteogenic differentiation of C3H10T1/2 cellsThe expression of osteoblastic marker genes (Runx2, OCN and Osx) was dramatically elevated during osteogenic differentiation of murine C3H10T1/2 cells. However, endogenous miR-144-3p decreased gradually during osteogenic induction, and reached its lowest expression level after induction for 21 days, suggesting that miR-144-3p may participate in regulating the osteogenic differentiation of C3H10T1/2 cells.We transiently transfected C3H10T1/2 cells individually with miR-144-3p mimics, miR-NC. And then osteoblast differentiation was induced. MiR-144-3p over-expression significantly decreased osteogenic differentiation, as indicated by ALP and ARS staining and quantification. Moreover, the qRT-PCR and western blotting results showed that miR-144-3p-transfected cells exhibited lower expression levels of osteogenic marker genes than the miR-NC-transfected cells. In contrast, after transfection of miR-144-3p inhibitor, the expression levels of osteogenic marker genes were up-regulated, which was consistent with the ALP and ARS staining and quantification results. These data suggested that miR-144-3p inhibited osteogenic differentiation in MSCs.The possible targets for miR-144-3p were predicted using microRNA.org, TargetScan and miRDB, and we found that Smad4 may be one of miR-144-3p’s target genes. Dual luciferase reporter assays, qRT-PCR and western blotting analysis further confirmed that miR-144-3p directly targeted the 3’UTR of Smad4 mRNA and regulated its expression by post-transcriptional inhibition.We examined the effects of Smad4 gene on osteoblast differentiation by transfecting the Smad4 over-expression plasmid and small interfering RNA into C3H10T1/2 cells. Interestingly, over-expression of Smad4 resulted in promotion of osteoblast differentiation. In contrast, the Smad4-knockdown cells exhibited a reduced potential to differentiate into osteoblasts. To further investigate the relationship between miR-144-3p and Smad4, we co-transfected miR-144-3p inhibitor with si-Smad4 into C3H10T1/2 cells and then performed osteogenic differentiation. We found that inhibition of miR-144-3p could no longer enhance the osteogenesis of C3H10T1/2 cells after Smad4 knockdown. From a sidewise approach, we demonstrated that deletion of Smad4 blocks the effects of the miR-144-3p inhibition, further indicating that miR-144-3p regulates the osteoblast differentiation of C3H10T1/2 cells through directly targeting Smad4.2. Effects of miR-144-3p on the adipogenic differentiation of C3H10T1/2 cellsThe expression of adipogenic marker genes (C/EBPα, C/EBPβ, PPARγ and AP2) was dramatically elevated during adipogenic conversion of murine C3H10T1/2 cells. Meanwhile, endogenous miR-144-3p increased gradually during adipogenic induction, and reached its peak after induction for 8 days, suggesting that miR-144-3p may participate in regulating the adipogenic differentiation of C3H10T1/2 cells.We transiently transfected C3H10T1/2 cells individually with miR-144-3p mimics, miR-NC. And then adipogenic differentiation was induced. MiR-144-3p overexpression significantly promoted adipogenic differentiation, as indicated by Oil red staining. Moreover, the qRT-PCR and western blotting results showed that miR-144-3p-transfected cells exhibited higher expression levels of adipogenic marker genes than the miR-NC-transfected cells. In contrast, after transfection of miR-144-3p inhibitor, the expression levels of adipogenic marker genes were down-regulated, which was consistent with the Oil red staining results. These data suggested that miR-144-3p enhanced adipogenic differentiation in MSCs.The possible targets for miR-144-3p during adipogenesis were predicted using microRNA.org, TargetScan and miRDB, and we found that bmp-3b may be one of miR-144-3p’s target genes. Dual luciferase reporter assays, qRT-PCR and western blotting analysis further confirmed that miR-144-3p directly targeted the 3’UTR of bmp-3b gene and regulated its expression both by transcriptional and post-transcriptional inhibition.3. Effects of miR-144-3p on the proliferation and apoptosis of C3H10T1/2 cellsWe transiently transfected C3H10T1/2 cells individually with miR-144-3p mimics and miR-NC. And then real-time cell analysis was performed. MiR-144-3p over-expression significantly inhibited cell proliferation, as indicated by higher cell index than the negative contrl group. In contrast, after transfection of miR-144-3p inhibitor, the proliferation of C3H10T1/2 cells was significantly promoted.Flow cytometry analysis further confirmed that miR-144-3p negatively regulated the proliferation of C3H10T1/2 cells. After transfection of miR-144-3p mimics, C3H10T1/2 cells were arrested in G0/G1 phase, whereas miR-144-3p inhibitor reversed this process. Therefore, miR-144-3p inhibited the proliferation of C3H10T1/2 cells through arresting them in G0/G1 phase.As we have confirmed that miR-144-3p directly targeted Smad4, we further investigated the function of Smad4 on cell proliferation in C3H10T1/2 cells. Interestingly, over-expression of Smad4 resulted in promotion of cell proliferation in C3H10T1/2 cells. In contrast, si-Smad4 reduced the proliferation of C3H10T1/2 cells. To further investigate the relationship between miR-144-3p and Smad4, we co-transfected miR-144-3p inhibitor with si-Smad4 into C3H10T1/2 cells and then performed cell proliferation assays. We found that inhibition of miR-144-3p could no longer enhance the proliferation of C3H10T1/2 cells after Smad4 knockdown. Thus, we demonstrated that deletion of Smad4 blocks the effects of miR-144-3p inhibition, further indicating that miR-144-3p regulates the proliferation of C3H10T1/2 cells through directly targeting Smad4.To test the function of miR-144-3p on cellular apoptosis in C3H10T1/2 cells, we transiently transfected C3H10T1/2 cells individually with miR-144-3p mimics (or miR-144-3p inhibitor) and their respective controls. And then flow cytometry analysis was performed. The flow cytometry results demonstrated that neither over-expression nor inhibition of miR-144-3p had any effects on the cellular apoptosis of C3H10T1/2 cells. Meanwhile, we found that miR-144-3p over-expression had no effcts on protein expression of apoptosis-related genes. Therefore, miR-144-3p did not affect the apoptosis of C3H1OT1/2 cells.In summary, we can conclude from the above study: ① miR-144-3p inhibited the osteogenic differentiation of C3H10T1/2 cells through directly targeting Smad4, which could promote osteoblast differentiation of C3H10T1/2 cells; ② miR-144-3p enhanced the adipogenic potential of C3H10T1/2 cells by targeting bmp-3b; ③ miR-144-3p inhibited the proliferation of C3H10T1/2 cells by targeting Smad4, which could promote the proliferation of C3H10T1/2 cells; ④ miR-144-3p had no effects on cellular apoptosis of C3H10T1/2 cells. |
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