ObjectiveTo investigate the specific microRNA (miRNA) in osteogenic and chondrogenic differentiations of C3H10T1/2 cells. MethodsC3H10T1/2 cells were induced to differentiate into osteoblasts and chondrocytes.Specific miRNA more than 2 fold change and 2 average normalized probe signal between C3H10T1/2 and C3H10T1/2-derived osteoblast,and between C3H10T1/2 and C3H10T1/2-derived chondrocytes were screened out by miRNA microarray,and verified by real-time fluorescence quantitative PCR (RT-qPCR). ResultsAlkaline phosphatase expression of osteogenic induced group was significantly higher than that of control group at 7 days after induced (P<0.05).RT-qPCR results showed the expressions of Runx2,serine protease (Sp7),collagen type I,and osteopontin (OPN) genes were significantly increased at 7,14,and 21 days after induced when compared with before induced (P<0.05).Western blot results showed the expressions of Runx2,Sp7,collagen type I,and OPN proteins of osteogenic induced group were significantly higher than those of control group at 21 days after induced (P<0.05).The expressions of SOX9,collagen type Ⅱ,Aggrecan,and Has2 were significantly increased at 5,10,and 15 days after induced when compared with before induced (P<0.05).The expressions of SOX9,collagen type 2,Aggrecan,and Has2 proteins of chondrogenic induced group were significantly higher than those of control group at 15 days after induced (P<0.05).Totally,10 osteogenic and 3 chondrogenic miRNA more than 2 fold change and 2 average normalized probe signal were screened out by miRNA microarray.RT-qPCR results of these specific miRNAs were similar to microarray results except miR-455-3p. ConclusionSpecific miRNAs are screened out by microarray and it is a good foundation for the future study on miRNA functional verification and target gene prediction.
ObjectiveTo investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon. MethodsSmad5 3'-UTRluciferase vector (pmiR-RB-REPORTTM) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRTPCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. ResultsDual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P<0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P>0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P<0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. ConclusionSmad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.
ObjectiveTo investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9). MethodsHey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. ResultsC3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05). ConclusionExpression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.