miR-196a-5p inhibition decreased the osteogenic differentiation ability of WJCMSCs in vitro
To confirm the role of miR-196a-5p, we used the miR-196a-5p inhibitor to knock down miR-196a-5p expression in WJCMSCs. After 2 µg/mL puromycin selection for 3 days, the inhibition efficiency of miR-196a-5p was detected by realtime-RT-PCR (Figure 1A). Then WJCMSCs were induced by using of osteogenic-inducing medium to decide miR-196a-5p effect on osteogenic. After 5 days induction, the WJCMSCs ALP activity was dramatically decreased in the miR-196a-5p inhibition group compared with the Consh group (Figure 1B). After 2 weeks induction, Alizarin red staining (Figure 1C) and quantitative calcium results (Figure 1D) revealed that the mineralization ability of WJCMSCs was dramatically reduced in the miR-196a-5p inhibition group compared with the Consh group. Also, the osteogenic markers’ level were tested, western blot results showed that BSP, DSPP, DMP1, and OCN were significantly down-expressed at osteogenic inducted 7 day and 14 day (Figure 1E).
miR-196a-5p overexpression enhanced the osteogenic differentiation ability of WJCMSCs in vitro
To further investigate its effect for WJCMSCs osteogenic ability, we overexpress miR-196a-5p in WJCMSCs by miR-196a-5p mimics transfection. After 3 days of selection with 2 µg/mL puromycin, realtime RT-PCR was applied to test the overexpression efficiency of miR-196a-5p (Figure 2A). After osteogenic inducted for 5 days, the ALP activity of WJCMSCs showed considerably increased in the miR-196a-5p mimics group compared to the Consh group (Figure 2B). After 2 weeks induction in vitro, Alizarin red staining (Figure 2C) and quantitative calcium analysis (Figure 2D) showed the mineralization ability of WJCMSCs was considerably increased in the miR-196a-5p mimics group compared to the Consh group. Next, the results of western blot indicated the osteogenic markers’ level of BSP, DSPP, DMP1, and OCN were significantly up-regulated in the miR-196a-5p mimics group on day-7 and day-14 during osteogenic induction (Figure 2E).
miR-196a-5p promoted the calvarial bone defected repair ability of WJCMSCs in SD rat
Since miR-196a-5p overexpression enhanced the osteogenic differentiation ability of WJCMSCs in vitro, we hypothesized whether miR-196a-5p regulated bone regeneration in vivo. To test this hypothesis, we established a calvarial bone defect models for a 5mm diameter round which difficultly to heal in SD rats. To promote the healing potential of MSCs, WJCMSCs sheets were formed by using 20 µg/mL vitamin C induction 7 days (Figure 3A). The H&E staining revealed that compared to the Consh group, the miR-196a-5p mimics group formed a better two- or three-layered uniformly spread structure in whole cell sheets (Figure 3B).
Then, WJCMSCs sheets was transplanted in calvarial bone defect of SD rat. After 12 weeks, the gross morphological manifestation showed that calvarial bone defect significantly healed in the miR-196a-5p mimics group (Figure 3C). And micro-CT reconstructed image (Figure 3D) and quantitative analysis results (Figure 3E) showed that the miR-196a-5p mimics group formed more new bone tissue compared to the Consh group. The 20× objective view of H&E staining showed more mature new bone tissue was formed in the miR-196a-5p mimics group compared to the Consh group (Figure 3F; Black line showed).
The profiling of differentially expressed genes in miR-196a-5p overexpressing WJCMSCs and negative control group
To explore the mechanism that miR-196a-5p regulated osteogenic differentiation of WJCMSCs, microarray and bioinformatics analyses were performed. In general, totally 959 differentially expressed genes (DEGs) regulated by miR-196a-5p were revealed. Among which 34 genes were significantly upregulated (fold change ≥ 2.0, P value ≤ 0.05), and 925 genes were significantly downregulated (fold change ≤0.5, P value ≤ 0.05) (Additional files 6). Here, we used a heatmap to show the mainly DEGs between the miR-196a-5p mimics group and the control group in WJCMSCs (Figure 4A). Among these DEGs, we chosen CLDN11, IL6, ENPP1, SERPINB2, and VCAM1 to verify that the accuracy of microarray results was credibly (Figure 4B).
Also, GO terms analysis was performed to detected the significantly changed functions caused by the differentially expressed genes in WJCMSCs. The significantly top 3 GO terms of miR-196a-5p up-regulated DEGs in WJCMSCs mainly included in proteinaceous extracellular matrix (biological process; G0:0005578), extracellular matrix component (biological process; GO:0044420), fibrillar collagen trimer (biological process; GO:0005583); extracellular matrix structural constituent (cellular component; GO:0005201), growth factor binding (cellular component; GO:0019838), insulin-like growth factor binding (cellular component;GO:0005520); extracellular matrix organization (molecular function; GO:0030198), extracellular structure organization (molecular function; GO:0043062), negative regulation of cell motility (molecular function; GO:2000146) (Additional files 1). The significantly top 3 GO terms of miR-196a-5p down-regulated DEGs in WJCMSCs mainly included in chromosomal region (biological process; G0:0098687), nuclear envelope (biological process; GO:0005635), spindle (biological process; GO:0005819); ATPase activity (cellular component; GO:0016887), helicase activity (cellular component; GO:0004386), cell adhesion molecule binding (cellular component; GO:0050839); chromosome segregation (molecular function; GO:0007059), cell cycle checkpoint (molecular function; GO:0000075), sister chromatid segregation (molecular function; GO:0000819) (Additional files 2).
Pathway enrichment analysis was then performed to detect the significantly associated major pathways with differentially expressed genes regulated by miR-196a-5p. The KEGG analysis results showed that the ECM-receptor interaction pathway (KEGG:hsa04512), the amoebiasis pathway (KEGG:hsa05146), and the leishmaniasis pathway (KEGG:hsa05140) were more correlative with the miR-196a-5p up-regulated DEGs in WJCMSCs (Additional files 3), and the RNA degradation pathway (KEGG:hsa03018), the steroid biosynthesis pathway (KEGG:hsa00100), and the endocytosis pathway (KEGG:hsa04144) were more correlative with the miR-196a-5p down-regulated DEGs in WJCMSCs (Additional files 4). And a network was analysed to reveal the relationship between the significantly miR-196a-5p regulated DEGs associated pathways in WJCMSCs (Figure 5).
In addition to transcriptome microarray analysis, we also use websites miRDB (http://www.mirdb.org), miRwalk (http://mirwalk.umm.uni-heidelberg.de), and TargetScan (http://www.targetscan.org) to predict the targeted genes of miR-196-5p. The predicted targeted genes by at least two sites were included in the analysis. Interestingly, there are 241 predicted genes were got (Additional files 7). And only 19 predicted targeted genes were consistent with our microarray results (Additional files 8).
Deletion of SERPINB2 enhanced the osteogenic differentiation potential of WJCMSCs in vitro
Here, a significantly downregulated gene by miR-196a-5p, SERPINB2 drawn our interest. We tried to predict the possible binding sites of miR-196a-5p and SERPINB2 mRNA, but unfortunately, SERPINB2 mRNA does not contain the possible binding sites of miR-196a-5p, which suggested the miR-196a-5p regulation to SERPINB2 seem not by binding to the 3' untranslated region of SERPINB2 mRNA. Therefore, we further hypothesized that the key role of miR-196a-5p in regulating osteogenesis does not simply depend on its classical regulatory role. So, Therefore, we further investigated the effect of SERPINB2 on the osteogenesis function in WJCMSCs.
Firstly, lentiviral shRNA infection was performed to knock down the SERPINB2 expression in WJCMSCs. Real-time RT-PCR determined that about 80% knockdown efficiency was achieved (Figure. 5A). Then, the ALP activity was tested at day 5 by osteogenic induction, and the result showed that the ALP activity of the SERPINB2sh group was significantly increased compared to the Scramsh group in WJCMSCs (Figure 5B). The two weeks Alizarin red staining (Figure 5C) and the quantitatively calcium results (Figure 5D) showed that the mineralization ability of the SERPINB2sh group significantly increased compared to the Scramsh group in WJCMSCs.