3.1 Altered expression of Bmal1 and/or Per2 at the mRNA and protein levels after transfection with viral vectors.
Green and red fluorescence protein expression were detected by fluorescence microcopy, confirming the knockdown of Bmal1 and the knockdown/overexpression of Per2 (Figure 1A). The protein expression level was in accordance with gene expression level for each gene (Figure 1B, C).
3.2 The growth curve
The growth curve(Figure 2A)according to the OD490 values obtained through the MTT assay indicated the proliferation rates of the different groups. The proliferative activity of cells overexpressing Per2 increased compared to that of control cells, and accordingly, the survival rate of this group (group 5) was the highest among all groups. When we inhibited Bmal1 (group 2), Per2 (group 4) or both (group 7), the proliferation rate decreased significantly compared with that in the groups transfected with an empty viral vector (groups 3, 6, and 9) .
3.3 The proliferation rates of different groups evaluated by flow cytometry.
The percentage of cells distributed in the G0 phase, G1 phase, G2 phase, S phase and M phase reflects the cell activity to some degree. The flow cytometry results(Figure 2B) showed that cells overexpressing Per2 had the greatest proliferative activity, with the highest SPF value and PI staining rate. Cells with knockdown of Bmal1, Per2 or both exhibited a lower proliferation rate than their matched control groups.
3.4 Expression of classical Wnt/β-catenin signaling pathway-related genes.
The difference in the expression of Wnt-3a between the Bmal1 and Per2 knockdown group and the matched empty vector group was significant, specifically showing that suppressing Bmal1 and Per2 simultaneously increased the levels of Wnt-3a, c-myc1 and axin2. Knockdown of Bmal1 alone increased the expression of c-myc1 and axin2, and the effect on axin2 was increased with combined knockdown of Per2 (Figure 3A).
3.5 Expression level of Wnt/β-catenin signaling pathway-related proteins.
It was concluded that the levels of β-catenin, TCF-1, and P-GSK-3β (Figure 3B)increased under four different conditions in our experiment: Bmal1 knockdown, Per2 knockdown, Bmal1 knockdown/Per2 knockdown and Bmal1 knockdown/Per2 overexpression. The expression level in the Bmal1 knockdown/Per2 overexpression group was higher than that in the Per2 overexpression group, and the difference was statistically significant. The probable mechanism is that the expression of Per2 is promoted when Bmal1 is suppressed. The effects of Per2 overexpression were strengthened in the combination group, leading to increased expression of proteins related to the Wnt/β-catenin signaling pathway.
3.6 Changes in osteogenic marker gene expression in each group after osteogenic induction.
Our previous research explored the effect of Bmal1 knockdown, Per2 knockdown and Bmal1 knockdown/Per2 knockdown on the osteogenic differentiation ability of BMSCs; we found that Bmal1 and Per2 negatively and synergistically regulated this process.18 Therefore, we designed an experiment to assess the osteogenic differentiation capability of BMSCs in different groups, including Bmal1 knockdown, Per2 overexpression, and Bmal1 knockdown/Per2 overexpression groups, to verify the previous results.
3.6.1 ALP activity in each group after 7 days of osteogenic induction.
In the Bmal1 knockdown group, ALP activity, an early osteogenic differentiation marker, was enhanced compared with that in the empty control group (Figure 4D) but declined in the Per2 overexpression group. The activity of ALP in the Bmal1 knockdown/Per2 overexpression group was also elevated and was even higher than that in the Bmal1 knockdown group. There were no significant differences between the control groups (group 1, group 3, group 6, and group 9), which shows that viral transfection had no impact on ALP activity.
3.6.2 Expression of osteogenic differentiation-related genes.
The mRNA expression levels of Alp, Runx2 and Ocn in the Bmal1 knockdown group exceeded those in the control group but were lower than those in the Bmal1 knockdown/Per2 overexpression group. Bmal1 knockdown/Per2 knockdown had significantly stronger effects than empty control transfection after 7 days of osteogenic induction (Figure 4A-C). The differences in Ocn expression in each group detected 14 days later were in accordance with the result observed after 7 days of osteogenic induction (Figure 5A). The protein expression levels were still in accordance with the mRNA expression levels at these time points.
3.7 Changes in Rorα and Rev-erbα expression after alteration of Bmal1 and Per2 expression in BMSCs.
3.7.1 The gene expression of Rorα
In the four experimental groups, Bmal1 knockdown, Per2 knockdown, Bmal1 knockdown/Per2 knockdown and Bmal1 knockdown/Per2 overexpression, the expression of Rorα was remarkably higher than that in the corresponding empty control groups; in the Per2 overexpression group, the outcome was the opposite (Figure 5B). Inhibiting both Bmal1 and Per2 had a greater effect than inhibiting Per2 alone. To our surprise, the effect in the Bmal1 knockdown/Per2 knockdown and Bmal1 knockdown/Per2 overexpression groups was greater than that in the Bmal1 knockdown group. There were no significant differences among the blank control group and the other control groups transfected with empty vectors, indicating that viral transfection exerted no impact on the expression of Rorα. The protein and gene expression trends of Rorα were consistent.
3.7.2 The gene expression of Rev-erbα
In the four experimental groups, Bmal1 knockdown, Per2 knockdown, Bmal1 knockdown/Per2 knockdown and Bmal1 knockdown/Per2 overexpression, the expression of Rev-erbα decreased compared with that in the blank control group, but the difference between the Per2 knockdown group and the control group was not significant (Figure 5C). In addition, knockdown of both Bmal1 and Per2 led to a much lower expression level of Rev-erbα than did knockdown of Bmal1 or Per2 alone, verifying their synergistic effects. Overexpression of Per2 increased the expression of Rev-erbα, which contrasted with the effect of combined Bmal1 knockdown/Per2 overexpression. The protein expression trend of Rev-erbα in these groups was in accordance the mRNA expression trend.(Figure 5D,5E)
The ALP activity of Bmal1 knockdown group was enhanced compared with the control group, which indicated that Bmal1 had negative effect on osteogenic differentiation of BMSCs. The ALP activity decreased in Per2 overexpression group, which demonstrated Per2 could inhibit the osteogenic differentiation of BMSCs. Whether Per2 was overexpressed or suppressed, Bmal1 knockdown would facilitate bone mineralization.
3.9 Alizarin red staining
The results of alizarin red staining shows that the mineralized nodules were increased in Bmal1 knockdown and Per2 knockdown group compared with blank control. While inhibiting Bmal1 and Per2 simultaneously would enhance the positive effect.
3.8 ChIP-Seq results
3.8.1 Gene Ontology (GO) functional analysis
GO functional analysis results showed that Bmal1 and Per2 were mainly enriched in the protein binding molecular function category. The GO database was established by the Association of Gene Ontology, and GO analysis results are divided into three categories:21 molecular function, biological process and cellular components. The results obtained in vitro can be classified according to the results of the Bmal1 and Per2 ChIP-Seq experiments. Based on the identified regions in their genomes, these proteins have specific binding affinities and are mainly involved in regulating pathways related to cell proliferation, cell senescence, and osteogenic differentiation (Figure 6A, B).
3.8.2 Kyoto Encyclopedia of Genes and Genomes (KEGG)
KEGG analysis results revealed key genes related to BMAL1 and PER2 in the mouse genome. The constituents of the Wnt/β-catenin pathway were detected in the region 2000 bp away from the transcription site. The peak genes of Bmal1 in the Wnt/β-catenin pathway included Wnt2b, Smad3, Axin2, Fzd6, Tcf712, etc. Similarly, the peak genes in Per2 included Wnt1, Wnt6, Wnt7b, and Smad3. Furthermore, both Bmal1 and Per2 were notably enriched in the mTOR signaling pathway,22 Hippo signaling pathway and ubiquitin-mediated proteolysis pathway (P<0.05, Figure 6C, 6D), which means that the specific regulatory mechanisms may be coming into view.