Orthodontic Centre, West China College of Stomatology, Sichuan University, Chengdu, China.
State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China.
Mol Biol Rep. 2022 Jun;49(6):4485-4501. doi: 10.1007/s11033-022-07292-6. Epub 2022 Apr 6.
Bmal1 and Per2 are the core components of the circadian clock genes (CCGs). Bmal1 mice exhibit premature aging, as indicated by hypotrichosis and osteoporosis, with a loss of proliferation ability. The same occurs in Per2 mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. Thus, our study aimed to explore the effects and specific mechanism.
Lentiviral and adenoviral vectors were constructed to silence or overexpress Bmal1 or Per2 and MTT, flow cytometry, RT-qPCR, WB, immunohistochemistry, alizarin red staining and ChIP-Seq analyses were applied to identify the possible mechanism. The successful knockdown and overexpression of Bmal1/Per2 were detected by fluorescence microcopy. Flow cytometry found out that Bmal1 or Per2 knockdown resulted in G1-phase cell cycle arrest. RT-qPCR showed the different expression levels of Wnt-3a, c-myc1 and axin2 in the Wnt/β-catenin signaling pathway as well as the gene expression change of Rorα and Rev-erbα. Meanwhile, related proteins such as β-catenin, TCF-1, and P-GSK-3β were detected. ALP activity and the amount of mineral nodules were compared. ChIP-Seq results showed the possible mechanism.
Bmal1 and Per2, as primary canonical clock genes, showed synergistic effects on the proliferation and differentiation of BMSCs. They would inhibit the Wnt/β-catenin signaling pathway by downregulating Rorα expression or upregulating Rev-erbα expression, both of which were also key elements of CCGs. And this may be the mechanism by which they negatively regulate the osteogenic differentiation of BMSCs. Bmal1 and Per2 show synergistic effects in the proliferation of BMSCs. In addition, they play a synergistic role in negatively regulating the osteogenic differentiation ability of BMSCs. Bmal1 and Per2 may regulate the aging of BMSCs by altering cell proliferation and osteogenic differentiation through Rorα and Rev-erbα to affect Wnt/β-catenin pathway.
Bmal1 和 Per2 是昼夜节律钟基因（CCGs）的核心组成部分。Bmal1 小鼠表现出早衰，表现为毛发稀疏和骨质疏松，增殖能力丧失。Per2 小鼠也发生同样的情况，但程度较轻。然而，Bmal1 和 Per2 对增殖和成骨分化的影响是协同还是拮抗尚不清楚。因此，我们的研究旨在探讨其作用及具体机制。
构建慢病毒和腺病毒载体沉默或过表达 Bmal1 或 Per2，并应用 MTT、流式细胞术、RT-qPCR、WB、免疫组织化学、茜素红染色和 ChIP-Seq 分析来确定可能的机制。荧光显微镜检测到 Bmal1/Per2 的成功敲低和过表达。流式细胞术发现 Bmal1 或 Per2 敲低导致 G1 期细胞周期停滞。RT-qPCR 显示 Wnt/β-catenin 信号通路中 Wnt-3a、c-myc1 和 axin2 的表达水平不同，以及 Rorα 和 Rev-erbα 的基因表达变化。同时，检测到相关蛋白如β-catenin、TCF-1 和 P-GSK-3β。比较 ALP 活性和矿化结节数量。ChIP-Seq 结果显示可能的机制。
Bmal1 和 Per2 作为主要的经典时钟基因，对 BMSCs 的增殖和分化表现出协同作用。它们通过下调 Rorα 表达或上调 Rev-erbα 表达抑制 Wnt/β-catenin 信号通路，这也是 CCGs 的关键元素。这可能是它们负调控 BMSCs 成骨分化的机制。Bmal1 和 Per2 在 BMSCs 的增殖中表现出协同作用。此外，它们在负调控 BMSCs 成骨分化能力方面发挥协同作用。Bmal1 和 Per2 可能通过改变细胞增殖和成骨分化来调节 BMSCs 的衰老，通过 Rorα 和 Rev-erbα 影响 Wnt/β-catenin 通路。
