METHODS: The aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied.
RESULTS: Cyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo.
CONCLUSIONS: Downregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.
方法:通过分析微阵列图谱GSE100609鉴定骨质疏松症中的异常表达基因。收集骨质疏松患者和正常组的血清标本,通过实时定量聚合酶链反应(qRT-PCR)检测候选基因的mRNA表达。用地塞米松(DEX)处理小鼠颅骨成骨细胞MC3T3-E1细胞以体外模拟骨质疏松症。采用茜素红染色和碱性磷酸酶(ALP)染色相结合的方法测定MC3T3-E1细胞的基质矿化沉积。同时,成骨相关基因包括碱性磷酸酶(ALP)的表达,骨钙蛋白(OCN),骨桥蛋白(OPN),Osterix,通过qRT-PCR和Western印迹方法评估骨形态发生蛋白2(BMP2)。然后研究了候选基因对调节卵巢切除术(OVX)引起的骨质流失的影响。
结果:发现CyclinA1(CCNA1)在骨质疏松患者血清和骨质疏松模型细胞中显著上调,这符合生物信息学分析。DEX处理抑制MC3T3-E1细胞成骨分化能力,表现为茜素红染色强度降低,ALP染色强度,和ALP的表达水平,OCN,OPN,Osterix,和BMP2。抑制CCNA1对成骨的调节作用与DEX相反。然后,京都基因和基因组百科全书(KEGG)分析表明,与CCNA1负相关的基因在TGF-β信号通路中富集。TGF-β信号通路抑制剂部分逆转CCNA1抑制诱导的成骨作用.此外,抑制CCNA1减轻OVX小鼠体内骨量。
结论:CCNA1下调可激活TGF-β信号通路,促进骨形成,从而起到治疗骨质疏松症的作用。