MiR-3609通过靶向调控CCND1抑制成骨细胞分化介导骨质疏松症发生  

MiR-3609 mediates osteoporosis by inhibiting differentiation of osteoblast through targeting CCND1

作　　者：余照宇 陈文创 李勇[1,2] 鲁尧[1,2] YU Zhaoyu;CHEN Wenchuang;LI Yong;LU Yao(The Second Clinical Medicine College,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Guangdong Province Hospital of Traditional Chinese Medicine,Zhuhai Branch,Zhuhai 519015,China)

机构地区：[1]广州中医药大学第二临床医学院,广东广州510006 [2]广东省中医院珠海医院,广东珠海519015

出　　处：《中国骨质疏松杂志》2024年第8期1140-1146,共7页Chinese Journal of Osteoporosis

基　　金：珠海市科技创新局项目(2220004000345)。

摘　　要：目的通过生物信息学分析和体外实验结合的方式验证miR-3609在骨质疏松症发病中的可能作用机制,为骨质疏松症的治疗提供新靶点。方法通过miRDB、miRWalk、TargetScan三大miRNA靶点分析数据库进行miR-3609下游靶点分析。双荧光素酶实验验证miR-3609和下游靶基因之间的靶向关系,RT-qPCR实验和WB实验验证miR-3609对下游靶基因表达的影响及WB实验检测miR-3609对抗凋亡基因Bcl2和成骨相关蛋白Runx2、OPG表达的影响。碱性磷酸酶实验及茜素红实验检测分析miR-3609对成骨细胞成骨分化、矿化的影响。结果首先,数据库靶点预测结果显示,CCND1可能是miR-3609导致骨质疏松发病的潜在靶点。其次,双荧光素酶实验验证了二者之间的靶向关系:miR3609的激活会下调成骨细胞中的CCND1表达。同时,碱性磷酸酶和茜素红实验结果表明miR3609的激活抑制成骨细胞向成骨分化及矿化。此外,miR3609的激活下调了成骨细胞成骨相关蛋白Runx2、OPG的表达,下调了抗凋亡蛋白Bcl2的表达。结论miR-3609可以通过靶向抑制CCND1表达抑制成骨细胞成骨分化、矿化,这可能是其导致骨质疏松发病的机制,靶向抑制miR-3609有望成为治疗骨质疏松症的新方向。Objective To verify the mechanism of miR-3609 in the pathogenesis of osteoporosis through the combination of bioinformatics analysis and in vitro experiments,providing a new target for the treatment of osteoporosis.Methods The downstream target analysis of miR-3609 was performed using three miRNA target analysis databases,miRDB,miRWalk,and TargetScan.Dual luciferase assay was used to examine the targeting relationship between miR-3609 and downstream target genes.RT-qPCR assay and WB assay were used to verify the influence of miR-3609 in downstream target gene expressions.WB assay was used to detect the influence of miR-3609 in the expressions of anti-apoptosis gene Bcl2 and osteoblast-related proteins Runx2 and OPG.The effects of miR-3609 on osteogenic differentiation and mineralization of osteoblasts were analyzed with alkaline phosphatase assay and Alizarin red assay.Results We predicted its related targets through online database and find that CCND1 may be a potential target.Dual luciferase assay confirmed the targeting relationship between the two:activation of miR-3609 down-regulates CCND1 expression in osteoblasts.Alkaline phosphatase and Alizarin red assay showed that miR-3609 inhibited osteogenic differentiation and mineralization of osteoblasts.At the same time,we found that the activation of miR-3609 down-regulated the expression of osteoblast-related proteins Runx2 and OPG,and down-regulated the expression of anti-apoptotic protein Bcl2.Conclusion miR-3609 inhibits osteogenic differentiation and mineralization of osteoblasts through inhibiting the expression of CCND1,which may be the mechanism that miR-3609 leading to the onset of osteoporosis.Targeted inhibition of miR-3609 is expected to become a new direction of osteoporosis treatment.

关 键 词：骨质疏松症 miR-3609 CCND1 成骨分化 治疗靶点 

分 类 号：R681[医药卫生—骨科学]