Chen Biying, Yang Wen, Zhao Huiqing, Liu Kaihua, Deng Adi, Zhang Guo, Pan Kaixia
Department of Orthopaedics, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510530, P.R. China.
Department of Rheumatology and Immunology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510530, P.R. China.
Exp Ther Med. 2020 Feb;19(2):1042-1050. doi: 10.3892/etm.2019.8278. Epub 2019 Dec 4.
Osteoporosis (OP) is an age-related bone disease occurring worldwide. Osteoporotic fracture is one of the leading causes of disability and death in elderly patients. MicroRNAs (miRNAs/miRs) are key molecular regulatory factors in bone remodeling processes. The present study investigated the expression and mechanism of miR-135b-5p in patients with osteoporosis. The present results suggested that miR-135b-5p was upregulated in bone tissue fragments of patients with osteoporosis compared with the control patients. MC3T3-E1 cells were used to perform osteogenic differentiation induction. Reverse transcription-quantitative PCR and western blot assay were used to detect the mRNA and protein expression levels of the osteogenic markers osteocalcin (OC), Osterix and alkaline phosphatase (ALP). A specific kit was used for detecting ALP activity. The present results indicated that the mRNA expression levels of OC, Osterix and ALP significantly increased on the 7 and 14th day after osteogenic differentiation induction compared with the control group. Protein expression levels of OC, Osterix and ALP also increased on the 7 and 14th day after induction. ALP assay showed that ALP activity was significantly increased on the 7 and 14th day after induction. In addition, the present study found that miR-135b-5p was downregulated in MC3T3-E1 cells 7 and 14 days after osteogenic differentiation induction. The results of TargetScan analysis and dual luciferase reporter gene assay indicated that runt-related transcription factor 2 (RUNX2) was a direct target gene of miR-135b-5p. RUNX2 was upregulated in MC3T3-E1 cells on the 7 and 14th day after induction. Moreover, the present study found that compared with the osteogenic differentiation induction group, miR-135b-5p mimic significantly decreased OC, Osterix and ALP expression, and reduced ALP activity in MC3T3-E1 cells. However, these reductions were reversed following overexpression of RUNX2. The present results showed that miR-135b-5p mimic significantly reduced cell viability in MC3T3-E1 cells and induced cell apoptosis, and these effects were significantly reversed following RUNX2 overexpression. In summary, the present results suggested that miR-135-5p participated in the occurrence and development of osteoporosis via inhibition of osteogenic differentiation and osteoblast growth by targeting RUNX2. The present study suggested a novel potential target that may faciliate the treatment of osteoporosis, and further study is required to examine this possibility.
骨质疏松症（OP）是一种在全球范围内普遍存在的与年龄相关的骨病。骨质疏松性骨折是老年患者致残和死亡的主要原因之一。微小RNA（miRNA/miR）是骨重塑过程中的关键分子调节因子。本研究调查了miR-135b-5p在骨质疏松症患者中的表达及机制。目前的结果表明，与对照患者相比，骨质疏松症患者骨组织碎片中miR-135b-5p表达上调。采用MC3T3-E1细胞进行成骨分化诱导。采用逆转录定量PCR和蛋白质印迹法检测成骨标志物骨钙素（OC）、osterix和碱性磷酸酶（ALP）的mRNA和蛋白表达水平。使用特定试剂盒检测ALP活性。目前的结果表明，与对照组相比，成骨分化诱导后第7天和第14天，OC、osterix和ALP的mRNA表达水平显著升高。诱导后第7天和第14天，OC、osterix和ALP的蛋白表达水平也升高。ALP检测显示，诱导后第7天和第14天ALP活性显著升高。此外，本研究发现，成骨分化诱导后7天和14天，MC3T3-E1细胞中miR-135b-5p表达下调。TargetScan分析和双荧光素酶报告基因检测结果表明， runt相关转录因子2（RUNX2）是miR-135b-5p的直接靶基因。诱导后第7天和第14天，MC3T3-E1细胞中RUNX2表达上调。此外，本研究发现，与成骨分化诱导组相比，miR-135b-5p模拟物显著降低了MC3T3-E1细胞中OC、osterix和ALP的表达，并降低了ALP活性。然而，RUNX2过表达后这些降低作用被逆转。目前的结果表明，miR-135b-5p模拟物显著降低了MC3T3-E1细胞的活力并诱导细胞凋亡，而RUNX2过表达后这些作用显著逆转。总之，目前的结果表明，miR-135-5p通过靶向RUNX2抑制成骨分化和成骨细胞生长，参与了骨质疏松症的发生和发展。本研究提出了一个可能有助于治疗骨质疏松症的新潜在靶点，需要进一步研究来验证这种可能性。
