miR-200b-3p与DNA甲基转移酶-3a在骨关节炎软骨细胞中的表达及作用机制

Effects of miR-200b-3p and DNA methyltransferase-3a on the chondrocytes of osteoarthritis

目的探讨mi R-200b-3p靶向DNMT3A对骨关节炎软骨细胞增殖和凋亡的影响。方法 RT-PCR法检测mi R-200b-3p,DNMT3A,MMPs和COLⅡ在正常软骨组织和骨关节炎软骨组织中的表达;采用双荧光素酶和western bolt验证mi R-200b-3p和DNMT3A之间的靶向关系;构建过表达的mi R-200b-3p和DNMT3A真核细胞载体;q RT-PCR和western bolt检测MMPs和COLⅡm RNA及蛋白质在稳定转染的软骨细胞中的表达;通过细胞活力测定(MTS法)、团块培养、Hoechst 33342染色法评估细胞的增殖与凋亡情况。结果 q RT-PCR法及western bolt结果显示,mi R-200b-3p和COLⅡ在骨关节炎软骨组织中呈低表达,而DNMT3A和MMPs呈高表达;双荧光素酶和western bolt证实mi R-200b-3p和DNMT3A之间存在靶向关系;MTS法、团块培养及Hoechst 33342染色法结果证实,在mi R-200b-3p过表达的软骨细胞中,DNMT3A和MMPs的表达水平明显下调,COLⅡ的表达水平则明显上调,且细胞的生存能力增强,凋亡率降低(P均<0.05);在过表达DNMT3A的软骨细胞中,MMPs的表达水平明显上调,COLⅡ的表达水平明显下调,且细胞的生存能力减弱,凋亡率增加(P均<0.05)。结论 mi R-200b-3p可以抑制MMPs分泌和促进COLⅡ合成,并且可以通过抑制DNMT3A的表达来增强骨关节炎软骨细胞的生长与增殖。

Objective To investigate the effects of miR-200b-3p and DNA methyltransferase-3a （DNMT3A） on the proliferation and apoptosis of chondrocytes of osteoarthritis. Methods The expression levels of miR-200b-3p, DNMT3A, matrix metalloproteinases （MMPs） and collagen （ COL Ⅱ ） in normal cartilage and osteoarthritis tissues were determined by qRT-PCR. The targeting relationship between miR-200b-3p and DNMT3A was verified by the double fluorescence report system and western blot. The eukaryotic cell vectors overexpressing miR-200b-3p and DNMT3A were constructed, and transfected into chondrocytes. Then, the expressions of MMPs and COL Ⅱ in the chondrocytes were detected by qRT-PCR and western bolt, and the proliferation and apoptosis of the chondrocytes were evaluated by the MTS method, pellet culture and Hoechst 33342 staining, respectively. Results qRT-PCR and Western blot results showed that there were low expressions of miR-200b-3p and COL Ⅱ and high expressions of DNMT3A and MMPs in osteoarthritis tis- sues. The double fluorescence report system and western bolt results demonstrated that there was a targeting relationship between miR- 200b-3p and DNMT3A. The MTS method, pellet cuhure and Hoechst 33342 staining results showed that the significantly reduced ex- pressions of DNMT3A and MMPs, the significantly increased expression of COL Ⅱ, improved cell viability and decreased apoptosis were found in the chondrocytes overexpressing miR-200b-3p （ all of P〈0.05） , and that the significantly increased expression of MMPs, the significantly reduced expression of COL Ⅱ, reduced cell viability and increased apoptosis were found in the chondrocytes overex- pressing DNMT3A （ all of P〈0.05）. Conclusion MiR-20Ob-3p may improve the growth and proliferation of osteoarthritic chondrocytes by inhibiting the secretion of MMPs, promoting the synthesis of COL Ⅱ and inhibiting the expression of DNMT3A.