微小RNA-138-5p与软骨细胞增殖和自噬的关系

MicroRNA-138-5p regulates chondrocyte proliferation and autophagy

背景:软骨细胞自噬活性的异常会导致软骨组织退行性病变,引发骨关节炎。近年的研究发现mircoRNA在调控软骨细胞自噬过程中发挥了重要的作用,但其作用的分子机制还不太清楚。目的:探究微小RNA-138-5p(miR-138-5p)对软骨细胞增殖和自噬活性的调节及其作用机制。方法:体外培养SW1353软骨肉瘤细胞系,对照组细胞转染阴性对照miRNA;实验组细胞转染miR-138-5p模拟物。CCK-8法检测细胞增殖活性,荧光定量PCR实验检测基质金属蛋白酶1,3,13的mRNA表达水平;免疫荧光染色法检测LC3蛋白亚细胞定位,用TargetScan7.1在线工具预测miR-138-5p与SIRT1信使RNA(mRNA)的作用位点,用蛋白质免疫印迹实验检测自噬相关蛋白和AMPK信号通路蛋白的表达水平。结果与结论:①与转染阴性对照的细胞相比,转染miR-138-5p模拟物的细胞增殖活性明显下降,细胞内LC3蛋白点状定位减少,且SIRT1、LC3-Ⅱ、p-AMPK蛋白表达水平下调,p62蛋白表达水平上升,SIRT1 mRNA的3’UTR区域存在一个保守的miR-138-5p结合位点;②与转染对照miRNA的细胞相比,转染miR-138-5p模拟物的细胞中基质金属蛋白酶1,3和13的mRNA表达均有上升;③结果提示,miR-138-5p调控细胞自噬活性和细胞增殖,其作用机制与SIRT1/AMPK信号通路相关;细胞内上调表达的miR-138-5p会促进软骨细胞基质金属蛋白酶的分泌,表明miR-138-5p在骨关节炎疾病进展中发挥了重要的作用。

BACKGROUND:Abnormal autophagy in chondrocytes often leads to cartilage degeneration,thereby triggering osteoarthritis.Recent studies have found that microRNAs play an important role in chondrocyte autophagy;however,the molecular mechanism is yet unclear.OBJECTIVE:To investigate the role of microRNA-138-5p(miR-138-5p)in the regulation of chondrocyte proliferation and autophagy activities,and to reveal its mechanisms.METHODS:Chondrosarcoma cell line SW1353 were cultured in vitro and transfected with negative control miRNA or miR-138-5p mimic.Cell proliferation activity was measured by cell counting kit-8 assay,the expression of matrix metalloproteinases 1,3,and 13 mRNA was measured by fluorogenic quantitative PCR.The endogenous LC3 subcellular location was detected by immunofluorescence staining.The miR-138-5p and SIRTI mRNA target sites were predicted using TargetScan 7.1 online tool.Autophagy-related proteins and AMPK signal proteins were detected by immunoblotting assay.RESULTS AND CONCLUSION:Cells transfected with miR-138-5p mimic,compared with those transfected with negative control miRNA,showed lower proliferation activity,less LC3 puncta,and reduced expression of SIRT1,LC3-Ⅱ,p-AMPK,but increased protein expression of p62 and increased mRNA expression of matrix metalloproteinases 1,3,13.There was a conserved miR-138-5p binding site in the 3’UTR region of SIRT1 mRNA.To conclude,miR-138-5p regulates SW1353 cell autophagy and proliferation through the SIRT1/AMPK signaling pathway.The up-regulated expression of miR-138-5p promotes the secretion of matrix metalloproteinases from chondrocytes,indicating that miR-138-5p plays an important role in the progression of osteoarthritis.