MALAT1通过调控miR-146a减轻关节软骨细胞凋亡和基质降解的研究

MALAT1 attenuates articular chondrocyte apoptosis and matrix degradation by regulating miR-146a

为探究转移相关肺腺癌转录本1(metastasis-associated lung adenocarcinoma transcript 1,MALAT1)对miR-146a的调控作用及其对关节软骨细胞凋亡和基质降解的影响,采用RT-PCR检测临床软骨组织中miR-146a和MALAT1的表达,分析其变化的相关性;双荧光素酶报告基因试验检测miR-146a和MALAT1的靶向作用关系;之后分组并转染miR-146a mimics或MALAT1重组过表达载体,经过IL-1β处理,采用RT-PCR检测miR-146a表达,CCK-8检测细胞增殖水平,Hoechst检测细胞凋亡水平,Western blotting检测活化型半胱天冬酶3(cleaved caspase 3,cl-CASP3)、B细胞淋巴瘤因子2(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein, Bax)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP-13)、带有血小板凝血酶敏感蛋白结构域的解聚素金属蛋白酶5(A disintegrin and metalloproteinase with thrombospondin motifs 5,ADAMTS-5)、Ⅱ型胶原蛋白(collagenⅡ,COL2)、聚蛋白聚糖蛋白水平。结果显示,相比于普通关节软骨组织,骨关节炎(osteoarthritis, OA)关节软骨组织MALAT1相对表达水平升高(P<0.05),miR-146a相对表达水平降低(P<0.05),MALAT1与miR-146a表达呈负相关(r=-0.907 8,P<0.001);双荧光素酶报告基因试验显示,miR-146a和MALAT1存在靶向作用关系;相比于IL-1β组,miR-146a组细胞增殖水平降低,细胞凋亡率及cl-CASP3、Bax相对蛋白表达水平升高,Bcl-2相对蛋白表达水平降低,同时MMP-13和ADAMTS-5相对蛋白表达水平升高,COL2和聚蛋白聚糖相对蛋白表达水平降低(均P<0.01);MALAT1过表达可显著降低miR-146a的表达水平,提高细胞增殖活性并抑制其凋亡,同时降低MMP-13和ADAMTS-5相对蛋白表达水平,提升COL2和聚蛋白聚糖相对蛋白表达水平(均P<0.01)。该研究提示,MALAT1可通过靶向作用于miR-146a发挥对软骨细胞的抗凋亡作用。

To investigate the regulation of metastasis-associated lung adenocarcinoma transcript 1(MALAT1) on miR-146 a and its effects on apoptosis and matrix degradation of articular chondrocytes, RT-PCR was used to detect the expressions of miR-146 a and MALAT1 in clinical cartilage tissue samples, and their correlation was analyzed. Dual luciferase reporter assay was used to test the relationship between miR-146 a and MALAT1. Cartilage tissue cells were divided into several groups and transfected respectively with miR-146 a mimics or recombinant overexpression vector of MALAT1. After the treatment with IL-1β, cells were undergone the following examinations: expression of miR-146 a was detected by RT-PCR, cell proliferation was determined by CCK-8, cell apoptosis was observed by Hoechst, protein levels of cleaved caspase 3(cl-CASP3), B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), matrix metalloproteinase 13(MMP-13), A disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), collagen Ⅱ(COL2) and aggrecan were measured by Western blotting. The results showed that, compared with normal cartilage tissues, MALAT1 relative expression level was increased in osteoarthritis(OA) tissues(P<0.05), miR-146 a relative expression level was decreased(P<0.05), and MALAT1 expression was negatively correlated with miR-146 a expression(r=-0.907 8,P<0.001). Dual luciferase reporter assay showed that miR-146 a and MALAT1 could target reciprocally. Compared with IL-1β group, in miR-146 a group, the cell proliferation level was decreased, cell apoptotic rate was increased, and relative protein levels of cl-CASP3 and Bax were increased, relative protein level of Bcl-2 was decreased(P<0.01);meanwhile, the relative protein levels of MMP-13 and ADAMTS-5 were increased, and relative protein levels of COL2 and aggrecan were decreased(P<0.01). The overexpression of MALAT1 significantly decreased the expression of miR-146 a, increased the cell proliferation and inhibited the cell apoptosis(P<0.01);meanwhile, it decreased the relative protein levels of MMP-13 and ADAMTS-5, but increased the relative protein levels of COL2 and aggrecan(P<0.01). In conclusion, MALAT1 can target miR-146 a, exerting the anti-apoptosis effects on chondrocytes.