Zhang Ying, Wang Fuyou, Chen Guangxing, He Rui, Yang Liu
Center for Joint Surgery, Southwest Hospital, The Third Military Medical University (Army Medical University), 30 Gaotanyan Main St., Shapingba Dist., Chongqing, 400038 People's Republic of China.
Cell Biosci. 2019 Jul 1;9:54. doi: 10.1186/s13578-019-0302-2. eCollection 2019.
Many studies have reported that long noncoding RNAs (lncRNAs) could act as sponges for microRNAs (miRNAs) and play important roles in the regulation of osteoarthritis (OA). Yet, the underlying mechanisms of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OA are still unclear. Therefore, we aimed to explore the regulation mechanisms of MALAT1 in OA procession.
IL-1β treatment in chondrocyte was used to mimic OA in vitro. MALAT1, miR-150-5p and AKT3 expression levels were detected via qRT-PCR. The protein levels of AKT3, MMP-13, ADAMTS-5, Bax, Bcl-2, cleaved-PARP, collagen II and aggracan were measured by western blot. MTT assay was performed to detect cell proliferation ability. The apoptosis of chondrocytes was determined using flow cytometry and western blot. Luciferase assay and RNA immunoprecipitation (RIP) assays were used to confirm the relationship among MALAT1, miR-150-5p and AKT3.
In our study, MALAT1 and AKT3 were upregulated while miR-150-5p was downregulated in OA in vitro and vivo. The level of miR-150-5p was negatively correlated with that of MALAT1 or AKT3. More importantly, overexpression of MALAT1 promoted the expression of AKT3 by negatively regulating miR-150-5p. MALAT1 knockdown inhibited cell proliferation, promoted apoptosis, increased MMP-13, ADAMTS-5 expression and decreased collagen II, aggracan expression in IL-1β treated chondrocytes. MALAT1 upregulation or AKT3 overexpression enhanced proliferation, inhibited apoptosis and extracellular matrix (ECM) degradation, which was undermined by overexpression of miR-150-5p. By contrast, miR-150-5p depletion rescued the effect of MALAT1 downregulation or loss of AKT3 on IL-1β-stimulated chondrocytes.
MALAT1 was responsible for cell proliferation, apoptosis, and ECM degradation via miR-150-5p/AKT3 axis.
许多研究报道，长链非编码RNA（lncRNA）可作为微小RNA（miRNA）的海绵，并在骨关节炎（OA）的调控中发挥重要作用。然而，lncRNA转移相关肺腺癌转录本1（MALAT1）在OA中的潜在机制仍不清楚。因此，我们旨在探讨MALAT1在OA进程中的调控机制。
采用白细胞介素-1β（IL-1β）处理软骨细胞以在体外模拟OA。通过定量逆转录聚合酶链反应（qRT-PCR）检测MALAT1、miR-150-5p和蛋白激酶B3（AKT3）的表达水平。通过蛋白质印迹法检测AKT3、基质金属蛋白酶13（MMP-13）、含血小板反应蛋白基序的解聚素样金属蛋白酶5（ADAMTS-5）、凋亡相关蛋白Bax、Bcl-2、裂解的聚（ADP-核糖）聚合酶（cleaved-PARP）、胶原蛋白II和聚集蛋白聚糖的蛋白水平。采用MTT法检测细胞增殖能力。使用流式细胞术和蛋白质印迹法测定软骨细胞的凋亡情况。采用荧光素酶报告基因检测和RNA免疫沉淀（RIP）试验来证实MALAT1、miR-150-5p和AKT3之间的关系。
在我们的研究中，体外和体内OA模型中MALAT1和AKT3上调，而miR-150-5p下调。miR-150-5p的水平与MALAT1或AKT3的水平呈负相关。更重要的是，MALAT1的过表达通过负向调节miR-150-5p促进AKT3的表达。敲低MALAT1可抑制IL-1β处理的软骨细胞的增殖，促进凋亡，增加MMP-13、ADAMTS-5的表达，并降低胶原蛋白II、聚集蛋白聚糖的表达。MALAT1上调或AKT3过表达可增强增殖，抑制凋亡和细胞外基质（ECM）降解，而miR-150-5p过表达则削弱了这种作用。相反，敲低miR-150-5p可挽救MALAT1下调或AKT3缺失对IL-1β刺激的软骨细胞的影响。
MALAT1通过miR-150-5p/AKT3轴参与细胞增殖、凋亡和ECM降解。
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