Liu Wenzhou, Chen Yanbo, Zeng Gang, Yang Tao, Song Weidong
Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University Guangzhou 510000, Guangdong, China.
Department of Emergency, Sun Yat-sen Memorial Hospital, Sun Yat-sen University Guangzhou 510000, Guangdong, China.
Am J Transl Res. 2020 Dec 15;12(12):7953-7967. eCollection 2020.
To probe into the role and regulatory mechanisms of INSR in pathogenesis of osteoarthritis (OA).
KLF4 and INSR expression was detected in cartilage tissues of 40 OA patients and 10 controls using RT-qPCR. IL-1β-induced OA chondrocytes and anterior cruciate ligament transection (ACLT)-induced OA models were respectively constructed. After overexpressing or silencing KLF4 or INSR, flow cytometry assay was utilized to detect chondrocyte apoptosis. Furthermore, JAK2/STAT3, cartilage markers and OA-related markers were examined by western blot. Dual luciferase report and CHIP assay were carried out to verify the interactions between KLF4 and INSR, followed by functional gain and loss assay. INSR promoter methylation was assessed by MS-PCR.
Both KLF4 and INSR were down-regulated both in OA chondrocytes and cartilage tissues. Knockdown of KLF4 or INSR accelerated apoptosis of IL-1β-induced OA chondrocytes. However, overexpression of KLF4 or INSR ameliorated OA progression both in OA chondrocytes and OA mouse models. Moreover, INSR inactivated JAK2/STAT3 pathway in OA chondrocytes. Dual luciferase report and CHIP assay results confirmed that INSR was transcriptionally regulated by KLF4. As shown in MS-PCR results, INSR expression was mediated by DNA methylation in OA.
Our findings suggested that INSR, as a key regulator for OA, was regulated by transcription factor KLF4 and DNA methylation, thereby mediating the activation of JAK2/STAT3 signaling, which was considered as an underlying therapeutic target for OA.
探讨胰岛素受体（INSR）在骨关节炎（OA）发病机制中的作用及调控机制。
采用逆转录定量聚合酶链反应（RT-qPCR）检测40例OA患者和10例对照者软骨组织中KLF4和INSR的表达。分别构建白细胞介素-1β（IL-1β）诱导的OA软骨细胞模型和前交叉韧带横断（ACLT）诱导的OA模型。在过表达或沉默KLF4或INSR后，利用流式细胞术检测软骨细胞凋亡。此外，通过蛋白质免疫印迹法检测Janus激酶2（JAK2）/信号转导和转录激活因子3（STAT3）、软骨标志物和OA相关标志物。进行双荧光素酶报告基因检测和染色质免疫沉淀（CHIP）分析以验证KLF4与INSR之间的相互作用，随后进行功能获得和缺失分析。通过甲基化特异性聚合酶链反应（MS-PCR）评估INSR启动子甲基化。
KLF4和INSR在OA软骨细胞和软骨组织中均下调。敲低KLF4或INSR可加速IL-1β诱导的OA软骨细胞凋亡。然而，过表达KLF4或INSR可改善OA软骨细胞和OA小鼠模型中的OA进展。此外，INSR使OA软骨细胞中的JAK2/STAT3信号通路失活。双荧光素酶报告基因检测和CHIP分析结果证实INSR受KLF4转录调控。如MS-PCR结果所示，INSR表达在OA中由DNA甲基化介导。
我们的研究结果表明，INSR作为OA的关键调节因子，受转录因子KLF4和DNA甲基化调控，从而介导JAK2/STAT3信号的激活，这被认为是OA的潜在治疗靶点。
