环状 RNA HIPK3 低表达通过作为 miR-124 的海绵来调节 SOX8 促进骨关节炎软骨细胞凋亡。
Low expression of CircRNA HIPK3 promotes osteoarthritis chondrocyte apoptosis by serving as a sponge of miR-124 to regulate SOX8.
机构信息
Orthopaedics Department, Shapingba People's Hospital, Chongqing, China.
出版信息
Eur Rev Med Pharmacol Sci. 2020 Aug;24(15):7937-7945. doi: 10.26355/eurrev_202008_22476.
OBJECTIVE
CircRNA, a type of circular RNA, has recently been shown to be a potential target for osteoarthritis (OA). Circular RNA HIPK3 (CircHIPK3) is reported to be abnormally expressed in various disease tissues and affects the occurrence and development of the disease. However, the role and underlying mechanism of CircRNA HIPK3 in osteoarthritis are still unclear. The purpose of this study is to explore the effect of CircRNA HIPK3 on osteoarthritis and analyze its regulatory mechanism.
PATIENTS AND METHODS
We took human OA tissues, normal knee cartilage, human OA chondrocytes and normal chondrocytes as the research objects. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was used to detect CircHIPK3 expression level, its target gene miRNA 124 (miR-124) and downstream target molecule SOX8. Flow cytometry analysis was applied to discover the apoptosis of CircHIPK3 and miR-124 on OA cartilage in different transfection situations. Moreover, Western blot and RT-qPCR were used to detect the expression of caspase-3 in OA chondrocytes. The binding site of CircRNA HIPK3 and miR-124, miR-124, and SOX8 were verified by using Dual-Luciferase assay.
RESULTS
High expressed CircHIPK3 and low expressed miR-124 were found in OA tissues and OA chondrocytes. In addition, the Dual-Luciferase report showed CircHIPK3 acted as a sponge of miR-124 in OA chondrocytes. CircHIPK3 and miR-124 expression in OA tissue were confirmed to be negatively correlated. To our surprise, knocking down CircHIPK3 and transfected miR-124 mimics both inhibited the apoptosis of OA chondrocytes. Further experiments verified that the downstream target molecule of miR-124 was SOX8 in OA chondrocytes. Besides, miR-124 inhibitors reversed the knockdown of CircHIPK3 while si-SOX8 reversed the miR-124 inhibitors effect of apoptosis on OA chondrocytes.
CONCLUSIONS
Our results demonstrated that CircHIPK3 was significantly upregulated in OA cartilage tissue and cells. Low expression of CircHIPK3 promoted the apoptosis of OA chondrocytes by promoting miR-124 to suppress SOX8 expression. The molecular mechanism of CircHIPK3 in present study is expected to provide new ideas for the treatment of osteoarthritis.
目的
环状 RNA（circRNA）是一种环状 RNA，最近被证明是骨关节炎（OA）的潜在靶点。环状 RNA HIPK3（CircHIPK3）在各种疾病组织中表达异常，影响疾病的发生和发展。然而，CircRNA HIPK3 在骨关节炎中的作用及其潜在机制尚不清楚。本研究旨在探讨 CircRNA HIPK3 对骨关节炎的影响，并分析其调控机制。
方法
以人 OA 组织、正常膝关节软骨、人 OA 软骨细胞和正常软骨细胞为研究对象。采用实时定量聚合酶链反应（RT-qPCR）检测 CircHIPK3 表达水平及其靶基因 miRNA 124（miR-124）和下游靶分子 SOX8。采用流式细胞术分析不同转染情况下 CircHIPK3 和 miR-124 对 OA 软骨细胞凋亡的影响。此外，采用 Western blot 和 RT-qPCR 检测 OA 软骨细胞中 caspase-3 的表达。采用双荧光素酶报告实验验证 CircRNA HIPK3 与 miR-124、miR-124 与 SOX8 的结合位点。
结果
OA 组织和 OA 软骨细胞中高表达 CircHIPK3 和低表达 miR-124。此外，双荧光素酶报告显示 CircHIPK3 在 OA 软骨细胞中作为 miR-124 的海绵。OA 组织中 CircHIPK3 与 miR-124 的表达呈负相关。令人惊讶的是，敲低 CircHIPK3 并转染 miR-124 模拟物均可抑制 OA 软骨细胞的凋亡。进一步的实验证实，miR-124 在 OA 软骨细胞中的下游靶分子是 SOX8。此外，miR-124 抑制剂逆转了 CircHIPK3 的敲低，而 si-SOX8 逆转了 miR-124 抑制剂对 OA 软骨细胞凋亡的作用。
结论
本研究结果表明，CircHIPK3 在 OA 软骨组织和细胞中显著上调。低表达 CircHIPK3 通过促进 miR-124 抑制 SOX8 表达促进 OA 软骨细胞凋亡。本研究中 CircHIPK3 的分子机制有望为骨关节炎的治疗提供新的思路。
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