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CircFAM160A2 Promotes Mitochondrial Stabilization and Apoptosis Reduction in Osteoarthritis Chondrocytes by Targeting miR-505-3p and SIRT3.

ABSTRACT: Competitive endogenous RNAs (ceRNAs), as a newly identified regulating mechanism, have been demonstrated to play a crucial role in various human diseases. An increasing number of recent studies have revealed that circular RNAs (circRNAs) can function as ceRNAs. However, little is known about the role of circFAM160A2 in the pathological process of osteoarthritis (OA). This study is the first to examine the crucial role of the circFAM160A2-miR-505-3p-SIRT3 axis in osteoarthritis progression. miR-505-3p was selected from the interaction of a microRNA (miRNA) microarray comparing chondrocytes in OA and normal conditions and prediction results from TargetScan. RT-qPCR was performed to assess the expression of circFAM160A2, miR-505-3p, and SIRT3. A dual luciferase assay was used to validate the binding of circFAM160A2, miR-505-3p, and SIRT3. We used lentivirus and adeno-associated virus to establish in vitro and in vivo overexpression models. Western blotting, apoptosis assay, ROS detection assay, Safranin O staining, and CCK-8 assay were employed to assess the role of circFAM160A2, miR-505-3p, and SIRT3. We found that miR-505-3p was upregulated and circFAM160A2 was downregulated in OA. While overexpression of circFAM160A2 decreased the production of extracellular matrix (ECM) degrading enzymes and ameliorated chondrocyte apoptosis and mitochondrial dysfunction, inhibition of miR-505-3p could reverse the protective effect of circFAM160A2 on the OA phenotype both in vitro and in vivo. In conclusion, circFAM160A2 can promote mitochondrial stabilization and apoptosis reduction in OA chondrocytes by targeting miR-505-3p and SIRT3, which might be a potential therapeutic target for OA therapy.

SUBMITTER: Bao J

PROVIDER: S-EPMC8505077 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThis study explores LncRNA TM1-3P effects on the proliferation, apoptosis, and inflammatory response of fibroblasts in osteoarthritis (OA) and its underlying mechanism.MethodsBioinformatics was performed to analyze OA disease-related genes, miRNA profiles, and function. The targeted regulation of LncRNA TM1-3P and miR-144-3p, ONECUT2 and miR-144-3p were analyzed by dual luciferase reporter gene assay, RNA Binding Protein Immunoprecipitation (RIP), and RNA pull down. Histopathological morphology of the knee joint was observed by hematoxylin-eosin (HE) and Annona Red O/Fast Green. The expressions of mRNAs and proteins were detected by RT-qPCR, Western blot, and immunohistochemistry. Unpaired T test was used between groups, and the one-way analysis of variance of repeated measurement data was applied for multi-group comparison, following Tukey's post-test.ResultsONECUT2 and Smurf2 genes were significantly elevated in the osteoarthritis group compared with the normal group (P < 0.001, P < 0.001). Expressions of ONECUT2 and LncRNA TM1-3P were increased, and expression of miR-144-3p was decreased in interleukin (IL)-1β-induced human fibroblast synovial cells (hFSCs) (mRNA: 1.06 ± 0.24 vs. 3.29 ± 0.73, proteins: 0.22 ± 0.03 vs. 0.46 ± 0.22, 1.23 ± 0.22 vs. 3.76 ± 0.73, 1.06 ± 0.25 vs. 0.37 ± 0.13, P < 0.01, P < 0.001, P < 0.01, P < 0.05). Overexpression of miR-144-3p down-regulated the ONECUT2 expression, reduced cell proliferation, promoted apoptosis in hFSCs induced by IL-1β (mRNA: 0.89 ± 0.14 vs. 0.15 ± 0.01, P < 0.05; proteins: 0.46 ± 0.01 vs. 0.23 ± 0.01, P < 0.001; CCK8: 1.88 ± 0.07 vs. 1.65 ± 0.07; P < 0.05; EDU: 55.82 ± 1.44 vs 40.57 ± 2.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.0001). Overexpression of LncRNA TM1-3P up-regulated the expression of ONECUT2, promoted cell proliferation, and inhibited apoptosis (mRNA: 0.9 ± 0.09 vs 1.94 ± 0.12, P < 0.05; proteins: 0.61 ± 0.05 vs 0.76 ± 0.03, P > 0.05; CCK8: 2.07 ± 0.05 vs 2.47 ± 0.06; P < 0.01; EDU: 52.67 ± 1.17 vs 60.06 ± 3.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.001), which were reversed by the overexpression of miR-144-3p treatment (mRNA: 1.82 ± 0.07 vs 0.31 ± 0.07, P < 0.0001; proteins: 0.74 ± 0.02 vs 0.35 ± 0.01, P < 0.01; CCK8: 2.41 ± 0.01 vs 1.67 ± 0.02; P < 0.0001; EDU: 66.85 ± 2.86 vs 44.68 ± 1.97, P < 0.0001; apoptosis: 7.19 ± 0.19 vs 13.36 ± 0.53, P < 0.0001). Silencing LncRNA TM1-3P attenuated the injury of knee joint tissue, down-regulated the expression of ONECUT2, Smurf2, IL-1β, IL-6, TNF-α, and improved the expression of Rap1 in rats (0.71 ± 0.04 vs 0.48 ± 0.02, 0.68 ± 0.06 vs 0.36 ± 0.02, 0.74 ± 0.03 vs 0.49 ± 0.04, 0.78 ± 0.01 vs 0.54 ± 0.03, 0.68 ± 0.02 vs 0.4 ± 0.04, 0.24 ± 0.01 vs 0.4 ± 0.03, P < 0.05, P < 0.05, P < 0.05, P < 0.01, P < 0.01, P < 0.05).ConclusionLncRNA TM1-3P improved inflammation and damage of knee joints in OA rats through miR-144-3p/ONECUT2 axis, providing a new theoretical basis for gene therapy of OA.

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CircFAM160A2 Promotes Mitochondrial Stabilization and Apoptosis Reduction in Osteoarthritis Chondrocytes by Targeting miR-505-3p and SIRT3.

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