Dataset Information

CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-? in TMJ osteoarthritis.

ABSTRACT: Altered expression of circular RNAs (circRNAs) has been identified in various human diseases. In this study, we investigated whether circRNAs function as competing endogenous RNAs to regulate the pathological process of temporomandibular joint osteoarthritis (TMJOA). High-throughput sequencing of mRNA (RNA seq) was performed to detect the expression of circRNAs in TMJOA and control synovial tissues isolated from humans. The differentially upregulated circGCN1L1 (hsa_circ_0000448) in synoviocyte was validated in vitro and in vivo. Here we demonstrate the interactions between circGCN1L1 and both miR-330-3p and tumor necrosis factor-? (TNF-?) through bioinformatics predictions, luciferase report assays, and fluorescence in situ hybridization. mRNA expression profiles of TNF-?-stimulated synoviocyte showed that circGCN1L1 and p65 expressions were upregulated by TNF-?. Moreover, miR-330-3p was negatively correlated with TNF-? secretion. Further, we found that miR-330-3p directly targeted TNF and restrained the production of matrix-degrading enzymes (MMP3, MMP13, and ADAMTS4). Mechanistic studies unveiled that circGCN1L1 in TMJOA synovial tissues and cells may be associated with condylar chondrocyte apoptosis and synoviocyte hyperplasia. Moreover, intra-articular injection of shcircGCN1L1 alleviated TMJOA progression in rat models. Altogether, we elucidated the important roles of a novel circRNA, namely, circGCN1L1, which induced inflammation in TMJ synoviocytes and decreased anabolism of the extracellular matrix (ECM) through miR-330-3p and TNF-? gene. This circRNA may represent a potentially effective therapeutic strategy against TMJOA progression at an early stage.

SUBMITTER: Zhu H

PROVIDER: S-EPMC7181816 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-α in TMJ osteoarthritis.

Zhu Huimin H Hu Yihui Y Wang Chuandong C Zhang Xiaoling X He Dongmei D

Cell death & disease 20200424 4

Altered expression of circular RNAs (circRNAs) has been identified in various human diseases. In this study, we investigated whether circRNAs function as competing endogenous RNAs to regulate the pathological process of temporomandibular joint osteoarthritis (TMJOA). High-throughput sequencing of mRNA (RNA seq) was performed to detect the expression of circRNAs in TMJOA and control synovial tissues isolated from humans. The differentially upregulated circGCN1L1 (hsa_circ_0000448) in synoviocyte ...[more]

PMID: 32332704

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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.

Project description:To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor alpha (TNF-alpha) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-alpha protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-alpha, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation.

Dataset Information

CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-? in TMJ osteoarthritis.

Publications

CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-α in TMJ osteoarthritis.

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