Dataset Information

Circular RNA circ_0000423 regulates cartilage ECM synthesis via circ_0000423/miRNA-27b-3p/MMP-13 axis in osteoarthritis.

ABSTRACT: Circular RNA (circRNA) is related to many human diseases including osteoarthritis (OA). Our research purpose was to show that functional circRNAs have a role in the pathogenesis of OA, while also identifying potential circRNA that bind to miRNA-27b-3p. Microarray analysis was used to evaluate the expression of CircRNA in OA and normal cartilage. The role and functional mechanism of Circ_0000423 up-regulation were detected in OA and verified in vitro and in vivo. RNA transfection, qRT-PCR, Western blot analysis, immunofluorescence, and dual-luciferase assays were used to investigate the interaction between Circ_0000423 and miRNA-27b-3p in vitro. The roles of Circ_0000423 were discussed in vivo. Our results discovered 11 down-regulated circRNAs and 101 up-regulated circRNAs between control and OA tissues, and confirmed that Circ_0000423 an increase significantly in OA tissues by evaluating the different circRNAs expressions. Meanwhile, luciferase analysis confirmed Circ_0000423 can be directly targeted by miRNA-27b-3p and act as a miRNA-27b-3p sponge. Circ_0000423 can influence MMP-13 and collagen II expression by targeting miRNA-27b-3p expression as ceRNA in OA. Furthermore, AAV-shRNA-Circ 0000423 intra-articular injection slows the progression of OA by decreasing articular cartilage destruction and erosion, joint surface fibrosis, osteophyte formation, MMP-13 expression, and increasing collagen II expression in the articular cartilage of ACLT-induced OA mice model. These findings confirmed that the Circ_0000423-miRNA-27b-3p-MMP-13 axis could affect the pathogenesis of OA which might lead to a novel target for diagnostic molecular biological indicators and potential OA treatments.

SUBMITTER: Li X

PROVIDER: S-EPMC9085232 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Circular RNA circ_0000423 regulates cartilage ECM synthesis via circ_0000423/miRNA-27b-3p/MMP-13 axis in osteoarthritis.

Li Xing X Xie Chaofan C Xiao Fangjun F Su Haitao H Li Zhen Z Weng Jiaxian J Huang Yongming Y He Peiheng P

Aging 20220419 8

Circular RNA (circRNA) is related to many human diseases including osteoarthritis (OA). Our research purpose was to show that functional circRNAs have a role in the pathogenesis of OA, while also identifying potential circRNA that bind to miRNA-27b-3p. Microarray analysis was used to evaluate the expression of CircRNA in OA and normal cartilage. The role and functional mechanism of Circ_0000423 up-regulation were detected in OA and verified <i>in vitro</i> and <i>in vivo</i>. RNA transfection, q ...[more]

PMID: 35439733

Similar Datasets

Project description:ObjectiveSynovial fibrosis contributes to osteoarthritis (OA) pathology, but the underlying mechanisms remain unknown. We have observed increased microRNA-27b-3p (miR-27b-3p) levels in synovial fluid of patients with late-stage radiographic knee OA. Here, we investigated the contribution of miR-27b-3p to synovial fibrosis in patients with severe knee OA and in a mouse model of knee OA.MethodsWe stained synovium sections obtained from patients with radiographic knee OA scored according to the Kellgren/Lawrence scale and mice that underwent destabilization of the medial meniscus (DMM) for miR-27b-3p using in situ hybridization. We examined the effects of intraarticular injection of miR-27b-3p mimic into naive mouse knee joints and intraarticular injection of a miR-27b-3p inhibitor into mouse knee joints after DMM. We performed transfection with miR-27b-3p mimic and miR-27b-3p inhibitor in human OA fibroblast-like synoviocytes (FLS) using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) array, RNA sequencing, RT-qPCR, Western blotting, immunofluorescence, and migration assays.ResultsWe observed increased miR-27b-3p expression in the synovium from patients with knee OA and in mice with DMM-induced arthritis. Injection of the miR-27b-3p mimic in mouse knee joints induced a synovial fibrosis-like phenotype, increased synovitis scores, and increased COL1A1 and α-smooth muscle actin (α-SMA) expression. In the mouse model of DMM-induced arthritis, injection of the miR-27b-3p inhibitor decreased α-SMA but did not change COL1A1 expression levels or synovitis scores. Transfection with the miR-27b-3p mimic in human OA FLS induced profibrotic responses, including increased migration and expression of key extracellular matrix (ECM) genes, but transfection with the miR-27b-3p inhibitor had the opposite effects. RNA sequencing identified a PPARG/ADAMTS8 signaling axis regulated by miR-27b-3p in OA FLS. Human OA FLS transfected with miR-27b-3p mimic and then treated with the PPARG agonist rosiglitazone or with ADAMTS8 small interfering RNA exhibited altered expression of select ECM genes.ConclusionOur findings demonstrate that miR-27b-3p has a key role in ECM regulation associated with synovial fibrosis during OA.

Project description:ObjectiveAberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA.MethodsChondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors.ResultsIn IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b.ConclusionOur data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.

Project description:BackgroundThough circular RNAs (circRNAs) are the key regulators in tumor carcinogenesis, they remain largely unexplored in hepatocellular carcinoma (HCC).MethodsThe expression of RanGAP1-derived circRNAs (circ_0063531, circ_0063534, circ_0063513, circ_0063518, circ_0063507, circ_0063723) were evaluated in eight paired HCC and normal tissues, and the correlation between circRanGAP1 (circ_0063531) expression and clinicopathological characteristics in 40 HCC patients was determined. The association between miR-27b-3p and circRanGAP1 or NRAS was predicted using bioinformatics analysis. The expression of circRanGAP1, miR-27b-3p, and NRAS were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The potential oncogenic role of circ-RanGAP1 was assessed using CCK-8, colony formation, transwell assays in vitro, subcutaneous tumor mouse model, vein tail metastatic model, and orthotopically implanted intrahepatic HCC model in vivo. Luciferase reporter and RNA immunoprecipitation (RIP) assays were used to explore the binding site between miR-27b-3p and circ-RanGAP1 or NRAS. Protein expression was detected using western blotting. The localization of miR-27b-3p and circ-RanGAP1 was investigated using fluorescence in situ hybridization (FISH). The level of immune infiltration was assessed by bioinformatics analysis, flow cytometry, and orthotopically implanted intrahepatic HCC models.ResultsHere, we found elevated circRanGAP1 in the cells and clinical tissues of patients with HCC. Increased circRanGAP1 levels are associated with enlarged tumors and the advanced stage of TNM. CircRanGAP1 promotes the growth, migration, and HCC cell invasion, concurrently with the growth and metastasis of tumors in-vivo. Moreover, circRanGAP1 is mainly located inside the cytoplasm. Mechanistically, circRanGAP1 as an oncogene promotes HCC progression by miR-27b-3p/NRAS/ERK axis, furthermore, affects the infiltration level of tumor-associated macrophages probably by sponging miR-27b-3p. Immune infiltration analysis shows that NRAS is positively correlated with the levels of CD68+ tumor-associated macrophages in HCC samples and that NRAS and CD68 are related to the poor outcome of HCC.ConclusionThese results reveal that circRanGAP1 is a HCC oncogene that function by the miR-27b-3p/NRAS/ERK axis and regulates the infiltration levels of tumor-associated macrophages by sponging miR-27b-3p. Therefore, circRANGAP1/ NRAS axis may be an important potential treatment target against HCC.

Project description:Oxaliplatin (OXA) resistance is the major obstacle to the anticancer effects of chemotherapy in colorectal cancer (CRC) patients. MicroRNAs (miRNAs) play an important role in the chemoresistance of various tumors. Our objective is to clarify the underlying mechanism of miRNAs in chemoresistance and provide a potential strategy to improve the response of CRC patients to chemotherapeutics. Methods: MiRNA microarray and Real-time PCR were performed to compare changes in miRNA expression between oxaliplatin-resistant and the parental cells. CCK8, apoptosis assay, immunofluorescence and xenograft studies were used to elucidate the impact of miR-27b-3p on regulating chemoresistance. Luciferase reporter assay and western blot were carried to assess the regulatory role of miR-27b-3p in ATG10 expression. The effects of miR-27b-3p and ATG10 on autophagy were investigated by GFP-LC3 fluorescence microscopy, transmission electron microscopy, and western blot. ChIP assay and luciferase assay were performed to test the c-Myc's occupancy on the miR-27B promoter. Results: We observed that miR-27b-3p expression was significantly downregulated in oxaliplatin-resistant cell lines (SW480-OxR and HCT116-OxR) compared to the corresponding parental cell lines and that miR-27b-3p expression was positively correlated with disease-free survival (DFS) time in colorectal cancer patients. MiR-27b-3p could sensitize colorectal cancer cells to oxaliplatin in vitro and in vivo. Under oxaliplatin treatment, chemoresistant cells showed a higher autophagy level than parental cells. Moreover, we also identified that miR-27b-3p inhibited the expression of ATG10 at the posttranscriptional level, thus inhibiting autophagy. Further study demonstrated that c-Myc can inhibit the expression of miR-27b-3p via binding to the promoter region of miR-27B gene. Conclusions: Our study identifies a novel c-Myc/miR-27b-3p/ATG10 signaling pathway that regulates colorectal cancer chemoresistance. These results suggest that miR-27b-3p is not only a potential indicator for evaluating efficiency of chemotherapy, but also a valuable therapeutic target for CRC, especially for patients with chemoresistance.

Dataset Information

Circular RNA circ_0000423 regulates cartilage ECM synthesis via circ_0000423/miRNA-27b-3p/MMP-13 axis in osteoarthritis.

Publications

Circular RNA circ_0000423 regulates cartilage ECM synthesis via circ_0000423/miRNA-27b-3p/MMP-13 axis in osteoarthritis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets