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Silencing long non-coding RNA NEAT1 attenuates rheumatoid arthritis via the MAPK/ERK signalling pathway by downregulating microRNA-129 and microRNA-204.

ABSTRACT: The participation of long noncoding RNAs (lncRNAs) and microRNAs (miRs) in the progression of rheumatoid arthritis (RA) is a key area of investigation. The current study aimed to investigate the action of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in fibroblast-like synoviocyte (FLS) proliferation and synovitis in RA. A rat model of RA was established. LncRNA NEAT1 expression in the synovial tissues of patients with RA and FLSs from the RA rat model was determined using RT-qPCR. Next, dual luciferase reporter gene assay was applied to investigate the relationship between miR-129/204 and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK). A putative binding relationship between miR-204 and lncRNA NEAT1 was evaluated by RIP assay, and miR-129 promoter methylation was determined using MSP. After the expression of lncRNA NEAT1, miR-129 or miR-204 was altered in FLSs, the extent of ERK1/2 phosphorylation was assessed. In addition, FLS synovitis and proliferation were determined by ELISA and EdU assay, respectively. In RA rats, lncRNA NEAT1 was silenced and miR-129/miR-204 was overexpressed to explore their roles in vivo. LncRNA NEAT1 was upregulated, while miR-129 and miR-204 were downregulated in RA synovial tissues and FLSs. MAPK1 was target gene of both miR-129 and miR-204. LncRNA NEAT1 bound to miR-204 and promoted miR-129 promoter methylation. Silencing lncRNA NEAT1 or overexpressing miR-129/miR-204 enhanced miR-129/miR-204 expression, but reduced the extent of ERK1/2 phosphorylation, proliferation of FLSs, and synovitis in RA. Collectively, silencing lncRNA NEAT1 promoted miR-129 and miR-204 to inhibit the MAPK/ERK signalling pathway, reducing FLS synovitis in RA.Abbreviations: ACR: American College of Rheumatology; ELISA: Enzyme-linked immunosorbent assay; ERK: extracellular signal-regulated kinase; FLS: fibroblast-like synoviocyte; GADPH: glyceraldehyde-3-phosphate dehydrogenase; HRP: horseradish peroxidase; IFA: Incomplete Freund's Adjuvant; lncRNAs: long noncoding RNAs; MSP: Methylation-specific PCR; NC: negative control; NEAT1: nuclear paraspeckle assembly transcript 1; OD: optical density; RA: rheumatoid arthritis; RIPA: Radio Immunoprecipitation Assay; RLU: relative light units; RT-qPCR: reverse transcription quantitative polymerase chain reaction; UTR: untranslated region.

SUBMITTER: Chen J

PROVIDER: S-EPMC8078539 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Laryngeal squamous cell carcinoma (LSCC) is a highly invasive malignant tumor in the head and neck area. As an oncogene, long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) promotes cell proliferation, migration and invasion several types of cancer. The present study aimed to reveal the effects of NEAT1 on the progression of LSCC. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect relative mRNA expression levels of NEAT1, microRNA (miR)-204-5p and semaphorin (SEMA) 4B. Kaplan-Meier analysis was used to analyze overall survival times. RNA in-situ hybridization (ISH) exhibited the distribution of NEAT1 and miR-204-5p in tissues. RNA fluorescence ISH was used to analyze the distribution of NEAT1 and miR-204-5p in the cells. Western blot analysis was used to detect the expression level of target proteins. Cell viability was analyzed using a MTT assay, while flow cytometry was used to determine cell apoptosis. Wound healing and Transwell invasion assays were used to value cell migration and invasion, respectively. RNA immunoprecipitation assay, bioinformatics prediction and a dual luciferase reporter assay were used to analyze the target relationship. The RT-qPCR results showed that NEAT1 was highly expressed and miR-204-5p had decreased expression in LSCC tissues and cells compared with that in the normal tissue and the 16HBE-14o cell line, respectively. Knockdown of NEAT1 using small interfering (si) RNA and overexpressed miR-204-5p both effectively inhibited the proliferation, migration and invasion of LSCC cells. Besides, further experiments revealed that miR-204-5p was a target of NEAT1. At the same time, silenced miR-204-5p reversed the anti-tumor effects of si-NEAT1. In addition, SEMA4B was targeted by miR-204-5p in LSCC cells and upregulated SEMA4B weakened the antitumor effects of miR-204-5p in LSCC cells. NEAT1 regulated the expression of SEMA4B by targeting miR-204-5p in LSCC cells. Overall, NEAT1 promoted the proliferation and invasion of LSCC cells by regulating the miR-204-5p/SEMA4B axis.

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Silencing long non-coding RNA NEAT1 attenuates rheumatoid arthritis via the MAPK/ERK signalling pathway by downregulating microRNA-129 and microRNA-204.

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