LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

Y. Zhou, T. Mu

Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China. zhouyu2021@126.com

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• Oncology

OBJECTIVE: To investigate the mechanism by which LINC00958 affects osteosarcoma progression through miR-4306/CEMIP axis.

PATIENTS AND METHODS: The microarray data (GSE66673) for gene expression in osteosarcoma cells were obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed genes were analyzed by bioinformatics tools. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression levels of LINC00958, miR-4306, and CEMIP in osteosarcoma tissues and cell lines. Western blot was performed to detect the expression levels of CEMIP. Subcellular fractionation analysis and RNA Fluorescence in situ hybridization (FISH) assay were performed to analyze the subcellular localization of LINC00958. The target relationship between LINC00958, CEMIP, and miR-4306 was verified by public bioinformatics database and dual-luciferase reporter assay. RNA immunoprecipitation (RIP) assay was performed to detect LINC00958 and miR-4306 bound to AGO2. The biological functions of LINC00958 and miR-205 on proliferation, cell cycle, apoptosis, migration, and invasion of osteosarcoma cells were evaluated by gain-of-function and loss-of-function experiments. Tumorigenic and metastatic abilities of cells in vivo were detected by xenograft tumor experiments and tumor metastasis assays in nude mice. Correlation between miR-4306 and LINC00958 or CEMIP expression in osteosarcoma tissues was analyzed using Pearson correlation analysis. Kaplan-Meier analysis was performed to assess the relationship between LINC00958 expression and overall survival of osteosarcoma patients.

RESULTS: LINC00958 expression levels significantly increased in osteosarcoma tissues while miR-4306 expression levels significantly decreased, and the expression of these two genes was negatively correlated. Subcellular fractionation analysis and RNA FISH assay demonstrated that LINC00958 was mainly localized in the cytoplasm. Dual-luciferase reporter gene assay verified that LINC00958 competitively bound miR-4306 and repressed its expression. Silencing of LINC00958 inhibited proliferation, cell cycle, metastasis, and invasion of osteosarcoma cells while inducing cellular apoptosis. The introduction of miR-4306 inhibitors reversed the tumor-suppressing effect of silencing LINC00958. miR-4306 binds to CEMIP and suppressed its expression. Xenograft tumor experiments and tumor metastasis assays in nude mice demonstrated that silencing LINC00958 inhibited osteosarcoma cells’ growth and metastasis while inhibiting miR-4306 reversed this effect. Kaplan-Meier analysis showed that high expression of LINC00958 was significantly associated with poor prognosis of osteosarcoma patients.

CONCLUSIONS: LINC0095 promotes tumorigenesis and metastasis in osteosarcoma by competitively inhibiting miR-4306 expression, leading to elevated expression of CEMIP.

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