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FBXO2 modulates STAT3 signaling to regulate proliferation and tumorigenicity of osteosarcoma cells.

ABSTRACT: BackgroundOsteosarcoma (OS) is the most common primary bone malignancy in children and adolescents, and hyperproliferation of cells is a major problem of OS. FBXO2 belongs to the family of F-box proteins, and is a substrate recognition component of the Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase complex with specificity for high-mannose glycoproteins. The aim of the present study was to investigate the critical role of FBXO2 in OS cells.MethodsThe protein and mRNA expression levels of FBXO2 in clinic OS patients were measured by quantitative real time-polymerase chain reaction (qRT-PCR), Western blot and Immunohistochemical (IHC) staining assays, respectively. The FBXO2 overexpression model was constructed by retro-virus transfection in OS cells. FBXO2 knockout (KO) cells were generated by Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) assay. Cell counting and colony formation assays were used to analyze the effect of FBXO2 on the biological function of OS cells. FBXO2 KO cells were injected into nude mice to observe tumor growth in vivo. The interaction between FBXO2 and IL-6 was detected by immunoprecipitation. Luciferase assay was used to determine the transcriptional activity of STAT3.ResultsHere, we show that FBXO2 is significantly up-regulated in clinical OS samples compared to adjacent normal tissues. Ectopic expression of FBXO2 leads to increased OS cell proliferation and colony-forming ability, while FBXO2 knockout by CRISPR-Cas9-based gene editing has the opposite effect. In addition, the glycoprotein recognition activity of FBXO2 is required for its biological function in OS. In vivo experiments showed that FBXO2 knockout greatly impaired the tumorigenicity of OS cells in nude mice. At the molecular level, we found that knocking out FBXO2 can significantly inhibit STAT3 phosphorylation and downstream target gene expression through IL-6R stabilization.ConclusionTogether, these results indicate that FBXO2 promotes OS development by activating the STAT3 signaling pathway, suggesting that FBXO2 may be a new target for OS treatment.

SUBMITTER: Zhao X

PROVIDER: S-EPMC7296666 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Project description:BackgroundN6-methyladenosine (m6A) is the most common and abundant mRNA modification and it plays crucial roles in many biological processes. However, as a key RNA demethylase, alkylation repair homolog protein 5 (ALKBH5) has not been well studied in human osteosarcoma. The present study sought to explore ALKBH5-mediated m6A modification and the underlying mechanisms in human osteosarcoma.MethodsThe expression of ALKBH5 and its correlation with clinicopathological features were examined by bioinformatics analysis and tissue microarrays. Cellular proliferation was detected by CCK8 assays. Cell cycle and apoptosis were analyzed by TUNEL and Flow cytometry assay. Finally, investigation of the regulatory mechanism of ALKBH5 in human osteosarcoma was performed by MeRIP assay, RNA-sequencing, dual luciferase reporter assay, RNA pull-down and RNA stability assay. Tumor xenograft models were established for in vivo experiments.FindingsOur data showed that low expression of ALKBH5 was associated with worse overall survival for osteosarcoma patients. Reducing m6A mRNA levels in human osteosarcoma cells through ALKBH5 up-regulation lead to cell proliferation inhibition, cell apoptosis and cycle arrest. We identified SOCS3, a negative regulator of STAT3, as a downstream target of ALKBH5-mediated m6A modification. And the m6A modified SOCS3 mRNA was recognized by YTHDF2, which promotes the decay of SOCS3. Mechanistically, our data revealed that ALKBH5 inactivated STAT3 pathway by increasing SOCS3 expression via an m6A-YTHDF2-dependent manner.InterpretationM6A methylation is rising as a pathway affecting tumorigenicity and tumor progression. Our findings illuminate the clinical significance of ALKBH5-mediated m6A modification in human osteosarcoma and the regulatory mechanisms underlying tumor proliferation and growth, suggesting that ALKBH5 is a potential biomarker for treatment in human osteosarcoma.FundingThis work was supported by and Science and Technology foundation of Hubei, China (Grant No.2017CFB762); the Tongji hospital foundation (Grant No.2201103013); and the National Natural Science Foudation of China (No.82002849).

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FBXO2 modulates STAT3 signaling to regulate proliferation and tumorigenicity of osteosarcoma cells.

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