| Purpose:To study the expression of METTL3 gene in retinoblastoma cells line Y79 and its cisplatin resistant cells line Y79/DDP.And then explore the role of METTL3 and its potential molecular mechanism in Y79/DDP,in order to provide insights for the cisplatin resistance in clinical treatment of human retinoblastoma.Methods:Real-time PCR and Western Blot were performed on ARPE-19(human normal retinal pigment epithelial cells),Y79 and Y79/DDP cell lines to certified that METTL3 was a differentially expressed gene.A lentiviral vector with overexpression or knockdown of METTL3 gene were transfected into Y79 to construct stable expression cell lines.And then use real-time PCR and Western Blot experiments to detected their m RNA and protein expression levels.MTT assay was used to measuring the effects of proliferation in Y79 cells with METTL3 overexpression or knockdown which were deal with different concentrations of cisplatin.The target genes of METTL3 in Y79 were determined by Me RIP-seq and Me RIP-q PCR,and the regulation of METTL3 on related target genes was further detected by Real-time PCR,Western Blot and flow cytometry in vitro.Results:In this study,we found that the expression of METTL3 was upregulation in Y79 and Y79/DDP cells.Overexpression of METTL3 in Y79 cells promoted proliferation and increased its cisplatin resistance,while cell growth and resistance to cisplatin was inhibited in METTL3 knockdown Y79 cells.Mechanism studies have shown that METTL3 can promote cisplatin resistance of Y79 by targeting MDM2 or platinum-resistance related gene BCL2 which can inhibit the apoptosis of Y79 cells.Conclusion:Our results indicated that METTL3 plays an important role in the chemotherapy resistance of retinoblastoma,and it can regulate the drug resistance of Y79 cells to cisplatin by targeting MDM2 and BCL2 which were platinum-resistance related genes.Thus,METTL3 may serve as a potential diagnostic biomarker and therapeutic target for retinoblastoma. |
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