A Study On The Role Of Methyltransferase-like 3 In Growth And Lymphatic Metastasis Of Oral Squamous Cell Carcinoma And Its Mechanism

Background and objectiveOral squamous cell carcinoma(OSCC)is the most common malignant tumor in head and neck region.Advanced OSCC commonly present poor prognosis,characterized by uncontrollable proliferation and aggressive lymphatic metastasis.Despite of the progress in OSCC treatments,the overall survival of OSCC patients remains pessimistic.it is of pivotal urgency to develop novel biomarker as potent prognostic indicator and effective therapeutic target.N6-methyladenosine(m6A)is the most abundant modification in mRNA and ncRNAs of eukaryotic species,which mediates tumor development via m6A-dependent RNA regulation.Methyltransferase-like 3(METTL3),as the core catalytic subunit of m6A RNA methylation,was reported to serve as oncogene in many tumors.However,it requires further efforts to make it clear how METTL3 works in OSCC growth and metastasis.In this study,we investigated the function and underlying mechanism of METTL3 in the development of OSCC,and aimed to explore a novel promising diagnostic and therapeutic biomarker.Methods and results1.The m6A methylation level of 13 pairs of OSCC samples and their adjacent normal tissues was detected by m6A methylation colorimetric quantification,revealing that OSCC tissues presented higher m6A methylation level.2.The METTL3 expresison level of OSCC and adjacent normal tissues was analyzed by qPCR and immunohistochemistry,and the result showed that METTL3 was upregulated in OSCC samples and positively correlated with m6A methylation level.3.The expression of METTL3 in 94 OSCC samples was detected by immunohistochemistry,proving that high METTL3 expression was correlated with higher T stage,cervical lymphatic metastasis rate,and lower overall survival rate.4.In OSCC cell lines,siRNA was used to inhibit the expression and activity of METTL3.CCK8,Ki67 flow cytometry,wound healing and Trans well assays demonstrated that cell proliferation,migration and invasion abilities were significantly reduced.5.The subcutaneous xenograft tumor model and orthodontic xenograft tumor model revealed that METTL3 depletion inhibited growth and cervical lymphatic metastasis OSCC cells.6.Through exploration of meRIP and transcriptome sequencing,which was furtherly verified by meRIP-qPCR,SLC7A11 was found to be regulated by METTL3.Overexpression of SLC7A11 could restore the suppressed proliferation and metastatic abilities caused by METTL3 inhibition in OSCC.7.In OSCC cell lines,RNA stability and RIP-qPCR assays demonstrated that IGF2BP2 stabilized SLC7A11 mRNA via m6A dependent manner.8.In OSCC cell lines,qPCR and western blot showed that METTL3 and SLC7A11 exprssion were inhibited after triptolide treatment.CCK8,Ki67 flow cytometry,wound healing and Transwell assays presented that cell proliferation,migration and invasion abilities were significantly reduced by triptolide.9.The subcutaneous xenograft tumor model and orthodontic xenograft tumor model confirmed the anti-tumor effect of triptolide.Conclusions and novelties1.METTL3 is highly expressed in OSCC and associated with poor prognosis.2.METTL3 enhances the SLC7A11 stability through an m6A-IGF2BP2 dependent manner,indicating a novel m6A regulatory mechanism of SLC7A11.3.Triptolide exerts anti-tumor effects in OSCC cells through regulating METTL3SLC7A11 axis,revealing the therapeutic value of METTL3 in OSCC.