| ObjectivesIn recent years,there is increasing evidence that RNA methyltransferase METTL3 plays an important role in carcinogenesis as a methyltransferase,and in the present study,METTL3 plays a pro-carcinogenic role in hepatocellular carcinoma,lung cancer and esophageal cancer.Therefore,searching for METTL3 inhibitors and exploring their possible molecular mechanisms of action can provide new ideas for clinical targeting of tumors.Since there is only one report on METTL3 inhibitors,and this report only demonstrated the inhibition of METTL3 protease function by this inhibitor extracellularly,while it has not yet explored the effect of this small molecule on tumors at the cellular level.Therefore,the aim of this work is to screen possible METTL3 small molecule inhibitors from the molecular library relying on METTL3 protein structure,and to investigate the effect and mechanism of its action on tumors.Methods1.Based on the three-dimensional structure of METTL3 protein,small molecules that may bind to the functional domain of METTL3 protein were screened from commercial molecular databases based on the affinity of the molecules to METTL3 protein.2.To validate the effectiveness of METTL3 inhibitors by RT-qPCR assays in the colon cancer cell line SW620 after co-incubation with small molecule inhibitors to detect the expression of SOX2,a downstream target of METTL3 that has been reported to be validated.3.The specificity of METTL3 inhibition by this small molecule was verified by RT-qPCR assay to detect the expression of METTL3 in colon cancer cell line SW620 treated with different concentrations of the inhibitor.4.The overall methylation level of RNA after inhibitor treatment was measured by the quantitative overall methylation level assay kit to verify the inhibition of METTL3 methyltransferase enzyme function by the small molecule inhibitor.5.CCK8 assay to detect the effect of inhibitors on tumor cell proliferation.6.Scratch assay and transwell assay to verify the effect of inhibitors on the migration ability of tumor cells.7.Detection of METTL3 expression in colon cancer tissues and paracancer tissues by RT-qPCR.8.Analysis of the TCGA database to analyze the expression of METTL3 in colon cancer cancer species and the relationship between METTL3 expression and survival of colon cancer patients.Result1.5 potential small molecules were screened from the molecular library depending on their affinity for binding to METTL3 protein.2.Small molecule 280 can inhibit the expression of SOX2 mRNA.3.The inhibition of SOX2 mRNA expression by small molecule 280 isconcentration-dependent,and the inhibition is specific.4.Small molecule 280 can reduce the overall methylation level.5.Small molecule 280 can inhibit tumor cell proliferation.6.Small molecule 280 can inhibit the migration of tumor cells.7.METTL3 expression was higher in colon cancer tissues than in paracancerous tissues.8.Analysis of data from TCGA database showed that METTL3 was highly expressed in a variety of cancer types including head and neck squamous carcinoma,lung cancer and colon cancer,and was associated with high METTL3 expression and poor prognosis in patients with head and neck squamous carcinoma and patients with hepatocellular liver cancer.Conclusion1.Small molecule 280 inhibited the expression of SOX2 mRNA,a downstream target of METTL3,and the inhibition of this downstream target by small molecule 280 was specific.2.Small molecule 280 inhibits the enzymatic catalytic function of METTL3 protein and reduces the overall intracellular methylation level.3.Small molecule 280 exhibits inhibition of tumor cell proliferation and migration.4.Clinical specimen assays showed high expression of METTL3 in colon cancer tissues.5.METTL3 is highly expressed in most cancers such as colon cancer and lung cancer.And METTL3 high expression is associated with poor prognosis in patients with head and neck squamous carcinoma and patients with hepatocellular liver cancer. |
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