Study On The Mechanism Of RNA Methylase METTL3 Promoting The Occurrence And Development Of Colorectal Cancer And The Intervention Of Huangqin Decoction And Its Medicinal Components

ObjectiveTo investigate the role and regulatory mechanism of RNA methylated m6A modification and modification enzyme in colorectal cancer.To investigate the therapeutic effect and mechanism of Huangqin Decoction and its medicinal components in the treatment of colorectal cancer.Methods1.First,The level of m6A in colorectal cancer tissues,adenoma tissues and paracancerous tissues was detected by the RNA methylation m6A modified quantitative detection kit,and then the level of m6A was further verified by immunohistochemistry and immunofluorescence.Then,based on tissue chip technology,Kaplan-Meier survival analysis and Cox regression were used to evaluate the relationship between m6A level and patients’ survival.Furthermore,the key enzyme METTL3 for RNA methylation of m6A modification was screened by real-time quantitative PCR.Immunohi stochemi stry and immunofluorescence were used to detect the expression level of METTL3 in colorectal cancer tissues,adenoma tissues and paracancerous tissues.Kaplanmeier survival analysis and Cox regression were used to evaluate the relationship between metTL3 expression level and survival time of patients.Then,the expression level was verified in different cell lines of colorectal cancer by real-time quantitative PCR and Western Blot.2.From METTL3 impact on the biological functions of colorectal cancer cells,and utilization of colorectal cancer cell lines HCT116,SW620 build METTL3 silence cell lines,by the CCK8 method and scratch test method and Transwell method for colorectal cancer cell line proliferation,migration,invasion and other biological function and the effect of further by sequencing m6A RNA methylation precipitation(MeRIP-seq)screening METTL3 CRB3 target genes,further study CRB3 influence on colorectal cancer cell biology function.3.The expression levels of METTL3 and CRB3 in the subcutaneous graft tumor model of SW620 nude mice with colorectal cancer cells treated with Huangqin Decoction were detected by Western Blot and immunofluorescence.Then,TCMNPAS database was used to screen the active ingredients of Huangqin Decoction,and molecular docking was conducted with METL3.The effect of Huangqin Decoction on the proliferation of colorectal cancer cell lines HCT116 and SW620 was detected by CCK-8 method.Western Blot,luciferase reporter gene and other methods were used to detect the effect of the active component on the expression of METTL3 protein.Results1.Compared with paracancerous tissues,the level of RNA methylated m6A was significantly increased in both colorectal cancer tissues and adenoma tissues,and survival analysis suggested that patients with low m6A level had a longer survival(P=0.001),and high levels of RNA methylated m6A were independent risk factors for death in colorectal cancer patients.Compared with paracancer tissues,METTL3 was highly expressed in colorectal cancer and adenoma tissues,and survival analysis suggested that patients with low METTL3 expression had a longer survival time(P=0.01),and high METTL3 expression was an independent risk factor for death in colorectal cancer patients.Compared with normal intestinal mucosal cells FHC,METTL3 expression was significantly increased in different colorectal cancer cells(HCT116,HT29,SW620 and SW480).2.METTL3 silencing cell lines were successfully constructed using colorectal cancer cell lines HCT116 and SW620,and the results showed that silencing METTL3 significantly inhibited the proliferation,wound healing,migration and invasion of HCT116 and SW620 cells.Silencing METTL3 significantly promoted the expression of CRB3.Compared with paracancerous tissues,CRB3 expression levels were significantly decreased in colorectal cancer tissues and adenoma tissues.The expression of CRB3 was positively correlated with OS and DFS in patients with colorectal cancer.Silencing CRB3 significantly promoted the proliferation,scratch healing,migration and invasion of HCT116 and SW620 scratch in colorectal cancer cells,but had no significant effect on proliferation.3.The expression of related proteins was detected in the tumor tissue of SW620 subcutaneous graft model of colorectal cancer cells treated with Huangqin Decoction,and the results showed that the expression of METTL3 was decreased and the expression of CRB3 was increased in Huangqin Decoction group compared with the normal saline group.TCMNPAS database and molecular butting method were used to screen out the active components such as squalene,hbaicalin,baicalin and betulinic acid in Huangqin Decoction.Betulinic acid had the most obvious inhibitory effect on the proliferation of colorectal cancer cell lines HCT116 and SW620,and betulinic acid could inhibit the expression of METTL3.Conclusions1.The modification level of RNA methylated m6A was significantly increased in adenoma and colorectal cancer,The high level of RNA methylation m6A modification was an independent risk factor for death in colorectal cancer patients.METTL3 was highly expressed in adenoma and colorectal cancer,and high METTL3 expression is an independent risk factor for death in colorectal cancer patient.2.METTL3 regulates the occurrence and development of colorectal cancer through the m6A-CRB3 axis.3.Huangqin Decoction in the treatment of colorectal cancer may be related to METTL3.