Study On The Role And Molecular Mechanism Of MiR-124/METTL16 Axis In The Occurrence And Development Of Colorectal Cancer

Objectives: Colorectal cancer is a common digestive tract tumor and the third most common malignant tumor and the second leading cause of cancer death in the world,especially in the incidence and mortality rate among young people and developing countries,which seriously affects human health.Studies have shown that the development and progression of colorectal cancer is a hereditary disease caused by genetic and epigenetic changes,and is regulated by multiple molecular signaling pathways.Therefore,exploring the molecular mechanisms of colorectal cancer development and progression is beneficial for understanding the pathophysiology and research on targeted treatment of colorectal cancer.m6 A methylation modification is an epigenetic transcription modification at the RNA level(including m RNA and nc RNA),affecting the transfer,cleavage,degradation,etc.It has been a hot topic in colorectal cancer research in recent years and plays an important role in the development and progression of colorectal cancer. Research shows that METTL 16 can act as a "writer" or "reader" to regulate RNA m6 A methylation,and can also affect cellular biology in a non-m6 A dependent manner.However,its impact on tumors,especially colorectal cancer,is still unclear.Therefore,standing on METTL 16,we will explore the effect of m6 A methylation transferase METLL16 on the development and progression of colorectal cancer and further investigate the upstream or downstream regulatory factors of METTL16.Some studies have found that m6 A methylation regulators are regulated by various small molecule RNAs such as miRNAs,lnc RNAs,etc.,and these studies mainly focus on the study of methylation enzyme METTL 13.However,the molecular study of the micro RNA/METTL16 axis in colorectal cancer has not yet been reported.Based on this,we will focus on the methylation transferase METTL16 and deeply study the molecular mechanism of the interaction between micro RNA and METTL16 and its impact on the development and progression of colorectal cancer,providing a theoretical basis for the mechanism research and targeted therapy of colorectal cancer.Methods:1.Collection of human Colorectal Cancer Tissue Samples 10 colorectal cancer tissue samples and adjacent normal tissue samples were collected at the Digestive Endoscopic Center,Second Affiliated Hospital of Kunming Medical University,and each patient was pathologic confirmed to have colorectal cancer.2.The expression of METTL16 in colorectal cancer tissues The expression data of METTL16 in colorectal cancer was downloaded from the TCGA database,and the difference in expression of this data was initially explored using R language.The expression and distribution of METTL16 in colorectal cancer tissues were detected using immunohistochemistry method.3.The impact of METTL16 on the biological characteristics of colorectal cancer cells. The expression effect of METTL16 over-expression plasmid and METTL16 si RNA transfection will be detected using fluorescence quantitative PCR(q-PCR)and WB methods.Methods such as CCK8 assays,Wound-healing assays,Transwell assays are used to examine the changes in proliferation,migration and invasion abilities of colorectal cancer cell lines RKO and HCT116 after transfection with METTL16 overexpression plasmids or METTL16 si RNA4.Expression of miR-124 in Colorectal Cancer Tissues Real-time fluorescence quantitative PCR(q-PCR)was used to detect the expression of miR-124 in human colorectal cancer tissues.5.The impact of miR-124 on the Biological Characteristics of Colorectal Cancer Cells Using fluorescence quantitative PCR(q-PCR)to detect the expression of miR-124 mimics and inhibitors after transfection in colorectal cancer cells;Using CCK8 assay,Wound-healing assay,and Transwell invasion assay to examine the changes in cell proliferation,migration,and invasion abilities in colorectal cancer cell lines RKO and HCT116 after transfection with miR-124 mimics and inhibitors6.miR-124 downregulating METTL16 affects the biological functions of colorectal cancer cells. The CCK8 assays,cell woundhealing assays,Transwell assays,etc.,were used to detect changes in cell proliferation,migration,and invasion abilities of the colorectal cancer cell lines RKO and HCT116 after co-transfection with miR-124 mimics and over-expression plasmids of METTL16,or miR-124 mimics and METTL16-NC or METTL16 over-expression plasmids and mimics-NC.7.Study of the Correlation between miR124 and METTL16 Pearson correlation analysis was used to analyze the correlation between the expression of miR-124 in colorectal cancer tissues and the expression of METTL16;Target Scan database was used to predict the targeting relationship between miR-124 and METTL16(human);Dual-luciferase reporter assay was used to verify whether miR-124 directly targets the 3’UTR of METTL16 m RNA.Results:1.METTL16 is highly expressed in colorectal cancer tissues,while miR-124 is lowly expressed in colorectal cancer tissues. The results of q-PCR analysis,immunohistochemical detection,and TCGA database analysis on 10 sets of colorectal cancer tissues showed that miR-124 was significantly lower in colorectal cancer tissues than in normal tissues with statistical significance(P < 0.05),while METTL16 was highly expressed in colorectal cancer tissues(P < 0.05).Patients with colorectal cancer with obvious high expression of METTL16 had a poorer prognosis than those with low expression of METTL16(P <0.05).2.Promotion of Colorectal Cancer Cell Proliferation,Migration and Invasion by METTL16 Through cellular experiments,the METTL16 overexpression plasmid and METTL16 si RNA interference plasmid were transfected into colorectal cancer cell lines RKO and HCT116 to test the effect of METTL16 on the biological characteristics of colorectal cancer cell lines.The results showed that overexpression of METTL16 promoted the proliferation,migration,and invasion of colorectal cancer cells,and interference with METTL16 suppressed the proliferation,migration,and invasion of colorectal cancer cells.These results indicate that METTL16 promotes the development of colorectal cancer.3.miR-124 Suppresses Colon Cancer Cell Proliferation,Migration,and Invasion Through constructing miR-124 mimics/inhibitors and transfecting into colorectal cancer cell lines RKO and HCT116,the effect of miR-124 on the biological characteristics of colorectal cancer cells was tested.The results showed that the miR-124 mimics group suppressed colorectal cancer cell proliferation,migration,and invasion,while the miR-124 inhibitor group promoted colorectal cancer cell proliferation,migration,and invasion,with statistically significant differences.These results indicate that miR-124 suppresses the development and progression of colon cancer."4.miR-124 Suppresses the Oncogenic Effect of METTL16 on Colorectal Cancer By constructing the overexpression of METTL16,miR-124 mimics/overexpression of METTL16,and miR-124 overexpression groups transfected into colorectal cancer cell lines RKO and HCT116,the effect of the miR-124/METTL16 axis on the biological characteristics of colorectal cancer cells was examined.The results showed that the overexpression of miR-124 downregulated the oncogenic effect of METTL16 on colorectal cells,including promotion of cell growth,migration and invasion.These results indicate that miR-124 regulates the oncogenic function of METTL16 in colorectal cancer.5.miR-124 cannot directly bind to the 3’ UTR region of METTL16 m RNA miR-124 expression is negatively correlated with METTL16 expression in human tissues through Pearson correlation analysis.Bioinformatics methods were then used to predict the binding target between miR-124 and METTL16 m RNA,and further verified by dual luciferase assays.The results showed that miR-124 cannot directly bind to the 3’ UTR region of METTL16 m RNA.Conclusion:This study systematically explored the role of miR-124 in downregulating METTL16’s tumor-promoting effect on colorectal cancer.It also investigated the interaction between the m6 A methyltransferase METTL16 and micro RNA,providing new insights into the regulation of RNA methylation modification and the molecular mechanism of METTL16 in colorectal cancer,as well as its interaction with micro RNA.The findings offer a basis for the molecular study of METTL16 in colorectal cancer and targeted drugs.