The Study On The Role Of MiR-372-3p In Inhibiting Colon Cancer Cell Proliferation By Targeting MAP3K2

Colon cancer(CRC)is one of the most common digestive tract malignancies and the third leading cause of cancer death worldwide.Recently,through comprehensive treatment strategies such as surgery,chemotherapy,radiotherapy,and biological therapy for colon cancer,although the incidence and mortality of colon cancer have continued to decline,the incidence of colon cancer has increased trend in developing countries.In fact,the occurrence-development process of colon cancer is affected by many factors,and it is a complex process involving multiple genes and multiple steps.Therefore,there is an urgent need to explore better cancer-related biomarkers and targeted drugs for the treatment of colon cancer.In other words,the research on the molecular mechanism of colon cancer pathogenesis is crucial for the diagnosis and treatment of colon cancer.At present,miRNAs and their target genes have attracted much attention as biomarkers or therapeutic targets for tumor therapy,and have become promising tumor predictors.Mi RNAs are a family of 18-25 nucleotide long endogenous small noncoding RNAs that negatively regulate the post-transcriptional level of genes by binding to the 3 ′-UTR targeting m RNA.A large number of research data have shown that miRNAs are involved in various biological processes in cells,including cell proliferation,differentiation,cycle,migration,invasion and apoptosis.In tumor cells,miRNAs can function as oncogenic factors or tumor suppressors,depending on the genes targeted for regulation.In this paper,by detecting and analyzing the expression level of miR-372-3p in primary colon cancer tissues and its correlation with clinical indicators and tumor pathological indicators,we found that miR-372-3p was negatively regulated MAP3K2 in colon cancer SW480 cells,it was clarified that miR-372-3p can target and regulate the expression level of MAP3K2,thereby inhibiting colon cancer cell proliferation,and miR-372-3p/MAP3K2 may be involved in the regulation of MAP2K5(MER5)-ERK5/ BMK1 signaling pathway.The main work and results of this paper are as follows:1.153 frozen tissue samples,including primary colon cancer tissue and matched normal colon tissue or tumor-adjacent tissue,were collected,and real-time quantitative polymerase chain reaction(RT-q PCR)technology was used to detect and assess the expression levels of miR-372-3p.Statistical analysis showed that compared with normal tissues,the expression of miR-372-3p in tumor tissues was significantly decreased.At the same time,the expression of miR-372-3p in the adjacent tissues of the tumor also showed a significant downward trend,suggesting that the expression of miR-372-3p began to decline in the early stage of colon cancer,and it may become a predictive indicator for early diagnosis of colon cancer.Statistical analysis also found that the low expression of miR-372-3p in colon cancer tissue was closely related to clinicopathological stage and lymph node metastasis;2.Tissue samples from all primary colon cancer patients were histopathologically sectioned and immunohistochemically stained for tumor-associated factors,including MLH1,MSH2,MSH6,PMS2,p53,Ki67,and HRE-2.Correlation statistical analysis found that the expression of Ki67,a related antigen of proliferating cells,was significantly increased in most colon cancer tissues with low expression of miR-372-3p,suggesting that the expression level of miR-372-3p affects the proliferation process of colon cancer cells;3.The UALCAN website was used to predict and screen candidate target genes with miR-372-3p complementary sequence in the 3′-UTR region of m RNA.Combined with published literature,the expression levels of HDAC4,p21,Wee1 and MAP3K2 were finally detected.The test results showed that in addition to p21,the expression levels of HDAC4,Wee1 and MAP3K2 in colon cancer tissues were significantly different from those in the matched normal tissues,in which the expression level of Wee 1 was significantly lower,while the expression levels of HDAC4 and MAP3K2 were high.The correlation analysis found that only the low expression of miR-372-3p was correlated with MAP3K2,suggesting that there may be a negative regulatory relationship between miR-372-3p and MAP3K2;4.In order to verify the above detection and analysis results,we first constructed a plasmid capable of expressing miR-372-3p in mammalian cells using the pm R-m Cherry vector,and confirmed miR-372-3p in colon cancer cells SW480 can inhibit the viability and colony formation ability of colon cancer cells using MTT,CCK-8 and colony formation experiments,validating our hypothesis that miR-372-3p affects colon cancer cell proliferation in our tissue samples.At the same time,it was found that overexpression of miR-372-3p can dose-dependently inhibit the m RNA and protein expression levels of MAP3K2,suggesting that miR-372-3p has a negative regulatory effect on MAP3K2;5.In order to clarify the above speculation,two miR-372-3p-binding target sequences were predicted and found in the MAP3K2-3′-UTR region through the UALCAN website,and the p MIR-REPORT-Luc vector was used to construct MAP3K2-3′-UTR containing two target sequences luciferase reporter gene plasmids(including target sequence mutants),and then examined the effect of miR-372-3p on dual-luciferase activity in SW480 colon cancer cells.As expected,miR-372-3p dose-dependently inhibited p MIR-MAP3K2-3’-UTR luciferase activity,whereas this effect was abolished by mutating the binding site of miR-372-3p,confirming that miR-372-3p by binding to the target sequence in the MAP3K2-3’-UTR region,it further regulates the expression level of MAP3K2 and its intracellular function.Finally,we found that overexpression of miR-372-3p could simultaneously inhibit the expression level of MAP2K5(MER5)in cells,suggesting that miR-372-3p/MAP3K2 may be involved in the regulation of MEK5-ERK5/BMK1 signaling pathway.In summary,in this thesis,primary colon cancer tissue samples and colon cancer SW480 cell line were used as the research objects,and it was confirmed that miR-372-3p was significantly low-expressed in colon cancer tissues,while the low-expression of miR-372-3p was significantly lower in colon cancer tissues.It was closely related to the expression levels of Ki67 and MAP3K2 in tissues.Cell experiments were used to clarify that miR-372-3p negatively regulates the expression and biological function of MAP3K2 by directly binding to the target sequence in the MAP3K2-3 ′-UTR region.This study provides new ideas for the subsequent development of early genetic diagnosis and targeted drug therapy for colon cancer.