The Function And Mechanism Study Of MiR-433-3p Inhibiting Esophageal Squamous Cell Carcinoma Proliferation By Targeting PAK4

Background and Objective Esophageal cancer(EC)is a very common malignant gastrointestinal tumor that origin from the human esophageal epithelium.The 5-year survival rate of patients is only 15%,and the morbidity and mortality rates of this disease are relatively high.The incidence of EC in our country is even higher.Esophageal squamous cell carcinoma(ESCC)is the most common pathological type of EC.Due to the lack of effective targeted drugs and effective treatment strategies,the current clinical therapeutic effect and prognosis of ESCC are still poor.Therefore,exploring new mechanisms of action in the occurrence and development of ESCC and using it as a target for the diagnosis and treatment of ESCC has important clinical significance and value.MiRNA is a non-coding small RNA molecule whose biological function is to accelerate the degradation of mRNA or inhibit the translation process of mRNA.It usually plays an important role in negatively regulating gene expression at the post-transcriptional level.Changing the expression of miRNA in cells can affect the expression of oncogenes and tumor suppressor genes,thereby affecting tumor cell proliferation and apoptosis,cell invasion and migration and other abilities.Studies have shown that the various expression patterns of miRNAs in different tumor types indicate that they may be extremely potential biomarkers and can be used as potential diagnostic and therapeutic targets for cancer.PAK4(p21-activated protein kinase 4)participates in a variety of cell biological processes in organisms,and is closely related to the occurrence and progression of tumors,and is highly expressed in a variety of cancer types.The previous research of our group has proved that PAK4 is highly expressed in ESCC and promotes the proliferation,migration and invasion of ESCC by regulating the phosphorylation state of its downstream protein LASP1.However,why PAK4 is highly expressed in ESCC and its regulatory mechanism are still unclear.This study aims to further search for the upstream regulatory factors of PAK4,explore its influence on the expression of PAK4 and the progression of ESCC,and clarify its potential molecular mechanism,provide some experimental evidence and theoretical support for the clinical prevention and treatment of esophageal squamous cell carcinoma.Methods1.Screening of miRNAs targeting PAK4 Used four online databases and the expression of miRNA in the database to find potential miRNAs targeting PAK4,constructed the wild-type and mutant type reporter gene vectors,and used luciferase reporter gene experiments to verify whether the two can be combined with each other.2.Exploring the expression of miR-433-3p in ESCC tissues and cells Customized the miR-433-3p probe,used in situ hybridization(ISH)to detect the expression of miR-433-3p in ESCC and adjacent tissues,and analyzed the effect of the expression level of miR-433-3p on the clinical stage and survival rate of ESCC.Carried out cell culture,extracted RNA and used RT-qPCR technology to detect the expression level of miR-433-3p in human normal esophageal epithelial cells and ESCC cell lines.3.Exploring the effect and mechanism of miR-433-3p on PAK4 expression and ESCC cell proliferation The miR-433-3p mimics was transfected into ESCC cells to overexpress it,and the miR-433-3p mutant plasmid was transfected to knockdown miR-433-3p.RT-qPCR and Western blot experiments were used to detect the transfection efficiency and the effect of miR-433-3p on the expression of PAK4 mRNA and protein.The cell proliferation experiments,plate cloning experiments,and soft agar experiments were used to observe the effects of knockdown and overexpression of miR-433-3p in ESCC cells on cell proliferation.Used WB experiments to detect the expression of PAK4 and its downstream proteins ERK,AKT and phosphorylated protein in cells after knockdown or overexpression of miR-433-3p.Used cell rescue experiments and perform the above cell phenotype and WB experiments to further verify the effect of miR-433-3p on PAK4 expression and ESCC cell proliferation.4.Exploring the mechanism that affects the expression of miR-433-3p Used biological information softwares to find the host gene of miR-433-3p.Taked the host gene as the research object,predicted the Cp G islands in the promoter region and used pyrosequencing technology to detect the methylation level of ESCC cells to analyze the molecules mechanism that affects the expression of miR-433-3p.Results1.MiR-433-3p can inhibit the fluorescence intensity of the wild-type PAK4 expression vector,but it has no obvious effect on the fluorescence activity of the mutant PAK4 vector expression,indicated that PAK4 is the target of miR-433-3p.2.Compared with normal esophageal epithelial tissues and cells,miR-433-3p is low expressed in ESCC,and its low expression is related to the poor prognosis of patients.3.Overexpression of miR-433-3p in cells can inhibit PAK4 expression at mRNA and protein levels,and suppress ESCC cell proliferation,clone formation and anchor-independent growth ability,and also repress the expression of p-ERK and p-AKT.Knockdown of it promotes PAK4 mRNA and protein expression, cell proliferation and clone formation,anchor-independent growth ability,and promotes the expression of p-ERK and p-AKT.Overexpression of PAK4 can reverse the inhibitory effect of miR-433-3p on PAK4 expression and ESCC cell proliferation.4.The methylation level of the RTL1 gene promoter in ESCC cells is higher,indicated that the hypermethylation state of the promoter can inhibit the expression of miR-433-3p.Inhibition of gene methylation can increase the expression of miR-433-3p.Conclusions Hypermethylation of RTL1 promoter leads to low expression of miR-433-3p in esophageal squamous cell carcinoma.Low expression of miR-433-3p in ESCC promotes ESCC proliferation by upregulating PAK4 to activate ERK/AKT signaling pathway.