The Research About MiR-20a-5p Targeting WTX Promotes Gastric Cancer Progress Through PI3K/AKT/mTOR Signaling Pathway

Gastric cancer(GC)is one of the most common malignant tumors of digestive tract,and the incidence of morbidity is the fourth highest in the world.In China,both the incidence of morbidity and mortality of GC are the second highest.Because of its concealment,unclear molecular mechanism,rapid progress and poor prognosis,it is usually in the middle and late stages when the diagnosis is clear.WTX(Wilms Tumour gene on X chromosome),also known as FAM123B,Amer1,is the first tumor suppressor gene located on the X chromosome discovered in the Wilms tumor.Previous studies have shown that WTX is also associated with sclerosing osteopathy,but its role in other tumors is unclear.In our previous study,we found that WTX was low expression in GC and many other tumors,but its clinical significance and molecular mechanism were unknown.Elucidate the role and molecular mechanism of WTX in the progression of GC,which may provide a new theoretical basis and molecular target for the prevention and treatment of GC.MicroRNA(miRNA)is a class of small non-coding molecules and single stranded RNA that regulate the post transcriptional level of genes.Studies show that at least 60%of the genes in the human body are regulated by miRNA.Whether WTX gene inactivation is regulated by miRNA has not been reported in GC.The role and mechanism of MiRNA-WTX regulatory axis in gastric carcinogenesis are unclear.Based on the above problems,this study intends to explore the clinical and pathological significance of WTX in gastric carcinoma,the function of cell biology and its mechanism,the screening of miRNA targeting WTX and its biological function,in order to reveal the function and clinical value of WTX in gastric cancer.Objective(1)To study the expression level of WTX in GC and analyze its clinical significance.(2)To explore the effect of WTX on the biological behavior of GC cells and reveal the molecular mechanism.(3)Screening and identifying miRNAs targeting WTX and analyzing its clinical significance and biological function.(4)Construct miRNA-WTX-downstream signaling pathway axis and elucidate its mechanism and clinical value.Method:(1)The expression and clinicopathological significance of WTX in GC tissue samples were analyzed by qRT-PCR,Western Blot and immunohistochemistry,and the public database resources were used to analyze and verify it.(2)The effects of WTX on the biological function of GC cells were detected by CCK-8,flat clones,Transwell and scratch healing,and the effects of WTX on the tumor growth,tumor formation and metastasis were analyzed by the subcutaneous tumor and gastric orthotopic transplantation model in nude mice.(3)Gene expression chip was used to screen the genes regulated by WTX in gastric cancer cells.The downstream pathway was screened by KEGG pathway enrichment analysis,and the co-immunoprecipitation and protein expression analysis were performed to preliminarily verify it.(4)Using miRNAs chip to analyze and screen miRNAs targeting WTX.Through the analysis and verification of the dual luciferase reporter system,qRT-PCR,in situ hybridization(ISH),Western Blot,immunofluorescence(IF),and the data resources of GEO and TCGA in the public database,the miRNA of the targeted WTX and its expression and clinical significance in GC were clearly defined.(5)The cellular biological function of miRNA targeting WTX was detected by CCK8,plate clone formation,scratch healing and Transwell cell migration assay.The effects of miRNA on the regulation of WTX expression,tumor formation,tumor proliferation and cell cycle were analyzed by subcutaneous tumor bearing experiment,immunohistochemistry and flow cytometry in nude mice.(6)Using TCGA database resources for bioinformatics analysis,and using Western Blot,immunohistochemistry and immunofluorescence assay,the regulation mode and molecular mechanism of the axis of miRNA-WTX-downstream signaling pathway in GC were studied.Result(1)WTX is generally low expression in GC,and is closely related to cancer differentiation,lymph node metastasis,histological type and clinical prognosis.(2)WTX can inhibit proliferation,migration,clone formation and tumorigenesis of GC cells by binding to PI3K and negatively regulating PI3K/AKT/mTOR signaling pathway.(3)MiR-20a-5p targets WTX,which promotes the progression of GC.(4)MiR-20a-5p promotes the proliferation,migration,cloning and tumorigenesis of GC cells,and promotes the transformation of GC cells from stage G1 to S and inhibits cell apoptosis.(5)MiR-20a-5p promotes the progression of GC by targeting WTX to regulate PI3K/AKT/mTOR signaling pathway.conclusionWTX plays the function of tumor suppressor gene in GC.MiR-20a-5p targeting WTX promotes cell cycle and inhibits apoptosis through PI3K/AKT/mTOR pathway,thus promoting the proliferation of GC cells.MiR-20a-5p/WTX/PI3K/AKT/mTOR signaling pathway may be a potential clinical diagnostic molecular marker and therapeutic target.