Let-7b-5p Targeting NKD1 Regulates Wnt/β-catenin And APC Ubiquitination To Inhibit Colon Cancer Progression And Its Mechanism

Objective:The hub Micro RNA(miRNA)regulating Naked Cuticle Homolog 1(NKD1)were screened based on bioinformatics,and the regulation of let-7b-5p targeting NKD1 on colon cancer proliferation,migration and invasion was confirmed through experimental verification.To explore the signaling pathway and molecular mechanism of let-7b-5p targeting NKD1 in colon cancer,providing new ideas and research basis for targeted therapy of colon cancer.Methods:1.miRNA molecules that can target NKD1 were screened by weighted gene coexpression network analysis,gene differential expression analysis and miRWalk online website.2.Bioinformatics was used to analyze the expression differences of let-7b-5p in different cancer species based on the TCGA database sequencing data,and quantitative real-time PCR(q PCR)was used to detect the expression of let-7b-5p in clinical samples of colon cancer and different colon cancer cell lines.3.The binding sites of NKD1 and let-7b-5p were predicted by targetscan website and verified by double fluorescein reporting assay.4.MTT,Ed U,wound healing and Transwell assays were used to detect the effects of let-7b-5p targeting NKD1 on the proliferation,migration and invasion of colon cancer cells.5.Gene set enrichment analysis and single sample gene set enrichment analysis were used to determine the correlation between the enrichment scores of Wnt/β-catenin signaling pathway and NKD1 expression.The regulation of let-7b-5p targeting NKD1 on Wnt/β-catenin pathway was analyzed by western blotting and immunofluorescence assay.6.The effect of let-7b-5p targeting NKD1 on ubiquitination level of Adenomatous Polyposis Coli(APC)was analyzed by co-immunoprecipitation and western blotting.7.The GEPIA website was used to analyze the correlation between the expression of NKD1 and ubiquitin-specific protease 15(USP15),and the regulation of NKD1 on the binding of APC and USP15 was analyzed by co-immunoprecipitation.Results:1.Bioinformatics analysis and dual fluorescein reporting systems confirmed let-7b-5p as a NKD1-targeting miRNA.2.let-7b-5p is relatively low expressed in colon cancer tissues and cells,and can inhibit the proliferation,migration and invasion of colon cancer by targeting NKD1.3.Gene set enrichment analysis showed that the expression level of NKD1 was positively correlated with Wnt/β-catnin enrichment scores(R=0.56,P<2.2e-16)in colon cancer.let-7b-5p inhibits Wnt/β-catenin signaling activity by targeting NKD1.4.In addition,NKD1 can bind to APC,a Wnt pathway inhibitor,to promote ubiquitination degradation of APC,which can be inhibited by let-7b-5p.5.Gene expression correlation analysis showed that NKD1 was negatively correlated with the expression of APC deubiquitination enzyme USP15(R=-0.25,P=3.9e-05),which was confirmed by western blotting.In addition,NKD1 can reduce the expression of USP15 protein and inhibit the binding of USP15 to APC.Conclusion:let-7b-5p targeted NKD1 to inhibit the proliferation,migration,and invasion of colon cancer and suppressed the ubiquitination degradation of APC as well as the activity of Wnt/β-catenin pathway.In addition,NKD1 can inhibit the expression of the deubiquitinating enzyme USP15 and the binding of USP15 to APC.In conclusion,the regulatory role of let-7b-5p/NKD1 on colon cancer progression demonstrates its potential research and therapeutic value.