| Objective:1.To analyze and validate the cellular function,and immune efficacy of the INHBA gene and its role in the TGF-β/Smad signaling pathway-induced colorectal carcinogenesis and development.2.To explore the molecular mechanism of upstream and downstream regulation of HNF1A-AS1,miR-214-5p and INHBA in colorectal cancer,and provide new theoretical basis for the diagnosis and treatment of colorectal cancer.Methods:1.Screening out the target gene INHBA,and verifying its role in the progression of colorectal cancer and its relationship with the response to immunotherapy.In this part,we performed gene differential expression analysis of the transcriptome,qPCR,Western blot,and IHC to detect colorectal cancer clinical samples,qPCR and Western blot to screen cells,construct INHBA interfering/overexpressing stable transgenic strains and detect the cellular function of regular transgenic strains,qPCR and Western blot to detect the cellular part of each stable transgenic strain of TGF-β/The cellular process of INHBA gene and its role in the development of colorectal cancer induced by TGF-β/Smad signaling pathway were examined by qPCR and Western blot.In addition,we explored the expression level of INHBA in tumor of patients with effective and ineffective neoadjuvant immunotherapy,and explored the correlation between INHBA and the efficacy of immunotherapy.2.Explore the mechanism of HNF1A-AS1,miR-214-5p and INHBA regulating colorectal cancer progression through ceRNA network.The expression of HNF1A-AS1,miRNA-214-5p,and INHBA in the pan-and colorectal cancer,the correlation between HNF1A-AS1 and immune-related genes or miRNAs and the target genes of hsa-miR-214 were investigated by using TCGA,miRDB,miRWalk,and TargetScan databases;the expression of HNF1A-AS1,miRNA-214-5p and INHBA in the pan-and colorectal cancers were analyzed by using dual luciferase assay to detect the relationship between miR-214-5p and INHBA,HNF1A-AS1 and miR-214-5p;miR-214-5p and HNF1A-AS1 suppression/overexpression stable transgenic strain construction and stable transgenic cell function assay and miR-214-5p and INHBA expression;by miR-214-5p and OEINHBA cell adhesion assay by Western blot to detect the expression of each gene of TGF-β/smad signaling pathway.And the effects of HNF1A-AS1 and INHBA on tumorigenesis and progression were detected by subcutaneous tumorigenesis and data monitoring;the impact of HNF1A-AS1 and INHBA on tumor microstructure were detected using HE staining of tumor tissues;the expression of miR-214-5p,HNF1A-AS1,and INHBA in tumor tissues were detected by qPCR,and the expression of miR-214-5p,HNF1A-AS1 and INHBA were detected by Western blot or IHC to see the expression of TGF-β/smad signaling pathway-related genes in tumor tissues.Results:1.It is confirmed the role of target gene INHBA in promoting the progression of colorectal cancer and the relationship between immune therapy response.The transcriptome analysis revealed that INHBA was highly expressed in colorectal cancer and played an essential role by mediating the TGF-β signaling pathway.The expression of various genes of the TGF-β/Smad signaling pathway in each stable transfectant indicated that INHBA could play an inductive role in the TGF-β/Smad signaling pathway.In addition,bioinformatics analysis results demonstrated that compared with patients with colorectal cancer who had poor immunotherapy effect,the expression of INHBA in patients with effective immunotherapy was significantly lower.2.It is verified that HNF1A-AS1,miR-214-Sp and INHBA can regulate the progression of colorectal cancer through ceRNA network.Bioinformatics analysis and luciferase assay identified INHBA as a target gene of miR-214-5p and miR-214-5p as HNF1A-AS1.miR-214-5p and HNF1A-AS1 repression/overexpression stable transgenic strain construction and functional assay of regular transgenic cells revealed that miR-214-5p and HNF1A-AS1 play an essential role in cell proliferation,The expression of genes in the TGF-β/Smad signaling pathway in each of the stable transfectants indicates that miR-214-5p and HNF1A-AS1 can play a regulatory role in the expression of genes in the TGF-β/Smad signaling pathway.Subcutaneous tumorigenesis of HNF1A-AS1 and INHBA and data monitoring results show that HNF1A-AS1 and INHBA overexpression could significantly affect tumor development;interference or overexpression of HNF1A-AS1 and INHBA could dramatically affect the number of tumor cells in tumor tissues;qPCR assay results of miR-214-5p,HNF1A-AS1 and INHBA expression in tumor tissues showed that the expression levels of miR-214-5p were significantly correlated with The western blot or IHC assay results of TGF-β/smad signaling pathway-related gene expression in tumor tissues showed that INHBA and HNF1A-AS1 could regulate tumorigenesis and progression by regulating the expression of TGF-β/smad signaling pathway-related genes in colorectal tumor tissues.Conclusions:1.Compared with adjacent tissues,INHBA and HNF1A-AS1 are highly expressed in colorectal cancer tissues,while miR-214-5p is low.2.INHBA is the target gene of miR-214-5p,while miR-214-5p is the downstream target of HNF1 A-AS 1;There is a ceRNA regulatory relationship between HNF1A-AS1,miR-214-5p and INHBA.3.HNF1A-AS1/miR-214-5p/INHBA axis can regulate TGF-β/Smad signaling pathway through ceRNA pathway promotes the invasion and migration of colorectal cancer.4.The expression level of INHBA was correlated with the degree of differentiation,depth of invasion(T stage),lymph node metastasis(N stage),distant metastasis(M stage),TNM stage and MMR status of colorectal cancer.Meanwhiles,the high expression of INHBA may reduce the efficacy of immunotherapy in patients with colorectal cancer. |
PDF Full Text Request