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The Effect Of HOXC8 On The Biological Characteristics Of Gastric Cancer Cell Lines And Its Related Mechanism

Posted on:2022-02-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:X H SongFull Text:PDF
GTID:1524306551474104Subject:Gastrointestinal Surgery
Abstract/Summary:PDF Full Text Request
Objective:Recent studies have shown that members of several HOX gene families,including HOXC8,are expressed aberrantly in some human tumors and play an important role in promoting or inhibiting tumor progression.Exploring the role of HOXC8 gene in tumor progression is of great significance for the diagnosis and treatment of tumors.However,the role of HOXC8 in gastric cancer is still unclear.The purpose of this study is to explore the effect of HOXC8 on the biological characteristics of gastric cancer cells and the clinical significance of HOXC8 expression in gastric cancer patients,and to further study the mechanism of its function.Materials and Methods:The expression of HOXC8 in gastric cancer tissue samples after gastrectomy in our hospital were tested by quantitative PCR and immunohistochemistry respectively.The relationship between HOXC8 expression and clinicopathological features and prognosis of gastric cancer was analyzed.HOXC8 differentially expressed gastric cancer cell model was constructed by lentivirus transfection.The effects of HOXC8 on the biological characteristics of gastric cancer cells were studied by CCK8 assay,clone formation assay,invasion and migration assay,wound healing assay,apoptosis assay,cell cycle assay and subcutaneous tumor transplantation assay in mice.To further study the possible mechanism of HOXC8,transcriptome sequencing was performed on the gastric cancer cells with differentially expressed HOXC8,and bioinformatics was used to compare the differentially expressed genes(DEGs)between the two groups.The GO,KEGG and Reactome database were used for annotation and enrichment analysis of DEGs.Quantitative PCR,Western Blot(WB)and chromatin immunoprecipitation(Ch IP)assay were used to screen and verify the downstream genes that HOXC8 might act on,and si RNA transfection,CCK8 assay,clone formation assay and invasion and migration assay were used to explore the function of downstream genes.Results:Compared with adjacent tissues and normal gastric mucosa cells,HOXC8 was low expressed in gastric cancer tissues and gastric cancer cell lines.In clinical samples,the results of immunohistochemistry showed that the expression level of HOXC8 in gastric cancer tissue samples was negatively correlated with the clinicopathological characteristics of gastric cancer,such as p N stage(P=0.009),degree of differentiation(P=0.017)and p TNM stage(P=0.019).Multivariate regression analysis suggested that HOXC8 expression(P=0.003,HR=3.044,95%CI[1.470,6.304])was an independent prognostic factor for gastric cancer.Quantitative PCR results showed that HOXC8 expression was negatively correlated with tumor size(P=0.005),gross classification(P=0.038),p N staging(P<0.001)and p TNM staging(P=0.002).Multivariate regression analysis showed that HOXC8 expression was an independent prognostic factor for gastric cancer before(P=0.001,HR=1.873,95%CI [1.283,2.735])and after(P=0.001,HR=2.078,95%CI [1.335,3.235])propensity score matching.In addition,both immunohistochemical and quantitative PCR results showed that HOXC8 was related to the prognosis of gastric cancer,and the overall survival rate of patients with low HOXC8 expression was significantly lower than that of patients with high HOXC8 expression(P<0.001).Cell function experiments showed that overexpression of HOXC8 inhibited invasion,migration,clone formation,proliferation in vitro and tumor formation in vivo,and promoted apoptosis of gastric cancer cells,while knocking down HOXC8 has the opposite effect.After analysis of differential gene expression,1560 DEGs were identified,of which 1006 were up-regulated and 554 were down-regulated.The GO functional enrichment analysis showed that the main biological functions of DEGs were extracellular matrix and structural components.The enrichment analysis results of KEGG and Reactome signaling pathways showed that the most representative signaling pathways of the two were the EMC-receptor interaction and extracellular matrix organization,respectively,suggesting that HOXC8 may be involved in the regulation of extracellular matrix.Quantitative PCR and WB verification of the first20 differential genes enriched in the extracellular matrix showed that HOXC8 expression was significantly correlated with SPP1 expression,and CHIP assay and Quantitative PCR confirmed that HOXC8 was bound to SPP1 promoter.The si RNA targeting SPP1 was used to transfect gastric cancer cells and HOXC8 knockdown gastric cancer cells.The results showed that the down-regulated expression of SPP1 reduced the ability of proliferation,clone formation,invasion and migration of gastric cancer cells in vitro.In addition,the down-regulation of SPP1 expression can reverse the promoting effects of HOXC8 knockdown on the proliferation,clone formation,invasion and migration of gastric cancer cells in vitro.Conclusion:HOXC8 expression is correlated with the clinicopathological characteristics of gastric cancer and is an independent prognostic risk factor for gastric cancer.HOXC8 inhibited the proliferation,invasion and migration of gastric cancer cells,and promoted cell apoptosis.HOXC8 may regulate the proliferation,invasion and migration of gastric cancer cells through SPP1.
Keywords/Search Tags:gastric cancer, gastrectomy, HOXC8, biological characteristics, prognosis
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