Effects Of Prognostic-related Genes On Proliferation, Migration And Metabolism In Hepatocellular Carcinoma

Chapter 1: Effects of KCTD 7 gene on prognosis,proliferation,i cell cycle and apoptosis in hepatocellular carcinomaLiver cancer is a malignant tumor with strong aggression and poor prognosis.Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer,accounting for 90%.At present,the pathogenesis of liver cancer has not been thoroughly studied,and the main treatment methods are surgery and chemotherapy.The KCTD7 gene is mainly associated with neurological diseases,and there is little research on tumors.Therefore,observing the role of KCTD7 gene in HCC is of great significance for clinical HCC treatment.Objects:In this study,the human HCC cell line Hep3B and HepG2 used bioinformatics to analyze the expression and prognosis of KCTD7 in HCC,studied the effect of KCTD7 on HCC,and initially explored the possible mechanism of action through in vitro cell experiment,so as to provide new ideas for the clinical treatment of HCC.Methods:(1)The RNA sequencing results and clinical information of HCC patients were downloaded from the TCGA database,and the expression level of KCTD7 gene and its correlation with prognosis in HCC patients were analyzed by bioinformatics.(2)Univariate and multivariate Cox regression methods were used to analyze the relationship between clinical information and KCTD7 expression level and the survival of HCC patients.(3)Gene enrichment analysis was used to predict the pathways that KCTD7 gene may regulate in HCC.(4)The Tet-on-induced lentiviral expression vector system was used to construct an HCC cell line that induced knockdown KCTD7 gene expression by doxycycline(DOX).(5)The effect of knockdown KCTD7 gene expression on the proliferation of HCC cells was detected by live-cell counting.(6)The effect of knockdown KCTD7 gene expression on HCC cell cycle and apoptosis was detected by Western Blot method.(7)Whether KCTD7 gene can affect HCC cell cycle was detected by flow cytometry,and the specific mechanism of knockdown KCTD7 gene expression blocking cell cycle was explored.(8)Caspase 3 activity experiments were used to detect whether KCTD7 gene affected apoptosis in Hep3B cells.(9)Single-sample gene enrichment analysis was used to compare the immune infiltration between the high-expression group and the low-expression group of KCTD7.Results:(1)Compared with normal tissues,KCTD7 gene is highly expressed in liver cancer tissues.The results of survival prognosis analysis showed that the low expression group had a better prognosis than the high expression group of KCTD7 gene.(2)Univariate and multivariate Cox regression analysis showed that the expression level of KCTD7 gene was an independent risk factor affecting the overall survival of HCC patients.(3)GSEA results showed that KCTD7 gene was associated with a variety of tumor signaling pathways.(4)The results of live cell count showed that knocking down the KCTD7 gene expression could inhibit the proliferation of HCC cells.(5)Western Blot results showed that knocking down the expression of KCTD7 gene could reduce the activity of CDK6 protein and increase the activity of P21 protein in HCC cells,indicating that knocking down KCTD7 gene expression affected the HCC cell cycle and may block it in the G0/G1 phase.(6)The results of flow cytometry showed that after knocking down the expression KCTD7 gene,there were more cells in the G1 phase,indicating that the knockdown of the KCTD7 gene expression could block HCC cells in the G0/G1 phase.(7)After knocking down the expression of KCTD7 gene through the detection of cell Caspase 3 activity,the activity of Caspase3 in Hep3B cells was significantly increased.(8)Western Blot results showed that knocking down the expression of KCTD7 gene could increase the activity of Cleaved PARP1 proteins in cells,indicating that the KCTD7 gene could inhibit apoptosis of Hep3B cells.(9)In the tumor microenvironment,the high expression of KCTD7 gene was positively correlated with activated CD4+T cells,central memory CD4+T cells and natural killer cells.Conclusions:(1)KCTD7 is highly expressed in HCC tissues and affects prognosis,which can be used as a prognostic marker for HCC.(2)KCTD7 has a positive regulatory effect on the proliferation of HCC cells.(3)Knocking down the expression of KCTD7 can block HCC cells in the G0/G1 phase,thereby inhibiting proliferation.(4)KCTD7 has an inhibitory effect on the apoptosis of HCC cells.(5)Starting from bioinformatics methods,combined with in vitro experimental analysis,the possible mechanism of action of KCTD7 on HCC can be preliminarily explored.Chapter 2;Effects of ATP6V0 B on the proliferation,migration,and metabolism in hepatocellular carcinomaHepatocellular carcinoma(HCC)is the highest type of primary liver cancer,up to 90%,and is a common cancer worldwide.Surgery and liver transplantation are the most common forms of HCC,but the prognosis is poor and the rate of recurrence is high.In recent years,the incidence and mortality of HCC have increased.V-ATPase hydrolyzes ATP and is responsible for proton transport,and J7P6F05 is a subunit of V-ATPase.At present,the molecular mechanism through v/hich ATP6V0 B regulates the occurrence and development of HCC is not clear,so it is of great significance to study its potential regulatory mechanism for the subsequent search for new HCC therapeutic targets.Objective:In this study,the human HCC cell line Hep3 B was used to analyze the expression and prognosis of ATP6V0 B in HCC,the effect of ATP6V0 B on HCC was studied,and its effects on HCC cell proliferation,migration and metabolism were preliminarily explored through in vitro experiments,which together provided new ideas for the clinical treatment of HCC.Methods:(1)Bioinformatics was used to analyze the expression level of ATP6V0 B gene in HCC patients and its influence on prognostic survival.(2)The Tet-on-induced lentiviral expression vector system was used to construct an HCC cell line that induced knockdown ATP6V0 B gene expression by doxycycline(DOX)?and the expression level of ATP6V0 B in the constructed cell line was detected by real-time PCR.(3)The effect of knockdown ATP6V0 B gene expression on the proliferation of Hep3 B cells was detected by live-cell counting.(4)The effect of knockdown ATP6V0 B gene expression on Hep3 B cell migration was detected by scratch and Transwell experiments.(5)The glycolytic pathway activation of each HCC patient in the TCGA database was analyzed by GSVA method,and the expression of ATP6V0 B was analyzed in relation to the activation of glycolytic pathway.(6)Changes in the knockdown of the ATP6V0 B gene expression relative to the glycolytic pathway-related targets in Hep3 B cells were detected by quantitative real-time PCR analysis.(7)The effect of knockdown ATP6V0 B gene expression on Hep3 B cell metabolism was detected by lactic acid content detection kit.Results:(1)The ATP6V0 B gene is highly expressed in HCC tissues compared with normal tissues;The high expression of ATP6V0 B will lead to poor prognostic survival.(2)The real-time PCR results showed that the expression level of ATP6V0 B gene in the constructed cell line was significantly down-regulated.(3)The results of live cell count showed that knocking down the ATP6V0 B gene expression could inhibit the proliferation of Hep3 B cells,(4)The results of scratch test and Transwell experiment showed that knocking down ATP6V0 B gene expression could inhibit Hep3 B cell migration.(5)The results of GSVA analysis showed that the expression of ATP6V0 B may be significantly positively correlated with the degree of glycolytic pathway activation,and the glycolytic pathway was activated in patients with HCC with high ATP6V0 B expression.(6)The real-time PCR results showed that the knockdown of ATP6V0 B gene expression was able to decrease the expression of HK2 and LDHA genes in the glycolytic pathway of Hep3 B cells.(7)The lactic acid content test results showed that knocking down the expression of ATP6V0 B gene could reduce the glycolytic metabolite: lactic acid.Conclusions:(1)In this study,the use of bio informatics detected that ATP6V0 B was highly expressed in HCC tissues,and patients with high expression had worse prognostic survival,which indicated that ATP6V0 B could be used as a prognostic marker for HCC.(2)Using the HCC cell line constructed to knock down the expression of ATP6V0B9 it was verified that the expression of knockdown ATP6V0 B had an inhibitory effect on the proliferation of HCC cells.(3)Knockdown the expression of ATP6V0 B inhibited the migration and invasion ability of HCC cells.(4)The expression of knockdown ATP6V0 B inhibited the glycolytic activity of Hep3 B cells by affecting the expression of some genes and metabolites in the glycolytic pathway,making the cells unable to maintain the acidic microenvironment.