| Objective:Objective to explore the relationship between S100A9 gene and protein expression and clinical prognosis of hepatocellular carcinoma(HCC)by detecting S100A9 gene and protein expression in HCC tissue samples;Objective to study the effect of S100A9 on the biological function of hepatoma cells,and to analyze the molecular pathway that S100A9 may be involved in the regulation of hepatoma progression combined with GSEA.Method:1.Total RNA was collected from HCC tissues and adjacent normal liver tissues.The gene expression of S100A9 in HCC tissues and adjacent normal liver tissues was detected by QRT PCR.The fresh specimens of HCC and adjacent normal liver tissues were made into paraffin embedded blocks.The protein expression of S100A9 in HCC and adjacent normal liver tissues was detected by immunohistochemistry.2.Chi square test to understand the relationship between S100A9 gene level and clinical data of liver cancer.Kaplan Meier method was used to investigate the effect of S100A9 gene and protein expression level on RFS(Disease-Free Survival)and OS(Overall Survival)of patients with hepatocellular carcinoma,and the differences of RFs and OS between the two groups were compared.At the same time,according to S100A9 Protein expression level,the nomogram prediction model of postoperative survival rate was established.3.To study the effect of S100A9 on the biological function of human hepatoma cell lines Hep G2 and Huh-7.Lentivirus mediated gene silencing and overexpression techniques were used to construct stable cell lines.QRT PCR and WB were used to detect the expression of S100A9 to determine the transfection efficiency.4.To investigate the effects of S100A9 gene overexpression or silencing on the biological behavior of the cells.CCK8 cell proliferation assay was used to detect the proliferation ability of hepatoma cells,plate clone formation assay was used to understand the clonogenic ability of hepatoma cells,scratch assay was used to analyze the lateral migration ability of hepatoma cells,and Transwell invasion migration assay was used to detect the chemotaxis and migration ability of hepatoma cells.5.Download the gene set of TCGA liver cancer database,and divide the gene set of tcga-lihc into two groups according to the median expression level of S100A9 gene.Use gene enrichment to analyze the molecular pathways that S100A9 may participate in the regulation of liver cancer progression.Result :1.The relative expression of S100A9 gene in HCC tissues was significantly higher than that in adjacent normal liver tissues(P < 0.05).It was further found that the relative expression of S100A9 gene in early recurrence patients was significantly higher than that in non early recurrence patients(P <0.05).2.The relative expression of S100A9 gene in liver cancer tissue was analyzed with clinical data.It was found that the high expression of S100A9 in patients with liver cancer was closely related to MVI,incomplete tumor envelope,b/c stage of BCLC,Ki67 ≥ 50% and early recurrence after operation.The results of single factor and multi factor showed that the high expression of S100A9 was an independent risk factor affecting the total survival time and tumor free survival time of patients with liver cancer.Log rank test showed that the tumor free survival rate and total survival rate of S100A9 high expression group were significantly lower than that of S100A9 low expression group.Further,the ROC curve suggested that S100A9 Protein expression level has the clinical significance of predicting the early recurrence of liver cancer after operation.The total survival time and tumor free survival time of the patients in the high expression group of S100A9 were significantly lower than that of the low expression group,which was in line with the trend of QRT PCR.We further determined the independent risk factors that affect the survival time of patients according to Cox regression model,and successfully established the survival chart.The predictive effectiveness of survival line chart was tested by area under ROC curve,calibration curve and clinical decision curve.Generally speaking,the model has better prediction.It is suggested that the high expression of S100A9 in patients with liver cancer is closely related to its clinical adverse prognosis,and it is expected to be a potential biomarker for evaluating the prognosis of HCC.3.Using lentivirus transfection technology,Hep G2 cell lines stably overexpressing S100A9 and Huh-7 cell lines stably silencing S100A9 were constructed.In Hep G2 cells,the relative expression of S100A9 in the experimental group transfected with p-s100a9 was significantly higher than that in the control group transfected with p-nc(P < 0.05).In Huh-7 cells,the relative expression of S100A9 in sh-s100a9 transfected group was significantly lower than that in SH NC transfected control group(P < 0.05).The expression of green fluorescent protein was 100% under fluorescence microscope.Therefore,cell lines stably expressing and silencing S100A9 were successfully constructed.4.In Hep G2 cells,CCK8 experiment results showed that the proliferation ability of S100A9 overexpression group was significantly higher than that of control group at 72 h and 96 h,P < 0.05.In the plate clone formation experiment,the clone formation rate of the over expression S100A9 cell group was significantly higher than that of the control group(P < 0.05).In Transwell invasion test,the number of cells passing through Transwell chamber in S100A9 overexpression group was more than that in control group(P < 0.05).In Transwell migration experiment,the number of cells passing through Transwell chamber in S100A9 overexpression group was more than that in control group(P < 0.05).In cell scratch test,the scratch mobility of S100A9 overexpression group was significantly higher than that of control group(P < 0.05).5.The results in Huh-7cell group were similar to those in Hep G2 cell group.The results of CCK8 experiment showed that the proliferation ability of S100A9 cells silenced at 24 h,48h,72 h and 96 h was significantly weaker than that of control cells(P < 0.05).In the plate clone formation experiment,the clone formation rate of S100A9 cell group was significantly lower than that of the control group(P < 0.05).In the Transwell invasion test,the number of cells passing through the Transwell chamber in the silenced S100A9 cell group was less than that in the control group(P < 0.05).In the Transwell migration experiment,the number of cells passing through the Transwell chamber in the silenced S100A9 cell group was less than that in the control group(P < 0.05).In cell scratch test,the scratch mobility of S100A9 cell group was significantly lower than that of control group(P < 0.05).6.GSEA results showed that S100A9 high expression gene set was enriched in cell adhesion,cytokines,inflammatory chemokines and immune response signal pathways,while S100A9 low expression gene set was enriched in fatty acid,amino acid and other metabolic activity related signal pathways.Conclusion : 1.S100A9 was highly expressed in both gene and protein levels in HCC tissues,and the expression of S100A9 in patients with early postoperative recurrence was higher than that in patients without early postoperative recurrence.2.Through the detection of S100A9 Protein Expression in cancer,we successfully established a nomogram model to predict the postoperative survival rate of patients,which can more effectively predict the possibility of early recurrence of liver cancer after operation.3.In vitro,S100A9 expression is positively correlated with the proliferation,cloning,invasion and migration of hepatoma cells.4.S100A9 may promote the malignant progression of HCC by regulating cytokines,inflammatory chemokines and immune response related pathways.Therefore,S100A9 is expected to become a tumor biomarker to evaluate the clinical prognosis and predict the early recurrence of HCC. |
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