Expression And Clinical Significance Of Replication Factor C4 In Hepatocellular Carcinoma

Objective: The expression of replication factor C4(RFC4)in hepatocellular carcinoma(HCC)tissue was studied using bioinformatics and immunohistochemistry(IHC),and the impact of changes in its expression on patient prognosis was investigated.The mechanism of RFC4 action in HCC,as well as its relationship with immune cell infiltration,were also investigated.The goal is to provide a scientific foundation for investigating early detection and prognostic markers of liver cancer.Methods: First of all,The Cancer Genome Atlas(TCGA)database was used to obtain high-throughput sequencing LIHC transcriptome data:(1)Using operator receiver characteristic(ROC)analysis,determine whether RFC4 mRNA expression differs between HCC and normal liver tissues,and assess the diagnostic value of the RFC4 molecule in HCC.(2)Determine whether RFC4 mRNA expression correlates with clinicopathological features.(3)Using SPSS 26.0 software,a Kaplan-Meier survival analysis was performed,followed by a bilateral-Log-rank test to compare the difference p values of survival rates among different groups,and finally,a survival curve was plotted using Graph Pad Prism 8 software.The RFC4 molecule’s prognostic value in HCC was then assessed using single-factor and multi-factor COX regression analysis.(4)The LIHC transcriptional group’s data were analyzed in R using co-expression.The threshold for screening differentially expressed genes was | Pearson’s r | > 0.7 and | Spearman’s r | > 0.7,P< 0.001.After extensive screening,188 RFC4-related genes were identified and organized into a gene set.R was used to examine Gene Ontology Molecular Function(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.(5)A Wilcoxon rank sum test was employed to contrast immune cell infiltration between RFC4 high and low-expression groups,while single-sample gene set enrichment analysis(ss GSEA)was utilized to contrast immune cell infiltration between RFC4 high and low-expression groups.Confirmation of the connection between RFC4 expression and immune cell infiltration was achieved through the Spearman correlation method.Finally,120 cases of HCC and their corresponding paracancerous tissue samples,clinical information,and follow-up data were collected for experimental verification:(1)RFC4 expression was detected using the IHC method.(2)A statistical examination was conducted to explore the correlation between RFC4 expression levels and clinicopathological features.Results: Analysis of the TCGA database showed that mRNA expression of RFC4 was notably higher in HCC tissues than in normal liver tissues.After analyzing the ROC curve,the Area under CURVE(AUC)attained a 0.974(95% confidence interval [Confidence,CI] = 0.948-0.989)result.(2)There were statistically significant correlations between RFC4 mRNA expression and T stage(P = 0.003),differentiation degree(P = 0.001),clinical stage(P = 0.002),and survival status(P = 0.020).(3)According to the results of the survival study,patients with high expression of RFC4 had a significantly worse overall survival rate than those with low expression(P=0.0058).T stage(P<0.001),M stage(P=0.028),clinical stage(P<0.001),and expression of RFC4(P=0.006)were risk factors that had an impact on patients’ prognoses,according to Cox univariate analysis.According to a Cox multivariate analysis,RFC4 expression had a statistically significant effect on patient survival time(P=0.035),and patients with high RFC4 expression had a 1.499-fold higher probability of dying than patients with low RFC4 expression.Other factors did not significantly affect the duration of survival(P>0.05).(4)Go analysis revealed that the co-expression gene of RFC4 primarily contributed to biological process(BP)activities such as cell mitosis,chromosome synthesis,DNA replication,and others;In terms of molecular function(MF),it is primarily involved in the catalytic reaction in the process of DNA replication and the activities that need to consume ATP.It is primarily found at the centromere,spindle,and replication fork of chromosomes.The KEGG signal pathway enrichment analysis revealed that RFC4 and its co-expression genes were primarily enriched in a number of signal transduction pathways,including the cell cycle,oocyte meiosis,progesterone-mediated oocyte maturation,and cell aging,among others.The relationship with the cell cycle signal pathway(count=25)and difference(P<0.05)were the most obvious among these.(5)Immune infiltration analysis revealed that four common immune cells(activated dendritic cells,macrophages,mast cells,and follicular helper T cells(Tfh))were statistically significant among the RFC4 low expression groups(P<0.05),with activated dendritic cells,macrophages,and Tfh in the RFC4 high expression group having higher infiltration levels of immune cells and mast cells in the RFC4 low expression group having higher infiltration levels of Furthermore,in the RFC4 high-expression group,the activation level of the type II interferon(IFN-GAMMA)response pathway was significantly lower,and the difference was statistically significant(P <0.001).The IHC test results revealed that:(1)RFC4 expression in HCC and adjacent tissues were significantly different(P<0.001).(2)RFC4 protein expression in HCC patients is related to HCC differentiation(P<0.001),clinical stage(P = 0.027),and the immunohistochemical index ki-67(P<0.001),but not to other clinicopathological indexes.Conclusion:(1)RFC4 has the potential to be employed as a molecular marker for the diagnosis of HCC because it is much higher expressed in HCC tissues compared to paracancerous tissues.(2)RFC4 expression in HCC is associated with tumor differentiation,clinical stage,and a high ki-67 proliferation index,implying that RFC4 is involved in HCC malignant progression.(3)The expression of RFC4 is linked to a poor outlook for those with HCC,and its gene expression is an independent risk factor for the same.As a molecular indicator,RFC4 can be utilized to assess the prognosis of HCC patients.(4)RFC4 co-expression genes are highly enriched in a variety of biological pathways and may influence the occurrence and progression of HCC via these pathways.(5)RFC4 may influence the abundance of immune cells infiltrating HCC,altering the immune microenvironment and influencing the occurrence and progression of HCC.