The High Expression Of H2AFY In Liver Cancer Promotes The Proliferation Of Liver Cancer And Activates The Autophagy Process Of Hepatocellular Carcinoma

Purpose: Hepatocellular Carcinoma(HCC)is a common and significant primary malignant tumor.It is not only the sixth most common cancer,but also the third most common cause of cancer-related deaths.With the increasing incidence and mortality of liver cancer,it has become a globally recognized health problem.Although the specific mechanisms and treatments for the occurrence and development of liver cancer have been continuously improved,a complete cure has never been achieved in the treatment of the disease.The H2AFY(H2A histone menber Y,H2 A histone family member Y)gene encodes a histone protein that has nothing to do with replication.It belongs to the histone H2 A family.It mainly replaces a subset of the traditional H2 A histone protein in the nucleosome,inhibits transcription and Participating in stable X chromosome inactivation and other aspects play an important role.In this study,we will focus on exploring the important role of the target gene H2 AFY in the occurrence and development of liver cancer from the three aspects of bioinformatics,cells and tissues,and the related mechanisms involved.Methods: We selected the target gene H2 AFY from three online databases of ONCOMINE,GEPIA and Kaplan-Meier Plotter and verified its differential expression between normal tissues and liver cancer tissues and analysis of its correlation with poor prognosis.Then,we download different gene expression matrices from the TCGA and GEO databases.More reliable,we use our own coding program on the Rstudio software to realize the statistical analysis of the gene expression matrix.In the TCGA data set,GSE14520 data set,GSE76427 data set and GSE6764 data set,the single-factor T test was used to compare the expression levels of H2 AFY in normal tissues and liver cancer tissues.Graph Pad Prism 8 software was used to complete the analysis and multi-sample analysis of variance to compare H2 AFY Relevance in clinical characteristics.The survival and prognosis analysis of H2 AFY in liver cancer was analyzed using packages such as "survival","survminer" and "ggplot2" in the Rstudio software and the survival curve and ROC curve of H2 AFY in liver cancer were drawn.q RT-PCR and western blotting experiments verify the difference in the expression level of H2 AFY in liver cancer cells and normal cells at the cell level and tissue level.Cell plate cloning experiment and CCK-8 experiment were used to detect the effect of hepatocarcinoma cell lines SMMC-7721 and Hep G2 on the proliferation of hepatocellular carcinoma cells after interfering with the expression of H2 AFY.Scratch experiment and transwell experiment examined the effect of SMMC-7721 on the migration ability of liver cancer cells after interfering with the expression of H2 AFY.Finally,the GSEA algorithm is used to predict the cellular functions and signal pathways that H2 AFY may participate in the development of liver cancer.According to the prediction results,we analyzed the correlation expression of H2 AFY and autophagy marker proteins LC3 and p62;through western blotting experiments,immunohistochemistry and fluorescence immunoassays Double staining experiments verify the effect of H2 AFY on autophagy of liver cancer.Results: First of all,we found that H2 AFY is overexpressed in liver cancer(Figure 1BF),which has a significant correlation with the clinical characteristics of liver cancer(Figure 2E-H),and as a tumor-promoting gene,it significantly shortens the survival time of liver cancer patients(Figure 2A-D).The liver cancer cell line SMMC-7721 interfered with the expression of H2 AFY,the cell plate cloning experiment(Figure 3A)and the CCK-8 experiment(Figure 3C)showed that the proliferation ability of liver cancer cells in the experimental group was significantly lower than that of the control group.The cell plate cloning experiment of Hep G2(Figure 3B)and CCK-8 experiment(Figure 3D)results are consistent with the previous.Scratch(Figure 4A)and transwell(Figure 4B)detect SMMC-7721 after interfering with H2 AFY expression.The results show that H2 AFY has no significant effect on the migration ability of liver cancer cells.The results of bioinformatics analysis showed that H2 AFY activetied the PI3K-AKT-MTOR signaling pathway(Figure 5A-B),and was positively correlated with the expression level of LC3 B and P62(Figure 5D-E)in liver cancer,and negatively correlated with LC3A(Figure 5C).Western blotting experiments,immunohistochemistry and fluorescence immunostaining experiments have verified that LC3 is highly expressed in liver cancer,and p62 is low in liver cancer,and both bind to H2 AFY in the cytoplasm and affect the autophagy function of liver cancer.Conclusion: In this study,we selected the target gene H2 AFY based on bioinformatics methods and verified that this gene is overexpressed in liver cancer,which is significantly related to the poor prognosis of liver cancer patients.Subsequently,molecular biology experiments verified that H2 AFY promotes the proliferation of liver cancer cells and enhances the autophagy process of liver cancer cells,and plays a role in promoting cancer in the occurrence and development of liver cancer.We believe that H2 AFY has the potential to become a new tumor surface marker,which plays a key role in the diagnosis,treatment and prognosis of liver cancer,and provides more basic research results and drug development directions for clinical diagnosis and treatment of liver cancer.