Effect Of HHLA2 Protein On Clinical Prognosis And Tumor Microenvironment Of Hepatocellular Carcinoma

Human endogenous retrovirus H long terminal repeat-associating 2(HHLA2)is a newly discovered member of the B7 family.HHLA2 has co-stimulation and co-suppression effects on T cell function,which can stimulate or inhibit the proliferation of T cells and the production of cytokines.In addition,HHLA2 can also promote angiogenesis and promote tumor growth and metastasis.A number of studies have confirmed that HHLA2 is highly expressed in various malignant tumor tissues,and related to tumor progression and metastasis.In this study,the bioinformatics method was first used to analyze the gene expression chip of the hepatocellular carcinoma(HCC)tissue in the GEO database to find the significant differential expression of HHLA2 in HCC and the potential signaling pathway;then the preliminary research of the research group was confirmed that HHLA2 is highly expressed in HCC cell line Hep G2 and the clinical HCC tissues(n = 55);and its expression is positively correlated with clinical stage,tumor differentiation and tumor invasion of adjacent tissues.In order to further study the relationship between HHLA2 and the malignant biological characteristics of HCC,and its impact on clinical prognosis and tumor microenvironment,we explore its potential value as a molecular marker and immunotherapy target for predicting the prognosis of HCC.We used immunohistochemistry to detect the expression of HHLA2 protein in 202 HCC patients' cancer tissues and its relationship with clinicopathological characteristics and survival prognosis;then we constructed a HHLA2 gene silencing cell model to explore effects of malignant biological behaviors such as cycle distribution,apoptosis,invasion and metastasis of HHLA2 on HCC cells.Finally,the effects of HHLA2 protein and immune microenvironment on the prognosis of HCC are discussed.PART 1 Expression of HHLA2 Protein in Hepatocellular Carcinoma and Its Impact on Clinical Prognosis Objective:Bioinformatics analysis was used to preliminary analyze the differential expression of HHLA2 in HCC and potential signaling pathways,and to explore the expression level of HHLA2 protein in HCC and its relationship with clinicopathological characteristics and prognosis.Methods:1.Use R language software package to analyze the bioinformatics data of the gene expression chip GSE33006 of the HCC tissue in the GEO database,draw a heat map of differentially expressed genes and analyze the GO pathway of gene function annotation.2.Immunohistochemical methods were used to detect the expression of HHLA2 in 202 HCC tissues and 50 normal control liver tissues,and to analyze the relationship between HHLA2 expression level and various clinical pathological factors of HCC and its impact on clinical prognosis.Results:1.The results of GO pathway analysis of the significantly differentially expressed genes(including HHLA2)in the GSE33006 chip showed that the significantly differentially expressed genes including HHLA2 participated in immune response,chemokine-mediated signaling pathway and extracellular matrix structural constituent.2.Among 202 HCC cases,103 cases(51%)had high HHLA2 protein expression and 99 cases(49%)had low expression.There was almost no expression of HHLA2 protein in the normal liver tissues.3.The expression level of HHLA2 protein was significantly positively correlated with CNLC stage,degree of tumor differentiation,number of tumors,microvascular invasion,liver capsule invasion and serum GGT levels.There was no correlation with gender,age,hepatitis B virus infection,HBV-DNA copy number,serum AFP and AST levels,tumor diameter,satellite nodules,liver cirrhosis,P53 and Ki67 positive rates(P > 0.05).4.Survival analysis showed that the DFS and OS in high HHLA2 expression group were significantly worse than that in HHLA2 low expression group(P < 0.001).5.The results of the COX proportional hazard regression model showed that microvascular invasion,tumor size,number of tumors,P53 positive rate,Ki67 positive rate and the expression of HHLA2 are related to the prognosis of HCC patients and are independent predictors for HCC prognosis.Conclusions:1.HHLA2 may be related to the immune microenvironment of hepatocellular carcinoma.2.The high expression of HHLA2 in hepatocellular carcinoma is closely related to the tumor CNLC stage,tumor differentiation,tumor number,microvascular invasion,liver capsule invasion and serum GGT level.This suggests that the hepatocellular carcinoma has a more malignant biological behavior.3.The clinical prognosis of HCC with high expression of HHLA2 is worse than that with low expression of HHLA2,indicating that it is an important indicator affecting the poor prognosis of hepatocellular carcinoma.4.High expression of HHLA2 is an independent prognostic factor that affects the overall survival of patients with hepatocellular carcinoma.Therefore,HHLA2 protein can be used as an important marker to predict the prognosis of patients with hepatocellular carcinoma.PART 2 Effect of HHLA2 on Malignant Biological Behavior of Hepatocellular Carcinoma Cells Objective:To investigate the effect of silencing HHLA2 gene on HCC cell line Hep G2 in the proliferation,adhesion,cell cycle distribution,apoptosis,invasion and metastasis and other malignant biological behaviors,and to initially clarify its biological function.Methods:1.Lentiviral-mediated RNAi technology was used to construct a stable HHLA2 gene silencing cell model of Hep G2 cells.Using RT-PCR and Western Blot technology to screen the sh RNA fragments with the best effect of silencing HHLA2 gene.2.CCK-8 method was used to detect the proliferation and adhesion ability.3.Flow cytometry was used to detect the cycle distribution and apoptosis.4.Cell scratch test and Matrigel-transwell invasion test method were used to observe the migration and invasion ability.Results:1.Three groups of recombinant lentiviral vectors including HHLA2-sh RNA-1,HHLA2-sh RNA-2,and HHLA2-sh RNA-3 were successfully constructed.By RT-PCR and Western blot,the relative m RNA expression of the HHLA2-sh RNA-1 group was the lowest(P = 0.006),and the level of HHLA2 protein expression was decreased mostly(P < 0.0001),so the HHLA2-sh RNA-1 plasmid lentivirus was used as the experimental group Hep G2-KD for subsequent experiments.2.The results of CCK-8 method showed that the cell proliferation capacity of the Hep G2-KD group began to decrease at the third day,and the difference was statistically significant compared with the Hep G2-NC group(P = 0.018);the number of adherent cells in the Hep G2-KD group was significantly more than the Hep G2-NC group(P = 0.0017).3.The apoptosis level of Hep G2-KD group was increased significantly(P< 0.0001),the number of G0/G1 phase and S phase was increased,and the number of G2/M phase was decreased.4.The scratch test results showed that the migration rate of Hep G2 cells at48 h was significantly less than the Hep G2-NC group(P = 0.0279);Matrigel-transwell experiments showed that the number of cells that migrated in the Hep G2-KD group decreased significantly.Conclusions:Inhibition of HHLA2 gene can significantly increase hepatocellular carcinoma cell adhesion ability and promote cell apoptosis,at the same time reduce cell proliferation ability,migration and invasion ability and induce cell cycle arrest in G1/S phase,suggesting that HHLA2 is involved in the occurrence and development of hepatocellular carcinoma.PART 3 Study on Correlation between HHLA2 and Tumor Immune Microenvironment of Hepatocellular Carcinoma Objective:Using bioinformatics methods to analyze the potential signaling pathways of HHLA2 in hepatocellular carcinoma,explore the effects of HHLA2 protein and immune microenvironment on the prognosis of hepatocellular carcinoma,and provide an experimental basis for finding new targets and new strategies of HCC immunotherapy.Methods:1.Linked Omics database was used to analyze genes related to HHLA2 transcriptional expression in HCC,and GO function and KEGG enrichment analysis were performed based on the above co-expressed genes.2.HE and immunohistochemistry were used to detect the degree of TILs infiltration and the expression of PD-L1 in 202 HCC patients.3.Kaplan-Meier survival curve was used to analyze the correlation of TILs infiltration degree,PD-L1 protein expression and survival prognosis of HCC patients.4.Spearman rank correlation analysis was used to analyze the correlation of HHLA2 expression and TILs infiltration degree and PD-L1 expression.5.Construct a subtype of HCC immune microenvironment based on the expression level of HHLA2 and the degree of TILs infiltration,and analyze its impact on the prognosis of HCC.Results:1.A total of 19922 differentially expressed genes related to HHLA2 in HCC were obtained by analysis Linked Omics.From the volcano map,the three genes most closely related to HHLA2 were TFF2,FABP6,and PPP1R14 D.2.GO function analysis shows that it is mainly involved in immune-related biological processes such as immune response-activating cell surface receptor signaling pathway,immune response-regulating cell surface receptor signaling pathway,etc.;KEGG pathway analysis shows that Immune-related pathways include Fc gamma R-mediated phagocytosis,TNF signaling pathway and T cell receptor signaling pathway.3.Among 202 HCC cancer tissues,31 cases(15.3%)had minimal TILs infiltration,107 cases(53.0%)had intermediate TILs infiltration,and 64 cases(31.7%)had high TILs infiltration;There were 66 cases(32.7%)with positive expression of PD-L1 protein and 136 cases(67.3%)with negative expression of PD-L1 protein.PD-L1 is rarely expressed in normal liver tissues.4.Survival curve analysis found that the DFS and OS of HCC patients with high and intermediate TILs infiltration were significantly longer than that of HCC patients with minimal TILs infiltration(P < 0.001).5.Survival curve found that there was no statistically significant difference DFS between HCC patients with PD-L1 positive and PD-L1negative(P = 0.259);The OS of HCC patients with PD-L1 positive was significantly worse than the median survival of PD-L1 negative patients(P =0.023).6.Spearman rank correlation analysis showed that there was a significant correlation between HHLA2 and TILs infiltration(P = 0.028);However,there was no significant correlation between HHLA2 expression and PD-L1expression(P = 0.057).Survival analysis found that in HCC patients with minimal and intermediate TILs infiltration,high expression of HHLA2 had worse overall survival;for overall survival of HCC patients with high TILs infiltration,there was no different between high and low expression of HHLA2.7.Based on the expression of HHLA2 and TILs infiltration,the HCC immune microenvironment is divided into four types: type I is HHLA2-high TILs+(38.1%),type II is HHLA2-low TILs-(5.0%);type III is HHLA2-high TILs-(12.9%);Type IV is HHLA2-low TILs+(44%).The median OS of type? patients was 49.5 months and the type ? patients was 51.3 months,which were significantly longer than those of type ? and type ?(median OS was24.2 months and 7.6 months,respectively)(P < 0.001).The DFS of type ?and type ? patients(median DFS were 15.8 months and 49.2 months respectively)were significantly longer than those of type ? and type ?(P <0.001).Conclusions:1.Bioinformatics analysis shows that HHLA2 may participate in and affect the immune microenvironment of hepatocellular carcinoma through the biological process of immune response and Fc gamma R-mediated phagocytosis,TNF signaling pathway and T cell receptor signaling pathway.2.The expression of HHLA2 in the immune microenvironment of hepatocellular carcinoma was significantly correlated with TILs infiltration.With the TILs infiltration increasing,it can inhibit the effect of high HHLA2 expression on the poor prognosis of patients with hepatocellular carcinoma.3.HHLA2-high TILs+ and HHLA2-low TILs+ are good immune subtypes in the immune microenvironment of hepatocellular carcinoma,suggesting a better survival prognosis for patients with TILs infiltration in hepatocellular carcinoma.