The Role And Mechanism Of ZKSCAN3 In Hepatocellular Carcinoma

Background and objectiveThe hepatocellular carcinoma(HCC)accounts for 80～90%of the primary liver cancer and it is the fourth leading cause of cancer related death worldwide.The incidence and death rates of HCC have been rising for decades,with about 800,000 new cases occurring each year.HCC will cause the death of 1 million people a year worldwide by 2030.The development of hepatocellular carcinoma is associated with multiple environmental and genetic risk factors,such as chronic infection with hepatitis B virus and hepatitis C virus,excessive alcohol consumption,consumption of aflatoxin-contaminated foods,non-alcoholic fatty liver disease,diabetes,obesity,smoking,and hereditary hemochromatosis et al.The early stage of HCC is asymptomatic or the symptoms are ambiguous,which greatly delays the timely diagnosis.Most patients of HCC are in the middle or advanced stages at the time of initial diagnosis.Patients with advanced HCC are not suitable for therapeutic surgery,and treatment options for patients with advanced HCC are limited in terms of availability and effectiveness.The clinical treatment of HCC mainly includes surgical resection,radiofrequency ablation,radiotherapy,chemotherapy,liver transplantation,et al.The current clinical treatment methods of HCC have made significant progress compared to the past.However,some treatment methods are still defective.For example,conventional treatments often fail to clear the tumor completely.Adverse reactions related to radiotherapy and chemotherapy also seriously affect the quality of life and immune function of HCC patients.Organ shortage remains a major constraint to the application of liver transplantation in the treatment of HCC.The prognosis of patients with HCC has not been significantly improved.The recurrence rate of HCC is high and the survival time of patients is short.The 5-year survival rate of patients with advanced HCC is less than 20%.In order to improve the therapeutic effect and prognosis of HCC,it is necessary to continuously improve and explore the treatment of HCC.In the treatment research of HCC,searching new therapeutic targets is one of important contents.More and more potential therapeutic targets are found and researched.ZKSCAN3 belongs to the KRAB zinc finger protein family,which is the largest family of transcriptional regulators in higher vertebrates that regulate a variety of cellular processes,including proliferation,apoptosis and tumor development.It has been reported that the expression of ZKSCAN3 increased in a variety of tumor tissues.It can promote the proliferation,migration and invasion of a variety of tumor cells.It promotes the progression of multiple tumors by inducing the expression of target genes such as Integrin β4,VEGF,Cyclin D2,NF-κB et al.Studies have shown that ZKSCAN3 can be used as a potential therapeutic target of breast cancer and bladder cancer.So,can ZKSCAN3 be used as a potential therapeutic target for HCC?Currently,there are few studies in this field.This study will preliminarily explore the role and mechanism of ZKSCAN3 in HCC,and provide a basis for whether ZKSCAN3 can be used as a potential therapeutic target for HCC.Methods1.Gene microarray and RNA sequencing data sets of HCC were collected from public biomedical database.The expression difference of ZKSCAN3 mRNA in HCC tissues and non-HCC tissues,the relationship between ZKSCAN3 mRNA and clinical characteristics of patients,the diagnostic ability of ZKSCAN3 mRNA for HCC tissue and non-HCC tissue,and the influence of ZKSCAN3 mRNA on the prognosis of HCC patients were analyzed by bioinformatics method.2.The tumor tissues and paracancerous tissues and clinical data of 250 patients of HCC were collected from the hepatobiliary surgery department of the hospital.Then the level of ZKSCAN3 mRNA in these clinical tissue samples was detected by RT-qPCR and the expression difference of ZKSCAN3 mRNA in cancer tissue and paracancerous tissue was analyzed.The correlation between the expression level of ZKSCAN3 mRNA and the clinical characteristics of the patients was analyzed.3.Both shRNA-blank lentivirus and shRNA-ZKSCAN3 lentivirus were transfected into SK-Hepl and Huh7 cell lines,and the effects of silencing ZKSCAN3 on the proliferation,migration,invasion,apoptosis and cell cycle of these two cell lines were detected.4.RNA sequencing was performed on SK-Hep1 cells and Huh7 cells that stably expressed shRNA-blank and shRNA-ZKSCAN3,and the low expression genes induced by ZKSCAN3 silencing in SK-Hep1 cells and Huh7 cells were obtained.The intersection of the two groups of low expression genes was performed,and then GO analysis,KEGG analysis and protein interaction network analysis were performed to obtain the hub genes.The binding of ZKSCAN3 to the hub gene promoter was analyzed by Cristrome data browser,and the correlation between ZKSCAN3 and the expression of the hub gene was analyzed by GEPIA database.The target gene of ZKSCAN3 was inferred from the hub genes.Results1.Bioinformatics analysis showed that the level of ZKSCAN3 mRNA was significantly increased in HCC tissue compared with non-cancer liver tissue.There was no significant difference in the level of ZKSCAN3 mRNA between HCC tissues of BCLC stage 0 and non-cancer tissues,while the level of ZKSCAN3 mRNA in HCC tissues of BCLC stage A,B and C was significantly higher than that in non-cancer tissues,and the increase degree of BCLC stage C was greater than that of BCLC stage A.ZKSCAN3 mRNA showed moderate diagnosibility between HCC tissues and non-cancerous tissues.High expression of ZKSCAN3 mRNA was associated with poor prognosis of HCC patients.2.Clinical tissue samples were detected and show that compared with paracancerous tissue,the expression of ZKSCAN3 mRNA in HCC cancer tissue was significantly increased.Compared with the low ZKSCAN3 mRNA expression group,the overall survival time of patients from the high expression group was significantly decreased.Both of them were consistent with the results of bioinformatics analysis.The expression of ZKSCAN3 mRNA was negatively correlated with the age and serum level of AFP of patients.3.SK-Hep1 cells and Huh7 cells both were transfected with shRNAZKSCAN3 lentivirus,and the expression of ZKSCAN3 mRNA in the two kind of cells was both significantly decreased,which indicating high silencing efficiency of shRNA-ZKSCAN3.Silencing ZKSCAN3 could inhibit the proliferation of SKHep1 cells and Huh7 cells,inhibit the migration of SK-Hep1 cells,inhibit the invasion of Huh7 cells,promote the apoptosis of SK-Hep1 cells and Huh7 cells,increase the percentage of G0/G1 phase cells and decrease the percentage of S phase cells in SK-Hep1 cells.4.The results of RNA sequencing showed that silencing ZKSCAN3 in SKHep1 cells resulted in low expression of 996 genes and silencing of ZKSCAN3 in Huh7 cells resulted in low expression of 4017 genes.The intersection of these two groups of genes resulted in 192 low expressed genes.GO analysis showed that the 192 low expressed genes in cells composition focus on cell membrane structure,in terms of biological process focused on cell signaling and cell response to the stimulus,focused on receptors in terms of molecular function.The KEGG analysis showed that the 192 low expressed genes were concentrated in RAS signaling pathway,synaptic pathway,ovarian hormone production pathway,and basal cell carcinoma pathway.Seven hub genes were identified from the 192 low expressed genes by analyzing the protein interaction network,which were GNB3,GRM2,APLN,GPER1,CCL28,S1PR2 and PYY.The CHIP-seq analysis showed that ZKSCAN3 had a binding peak signal with the promoter of APLN.The gene expression analysis showed that only the expression of APLN was positively correlated with the expression of ZKSCAN3.It was speculated that APLN was the target gene of ZKSCAN3.ConclusionsBoth bioinformatics analysis and clinical sample detection showed that the level of ZKSCAN3 mRNA in HCC tissues was significantly increased compared with that in non-cancerous tissues or paracancerous tissues,and the overall survival of patients with high expression of ZKSCAN3 mRNA was significantly decreased compared with those with low expression.Bioinformatics analysis showed that the level of ZKSCAN3 mRNA in HCC tissues of BCLC A,B and C was significantly higher than that in non-cancer tissues,and the degree of increase in BCLC C was greater than that in BCLC A.In terms of function,silencing ZKSCAN3 can inhibit the proliferation of SK-Hepl and Huh7 cells,migration of SK-Hepl cells,invasion of Huh7 cells,promote the apoptosis of SK-Hepl and Huh7 cells,and increase the percentage of G0/G1 phase cells and decrease the percentage of S phase cells in SK-Hepl cells.In terms of mechanism.APLN is speculated to be a target gene regulated by ZKSCAN3 in HCC.The mechanism of ZKSCAN3 to HCC may be that the increased expression of ZKSCAN3 in HCC induces transcription and increases the expression of APLN,thus promoting the occurrence and development of HCC.