FOXK1 Inhibits Autophagy Of Hepatocellular Carcinoma Cell To Induce EMT And Promote Migration And Invasion Of Tumor Via PI3K/AKT Signaling Pathway

Background:Cancer is one of the important factors affecting the world’s population mortality,according to World Health Organization（WHO）’s Global Cancer Statistical report released in 2018,liver cancer is the fourth leading cause of cancer-related death,ranking sixth among all the various cancers,and according to WHO estimates,more than 1 million patients will die of liver cancer by 2030.The incidence of liver cancer is continuously increasing,the case fatality rate is high,with the 5-year survival rate being only 18%.It is a major challenge for the medical profession to control the increasing incidences of liver cancer and improve the survival rate.The extensive use of sequencing databases and the network tools,the integration of bioinformatics data,gene expression,mutation and pathway interaction analysis have emerged as important tools in medical genomics research in recent years.Machine learning methods of personalized oncology have also contributed to the current trend of medical genomics research.The various bioinformatics tools for analyzing the human diseases are in great demand in the field of of medical genomics.In the past decade,bioinformatics methods have been widely used to interpret the sequencing data as well as to predict drug sensitivity（or drug resistance）and thus facilitate accurate drug selection.FOXK1 transcription factor has been found to be widely expressed in the various tissues and organs,and play an important role in the regulation of various cellular functions in the higher organisms.In mammalian species,the FOXK family consists of two distinct members,namely,the FOXK1 and the FOXK2,which can regulate cell proliferation,survival,skeletal muscle regeneration,myogenic differentiation,etc.The oncogenic effect of FOXK1 has been closely related with the promotion of cell proliferation and inhibition of apoptosis in a variety of cancers,such as colorectal cancer,gastric cancer,esophageal cancer,ovarian cancer lung cancer and glioblastoma.It has been established that FOXK1 can also function as a tumor promoter in liver cancer based on the findings of few previous reports.Thus,this study primarily aimed to explore the potential role of FOXK1 in regulating the progression of liver cancer and its underlying mechanism of actions as a putative tumor promoter.Aims:1.To analyze the potential role of FOXK1 in the primary liver cancer based on bioinformatics,and to predict the potential mechanisms through which FOXK1 can regulate the occurrence and development of LIHC;2.The immunohistochemical results of the HCC tissues microarray was used to further explore the clinical prognostic value of FOXK1 in patients with HCC and to validate the prediction of bioinformatics.3.To determine the impact of FOXK1 in affecting EMT and autophagy processes in HCC and its underlying mechanisms.Methods:1.Bioinformatics prediction:online databases were used to predict the difference of mRNA and protein expression of FOXK1 in HCC.Thereafter,the expression of FOXK1 was integrated with the corresponding clinical data.Univariate and multivariate Cox regression analysis was used to predict the clinical prognostic value of FOXK1 in patients with TCGA-LIHC,and a clinical predictive model was constructed.In addition,we have also analyzed the possible regulatory role of FOXK1 in the immune microenvironment of HCC and its predictive value for immunotherapy by ssGSEA,which aided in predicting the potential pathways involved in mediating the actions of FOXK1 in affecting the occurrence and development of HCC by GSEA,GO and KEGG enrichment analysis.2.Clinical sample validation:The protein level of FOXK1 in HCC tissues and its correlation with protein expression of PD-L1 and CTLA-4 was verified by tissue microarray of HCC.The prognostic value（OS and DFS）of FOXK1 in HCC was verified by univariate and multivariate Cox regression,and the clinical correlation between FOXK1 and HCC was studied by chi-square test.3.In vitro and in vivo experiments:Stable HepG2 cell lines were constructed by shNC and shFOXK1 lentiviral infection,and the effect of FOXK1 on EMT was measured by wound healing,transwell,and cell adhesion assays,immunofluorescence,RT-PCR as well as WB.Immunofluorescence,transmission electron microscopy,RT-PCR and WB were used to verify the effects of FOXK1 on HepG2 autophagy.The autophagy inhibitor chloroquine was added to explore the effect of FOXK1 inhibition induced autophagy on EMT in HepG2 cells by wound healing and cell adhesion assays as well as WB.The effect of FOXK1 on EMT and autophagy was verified by the subcutaneous tumor formation in nude mice in vivo.Results:1.Based on the results of related online databases including TCGA,GEO（GSE36376 and GSE112790）,TCGA+GTEx,and THPA,the expression of FOXK1 mRNA and protein in the liver cancer tissues was significantly higher than that in the normal or adjacent tissues.The area under ROC curve of FOXK1 for TCGA-LIHC diagnosis was 94.8%,the diagnostic sensitivity of FOXK1 was 83.96%while with a specificity at 98%,and the sensitivity of AFP for TCGALIHC diagnosis was only 50.53%and the specificity was 94%.According to the median of FOXK1 mRNA,patients were divided into high and low groups.K-M curve results showed that high FOXK1 expression was closely correlated with low OS,PFS and DFS in TCGA-LIHC patients.FOXK1 expression was positively correlated with the pathological stage,histological grade and the tumor size.Tumor recurrence rate of patients with high FOXK1 expression was significantly higher than patients with low FOXK1 expression.Univariate and multivariate Cox regression results suggested that FOXK1 was an independent risk factor for the prognosis of TCGA-LIHC patients,which was verified by ICGC database.2.FOXK1 was involved in regulating cell adhesion,apoptosis and endocytosis in HCC,and was closely related to the activation of ERBB,Wnt,NOTCH and mTOR signaling pathways.FOXK1 was significantly correlated with both autophagy（including SQSTM1,BECN1 and MAP1LC3B）and EMT-related genes（including CDH1,CDH2,VIM,MMP3 and MMP9）.GO and KEGG analysis showed that PI3K/AKT/mTOR signaling pathway was enriched in the effect of FOXK1 on EMT and autophagy of HCC.3.ssGSEA analysis results showed that abundant infiltration of CD4+T and dendritic cells was observed in FOXK1 high expression group,while the FOXK1 low expression group had significant infiltration of CD8+T,eosinophilic and neutrophil cells.No significant differences in the degree of infiltration of other immune cell types between the two groups.IPS results predicted that patients with low FOXK1 expression could be treated with anti-CTLA-4,whereas patients with high FOXK1 expression may be more sensitive to PI3K/AKT pathway inhibitors AZD8055,A-443654,AZD6482 and rapamycin.4.Immunohistochemical results of the tissues microarray contained 90 cases with HCC showed that FOXK1 expression was significantly up-regulated in the liver cancer tissues compared with adjacent tissues.In the HCC tissues,FOXK1 was positively correlated with the expression of PD-L1,and the survival rate and disease-free survival rate of patients with high expression of FOXK1 were significantly shortened.In addition,FOXK1 was positively correlated with pathological grade of HCC,number of cirrhotic nodules and serum AFP level.High FOXK1 can act as an independent risk factor for prognosis of HCC patients,regardless of overall survival rate or disease-free survival rate.5.Compared with the human normal liver cells L02,FOXK1 was significantly overexpressed in HepG2 and Huh7.Inhibition of FOXK1 expression reduced the migration and invasion potential of HepG2 and the cell adhesion ability.It also increased the expression of E-cadherin,and decreased the expression of N15 cadherin and Vimentin.It was found that the number of intracellular autophagosomes increased,levels of Beclin-1 and LC3II/LC3I were enhanced,but the level of P62 was decreased.And the p-Akt/AKT and p-PI3K/PI3K ratios were significantly decreased.Moreover,the expressions of N-cadherin,Vimentin and P62 in HepG2 cells were significantly up-regulated after the addition of autophagy inhibitor chloroquine,while expression of E-cadherin,Beclin-1 and LC3II/I were significantly inhibited.In the mouse xenotransplantation model,the tumor volume and weight of the nude mice immunized with LV-ShFOXK1 was significantly decreased,E-cadherin,Beclin-1 and LC3II/LC3I levels were increased,and N-cadherin,Vimentin and P62 expression was found to be decreased.Conclusion:1.Bioinformatics prediction:Based on online database and bioinformatics analysis,it was found that the expression of mRNA and protein of FOXK1 in HCC tissues was significantly higher than that in the normal liver tissues and adjacent paracancerous tissues.High FOXK1 might serve as an independent risk factor for poor prognosis in patients with TCGA-LIHC and can influence the malignant behavior of HCC cells through modulating both EMT and autophagy.2.Clinical verification:Immunohistochemical analysis of the liver cancer tissue microarray confirmed that the expression of FOXK1 was significantly higher in the liver cancer.The increased expression of FOXK1 was positively correlated with the pathological grade of the patients and the number of cirrhotic nodules present.The total survival time and tumor-free survival time of patients with high expression of FOXK1 was found to be significantly shorter.FOXK1 thus can be used as an independent predictor of prognosis in patients with liver cancer.3.In vitro and in vivo experiments:It was observed that inhibition of FOXK1 expression can induce autophagy of HepG2 cell line and inhibit autophagy induced by EMT,FOXK1 of tumor cells.Suppression of autophagy in turn can attenuate the inhibition of EMT.