STAT4 Genetic Polymorphism Is Involved In The Occurrence And Progression Of HCC By Regulating CYP2E1

Liver cancer,mainly referring to hepatocellular carcinoma?HCC?,is one of the most common malignant tumors in the world.It has the characteristics of high recurrence rate,high mortality rate,high malignancy and poor prognosis,and seriously threatens human life and health.The etiology of HCC includes factors such as chronic hepatitis B virus?HBV?infection,carcinogens,and metabolic diseases.In addition,genetic polymorphism as a intrinsic factor affects individual HCC susceptibility and prognosis.Signal transducer and activator of transcription 4?STAT4?is involved in various pathophysiological processes such as inflammation,immunity,and tumorigenesis.STAT4 can be activated by IL-12,regulate the release of inflammatory mediators by T cells and NK cells,and play an important role in cell proliferation,invasion and metastasis.Although literatures report that STAT4 rs7574865 genetic polymorphism is associated with HCC susceptibility,the effect of STAT4 genetic polymorphism on the occurrence and prognosis of HCC has not been fully understood.CYP2E1 is an important member of the cytochrome P450 enzyme family.It is not only involved in the metabolism of approximately 6%of drugs in the clinic,but also in the metabolism of carcinogens and inflammation.Studies have reported that CYP2E1 participates in the development of liver fibrosis,HCC,and tumors.Previous studies conducted by our group have confirmed that the increased metabolic activity of CYP2E1 is a susceptible factor for HCC,and as the activity increases,the severity of HCC increases.Other studies have shown that STAT1 and STAT3 can regulate the expression,transcription and post-translational modification of CYP2E1,but whether STAT4 is involved in the occurrence and progression of HCC by regulating CYP2E1is not clear.As the basis of the activities of cells and organisms,the structure and function of proteins directly affect the changes of physiological and pathological states of the body.And proteomics based on all proteins,quantitative and qualitative analysis of proteins,further revealed the functions of proteins,therefore,using proteomics technology to find proteins and pathways that involved in the regulation of liver cancer.Taking STAT4 and CYP2E1 as the research objects,we will explore that STAT4 genetic polymorphism are mainly involved in the HCC-related proteomic changes and possible molecular mechanisms through regulation of CYP2E1.Effect of STAT4 genetic polymorphism on HCC susceptibility and prognosis Genome-wide association study?GWAS?and meta-analysis have found that STAT4rs7574865 can significantly increase the risk of HBV-related HCC.Studies have also found that STAT4 is highly expressed in tumor tissues in lung cancer,gastric adenocarcinoma and colon cancer,suggesting that the occurrence of tumors is closely related to the expression of STAT4.Although literatures report that STAT4 rs7574865genetic polymorphism is associated with HCC susceptibility,the effect of STAT4genetic polymorphism on the occurrence and prognosis of HCC has not been fully understood.In this study,blood samples of 500 HBV-HCC hospitalized patients?HCC group?and 500 healthy people?Control group?were collected,and Sequenom Mass Array method was used to detect the SNP of STAT4 rs7574865.The results suggested that STAT4 rs7574865 was closely related to the susceptibility of HCC,and GG genotype was a risk factor.In HCC patients,the STAT4 expression level in the GG genotype was significantly higher than that in the GT,TT and GT+TT genotypes?all P<0.05?;In the control group,there was no significant affect in STAT4 expression levels between the various genotypes?all P>0.05?,suggesting that the STAT4 genetic polymorphism only affect the expression of STAT4 in HCC,but not in healthy people.Factors that affect the prognosis of patients are not only related to the characteristics of the tumor itself?such as the number,size,surrounding infiltration and metastasis?,clinical indicators and pathological grades,but also related to SNP locus mutations.Whether STAT4 genetic polymorphism is involved in the prognosis of HCC patients.The follow-up by telephone and outpatients were conducted after314 HCC postoperation patients every six months,and for approximately 42?56months.To further study the effect of STAT4 genetic polymorphism on prognosis of HCC patients.Kaplan-Meier survival curve analysis showed that the survival time of HCC patients carrying with GG genotype and the high STAT4 group were significantly shortened(P_log-rank<0.05),the risk of death increased,and the prognosis was poor.Therefore,STAT4 genetic polymorphism may cause STAT4 protein content change and affect prognosis of HCC.In short,STAT4 genetic polymorphism may cause changes in the expression of STAT4 in the peripheral blood of HCC patients,thereby affecting the occurrence and progression of HCC.Its detection may have certain significance for the early diagnosis and prognosis evaluation of liver cancer.The mechanism of STAT4 involving in the development of HCC by regulating CYP2E1 A total of 42 healthy liver specimens and 42 HCCpatients'liver fibrosis tissue specimens were collected.The results were further verified in liver tissue experiments,and found that the HCC group carrying with GG genotype had high STAT4 expression,increased risk of death,and poor prognosis?P<0.05?,suggesting that STAT4 rs7574865 GG genotype in human liver tissue may affect the prognosis of HCC.The experimental results of human serum are consistent.CYP2E1 was detected by HPLC-UV Metabolic activity,and its correlation with STAT4 expression was analyzed.Compared with the low STAT4 group,the high STAT4 group had significantly elevated CYP2E1 enzyme kinetic parameters(V_maxand Cl_int),suggesting that STAT4 is positively correlated with CYP2E1 metabolic activity?P<0.05?.The effect of STAT4 on Hep G2 and L02 cell proliferation,invasion,migration,and apoptosis,and its regulatory effect on CYP2E1 expression were investigated in cell experiments.After transfecting p Silencer3.0-STAT4/si RNA expression vector with L02 and Hep G2 cells,a series of in vitro experiments were carried out.In the si RNA-STAT4 group,the migration and invasion of L02 and Hep G2 cells were significantly weakened;the early apoptosis rate and late apoptosis rate in L02 cells and Hep G2 cells were significantly increased;the content of cells in G1/0 and G2/M phases increased,while the content in S phase decreased significantly.The m RNA levels and protein expressions of STAT4 and CYP2E1 in L02 and Hep G2cells were significantly reduced?P<0.01?in si RNA-STAT4 group,but the expression of STAT4 and CYP2E1 increased in IL-12 treated group?P<0.01?.Therefore,STAT4 is involved in the occurrence and progression of liver cancer by regulating the promoter region of CYP2E1,which provides a theoretical basis for finding new therapeutic targets for HCC,and provides new ideas for early prevention,early diagnosis and early treatment of liver cancer.Proteomic investigation on STAT4 involvment in the development of HCC by regulating CYP2E1 As the basis of the activities of cells and organisms,the structure and function of proteins directly affect the changes of physiological and pathological states of the body.Protein is the basis of biological activities,mainly involved in gene regulation,cell metabolism and signal transduction processes.Proteome refers to all the proteins expressed by the genome of a cell or tissue.In recent years,with the rapid development of proteomics,analyzing the composition and expression level of proteins from a holistic,dynamic and comprehensive perspective.Therefore,proteomics has become a hot research topic to find proteins and pathways involved in the regulation of cancer development..Therefore,Therefore,proteomics is helpful to elucidate the signaling pathways and possible mechanisms of STAT4 genetic polymorphism affecting the occurrence of HCC.34 normal liver tissue specimens and 42 HCC patients liver fibrosis tissue samples were taken and identified by Label-free quantification.The samples were divided into two groups with high and low expression according to the median of STAT4 protein expression,and 273 differential proteins were identified and screened for subsequent analysis,including of the 175 up-regulated proteins and 98down-regulated proteins.Based on the significance of the difference,the number of differential proteins with P<0.01,0.01?P<0.03,and 0.03?P<0.05 were 63,115,and 95,respectively.Screening for differential proteins directly related to STAT4 expression,104differential proteins screened by HCC group were also screened by STAT4 expression subgroup.Use GO classification,Gene Cards,NCBI Pub Med and other databases to classify proteins functionally:including inflammation and immunity,DNA damage repair,cell metabolism,signal pathway regulation,and antigen processing and presentation.A total of 18 differential proteins are included in the functional classification.And in the STAT4 high-expression subgroup,and one of the differential proteins that meet the CYP2E1 metabolic activity and have a cancer-promoting effect,namely the FGL2 protein,is selected from them.The enrichment analysis of the 273 differential proteins in the subgroup,GO analysis in the following three aspects shows that:in terms of cellular components,molecular function,and biological process.The KEGG pathway revealed that the signaling pathways involved in the immunity,drug metabolism cytochrome P450,and inflammation,etc.In order to further analyze the relationship between the differential proteins CYP2E1 and FGL2,we used human liver tissues and animal models to explore the regulation of FGL2 expression by CYP2E1 and its effect on the prognosis of HCC patients.the proteomics data displayed that the expression of FGL2 in HCC group were increased?P<0.0001?,and the results were verified by Western bolt?P<0.05?.It is suggested that the increase of FGL2 may be related to the development of HCC.In the HCC group,the group with high STAT4 expression had high expression of FGL2?P=0.0004?,suggesting that the increased expression of FGL2 may be related to the high expression of STAT4 in HCC;Kaplan-Meier survival curve shows that the survival time of HCC patients with high FGL2 expression was significantly shortened,and the prognosis was poor(P_log-rank=0.009),suggesting that FGL2 may affect the prognosis of HCC patients and promote the development of HCC.The area under the ROC curve was 0.760?P<0.0001?,suggesting that the protein FGL2 had potential clinical predictive value for HCC diagnosis.The correlation between the expression of FGL2 and the activity of CYP2E1?Vmax and Clint clearance?was analyzed in human HCC liver tisstue.In the HCC group,it was found that group with high V_maxand Cl_inthad high FGL2 expression and group with low V_maxand Cl_inthad low FGL2 expression,suggesting that CYP2E1metabolic activity increased FGL2 expression,and FGL2 expression was positively correlated with CYP2E1 activity?P<0.05?.Western blot results showed that the group with high CYP2E1 activity(V_maxand Cl_int)had high FGL2 expression?P<0.05?.Building orthotopic tumor xenograft model of mouse H22 cells,BALB/c male mice were randomly divided into sham group,model group and CYP2E1 specific inhibitor?SMI 12?treatment group.Western blot results showed that the expression of FGL2 in the model group was higher than that of the sham group?P<0.05?,the SMI 12 intervention group was lower than the model group?P<0.05?;cyp2e1 gene knockdown rat were identified by PCR,the expression of FGL2 in the cyp2e1^-/-homozygous group was lower than cyp2e1^+/+wild type group?P<0.05?,suggesting that CYP2E1 may up regulate FGL2 expression.In conclusion,we first explored the possible mechanism that STAT4 genetic polymorphism may affect the expression of FGL2 by regulating CYP2E1,thereby affecting the occurrence and development of liver cancer,providing a new theoretical basis for elucidating the mechanism of HCC occurrence and progression and discovering new therapeutic targets.Conclusion1.STAT4 genetic polymorphism may cause STAT4 protein content change and affect the occurrence and progression of HCC.2.STAT4 possible participates in the occurrence and progression of HCC by regulating CYP2E1.3.CYP2E1 regulates the expression of FGL2 in HCC patients.With the high expression of FGL2 in liver fibrosis tissue,having a poor prognosis and an increased risk of death.4.STAT4 genetic polymorphism is involved in the occurrence and progression of HCC,and its mechanism may be related to STAT4's regulation on CYP2E1 and further affect the expression of FGL2.