The Function And Mechanism Study Of DNASE1L3 In Hepatocellular Carcinoma

PartⅠ The expression of DNASE1L3 in hepatocellular carcinoma and its effect on the biological behavior of tumor cellsObject In order to explore the expression level of deoxyribonuclease(DNASE1L3)in hepatocellular carcinoma(HCC),analyze the relationship between the expression level of DNASE1L3 and the clinicopathological data as well as prognosis of patients,and observe the effect of DNASE1L3 on the biological behavior of tumor cells.Method Firstly,m RNA and protein levels of DNASE1L3 in liver cancer samples and adjacent tissues were detected by quantitative PCR(rt-q PCR)and western blot analysis.The clinical data of HCC patients downloaded from TCGA-LIHC were analyzed.The correlation between DNASE1L3 expression and the collected clinicopathological data of liver cancer patients was analyzed.Kaplan-meier analysis was performed on the overall and disease-free survival rates of 364 HCC patients from TCGA databas with different DNASE1L3 expression levels.Then we constructed DNASE1L3 overexpressed Huh7 and HCCLM3 cell lines,and DNASE1L3 knock-down Hep G2 cell lines.Transwell and wound healing assay were performed in these three cell lines to test cell invasive and migratory ability,cell proliferative capacity was examined by CCK8(Cell Counting Kit-8)and clone formation test.Flow cytometry analysis technology to detect DNASE1L3 effects on tumor cell apoptosis and cell cycle.Western blotting was used to analyze the effects of cell proliferation,apoptosis,cycle.We also established subcutaneous and hepatic tumors in nude mice to observe the antitumor effect of DNASE1L3 in vivo.In addition,the blood of 54 normal persons,72 patients with hepatocellular carcinoma were collected for the detection of DNASE1L3 in serum.Result The expression of DNASE1L3 was significantly decreased in HCC tissues,and the m RNA level of DNASE1L3 in tumor specimens of HCC patients was significantly correlated with pathological grade(P= 0.0062),tumor size(P= 0.0012),BCLC stage(P=0.045),and vascular invasion(P= 0.034).Kaplan-meier survival curve demonstrated that HCC patients with high DNASE1L3 had favorable prognosis.Overexpression of DNASE1L3 can inhibit cell proliferation in vivo and in vitro,and suppress cell migration and invasion.In addition,up-regulation of DNASE1L3 can also promote apoptosis and arrest cell cycle.ELISA results showed that serum DNASE1L3 was the highest in normal people,followed by patients with HCC.Conclusion DNASE1L3 is closely related to the occurrence and development of HCC,which can be used to evaluate the prognosis of liver cancer patients in the future,and regard as a new biomarker and therapeutic target for liver cancer.PartⅡ The role of DNASE1L3 in inhibiting glycolysis and the molecular mechanism of regulating glycolysis reprogramming of HCCObject To discuss the effect of DNASE1L3 on glycolysis of liver cancer cells,screen and identify the interacting protein of DNASE1L3,and analyze the molecular mechanism of DNASE1L3 in inhibiting glycolysis of tumor cells.Method We first carried out single gene GSEA enrichment analysis on DNASE1L3,and screened out the pathway with the most significant differences.Then we establishd DNASE1L3 stably-expressed HCCLM3 cell line,label-free protein quantitative proteomics analysis was performed to identify all of the differences in protein gene ontology(GO)function annotation analysis,protein adjacent class clustering(COG)analysis and the Kyoto encyclopedia(KEGG)gene and genome metabolic pathway analysis.Then,the central carbon metabolites of DNASE1L3 stably-expressed HCCLM3 cell lines were quantitatively analyzed to investigate whether DNASE1L3 had an inhibitory effect on glycolysis of HCC.The effect of DNASE1L3 on m RNA and protein levels of key enzymes in glucose metabolism was determined by RT-q PCR and western blotting.GSEA enrichment analysis and proteomics analysis indicated that DNASE1L3 was closely related to the JAK/STAT pathway,PTPN2 who is non-receptor type tyrosine-specific phosphatase acted as an inhibitor of JAK/STAT pathway,whose expression affected by DNASE1L3.Overexpressing DNASE1L3 can up-regulate PTPN2.Overexpressed plasmids expressing PTPN2 and hexokinase 2(HK2)were co-transfected in HCCLM3 cells,following immune co-precipitation(Co-IP)to verify the interaction between PTPN2 and HK2.In addition,proteins interacting with DNASE1L3 were screened out by protein spectrum assay,and then overexpressed plasmids expressing myc-DNASE1L3 and CEBPβ were co-transfected into HCCLM3 cells,and the interaction between the two was verified again by co-immunoprecipitation(co-ip)assay.Result Single gene enrichment analysis showed that DNASE1L3 was closely related to the JAK/STAT pathway and the glucose metabolism pathway.Subsequent proteomics and metabolomics results further confirmed that overexpression of DNASE1L3 inhibited the JAK/STAT pathway,reduced the production of glycolysis product including 3-phosphoglyceraldehyde,lactic acid,and promoted intermediates such as citric acid,p-ketoglutaric acid,succinic acid,and fumaric acid in Kreb’s cycle.In addition,upregulation of DNASE1L3 in HCC cell lines can inhibit the rate-limiting enzymes of glytolysis from m RNA and protein levels,including hexokinase 2(HK2),pyruvate kinase 2(PKM2),phosphofructokinase(PFK),etc.Besides,DNASE1L3 can upregulate PTPN2 to inhibit glycolysis.PTPN2 weakened the enzymatic activity of HK2 by binding and dephosphorylating HK2,thus inhibiting the tumor glycolysis.In addition,protein spectrum indicated DNASE1L3 may bind to CEBPβ,and coip assay confirmed their binding interaction.CEBPβ is a transcription factor upstream of p53,which can promote the expression of p53.P53 can directly activate TIGAR(t53-induced glycolysis and apoptosis regulator),whose coding protein can degrade fructose 2,6-diphosphate,which is the strongest activator of PFK1(6-phosphofructokinase 1),a key enzyme in the glycolysis pathway,thus further inhibiting glycolysis.Conclusion DNASE1L3 inhibited HCC glycolysis by up-regulating the JAK/STAT pathway inhibitor PTPN2,which can bind to hexokinase 2 and suppress the phosphorylation of HK2.In addition,DNASE1L3 can interact with CEBPβ and increase the expression of p53,which can directly activate the downstream target gene TIGAR expression and further inhibit glycolysis.PartⅢ Transcriptional factor ZNF384 negatively regulates the expression level of DNASE1L3 in liver cancer patientsObject To screen the upstream transcriptional factors of DNASE1L3 and how ZNF384 negatively regulates the low expression of DNASE1L3 in liver cancer tissues.Method We first predicted the potential transcriptional factors upstream of DNASE1L3 through the JASPAR and Gene Cards databases,two databases were taken to the intersection,and 13 transcription factors were found to be in common.Then we downloaded the m RNA sequencing data of these 13 genes from TCGALIHC database,analyzed the correlation between t these 13 genes and DNASE1L3 using computer R language.We then compared the expression levels of ZNF384 in the TCGA-LIHC database between tumor and non-tumor group,analyzed the relationship between high expression of ZNF384 and prognosis of liver cancer patients.Besides,we also investigated the relationship between ZNF384 expression level and gene copy number variation.In addition,we verified the interaction between ZNF384 and DNASE1L3 by CHIP-PCR and Luciferase reporter assay,constructed HBx overexpressed stable cell lines,and further explored the influence of HBx on the changes in the expression levels of ZNF384 and DNASE1L3.Result By analyzing the correlation between 13 genes and DNASE1L3 expression,we found the highest correlation between ZNF384 and DNASE1L3 expression,r=-0.46,so we inferred that ZNF384 could be a potential transcriptional factor of DNASE1L3.The expression level of ZNF384 in liver cancer group in TCGA-LIHC database was significantly higher than that in adjacent tissues,which may be related to the increased level of gene copy number variation,and kaplan-meier survival curve showed that HCC patients with high expression of ZNF384 had a poor prognosis.CHIP-PCR and Luciferase reporter assay confirmed that ZNF384 could inhibit the expression level of DNASE1L3 by directly binding to the promoter region of DNASE1L3.Moreover,HBx can also inhibit the expression of DNASE1L3 by upregulating ZNF384,thus promoting the occurrence of liver cancer.