Expression Of DEK Gene In Patients With Primary Hepatic Carcinoma And Phenotypic Analysis Of Liver Specific Dek Knockout Mice

Background:Primary hepatocellular carcinoma?PHC?is the fourth most common malignant tumor and the third leading cause of cancer death in China,which seriously threatens the life and health of our people.Because the recurrence rate and metastasis rate of PHC patients remain high after operation,it is of great significance to reveal the development mechanism of the disease for improving the prognosis of PHC.DEK protein is a structural protein involved in various basic nuclear processes,and has a high expression trend in many malignant tumors.Whether DEK gene plays a crucial role in primary hepatocellular carcinoma has not been clearly confirmed.The purpose of this project is to further reveal the relationship between DEK gene and primary hepatocellular carcinoma and the basic function of DEK gene by conducting clinical and animal experiments on the role of DEK gene in primary hepatocellular carcinoma.²Methods:1.Clinical samples of patients with primary hepatocellular carcinoma were collected and HE staining was used to verify that cancer tissue and adjacent tissues could be used for subsequent experiments.Western Blot and Real-Time PCR were used to detect the expression of DEK protein and DEK mRNA,immunofluorescence was used to detect the localization of DEK protein,and the correlation between DEK protein and clinicopathological factors was analyzed.2.Establishment of liver-specific DEK gene knockout mice model.The experiment was divided into DEK^fl/fl group,DEK^fl/fl/Alb-Cre group.HE staining was used to detect the pathological morphology of liver tissue in mice;Western Blot was used to detect whether DEK was knocked out at protein level;Real Time-PCR and PCR were used to detect whether DEK was knocked out at mRNA level,and immunofluorescence was used to detect whether DEK was knocked out.DEK^fl/fl mice and DEK^fl/fl/Alb-Cre mice were selected to record body weight and liver quality at 1 week,3weeks,5 weeks,7 weeks,and 9 weeks after birth,analysis of phenotype.Mice were sacrificed before and 4,7,14,21 and 28 days after hepatectomy to induce hepatic proliferation.The hepatic proliferation of mice was detected by immunofluorescence.3.Three DEK^fl/fll/fl mice and three DEK^fl/fl/Alb-Cre mice were selected to extract liver tissue for homogenization.The homogenate was sent to Shenzhen Health Time Gene Company for bioinformatics analysis?cluster analysis of differentially expressed genes,GO analysis and Pathway analysis?.Results:1.HE staining in patients with primary hepatocellular carcinoma confirmed that there were infiltration of cancer cells in the tumor tissues and no infiltration of tumor cells in the adjacent tissues.2.The results of Real-Time PCR in patients with primary hepatocellular carcinoma showed that the expression of DEK in tumor tissue was significantly higher than that in adjacent adjacent tissues?p<0.05?.3.Western Blot results of patients with primary hepatocellular carcinoma showed that the expression of DEK protein in tumor tissues was significantly higher than that in adjacent adjacent tissues.4.Immunofluorescence results of patients with primary hepatocellular carcinoma showed that DEK protein was mainly located in the nucleus.5.The expression of DEK was correlated with the occurrence of cirrhosis?p<0.0079?and the staging of G1/G2 and G3?p<0.0001?.6.Establishment of liver-specific DEK gene knockout mice model?1?Genotyping results showed that DEK^fl/fl mice and DEK^fl/fl/Alb-Cre mice all contained 387bp of DEK gene bands,while only DEK^fl/fl/Alb-Cre mice contained 272bp of Cre gene bands.?2?The knockout efficiency test showed that only 520bp deletion positive bands were found in the liver tissues of DEK^fl/fl/Alb-Cre mice,while no Deletion positive bands were found in DEK^fl/fl mice.?3?Western Blot results showed that DEK protein expression in liver tissue of DEK^fl/fl/Alb-Cre mice was significantly lower than that of DEK^fl/fll/fl mice,but there was no difference in DEK protein expression in lung and kidney of mice.?4?Real-Time PCR results showed that DEK expression in liver tissue of DEK^fl/fl/Alb-Cre mice was significantly lower than that of DEK^fl/fl mice?p<0.05?.?5?Immunofluorescence results showed that DEK red fluorescence appeared in the nuclei of all hepatocytes in DEK^fl/fl mice,but no DEK fluorescence signal was detected in DEK^fl/fl/Alb-Cre mice.7.the mice were sacrificed at different time points to weigh the mice's body weight and liver quality.The results showed that there was no significant change in body weight and liver quality between DEK^fl/fl/Alb-Cre mice and DEK^fl/fll/fl mice.?P>0.05?.8.The results of liver proliferation detection at different time points after lobectomy showed that the proliferation of liver tissue in DEK^fl/fl/Alb-Cre mice was significantly lower than that in DEK^fl/fl mice?p<0.05?,and the difference reached its peak seven days after lobectomy.9.The results of liver apoptosis detection at different time points after hepatectomy showed that there was no significant difference in liver apoptosis between DEK^fl/fl/Alb-Cre mice and DEK^fl/fl mice?p>0.05?.10.Results of Bioinformatics Analysis?1?Differentially expressed gene analysis showed that there were 58differentially expressed genes in DEK^fl/fl/Alb-Cre mice compared with DEKfl/fl mice?difference multiple?2;p<0.05?,of which 46 were up-regulated and 12 were down-regulated.Col7a1 is the most significant up-regulation,while Nlrp6 is the most significant down-regulation.?2?GO analysis showed that a total of 396 GO Termes were obtained,of which 223 were biological processes?56.31%,biological process,BP?,and the most significant ones were redox processes?GO:0055114?and cyclooxygenase P450 pathways?GO:0019373?.There are 84 cell components?21.22%,cellular component,CC?,and the most significant enrichment is cell projection?GO:0044995?,collagen trimer?GO:0005581?.There are 89 molecular function?22.47%,Molecular Function,MF?,and the most significant enrichment is oxidoreductase activity?GO:0016491?,monooxygenase activity?GO:0004497?.?3?The KEGG pathway analysis showed that the differentially enriched pathways of the differential genome were mainly metabolic pathways,steroid hormone biosynthesis pathways,retinol metabolic pathways,etc.The most abundant pathways were metabolic pathways,and a total of 7 up regulated differential genes were enriched.Conclusions:1.This study demonstrated that the expression of DEK protein and its mRNA in primary hepatocellular carcinoma was higher than that in adjacent adjacent tissues,and DEK protein was only expressed in the nucleus.2.The expression of DEK in primary hepatocellular carcinoma is related to the occurrence of cirrhosis and the G1/G2 and G3 stages of tumors,but not to sex,age and differentiation degree.3.In this study,we successfully constructed a liver-specific DEK gene knockout mouse model,and confirmed that DEK gene mainly affects liver proliferation.4.Cluster analysis of differentially expressed genes revealed a total of58 differentially expressed genes,of which 46 were up regulated by 12,Col7a1 was the most up regulated,and Nlrp6 was the most significant down regulation.5.GO analysis showed that the most enriched GO Term in biological processes and differential genes is a redox process.The most abundant GO Term in cell components is cell projection,and the most enriched GO Term in molecular function is oxidoreductase activity.The KEGG pathway analysis indicated that the major pathways involved in differentially expressed genes were metabolic pathways,steroid hormone biosynthesis pathways,and retinol metabolic pathways.