The Pathological Characteristics And Molecular Mechanisms Of The Digestive System Tumors Induced By JC Virus T Antigen

Objective: JC virus(John Cunningham virus,JCV)is a member of the polyoma virus family(family members also include SV40 and BK viruses),and its genome is circular double-stranded DNA.JCV mainly enters the human body through the digestive tract and respiratory tract,and is latent in the lymphatic tissue and urinary system.When the body's immunity is damaged,JCV replicates and breaks the nervous system in large quantities,causing progressive multifocal leukoencephalopathy(PML),or integrating into the human genome for tumorigenesis.Intravenous and intracranial injection of JCV can induce animal astrocytoma,glioblastoma,and neuroblastoma.Using its own promoter,JCV T antigen transgenic mice can develop pituitary adenoma,schwannomas,and medulloblastoma.Histopathologically,the positive rate or copy number of JCV DNA was higherin nasopharyngeal squamous cell carcinoma,esophageal cancer,gastric cancer,colon cancer,lung cancer and prostate cancer than normal tissues.The JCV T antigen can bind to the ?-Trcp 1/2 protein in the proteasome SCF through DpSGX(2-4)pS,which interferes with the ubiquitin-mediated degradation of ?-catenin protein and can also bind to ?-catenin to improve its protein stability and entrance into the nucleus,nuclear?-catenin promotes cell S-phase evolution by up-regulating c-myc and Cyclin D1 protein expression.Our previous results suggest that overexpression of JCV T antigen can cause lung cancer or lens tumors in transgenic mice.Here,we aimed to reveal the pathological characteristics and molecular mechanisms of JCV T antigen-induced digestive system tumors at the human,animal,cellular,and molecular levels,and to provideexperimental evidences for the prevention and treatment of JCV-related digestive system tumors.Methods1.We performed primary culture of mouse lens tumor cells with high expression of JCV T antigen.After mouse lens tumor cells were stably transfected with JCV T shRNA,qRT-PCR,Western blot and immunofluorescence were used to detect the m RNA and protein expression of JCV T antigen;MTT and PI staining were used to detect cell proliferation and cycle;APC and PI double staining was used to detect apoptosis;wound healing and transwell assays were used to detect cell migration and invasion;immunoprecipitation,silver staining,flight mass spectrometry and Western blot were used to detect interaction proteins;proteomics and Western blot were used to analyze differential signaling pathways and proteins.After human liver cancer,pancreatic cancer and mouse pancreatic cancer cells were stably transfected with pEGFP-N1-JCV T,Western blot was used to detect the expression of GFP and T antigen;MTT was used to detect cell proliferation;APC and PI double staining was used to detect apoptosis;transwell was used to detect cell migration and invasion.2.To activate T antigen expression in hepatocytes or pancreatic ductal epithelia,we crossed CAG-loxp-Lac Z T antigen mice with Pdx1-cre and Alb-cre mice,mice tail DNA PCR was employed to screen the positive mice with the existence of cre and T antigen,and target organ DNA PCR was employed to verify the successful activation of T antigen targeting LacZ;CT and gross were used to observe organ lesions in transgenic mice;HE staining was used to observe histopathological changes;immunohistochemistry was used to detect T antigen and tumor markers' expression;Western blot was used to detect the related protein expression in transgenic mouse tissues.3.We collected paraffin-embedded specimens and frozen tissues of liver cancer and pancreatic cancer tissues and corresponding normal tissues,DNA was extracted from paraffin specimens,PCR and qPCR were used to detect the positive rate and copy number of JCV T antigen DNA;protein was extracted from the fresh tissue,and western blot was used to detect the protein expression of JCV T antigen.Paraffin-embedded samples were used to prepare tissue microarrays,and immunohistochemistry and in situ PCR were used to detect JCV T antigen DNA and protein.Result:Part 1: The effect and molecular mechanisms of JCV T antigen on the malignant phenotype of tumor cells in the digestive system1.We successfully transfected JCV T shRNA into mouse lens tumor cells,evidenced by RT-PCR,Western blot and immunofluorescence(P <0.05);2.JCV T antigen knockdown inhibited cell proliferation,induced G2/M phase arrest and apoptosis,and reduced cell migration,invasion,glycolysis and aerobic oxidation capacity(P <0.05).3.JCV T antigen could bind to RPL19,?-catenin,?-Trcp,NF-?B and C/EBP,but not toRb and mainly affected protein synthesis and degradation,energy metabolism and adhesion signaling pathways(P <0.05).JCV T antigen knockdown increased the protein expression of ING2,ING5,p21,p53,Bax and p-p38;whereas suppressed the protein expression of CyclinD1,survivin,p-m TOR,VEGF,NF-?B,Akt and Bcl-2.4.In human liver cancer,pancreatic cancer and mouse pancreatic cancer cells,JCV T antigen overexpression increased cell proliferation,inhibited apoptosis,and promoted cell migration and invasion(P <0.05).Part 2: Pathological characteristics and molecular mechanism of digestive system tumors induced by JCV T antigen in transgenic mice1.We employed tail DNA PCR to screen the positive mice with the existence of cre and T antigen,and target organ DNA PCR to verify the successful activation of T antigen targeting LacZ.2.According to CT scanning,we found diffuse swelling liver in Alb-cre/T antigen transgenic mice,but not in wild-type mice.Grossly,there were white nodules in the liver,lung,spleen and abdominal cavity of the transgenic mice.Histologically,we also found primary hepatocellular carcinoma,and metastatic cancers into spleen,lung and peritoneum,which showed strong T antigen expression.In hepatocellular carcinoma,AFP,Hep-1,?-catenin,and Ki-67 proteins were positively expressed,but CEA and CK19negatively3.Compared with wild-type mice,liver tissues of Alb-Cre/T antigen transgenic mice showed a higher protein expression of T antigen,mTOR,p-m TOR,?-catenin,Rb,?-Trcp,NF-?B and survivin,While a lower protein expression of ING2,ING4,ING5 and Bax.4.According to CT scanning,there was a pancreatic tumor with necrosis and grossly irregular tumors in Pdx1-cre/T antigen mice.Additionally,white tumors were also found in the stomach and duodenum.Histologically,we observed the pancreatic ductal carcinoma,and squamous dysplasia in gastric forestomach,adenoma in gastric body and antrum and adenocarcinoma in duodenum,which showed strong T antigen expression.5.Compared with wild-type mice,pancreas tissue of Pdx1-cre/T antigen transgenic mice showed a higher protein expression of T antigen,?-catenin,Rb,?-Trcp and survivin,While a lower protein expression of ING2,ING4,ING5,Bax,p53 and p21.Part 3: The relationship between JCV T antigen and the occurrence of human liver cancerand pancreatic cancer1.PCR results showed that both JCV T antigen DNA integration in liver cancer and normal liver tissues,the positive rate of JCV T antigen DNA in liver cancer tissues was significantly lower than that in normal liver tissues(79.0% vs 84.7%,P<0.05);pancreatic cancer had the same results(76.0% vs 92.3%,P<0.05).2.In human liver cancer and pancreatic cancer,there was no significant difference in JCV T antigen DNA copy number between cancer and normal tissues(P> 0.05).JCV T antigen DNA can be detected in liver cancer and normal liver cell nuclei by in situ PCR.3.In human liver cancer and pancreatic cancer,JCV T antigen expression was stronger in cancer tissue than normal by western blot(P <0.05).Immunohistochemically,JCV T antigen was strongly expressed in the nucleus of liver cancer and pancreatic cancer cells,but no or weak immunoreactivity to JCV T antigen was observed in normal liver and pancreas cells.Conclusion:1.JCV T antigen regulates tumor cell proliferation,cycle,apoptosis,energy metabolism,migration and invasion by affecting protein synthesis and decomposition,WNT and NF-?B signaling pathways.JCV T antigen promotes the malignant transformation of the biological phenotypes of liver cancer and pancreatic cancer cells,and may play a role in the malignant evolution of digestive system tumors.2.JCV T antigen induces the digestive system tumors in transgenic mice,which provides a direct experimental basis for T antigens to participate in the development of digestive system tumors.JCV T antigen promotes the development of digestive system tumors by down-regulating tumor suppressor gene expression and activating WNT and other signaling pathways.3.JCV is involved in the occurrence of liver cancer and pancreatic cancer by intercalating into the genome of human liver and pancreas cells and expressing T antigen.The differential expression of JCV T antigen in normal and cancer tissues is because cancer cells are more conducive to the transcription and translation of JCV T antigen.