The Functional And Mechanistic Study Of PURA,RUVBL2 And CASP8 On Digestive Cancer Progression

Esophageal squamous cell carcinoma(ESCC)is one of the most aggressive gastrointestinal malignancies,ranking fourth leading cause of cancer-related death in China,which accounts for 90%of the cases.Despite recent progress in early detection and therapeutic efficacy,however,prognosis is hampered because of the high recurrence rates and the development of metastasis.Majority of patients are at advanced stages when first diagnosed and tumor recurrence and metastasis.The mechanisms promoting ESCC progression remain not clear,thus,it is crucial to illuminate the molecular mechanisms for the new diagnostic approaches and therapeutic modalities.Purine-rich element binding protein alpha(PUR?),a member of Pur family,which plays a critical role in the initiation of DNA replication,cell cycle regulation,transcription regulation and mRNA translation by specifically binding with DNA or RNA sequences.Many reports have shown that the mutation or the loss of PUR? expression involves in neurodevelopmental abnormality and contributes to initiation of PURA syndrome.The dysregulation of PURa has also been reported to be involved in the tumor proliferation of multiple cancers,however,the expression pattern and function of PURa in ESCC remain poorly understood.Thus,in the first to third parts of the study,we aim to investigate the potential role and mechanism of PURa in ESCC.We analyzed the PURA mRNA level in esophagus cancer based on the dataset of The Cancer Genome Atlas(TCGA),and performed an immunohistochemistry staining on ESCC tissues and its adjacent normal tissue to assess the expression of PUR?.Further to analyze the correlation between PURa expression and clinicopathological features or prognosis.To investigate the function of PUR? in ESCC,we generated the stable PURa overexpression and knockdown ESCC cells.The in vitro effects of PUR? on cell proliferation,migration and invasion abilities were explored by CCK-8,colony formation,wound healing and transwell assays.The in vivo effects of PURa on tumor growth and metastasis were detected by animal experiments.To gain further insight into the mechanism of PURa functions,the RNA sequencing(RNA-seq)was performed and analyzed,further qPCR,western blot and immunofluorescence assays were used to validate the expression of candidate pathways and genes.Besides,the luciferase reporter assay and Chromatin immunoprecipitation(ChIP)assay were conducted to explore the effect of PURa on regulating transcriptional activity of candidate gene and then the function of candidate gene was validated by rescue assay.Finally,an immunohistochemistry staining was performed on ESCC tissues to assess the correlation between PURa and Snail2 protein levels.The results showed that PURA mRNA and protein levels were highly expressed in ESCC tissues.High PURa expression was positively associated with lymph node metastasis and AJCC stage,and ESCC patients with high PURa expression had worse survival,indicating that PURa promotes ESCC progression and poor prognosis.Functional study showed that PURa promoted ESCC cell proliferation,migration and invasion in vitro and in vivo.The RNA-seq analysis implied that PURa induced epithelial-mesenchymal transition(EMT)and further validated by biological experiments.Mechanistically.PURa up-regulated the expression of Snail2 via directly binding to its promoter to activate its transcriptional activity.In addition,knockdown of Snail2 reversed PUR?-induced EMT and the enhancement of migration and invasion.Finally.PUR? expression was positively correlated with the Snail2 expression in ESCC tissues.In conclusion.this study indicates that PURa promotes Snail2 transcriptional activity to induce EMT during ESCC progression.Liver cancer is the most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide.Hepatocellular carcinoma(HCC)represents almost 90%of all primary liver cancer cases.However,the molecular mechanisms of HCC remain only partially understood.RuvB-like 2(RUVBL2)belongs to the conserved ATPases associated with various cellular activities(AAA+)protein subfamily.which is characterized by the presence of conserved Walker A and B motifs that are involved in ATP binding and hydrolysis.RUVBL2 was previously found to contribute to hepatocarcinogenesis.However,its expression,regulation and clinical significance have not been systematically evaluated in a large number of clinical samples.In the fourth part of the study,we performed a comprehensive analysis of RUVBL2 based on multiple datasets from 371 liver cancer patients of The Cancer Genome Atlas(TCGA).In addition.the aberrant signaling pathways caused by RUVBL2 overexpression were investigated.In this study.we demonstrated that promoter hypomethylation,copy number gain,MYC amplification and CTNNB1 mutation were all responsible for RUVBL2 overexpression in HCC.High levels of RUVBL2 mRNA were associated with shorter recurrence-free survival time(RFS)but not overall survival time(OS).Furthermore.high levels of RUVBL2 promoted carcinogenesis through the heat shock protein 90(HSP90)-cell division cycle 37(CDC37).AKT serine/threonine kinase(AKT)and mitogen-activated protein kinase(ERK/MAPK)pathways.In conclusion,the deregulation of RUVBL2 in HCC is influenced at the genomic.epigenetic and transcriptional levels.Our findings highlight the potential roles of RUVBL2 as a promising prognostic marker as well as a therapeutic target for HCC.In the previous study,we have found 5 SNPs at 2p33.1 with the risk of ESCC in a Chinese population with a case-control study.In the fifth part of study,we investigated the effect of rs10931936-T,the main locus located in deep intron of CASP8,on ESCC susceptibility.The results showed that non-coding susceptibility locus rs10931936-T modulates alternative splicing of CASP8 to produce a novel splice variant which encode a truncated protein Caspase-8X.It further regulates tumorigenesis of ESCC,which might provide new biomarkers for the early diagnosis and treatment of ESCC.