The Mechanism Of UBE2C In Pancreatic Ductal Adenocarcinoma Proliferation And Matastasis

Population growth and aging changes largely contribute to the total incidence and mortality rates of cancer,which has become a major public health problem.According to the epidemiological survey and statistical analysis,pancreatic ductal adenocarcinoma(PDAC)is one of the most lethal malignant tumors,hundred of thouand people die of pancreatic cancer wordwide annually,and its mortality rate have ranked fourth.PDAC which originates in glandular epithelium accounts for about 90% of pancreatic cancer.Although the diagnosis and therapeutic of pancreatic cancer developments,PDAC is a high degree of malignancy with a tremendously low surgical resection rate,a very poor prognosis,and its incidence rate is approaching its mortality rate.The reason is mainly because of pancreatic cancer is insidious onset and rapid progress.To date,not only are effective early diagnostic biomarkers lacking but also more than 90% of PDAC patients show advanced-stage disease with distal metastasis.Therefore,there is an urgent need to elucidate the underlying molecular mechanisms that regulate the development of pancreatic cancer to develop novel and effective therapeutic targets and strategies for PDAC.Ubiquitin-conjugating enzyme 2C(UBE2C)is a protein-coding gene located on human chromosome 20q13.12 that was originally cloned by Townsley.It is translated into a protein consisting of 197 amino acids and belonging to one of the E2 ubiquitin-conjugating enzyme family.Ubiquitin-conjugating enzyme 2C(UBE2C)is one of the ubiquitin-conjugating enzyme family in the ubiquitin-proteasome system that is taking part in regulating various cellular processes,such as cycle progression,antigen presentation,transcription and programmed cell death.Recent accumulating evidence suggests that UBE2 C is expressed at high levels in various cancerous cell lines and cancer tissues,including malignant breast carcinomas,liver cancers,lung cancers,gastric tumors,colorectal cancers and brain tumors.In gastric cancer,UBE2 C is overexpressed and can induce epithelial mesenchymal transition(EMT)through the Wnt/?-catenin and PI3K/AKT signaling pathways.Moreover,UBE2 C can enhance glioma cell proliferation and inhibit apoptosis in vitro.Other studies have demonstrated that UBE2 C overexpression is correlated with tumor cell proliferation and invasion in colorectal cancers.However,little is known about the molecular mechanisms of UBE2 C in PDAC patients.The study mainly investigated the role of UBE2 C in PDAC proliferation and migration.Associations between UBE2 C expression and clinicopathological characteristics(clinical stage,Differentiation grade,lymph node metastasis,perineural invasion)and survival were assessed using a tissue microarray based on a PDAC cohort.From the reasult of the clinicopathological characteristics,we will find that whether UBE2 C is an independent risk factor and a novel potential therapeutic target for pancreatic cancer.We found that UBE2 C expression level was closely associated with prognosis of PDAC patients in many cancer databases.We performed IHC to detect UBE2 C expression changes in PDAC patients and we found UBE2 C expression was markedly upregulated in pancreatic cancer tissues compared with normal pancreatic tissues.Furthermore,the high UBE2 C level was strongly correlated with clinical stage,lymph node metastasis and perineural invasion(p values were: p<0.001;p=0.02;p=0.01).Upregulated UBE2 C expression was correlated with poor prognosis and the survival curve of the high expression group was significantly lower than the low expression group(p <0.01).Then,we examined UBE2 C expression in several PDAC cell lines,including SW1990,Panc-1,Bx PC-3,CFPAC-1(CF),Hs766 t,As PC-1 and an immortalized human pancreatic ductal epithelial cell line(HPDE6-C7)by real-time PCR and Western blotting.We found that both the m RNA and protein expression levels of UBE2 C were upregulated in the PDAC cell lines.Furthermore,we detected UBE2 C expression in pancreatic tumor tissues and matched adjacent nontumor tissues.We found that both the m RNA and protein expression levels of UBE2 C were differentially elevated in the pancreatic cancer tissues compared with the matched adjacent nontumor tissues.To investigate the biological role of UBE2 C in PDAC development and progression,UBE2 C was knocked down using si RNA in Panc-1 and CFPAC-1 cells.According to the CCK-8 assay results,UBE2 C knockdown significantly suppressed the cell viability of PDAC cells,and the Ed U-positive rate was lower in the si UBE2 C group than in the negative control group.Then,we used flow cytometry to analyze which portion of the cell cycle is impacted when UBE2 C expression is knocked down in PDAC cells.Compared to the control,the knockdown of UBE2 C in Panc-1 and CFPAC-1 cells significantly increased the fraction of cells in the G0/G1 phase and decreased the fraction of cells in the S phase.We performed wound-healing and Transwell assays to investigate whether UBE2 C is an important factor that regulates the migration of PDAC cells.UBE2 C silencing decreased the wound-healing ability of PDAC cells.Similarly,the Transwell assay showed a lower migration ability in si UBE2C-treated cells than in the control cells.The RNA-seq analysis showed silencing UBE2 C by si RNA in CFPAC-1 cells;cyclin D1 and vimentin was downregulated by approximately 3.5-fold and 2.6-fold,and the major enriched pathways were the cell cycle pathway.Western Blotting examined the cell cycle associated proteins and EMT markers.We found that the expresssion of E-cadherin was increase,the expresssion of cycling D1,CDK6,CDK4,CDK2,Vimentin,N-cadherin and MMP2 were decrease in different degree.Tumor-bearing mice analysis found in CFPAC-1 cells UBE2 C depletion by lentivirus encode sh RNA significantly inhibited tumor growth in vivo.In this study,we preliminarily investigated the role of UBE2 C in PDAC proliferation and migration.We discovered that UBE2 C is highly expressed in human pancreatic cancer cell lines compared with an immortalized human pancreatic ductal epithelial cell line(HPDE6-C7).UBE2 C knockdown in Panc-1 and CFPAC-1 cells significantly suppressed proliferation and migration.UBE2 C knockdown induced cell cycle arrest at G1/S.The growth of subcutaneous tumor was significantly inhibited in nude mice.Overall,UBE2 C is involved in regulating the development and progression of pancreatic cancer;furthermore,it could serve as an effective diagnostic biomarker in the early stage and a prognostic biomarker in the advanced stage of pancreatic cancer,which suggests that UBE2 C may also have potential as a therapeutic target for PDAC.