The Effects Of EI24 On The Biological Behavor Of Pancreatic Cancer Cells And Related Mechanisms

Background:Pancreatic ductal adenocarcinoma(PDAC)is a malignancy characterized by genetic alteration,always caused by the accumulation of mutations in oncogenes and tumor suppressor genes.The EI24(etoposide-induced 2.4kb transcript)gene was found abnormally expressed in various tumors including breast carcinomas and cervical cancers,closely associated with prognosis,playing an important role in apoptosis,cell growth,invasion,metastasis and chemotherapy resistance.There is currently no studies about expression of EI24 in pancreatic cancer,with the role and molecular mechanism still enigmatic.Objectives:To validate the expression of EI24 in pancreatic cancer,analyze its relation with clinical parameters,investigate effects of EI24 on pancreatic cancer cell and the molecular mechanism,seek new biomarkers of diagnosis and prognosis and new therapy targets.Methods:1.We examined the expression of EI24 in tumor tissues and matched adjacent normal tissues by q RT-PCR and Western Blot.We further evaluated EI24 levels in tumor tissues from pancreatic cancer patients using immunohistochemistry and analyzed association of its expression with clinical parameters.2.We constructed stable transfected EI24 overexpressed and knockdown pancreatic cancer cells.We next explored effects of EI24 on cell proliferation,cell cycle and apoptosis by CCK8 assay,colony formation assay and flow cytometry in pancreatic cancer cells in vitro.Wound healing assay was conducted to detect cell migration.The expressions of relative molecules were studied by q RT-PCR,Western Blot and human apoptosis related antibodies array to investigate the molecular mechanism.3.For confirmation the function of EI24 on pancreatic cancer tumorigenesis in vivo,we subcutaneously performed tumor formation assay in Balb/c nude mice.Western Blot,immunohistochemical analysis and immunofluorescence analysis of the tumors were performed to study the molecular mechanism.Results:1.We demonstrated increased expression of EI24 in human PDAC compared with matched normal pancreatic ducts and found higher EI24 expression in moderately to well differentiated PDAC tissues compared with poorly differentiated tissues.2.Overexpression of EI24 suppressed cell proliferation both in vitro and in vivo,while knockdown of EI24 displayed reverse effects.The possible mechanism for this effect might involve EI24-mediated autophagy-related degradation of c-Myc,through regulating EI24/Beclin 1/p62/c-Myc signaling pathway.3.Overexpression of EI24 suppressed pancreatic cancer cell apoptosis,and knockdown of EI24 promoted cell apoptosis.This probably caused by interacting with apoptosis related protein.4.Neither overexpression nor knockdown of EI24 significantly affected migration ability of pancreatic cancer cells.Conclusions:EI24 was upregulated in human PDAC compared with matched normal pancreatic ducts and positively correlated with degree of differentiation.EI24-mediated autophagy-related degradation of c-Myc,through regulating EI24/Beclin 1/p62/c-Myc signaling pathway resulted in suppression of cell proliferation.EI24 inhibited pancreatic cancer cell apoptosis partially attributed to interacting with apoptosis related protein.There may be a crosstalk between autophagy and apoptosis,where EI24 might function as an important regulator,waiting for further study to reveal the deep mechanisms.