| BackgroundCholangiocarcinoma(CCA)originates in the epithelial cells of the bile duct wall,and its incidence is second in hepatobiliary malignancies only to hepatocellular carcinoma.Cholangiocarcinoma is divided into intrahepatic cholangiocarcinoma,hilar cholangiocarcinoma,and distal cholangiocarcinoma according to its site,and hilar cholangiocarcinoma is the most common type.Hilar cholangiocarcinoma(HCCA)is a type of epithelial malignant tumour that occurs in from the left and right hepatic duct to common hepatic duct.Its incidence has been increasing year by year in Asian countries.Surgical radical resection is the best treatment for patients at present.However,the resection rate is low and the recurrence rate is high disappointedly because of its insidious onset,Special anatomy and early metastasis.Because of the low sensitivity to chemoradiotherapy and the lack of effective treatment,it is very significant to explore the key molecules and new therapeutic targets for improving the prognosis of HCCA patients.The occurrence and development of tumour are closely related to cell cycle regulation disorder and apoptosis inhibition.Cyclin-dependent kinase(CDK)maintains cell cycle dynamics under the control of cyclin and CDK inhibitors.Once the complex regulatory networks which promote and inhibit each other among these regulators are abnormal,the imbalance between positive and negative regulatory factors will lead to unlimited proliferation of tumour cells.And cell proliferation is related to not only cycle disorders,uncontrolled proliferation,abnormal differentiation,but also apoptosis inhibition.Oncogene mutations often lead to the abnormal expression of apoptosis suppressor genes or pro-apoptotic genes,then the disorder of Cell monitoring program happen and the cells proliferate malignantly.EVI1(ecotropic virus integration site-1)is a oncogene located on human chromosome 3q26.As a retrovirus binding site,it was first found in AKXD rat myeloid malignant tumor DNA,and its encoded protein is a transcription factor with zinc finger domain,which plays an important role in normal tissue development,differentiation and tumorigenesis.In recent years,it has been found that EVI1 is not only closely related to the occurrence and development of hematological malignancies,but also plays an important role in other solid malignancies.For example,the expression of EVI1 was significantly increased in breast cancer,colorectal cancer,ovarian cancer,pancreatic cancer,prostate cancer and other tumours.And it was negatively correlated with the poor prognosis of patients.In these studies,EVI1 was found to promote tumour cell growth by regulating cell cycle,inhibiting cell apoptosis,and it also could promote tumour cell invasion.However,there isn't any report about the expression of EVI1 in hilar cholangiocarcinoma and the relationship between EVI1 and HCCA.PTEN(phosphotase and tensin homologous gene on chromosome 10)is one of the Mutated tumor suppressor genes in human cancers.It is very important to maintain normal cell differentiation,and PTEN deficiency can lead to cell proliferation,apoptosis inhibition and enhanced cell migration.The expression of PTEN is decreased in most solid tumours,but there is no report about PTEN in hilar cholangiocarcinoma.ObjectiveThe purpose of this study is to explore the expression of EVI1 in hilar cholangiocarcinoma,and analyze the relationship between the expression level of EVI1 and clinicopathological parameters in hilar cholangiocarcinoma.The effects of EVI1 on proliferation,cell cycle,apoptosis,invasion and migration of hilar cholangiocarcinoma cells were detected after changing the expression of EVI1.by knockdown and over-expression.And experiments were performed to further explore possible molecular mechanisms that EVI1 regulated cell proliferation,cell cycle,and apoptosis.These can provide theoretical basis for whether EVI1 can be a new marker for diagnosis and prognosis of HCCA and a new therapeutic targetMethodsThis study was carried out in three parts to study the expression and clinical value of EVI1 in hilar cholangiocarcinoma,the effect of EVI1 on the biological function of hilar cholangiocarcinoma cells,and the underlying molecular mechanisms that regulate the biological function of cancer cells.1.The expression of EVI1 in hilar cholangiocarcinoma and its clinical significance:The mRNA and protein expressions of EVI1 in fresh hilar cholangiocarcinoma and its paracancerous tissues were detected by real-time PCR and western blot.By constructing tissue microarray of 114 patients with hilar cholangiocarcinoma,the correlation between EVI1 and clinicopathological parameters of HCCA patients was analyzed,and the survival curve of HCCA patients was drawn.Cox univariate and multivariate survival analysis were used to choose the prognostic factors of HCCA patients.2.Effect of EVI1 on the biological function of HCCA cells:qRt-PCR and western blot were used to detect the expression of EVI1 in every CCA cell line,and the HCCA cell lines with high-expression and low-expression of EVI1 were selected out.CCK8 assay,clone formation assay and EdU assay were used to detect the effect of EVI1 knockdown and overexpression on the proliferation of HCCA cells.The effect of EVI1 on HCCA cell cycle was detected by flow cytometry.The effect of EVI1 on HCCA cell apoptosis was determined by flow cytometry and TUNEL assay.Wounding healing assay and Transwell test examined the effects of EVI1 on the migration and invasion ability of HCCA cells.The effect of EVI1 on the proliferation of HCCA cells in vivo were observed by subcutaneous tumorigenesis that the QBC939 cells knocked down EVI1 were injected into nude mice.3.The potential molecular mechanism of EVI1 regulating cell proliferation,cycle and apoptosis:mRNA expression of PTEN and EVI1 in fresh HCCA tissues were detected by qRt-PCR,and the expression results were analysed by the linear correlation and linear regression.Immunohistochemical staining was used to detect the protein expressions of PTEN and EVI1 in HCCA patients in TMA,and the linear correlation and linear regression analysis of both H-Score scores were performed.Western blot and remedial experiments were used to clarify the interaction among EVI1,PTEN and AKT pathway.And the potential mechanism that EVI1 regulates cell biological function was further elucidated.Results1.EVI1 was highly expressed in hilar cholangiocarcinoma,and its expression was significantly correlated with tumor tissue differentiation,tumor size,and prognosis of patients.High EVI1 expression was an independent factor of the poor prognosis of HCCA.2.EVI1 played an important role in the development of hilar cholangiocarcinoma.Knockdown or overexpression of EVI1 could inhibit or promote the proliferation of HCCA cells.Knockdown of EVI1 expression could inhibit the migration and invasion of HCCA cells.The effect of EVI1 on cell proliferation might be realized by influencing cell cycle and regulating cell apoptosis.The vivo experiments showed that knockdown of EVI1 expression could inhibit the growth of HCCA tumour.3.EVI1 could regulate the activity of PI3K/Akt pathway by inhibiting PTEN,promote Akt phosphorylation,and then affect the proliferation and apoptosis of hilar cholangiocarcinoma cells.PTEN/PI3K/Akt signaling pathway was involved in EVI1-mediated proliferation and apoptosis of hilar cholangiocarcinoma cells.Conclusion and Significance1.It was found for the first time that EVI1 was highly expressed in hilar cholangiocarcinoma and related to the poor prognosis of patients2.EVI1 regulated the activity of PI3K/Akt pathway by inhibiting PTEN,and promoted the expression of p-Akt.This promoted the proliferation,migration and invasion of HCCA cells and inhibited cell apoptosis in vitro,and promoted the growth of tumour in vivo. |
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