Expression Of NDRG2 Gene In Cholangiocarcinoma And Its Effect On The Growth Of Cholangiocarcinoma Cells

Objective:Cholangiocarcinoma,as a kind of malignant tumor,has poor prognosis because of its early metastasis and insensitivity to chemotherapy or radiotherapy.Many data show that Nmyc downstream regulatory gene 2(NDRG2)as a tumor suppressor gene is involved in the occurrence and development of a variety of cancers.However,its mechanism in cholangiocarcinoma is still unclear.Preliminary studies showed that NDRG2 was abnormally expressed in cholangiocarcinoma.Therefore,we speculate that the abnormal expression of NDRG2 may be related to the occurrence and development of cholangiocarcinoma.The purpose of this study was to investigate the biological role and mechanism of NDRG2 in the occurrence and development of cholangiocarcinoma.Methods:Bioinformatics analysis verified the differential expression of NDRG2.Quantitative Real-time PCR(qPCR)and Western blot assay were performed on samples of normal tissues and cholangiocarcinoma that had not received preoperative treatment.The expression levels of NDRG2 in the two types of surgically resected tissue samples were analyzed.The relationship between NDRG2 expression level and pathological factors in patients with clinical cholangiocarcinoma was investigated.Compared with normal bile duct epithelial cells,FRH0201,the bile duct cancer cell with the lowest expression level in the bile duct cancer cell line,was selected as the main research object in the intracellular experiment of this subject.Bile duct cancer cell FRH0201 with stable and high expression of NDRG2 was constructed by transfection with Plent-U6-GFP-Puro-NDRG2,and its expression level and transfection efficiency were verified by qPCR and Western blot assay,respectively.CCK-8 assay and clone formation assay were used to detect the effect of stable overexpression of NDRG2 on the growth ability of bile duct cancer cells and their negative control cells.Meanwhile,the constructed bile duct cancer cells with stable overexpression of NDRG2 and their negative control cells were used to detect the cell invasion and migration ability,respectively: scratch test,Transwell migration test and invasion test were used to detect the influence of overexpression of NDRG2 gene in bile duct cancer cells on cell migration and invasion ability.In addition,the intracellular functional experiments were completed to further investigate the regulatory effects of NDRG2 downstream epithelial-mesenchymal transition(EMT)related genes.QPCR and Western blot experiments were used to analyze the regulation effect of NDRG2 on the expression levels of EMT-related indexes E-cadherin and Vimentin compared with the control group.Results:First,the differential expression of NDRG2 in cholangiocarcinoma and normal tissues was confirmed by TCGA database analysis.Subsequently,qPCR and Western blot analysis results showed that,compared with the negative control group(normal bile duct epithelial tissue),NDRG2 was abnormally expressed in the bile duct carcinoma tissues collected before operation,which was lower than the expression level in normal tissues.Combined with clinicopathological analysis,NDRG2 level was negatively correlated with lymph node metastasis,vascular invasion,CA19-9 level and clinical TNM stage.The qPCR assay confirmed that the expression of various bile duct cancer cell lines,such as QBC939,FRH0201 and RBE,was lower than that of human intrahepatic bile duct epithelial cells,Hibepic.The expression of FRH0201 was the lowest among them.By establishing FRH0201-plent-NDRG2 bile duct cancer cells with stable overexpression of NDRG2,the proliferation experiment of bile duct cancer cells was completed,which confirmed that the overexpression of NDRG2 in bile duct cancer cells through p Lent carrier could inhibit the proliferation of cells.The migration and invasion experiments of bile duct cancer cells showed that the overexpression of NDRG2 in bile duct cancer cells could inhibit the migration and invasion of cells.Finally,qPCR and Western blot experiments showed that the expression of NDRG2 was correlated with the expression of EMT-related genes: overexpression of NDRG2 could increase the expression level of epithelial marker factor E-cadherin in EMT indicators,and decrease the expression level of interstitial marker factor Vimentin.Conclusion:In this study,we confirmed that NDRG2 was down regulated as a differential gene in cholangiocarcinoma.Overexpression of NDRG2 significantly inhibited the proliferation,migration and invasion of human cholangiocarcinoma cell line frh0201.The expression level of E-cadherin,an epithelial marker,was significantly up-regulated,while vimentin,an interstitial marker,was on the contrary The occurrence of T.Therefore,we believe that NDRG2 may be a potential new target for clinical treatment of cholangiocarcinoma.