| Extrahepatic cholangiocarcinoma(EHCC),primary bile duct cancer,is rare but highly malignant tumor.It is difficult to diagnose and treat.Its risk factorsinclude congenital choledochus cyst,cholelithiasis and primary sclerosing cholangitis,et al.The incidence of cholangiocarcinoma is increasing globally.However,we still known little about the mechanism of Cholangiocarcinoma.Therefore,it is necessary for us to expound the mechanisms of development,invasion and metastasis of EHCC.Recently,increasing number of studies has focused on a new class of small non-coding regulatory RNA molecules termed micro RNA.Micro RNAs consist of 21-25 nucleotides and regulate gene expression in a post-transcriptional manner by pairing with complementary nucleotide sequences in 3-UTRs of target m RNAs.Recent studies have shown that some mi RNAs play roles as tumor suppressors or oncogenes in cancers' depending on whether they specifically target tumor suppressor genes or oncogenes,respectively.Recently,mi R-221 has been shown to play an important role in the progress of breast cancer,gastric carcinoma,hepatic carcinoma,prostatic cancer,et al.However,the function of mi R-221 in EHCC is still not clear yet.PTEN(phosphatase and tensin homolog deleted on chromosome 10),is an anti-oncogene.It is also named as MMAC1(mutated in multiple advanced cancers,MMAC1),was found in 1997.It is the only one which could be phosphorylation of tumor suppressor genes so far,and also its anti-oncogene's role is depending on this character.PTEN was the most frequently mutated in human tumor suppressor genes except for the p53 gene which plays an important role in the development of malignant tumors,and is becoming one of the hot topics in the study of tumor suppressor genes.However,the function of PTEN in EHCC is still not clear yet.In the present study we found that mi R-221 promoted EHCC metastasis and invasion via targeting PTEN expression,moreover,mi R-221/PTEN formed a positive feedback loop with ?-catenin/c-Jun signaling pathway.In EHCC specimens and CC cell lines mi R-221 was upregulated.mi R-221 was associated with prognosis of extrahepatic cholangiocarcinoma patients.In EHCC patients and CC cell lines the expression of PTEN was down regulation.In extrahepatic cholangiocarcinoma cell lines QBC939 and Hu CCT1 PTEN was one of the downstream targets of mi R-221 in the study.Our data also demonstrated that ?-catenin signal pathway promoted mi R-221 through activating c-jun expression,while mi R-221 enhanced ?-catenin signal pathway induced-epithelialmesenchymal transition through targeting PTEN,hence forming a positive feedback loop in EHCC cell lines.In conclusion,our results indicated that mi R-221 induces EMT by targeting PTEN and forms a positive feedback loop with ?-catenin/c-Jun pathway in human EHCC.This study includes two parts.Part?: The expression of mi R-221 and PTEN in extrahepatic cholangiocarcinoma tissues and EHCC cell lines.The relationship between the expression of mi R-221 and PTEN was also demonstrated.(1)The expression of mi R-221 and PTEN in Cholangiocarcinoma and cell lines;(2)PTEN is one of the downstream target gene of mi R-221;(3)mi R-221 is one of the factors which affecting the prognosis of EHCC patients.Part?: mi R-221 gets the function of inducing EMT through targeting PTEN and forms a positive feedback loop with ?-catenin/c-Jun pathway in human EHCC cell lines:(1)?-catenin/c-Jun signaling pathway was involved in mi R-221 mediated EMT in EHCC;(2)?-catenin/c-Jun signal pathway promoted the expression of mi R-221 in extrahepatic cholangiocarcinoma cell lines QBC939 and Hu CCT1.Part? ?-catenin/c-Jun signaling pathway was involved in mi R-221 induced EMT and promoted invasion and migration in EHCC through suppressing the expression of PTENObjective: 1.To explore the expression of mi R-221?PTEN??-catenin and cJun in EHCC tissues and cell lines.2.To explore PTEN is one of the target gene of mi R-221 in EHCC cell lines.3.To explore mi R-221 induces EMT and promotes invasion and migration in EHCC through inhibition the expression of PTEN.Methods: 1.The expression of mi R-221,PTEN,?-catenin and c-Jun m RNA in EHCC tissues and normal human bile duct epithelium cells(HIBEC)and 4 cholangiocarcinoma cell lines(HCCC-9810,RBE,QBC939,Hu CCT1)were examined by real-time PCR.2.The experiments were consists of 5 groups:(1)The normal control group;(2)The negative control group(NC);(3)mi R-221 inhibitor group;(4)si PTEN group;(5)BIO group.Then rt-PCR,western blot,morphology analysis,Transwell was employed.Results: 1.In extrahepatic cholangiocarcinoma tissues and cholangiocarcinoma cell lines,mi R-221 was upregulated.mi R-221 was associated with the metastasis and prognosis of human extrahepatic cholangiocarcinoma patients.2.The expression of PTEN was reduced in both cholangiocarcinoma tissues and cholangiocarcinoma cell lines,and PTEN was one of the downstream targets of mi R-221 in the present study.3.mi R-221 got the function of enhancing ?-catenin signal pathway induced-EMT via suppressing PTEN expression.Conclusion: ?-catenin/c-Jun signaling pathway was involved in mi R-221 induced EMT and promoted invasion and migration in EHCC through suppressing the expression of PTEN.Part? ?-catenin/c-Jun signaling pathway was involved in mi R-221 induced EMT and forms a positive feedback loopObjective: 1.To explore ?-catenin/c-Jun signaling pathway was involved in mi R-221 induced EMT and promoted migration and invasion in EHCC.2.To explore ?-catenin/c-Jun signaling pathway induced mi R-221 expression and forms a positive feedback loop in EHCC.Methods: The experiments were consists of 5 groups:(1)The normal control group;(2)The negative control group(NC);(3)mi R-221 inhibitor group;(4)sic-Jun group;(5)BIO group.Then rt-PCR,western blot,morphology analysis,Transwell was employed.Results: Our data demonstrated that ?-catenin pathway promoted mi R-221 expression via activiating c-jun expression,and mi R-221 promoted ?-catenin signal pathway,and the epithelial-mesenchymal transition induced by ?-catenin signal pathway and enhanced migration and invasion,moreover,they form a positive feedback loop in extrahepatic cholangiocarcinoma cell lines.Conclusion: ?-catenin pathway promoted mi R-221 expression via the activation of c-jun,and mi R-221 enhanced ?-catenin pathway induced-EMT,promoted migration and invasion,hence forming a positive feedback loop with ?-catenin/c-jun signaling pathway in human EHCC cell lines. |
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