| Part I:The expression of Lnc-ATB/miR-200 c in cholangiocarcinoma and its clinical significanceObjective:To study the expression of Lnc-ATB and miR-200(miR-200a/b/c)in cholangiocarcinoma tissues and explore the correlation between the expression of them,and Preliminary analyze their clinical significance.Methods:The q RT-PCR method was used to detect the expression of Lnc-ATB and miR-200(miR-200a/b/c)in 30 cholangiocarcinoma tissues and corresponding non-cancer tissues.Analyze the relationship between of them,and determine the miR-200 that is most closely related to the expression of Lnc-ATB.The clinical significance was also analyzed by combining the clinicopathological data of the patients.Results : Lnc-ATB was highly expressed in cholangiocarcinoma tissues(3.807±0.907 VS 1.049±0.510,P <0.001),miR-200a(2.260±0.899 VS 3.099±1.062,p< 0.05),miR-200b(1.338±0.587 VS 2.268±0.510,P<0.001)and miR-200c(0.469±0.251 VS 1.452±0.410,P <0.001)all showed a low expression change,but miR-200 c showed the lowest expression.The high expression of Lnc-ATB in cholangiocarcinoma was related to the low expression of miR-200c(P <0.01).Patients with high expression of Lnc-ATB have a later TNM stage,larger tumor volume and more prone to have lymph node metastasis.However,patients with low expression of miR-200 c have a lateer TNM stage and are more prone to have lymph node metastasis.Conclusion: The expressions of Lnc-ATB was increased and miR-200 c was decreased in cholangiocarcinoma.The expressions of them are negatively correlated,indicating that Lnc-ATB may play an oncogene role,however miR-200 c perhaps play a role of tumor suppressor gene in cholangiocarcinoma Part II: The expression and function of Lnc-ATB/miR-200 c signal axis in cholangiocarcinoma cellsObjective:To study the expression and biological effect of Lnc-ATB/miR-200 c signal axis in cholangiocarcinoma cells in vitro.Methods:The q RT-PCR method was used to detect the expression of Lnc-ATB and miR-200 c in HUCCT1?RBE?TFK1?Huh-28 and BEC cells.The expression level of Lnc-ATB in HUCCT1 and RBE cells was knocked down by si RNA technology,and the expression level of Lnc-ATB and miR-200 c was detected by q RT-PCR.HUCCT1 cells were simultaneously transfected with plasmids that with wild-type or mutant psicheck2-Lnc-ATB binding site and miR-200 c mimic.48 hours after transfection,luciferase activity was determined by double luciferase reporter gene detection kit,Combined with bioinformatics analysis,the regulatory relationship and binding site between Lnc-ATB and miR-200 c were preliminarily determined.Three groups of cells with different expression levels of Lnc-ATB and miR-200 c were constructed by knockdown the expression of Lnc-ATB in HUCCT1 and RBE cells with si RNA technology and co-transfection of miR-200 c inhibitor.The three groups were single knockdown Lnc-ATB expression group(si RNA-Lnc-ATB group),si RNA-Lnc-ATB+cotransfected with miR-200 c inhibitor group and negative control group.The q RT-PCR method was used to detect the expression of miR-200 c in each group.The effects of Lnc-ATB/miR-200 c signal axis on the proliferation,apoptosis,cell cycle and cell migration of cholangiocarcinoma cells were analyzed by CCK8 cell proliferation experiment,flow cytometry,cell apoptosis experiments and Transwell experiment.Results:In vitro cell experiments showed that compared with normal BEC cells,the expression level of Lnc-ATB in cholangiocarcinoma HUCCT1?RBE?TFK1?Huh-28 cells were all significantly increased,however the expression of miR-200 c was relatively low.HUCCT1 and RBE cells were the most obvious,therefore,HUCCT1 and RBE cells were selected for si RNA interference experimenst.Si RNA interference experiments showed that knockdown of the expression of Lnc-ATB in HUCCT1 and RBE cells could increase the expression level of miR-200 c.And si RNA 1 interference effect is better than si RNA 2,so si RNA 1 sequence is used for follow-up test.Bioinformatics analysis showed that Lnc-ATB had potential miR-200 c binding site.Double luciferase analysis showed that miR-200 c mimic could significantly inhibit the luciferase activity of psicheck2-Lnc-ATB with wild-type miR-200 c binding site,but this phenomenon was not observed in psicheck2-Lnc-ATB with mutant miR-200 c binding site.So we predicted that miR-200 c was the target gene of Lnc-ATB in cholangiocarcinoma cells.It was found that knocking down Lnc-ATB expression of HUCCT1 and RBE cells cells with si RNA technology can increase miR-200 c expression,and promote tumor cell apoptosis and G0/G1 phase arrest,and inhibit the proliferation and migration ability.However,when miR-200 c inhibitor was cotransfected into the cells to down regulate the expression of miR-200 c,the inhibition of si RNA-Lnc-ATB on the proliferation,apoptosis,cell cycle arrest and migration of cholangiocarcinoma cells could be reversed.Conclusion:In cholangiocarcinoma cells,the expression of Lnc R-ATB is increased,and the expression ofmiR-200 c.is decreased.Lnc R-ATB is involved in regulating the growth and migration of cholangiocarcinoma cells by inhibiting the expression of miR-200 c.Part III: The molecular mechanism of Lnc R-ATB / miR-200 c signal axis regulating the growth and migration of cholangiocarcinomaObjective:To study the expression of Lnc-ATB/miR-200 c related signal pathway factors in cholangiocarcinoma cells,and explore the molecular biological functions of Lnc-ATB/miR-200 c.So as to provide theoretical guidance for further research.Method: Three groups of cells with different expression levels of Lnc-ATB and miR-200 c were constructed by knockdown the expression of Lnc-ATB in HUCCT1 and RBE cells with si RNA technology and co-transfection of miR-200 c inhibitor.The three groups were single knockdown Lnc-ATB expression group(si RNA-Lnc-ATB group),si RNA-Lnc-ATB+cotransfected with miR-200 c inhibitor group and negative control group(NC group).Western blotting were used to detect cell cycle-related proteins CCND1 and CDK2,apoptosis-related proteins BCL-2/Caspase-3,and EMT related signaling pathway factors ZEB1/2 and E-cadherin/N-cadherin expression.Results:Knockdown of Lnc-ATB expression in HUCCT1 cells in vitro can increase miR-200 c expression,thereby inhibiting the expression of cell cycle related proteins CCND1 and CDK2,promoting the expression of apoptosis related proteins BCL-2,and inhibiting the apoptosis related protein Caspase-3.The expression of ZEB1/2 protein was also inhibited,and resulted in an increase in E-cadherin protein and a decrease in N-cadherin expression.Hower when the HUCCT1 cells were treated with si RNA-Lnc-ATB +miR-200 c inhibitor could restore the expression of ZEB1 2,E-cadherin and N-cadherin.Conclusion:Lnc-ATB can regulate cholangiocarcinoma cell cycle,apoptosis and EMT related signal pathway factors in vitro by inhibiting miR-200 c expression,so as to promote the proliferation and migration of cholangiocarcinoma cells.Part IV: The role of Lnc-ATB/miR-200 c signal axis in the model of cholangiocarcinoma transplanted tumor in vivo Objective:To study the effect of Lnc-ATB/miR-200 c signal axis on the tumorigenesis of cholangiocarcinoma cells and the expression of related signal pathway factors in vivo.Preliminary to explore whether it may be a potential molecular target for the diagnosis and treatment of cholangiocarcinoma.Method:The lentivirus with RNAi-Lnc-ATB,miR-200 c inhibitor and negative control was designed and packed.The virus was first used to infect HUCCT1 cell line,and three groups of cells with long-term stable expression were obtained.Respectively single knockdown Lnc-ATB expression group,knockdown Lnc-ATB expression+ co-transfected with miR-200 c inhibitor group and negative control group(NC group).Five week BALB/c nude mice were randomly divided into three groups,5 mices in each group.And then established a nude mouse subcutaneous tumor transplantation model.The changes of tumor volume were observed and recorded every three days.After 3 weeks,the nude mice were killed to compare the tumor size.and explore the tumorigenic effects of Lnc-ATB/miR-200 c in nude mice.The expression of PCNA that is a cell proliferation marker was detected by immunohistochemical in tumor tissue of three groups of nude mice.Western blotting was used to detect the expression of CCND1/CDK2,Bcl-2/Caspase-3,ZEB1/2 and E-cadherin/N-cadherin.Results:Using lentivirus transfection technology to knock down the expression of Lnc-ATB in HUCCT1 cells,the subcutaneous transplanted tumor tissue of nude mice was small in size and accompanied with decreased PCNA expression,At the same time,stable co-transfection of miR-200 c inhibitor could reverse this result.Knockdown of Lnc-ATB expression in HUCCT1 cells by lentivirus transfection can reduce the expression of cell cycle related proteins CCND1 and CDK2,inhibit the expression of apoptosis related proteins Caspase-3 and ZEB1/2,and increase the apoptosis related protein BCL2 expression.Also the expression of E-cadherin protein was incresed and N-cadherin was decreased.However,when the si RNA-Lnc ATB and miR-200 c inhibitor was stably co-transfected to the HUCCT1 cells,the results can be restored.Conclusion:Lnc-ATB regulates cholangiocarcinoma cell cycle,apoptosis and EMT related pathway signal factors by inhibiting miR-200 c expression in nude mice,so as to promote the Proliferation and migration of cholangiocarcinoma cells.Lnc-ATB/miR-200 c may be a potential molecular target for the diagnosis and treatment of cholangiocarcinoma. |
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