| Objective:Through clinical research,function and molecular mechanism,evaluation of miR-181 b in diagnostic and prognostic value of bile duct carcinoma and to analyze its effect on bile duct cancer progress,explore its biological function involving molecular mechanism into full play.Methods:1、MiRNA microarray technology was used to analyze the differential expression profile of miRNAs in cholangiocarcinoma,and q RT-PCR was used to analyze the expression level of mir-181 b in cholangiocarcinoma tissues and adjacent tissues.2、 Clinical data of 189 patients with cholangiocarcinoma and 68 healthy volunteers were retrospectively analyzed.q RT-PCR analysis that miR-181 b in bile duct carcinoma patients and healthy controls the expression difference in serum miR-181 b,q RT-PCR analysis in patients with bile duct carcinoma tissue adjacent to carcinoma tissues and the expression difference of bile duct carcinoma in patients with carcinoma tissue,chi-square analysis and miR-181 b expression level in serum and clinical pathology in patients with the relevance,the ROC curve evaluation of serum miR-181 b diagnostic value in patients with bile duct cancer,the use of Kaplan-Meier method to establish the survival of patients with curve,COX regression analysis in the prediction of bile duct carcinoma independence in patient prognosis.3、Three bile duct cancer cell lines HUCCT1,HUH28,RBE and normal cell lines HIBEC were cultured,and the expression level of mir-181 b in the cells was detected by q RT-PCR.Mir-181 b mimics,mir-181 b inhibitor(inhibitors),and corresponding negative control mimic NC,inhibitor NC,and q RT-PCR were transfected into two bile duct cancer cell lines of HUCCT1 and HUH28 to verify the expression level of mir-181 b after transfection.The effect of mir-181 b expression after transfection regulation on the proliferation ability of cancer cells was analyzed by MTT method,and the effect of mir-181 b expression after transfection regulation on the migration and invasion ability of cancer cells was analyzed by Transwell method.In mouse experiments,the effects of mir-181 b on tumor tissue size and tumor size in mice were analyzed.The expression level of mir-181 b was positively correlated with tumor tissue size and tumor in mice(P<0.01,the difference was statistically significant).4、Platform using Target Scan(http://www.targetscan.org/vert＿72/)to predict the target genes of miR-181 b,use luciferase report experiments to verify this predicted target genes,further in the regulation of miR-181 b bile duct cancer detection PARK2 m RNA expression level,validation of bile duct carcinoma in miR-181 b targeted macro-control PARK2 hypothesis.The relationship between mir-181 b expression and PTEN/PI3K/AKT signaling pathway was analyzed.Results:1、Mi RNA microarray analysis showed that the upregulated multiple of mir-181 b in cholangiocarcinoma was the highest(Fold change=2.248),and the expression level of mir-181 b in the cancer tissues was significantly higher than that in the corresponding adjacent tissues(P<0.01,the difference was statistically significant).2、Retrospective analysis of 189 patients with bile duct carcinoma and the clinical data of 68 cases of healthy volunteers,miR-181 b in patients with bile duct carcinoma in serum expression quantity higher than healthy controls(P<0.01,the difference was statistically significant),miR-181 b in bile duct carcinoma in patients with carcinoma tissues express quantity is higher than the corresponding tissue adjacent to carcinoma(P<0.01,the difference was statistically significant),serum miR-181 b expression quantity and patients was significantly associated with lymph node metastasis and TNM stages(P<0.01,the difference was statistically significant).Serum mir-181 b was found to be a new method for early diagnosis of cholangiocarcinoma(AUC=0.828,sensitivity 76.2%,specificity 76.5%,the cut-off value of diagnosis was 0.835).The overall survival rate of patients with high mir-181 b expression was significantly lower than that of patients with low mir-181 b expression through kaplan-meier analysis(P<0.01,the difference was statistically significant),and COX regression analysis showed that mir-181 b was an independent prognostic factor for patients with cholangiocarcinoma(P<0.01,the difference was statistically significant).3、The expression levels of mir-181 b in the bile duct cancer cell line HUCCT1,HUH28 and RBE were higher than those in the normal cell line HIBEC(P<0.05,the difference was statistically significant);the expression levels of mir-181 b were significantly up-regulated after transfection of mir-181 b mimic into the two bile duct cancer cell lines HUCCT1 and HUH28(P<0.05,the difference was statistically significant);the proliferation capacity of the cancer cells was also significantly enhanced after MTT analysis of the up-regulated mir-181 b expression(P<0.05,the difference was statistically significant).Transwell analysis showed that the metastasis and invasion ability of cancer cells were significantly enhanced after the expression level of miRNA-181 b was up-regulated(P<0.05,the difference was statistically significant).4.Bioinformatics predicted that PARK2(parkin RBR E3 ubiquitin protein ligase)might be the potential target gene of mirna-181 b,and luciferase report experiment verified that mir-181 b could bind directly in the 3’-utr region of PARK2,and mir-181 b could significantly inhibit the expression level of its target gene PARK2 in cholangiocarcinoma(P<0.05,the difference was statistically significant).Mir-181 b can promote cholangiocarcinoma by inhibiting the expression of PTEN and promoting the expression of PI3K/AKT.Conclusion1.The expression of mir-181 b increased in the carcinoma tissues,serum and cell lines of patients with cholangiocarcinoma.Mir-181 b can diagnose cholangiocarcinoma and determine the prognosis of patients with cholangiocarcinoma.2.Mir-181 b promotes cell growth,migration,and invasion by targeting PARK2 and regulating the PTEN/PI3K/AKT signaling pathway3.The mir-181b/PARK2 axis may be a therapeutic target for the treatment of cholangiocarcinoma. |
PDF Full Text Request