The Expression And Function Study Of MicroRNA-138and RhoC In Cholangiocarcinoma

Cholangiocarcinoma, primary bile duct cancer, is rare but highly malignant tumor. It is difficulty to diagnose and treat. Its risk factors include congenital choledochus cyst, cholelithiasis and primary sclerosing cholangitis, et al. The incidence of cholangiocarcinoma is increasing globally. Recently, increasing number of studies have focused on a new class of small non-coding regulatory RNA molecules termed microRNA. MicroRNAs consist of19-25nucleotides and regulate gene expression in a posttranscriptional manner by pairing with complementary nucleotide sequences in3-UTRs of target mRNAs. Although at present the precise biological effects are not well understood, miRNAs appear to be crucial factors in diverse regulation pathways, including development, tumorigenesis, cell differentiation, proliferation, and apoptosis. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in cancers, depending on whether they specifically target tumor suppressor genes or oncogenes, respectively.Many studies showed that the downregulation of miR-138has been frequently observed in a variety of tumors. miR-138is able to target various genes such as G protein alpha inhibiting activity polypeptide2in tongue squamous cell carcinoma, cyclin D3in hepatocellular carcinoma, cyclin D1in nasopharyngeal carcinoma and so on. So far, the expression pattern of miR-138in cholangiocarcinoma and whether or not miR-138involving the progression of cholangiocarcinoma remains unknown.This article intended to study the relationship between miR-138and cholangiocarcinoma. we assumed that whether miR-138as a tumor potential cancer therapeutic target in colorectal cancer.Accordingly,the design of this experiment was as follows:1. To investigate the expression levels of miR-138in normal human Bile duct epithelium(HTBEC)and three cholangiocarcinoma cell lines with different malignant potential, and exploring the relationships between the expression level of miR-138.The changes of activity,apoptosis and metastasis were examined by MTT,FCM, Transwell, Cell scratch test.2.The relationship of miR-138and RhoC was revealed by Luciferase Reporter Assay Systerm3. Investigated the expression of miR-138and RhoC protein in cholangiocarcinoma and normal bile duct tissues to find out the relationship between clinical features and pathology in them. Part Ⅰ The expression and Effects of miR-138on biological characteristics of cholangiocarcinomaObjective1. To explore the expression pattern of miR-138in cholangiocarcinoma cell lines with different malignant potential.2. Research on the effects of high expression miR-138on the cell proliferation, apoptosis, migration and invasion ability in cholangiocarcinomaMethods1. The expression of miR-138in normal human bile duct epithelium cell(HIBEC))and three cholangiocarcinoma cell lines(HCCC-9810、RBE、QBC939)with different malignant potential were examined by real-time PCR.2. The experiments were consists of four groups:1)miR-138group;2) miR-138inhibitor group;3) The negative control group(NC);4) QBC939groups(Con). MTT,FCM, Transwell, Cell scratch test were employed to changes of proliferation,apoptosis, invasion and metastasisResults1.The miR-138expression level in HIBEC was much higher than in the three cancer cells(HCCC-9810、RBE、QBC939)(P<0.05). This downregulation of miR--138is associated with malignant potential of cholangiocarcinoma cell。 The expression of miR-138was lowest in QBC939cells, and this cell line was selected for further experiment.2. The expression of miR-138was highest in miR-138group, and lowest in miR-138inhibitor group. It is description that the transfection was successful.3. QBC939cell experiment Results:1) MTT assay shown that markedly retarded cell proliferation rates were observed in miR-138group cells compared to NC and Con groups (P<0.05).2) Fluorescence-activated cell sorting assay:the result showed that as comparing to the Con (4.22±0.65)%and NC (4.91±0.71)%group, the percentage of apoptosis cells in miR-138group (18.42±2.37)%was markedly increased(p<0.05);3) Tanswell invasion assay:Invasion of miR-138group was significantly inhibited, but Invasion of miR-138inhibitor group was significantly enhanced,(p<0.05);4) The cell wound-healing assay:At24h, the average distance between cells: miR-138inhibitor group((2.79±0.05)mm,was markedly decreased as Con (3.85±0.03) mm and NC (3.83±0.03)mm group (P<0.05);than the difference is not obvious in miR-138group, NC and Con group. At48h, the average distance between cells is obvious in miR-138group, NC and Con group.(P<0.05)Conclusion1.miR-138expression was shown to be greatly down-regulated in cholangiocarcinoma cells compared to normal human bile duct epithelium cells and this down-regulation is associated with malignant potential. The expression of miR-138was lowest in QBC939cells. 2. In cancer QBC939cells, ectopic expression of miR-138potently inhibits tumor cell proliferation, migration and invasion, meanwhile promotes apoptosis. Part Ⅱ Identify the relationship between MiR-138and RhoCObjective1. To find out the possible role of miR-138with its predicted target gene in cholangiocarcinoma by bioinformatics method,and identify it.2. To explore the expression pattern of target gene in cholangiocarcinoma.Methods1.Target gene prediction platform:PicTar, miRanda and RargetScan.we used the platform to find out the predicted target gene of miR-138.2.The relationship of miR-138and RhoC was revealed by Dual-Luciferase Reporter Assay Systerm.3. Real-Time PCR and Western Blot were used to explore the expressio of miR-138and RhoCResults1.we found out the target gene of miR-138:RhoC.2. Luciferase Reporter Assay Systerm:Luciferase figure:Con group(2.236±0.008), miR-138group(1.328±0.011)、inhibitor group(2.235±0.008). NC group(2.227±0.005) and NC inhibitor group(2.232±0.001)。 Comparing to the Con and NC group, The miR-138group was markedly decreased(p<0.05).3. Real-Time PCR:The expression level of RhoC mRNA in miR-138inhibitor group was markedly increased,compare with Con and NC group(p<0.05); than miR-138group was markedly decreased(p<0.05).4. Western Blot:The expression level of RhoC protein in miR-138inhibitor group was markedly increased,compare with Con and NC group(p<0.05); than miR-138group was markedly decreased(p<0.05).Conclusion1. RhoC is a target gene of miR-138.2.The expression of miR-138is significantly correlated with RhoC mRNA and RhoC protein Part Ⅲ Expression and clinical significance of miR-138and RhoC in CholangiocarcinomaObjective1.To explore the expression of miR-138and RhoC in normal bile duct and cholangioearcinoma.2.To analyze the relationship between miR-138and RhoC expression and the clinical pathological features in cholangiocarcinomaMethods1.The expression of miR-138was detected by RNA in situ hybridization in10cases of normal bile duct and76cases of cholangiocarcinoma2. The expression of RhoC was detected by immunohistochemistry in40cases of normal bile duct and76cases of cholangiocarcinoma.Results1.The positive rate of miR-138was42.1%(32/76) in cholangiocarcinoma, however, in normal bile duct, the positive rate was90.0%(9/10); There was a significant difference between the them.(P<0.05)2. The positive rate of RhoC was73.7%(56/76) in cholangiocarcinoma. but, in normal bile duct, the positive rate was20.0%(2/10); There was a significant difference between the them.(P<0.05)3. miR-138positive rate were28.1%,24.1%,16.7%in Poorly differentiated group,nodelymph metastasis group and distant metastasis group,whereas52.3%,55.3%,51.6%in well and moderately differentiated group,no lymphnode metastasis group and no distant metastasis group(P<0.05). RhoC positive rate were87.5%,96.6%,100%in Poorly differentiated group,nodelymph metastasis group and distant metastasis group,,whereas63.6%%,59.6%,68.8%in well and moderately differentiated group,no lymphnode metastasis group and no distant metastasis group(P<0.05).Conclusion1. The express of miR-138is less in cholangiocarcinoma than it in normal bile duct; however the express of RhoC is more in cholangiocarcinoma than it in normal bile duct;2. The positive expression of miR-138and RhoC had related with poorly differentiated,lymph node metastasis and distant metastasis.3. The positive expression of miR-138and RhoC are negative relationship in cholangiocarcinoma.