The Role And Mechanism Of MiRNA-145in Gallbladder Carcinoma

Part I Establishment of differential expression profile of miRNAs between normal gallbladder cells and gallbladder carcinoma cellsObjective:The main aim of this chapter is to compare the difference in expression profile of miRNAs between normal gallbladder cells and gallbladder carcinoma cells (GBC-SD).Methods:The normal gallbladder cell line and GBC-SD cell line were cultured for extraction of total RNA and then quantified to produce a chip. The signals were scanned using LuxScan10KA dual-channel laser scanner, microarray images were analyzed using luxScan3.0image analysis software, and the differentially expressed miRNAs were screened. miRNA-145was selected to be investigated. Then15gallbladder carcinoma samples were respectively obtained from gallbladder carcinoma patients and fresh paracancerous tissue for extraction of total RNA, and the expression of those RNAs were detected using Real-time PCR.Results:By comparison, miRNA chips were successfully established for normal gallbladder cells and gallbladder carcinoma cells.150miRNAs that were differentially expressed were screened, among which expressions of89miRNAs were up-regulated and expressions of61miRNAs were down-regulated in gallbladder carcinoma cells. Expression of miRNA-145was selected to be investigated and the results showed down-regulated expression in gallbladder carcinoma tissues.Conclusions:Analysis using miRNA chips revealed150differentially expressed miRNAs between normal gallbladder cells and gallbladder carcinoma cells, among which, expressions of89miRNAs were up-regulated and expressions of61miRNAs were down-regulated in gallbladder carcinoma cells. And miRNA-145showed down-regulated expression in both GBC-SD in vitro and fresh gallbladder carcinoma tissue. Part Ⅱ Biological function of hsa-miRNA-145in gallbladder cancer cell line GBC-SDObjective:The main aim of this chapter is to investigate the effects to proliferation, invasion and apoptosis of up-regulated miRNA-145expression of gallbladder cancer cells in vitroMethods:miR-hsa-miRNA-145expression vector was established and transfected into GBC-SD cell line, and then GBC-SD cells were collected. The group that transfected with empty vectors and blank control group were also setted. The proliferations in each group were detected using MTT method and clonogenic assay, and cell cycle distribution as well as cell apoptosis were observed using Flow Cytometry. The total protein were extracted in transfection group and blank control group to compare the difference in expression of caspase3(apoptosis-related protein) using Western blot. Besides, the invasion capacity was analyzed by setting up Transwell chamber.Results:hsa-miRNA-145over-expression vectors were successfully established. Compared to that in the group transfected with hsa-miRNA-145expression vector, MTT assay revealed30%cell growth inhibition (P<0.05) in empty vector transfection group and blank control group on the4th day after culture. Besides, we selected2000GBC-SD cells that were transiently transfected with hsa-miRNA-145expression vectors to compare their colony-forming activity with those of control cells, and the results indicated that GBC-SD cells formed much more cell clones. Analysis by Flow Cytometry indicated that the percentage of cells that were in G1and S phase were45.16%and31.93%respectively for the control cells, and the corresponding percentage for the hsa-miRNA-145overexpression cells were53.83%and46.17%. The percent of cells in DNA synthesis phase for hsa-miRNA-145overexpression cells higher than that for the control cells. Besides, compared with the control cells, Flow Cytometry revealed higher number of apoptotic cells that were double positive for both annexin V and PI in GBC-SD cells that were transiently transfected with hsa-miRNA-145expression vectors. The expression of caspase3(apoptosis-related protein) in miRNA-145over-expressed GBC-SD cells was significantly higher compared to the control cells. Transwell migration assay revealed lower percentage of cells that migrated through the chamber bottom for GBC-SD cells that transiently over-expressed hsa-miRNA-145when compared to the control cells.Conclusions:Over-expression of miRNA-145inhibited the growth of GBC-SD cells and the invasive ability of GBC-SD cells. Over-expression of miRNA-145exhibited no influence of cell cycle of GBC-SD cells but induced apoptosis of GBC-SD cells. PartⅢ Sequencing and validation for miRNA-145target genes which influence on gallbladder cancer cell line GBC-SDObjective:The main aim is to discuss the probable target genes of miRNA-145for regulating the functions of gallbladder cancer cell line.Methods:Above mentioned investigations had demonstrated obvious regulation by miRNA-145on cell apoptosis of gallbladder cancer cell line, we believed that DFF45was the probable target gene of miRNA-145. We intended to validate our prediction using Dual-Luciferase Reporter Assay System. Besides, we analyzed the differences in variations of mRNA and protein expressions of DFF45between gallbladder cancer cells that over-expressed miRNA-145and normal GBC-SD cells by Real-time PCR and Western blot.Results:When compared with control group, Real-time PCR revealed significantly down-regulated DFF45transcript level in cell lines that over-expressed miRNA-145, and the difference was statistically significant (P<0.05). Cell lines over-expressed miRNA-145had obviously lower concentration level of DFF45protein compared to that in blank control group or empty vector transfection group. Validation by Dual-Luciferase Reporter Assay System demonstrated that DFF45was the target gene of miRNA-145.Conclusions:Over-expression of miRNA-145led to down-regulated expression of DFF45in GBC-SD cell lines. miRNA-145inhibited expression of DFF45by targeting the CDS854site in DFF45mRNA. PartⅣ Clinicopathological significance of DFF45expression in benign and malignant lesions of the gallbladderObjective:To study on the expressive levels of DFF45and detect its clinicopathological significances in the benign and malignatnt lesions of gallbladder.Methods:EnVisiom immunohistochemical method for determining the expressions of DFF45was used in routinely paraffin-embedded sections of surgical resected specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyp (n=15), and chronic cholecystitis (n=35).Results:The positive rates of DFF45expression was significantly higher in gallbladder adenocarcinoma than that in peritumoral tissues (χ2=6.92, P<0.01), adenomatous polyp (χ2=4.49,P<0.05) and chronic cholecystitis (χ2=12.98, P<0.01). The positive rates of DFF45was significantly higher in the cases of well-differentiated adenocarcinoma, no-metastasis of lymph node, and no-invasiveness of regional tissues than those in the ones of poorly-differentiated adenocaarcinoma, metastasis of lymph node, and invasiveness of regional tissues in gallbladder adenocarcinoma (P<0.05or P<0.01). Unitivariate Kaplan-Meier analysis showed that decreased expression of DFF45(P=0.025) was associated with decreased overall survival. Multivariate Cox regression analysis showed that decreased expression of DFF45was an independent bad-prognostic predictor in gallbladder adenocarcinomaConclusions:The expression of DFF45might be closely related to the clinical biological behaviors and prognosis of gallbladder adenocarcinoma.