| Gallbladder cancers (GBC) are aggressive cancers with extremely poor prognosis. Surgery was recommended for treatment, while most patients were diagnosed at an advanced, inoperable stage with metastasis and invasion to other organs. Its clinical presentation is nonspecific and may include abdominal pain, weight loss, fever, and jaundice. The incidence of GBC was alarmingly increasing in recent years.Emerging evidence has shown that the abilities for tumor growth and propagation reside in a small population of tumor cells, termed cancer stem cells (CSCs) or tumor-initiating cells. These cells possess properties of self-renewal, differentiation potential, resistance to chemotherapy, and high tumorigenicity. It is generally considered that the identification of the CSCs could have a significant impact on the understanding of tumor biology and therapy. The newly developed "DNA microarray" technology was used to detect thousands of gene expression simultaneously, and some important cancer-related gene had been identified by DNA microarray. By using DNA microarray, we found the differentially expressed genes compared the normal cells from gallbladder and gallbladder cancer cell line (GBC-SD), and the CSC-related genes were screened by bioinformatics technology. The expressive levels of the CSC-related genes and their clinicopathological significances in the benign and malignant lesions of gallbladder were verified, and the gene function was revealed by using RNA interference. The research includes the following four parts:Part I Differential expression genes of normal gallbladder cells comparing to gallbladder cancer cellsObjectiveScreening the differential expressed genes between normal gallbladder cells and gallbladder cancer cells by DNA microarray, to give grounds for screening the cancer-stem-cell related genes in gallbladder cancer.MethodsGBC-SD cells are continuous passage cultured while normal gallbladder epithelial cells primary cultured, and their RNA was extracted respectively. Afterwards these RNA was labeled by fluore-scent probe and hybridization with the22K human genome array was performed. The image scanning was performed by LuxScan10KA dual Channel laser scanner, and the fluorescence intensity was analyzed to verifying the differential expression genes. All the data were analyzed by MAS2.0.Results2,288genes were verifyed between GBC-SD cells and normal gallbladder cells. There are814up-regulated genes and1474down-regulated genes in gallbladder cancer cells. The genes were involved in several molecular process such as cell cycle, cell proliferation, actin adjustment, cell apoptosis, etc. While the pathway analysis revealed the connection of these genes with MAPK pathway, Wnt pathway, TGF-β pathway, VEGF pathway, T-cell receptor pathway, ErbB pathway, Jak-STAT pathway etc.ConclusionsThe DNA microarray revealed the gene expression profiles of normal gallbladder cells and GBC-SD cells, and the differentially expressed genes were identified. Among them the ALDH1A3and GPX3genes may be correlated with cancer stem cells in gallbladd-er cancer, and further study was preceded in following steps. Part II The mRNA and protein expression of ALDH1A3and GPX3in gallbladder adenocarcinoma and chronic cholecystitisObjectiveDetect the mRNA and protein expression of ALDH1A3and GPX3in gallbladder cancer and chronic cholecystitis, and comfirm the results of DNA microarray.MethodsFresh tissue samples from gallbladder adenocarcinomas(n=15) and chronic cholecystitis(n=15) were collected, and the total RNA and protein were extracted. The expression of ALDH1A3and GPX3mRNA was evaluated by RT-PCR, while the expression of protein was evaluated by Western-blot. All data were statistically analyzed.Results1. The expression of ALDH1A3mRNA was significantly lower in chronic cholecystitis than that in gallbladder adenocarcino-ma (0.312±0.019vs1.255±0.042, P<0.01), while the GPX3mRNA was significantly higher in chronic cholecystitis than that in gallbla-dder adenocarcinoma (1.187±0.013vs0.107±0.015, P<0.01).2. The expression of ALDH1A3protein was significantly lower in chronic cholecystitis than that in gallbladder adenocarcino-ma (0.711±0.022vs2.121±0.226, P<0.01), while the GPX3protein was significantly higher in chronic cholecystitis than that in gallbla-dder adenocarcinoma.ConclusionsThe expression of ALDH1A3mRNA and protein was higher in gallbladder adenocarcinoma than that in chronic cholecystitis, and the expression of GPX3mRNA and protein was lower in gallblad-der adenocarcinoma than that in chronic cholecystitis. The results were in line with the DNA microarray. ALDH1A3and GPX3may be related with cancer stem cells, which may affect the carcinoge-nesis of gallbladder adenocarcinoma.Part Ⅲ Expression of ALDH1A3and GPX3in benign and malignant lesions of the gallbladder and their clinicopathological significance in GBCObjectiveDetect the expression of ALDH1A3and GPX3and reveal their clinicopathological significance in the benign and malignant lesions of gallbladder.MethodsThe expression level of ALDH1A3and GPX3was evaluated by En VisionTM immunohistochemistry in routinely paraffin-embedded sections from specimens of benign and malignant lesions of gallbla-dder, which include100gallbladder adenocarcinoma,46peritumor-al tissues,30adenoma,15adenomatous polyps and35chronic cho-lecystitis.Results(1) The positive rates of ALDH1A3expression was significan-tly higher in adenocarcinoma of gallbladder than that in peritumor-al tissues, adnoma, adenomatous polyps and chronic cholecystitis (P <0.01), while the positive rates of GPX3expression was significant-ly lower in adenocarcinoma than that in peritumoral tissues, adnoma, adenomatous polyps and chromic cholecystitis (P<0.05or P<0.01). The positive cases of ALDH1A3and/or negative GPX3in the be-nign lesions showed moderately-or severely-atypical hyperplasia in gallbladder epitheli.(2) The positive rates of ALDH1A3were significanctly low-er in the cases of well-differentiated adenocarcinoma, maximal dia-meter of mass<2cm, no-metastasis of lymph node, and no-invasiveness of regional tissues than those in the ones of poorly-differentiated adenocarcinoma, maximal diameter of mass>2cm, metastasis of lymph node, and invasiveness of regional tissues in gallbladder adenocarcinoma (P<0.05or P<0.01). In contrast, the expression of GPX3showed an opposite correlation in these cases (P<0.05or P<0.01).(3) The tumor size (p=0.03), lymph node metastasis (p=0.005), and invasiveness of regional tissues (p=0.002) were significantly associated with the average survival time in patients with GBC. The average survival time in ALDH1A3(+) or GPX3(-) patients was significantly lower than that having ALDH1A3(-) or GPX3(+)(p ALDHIA3=0.011and p GPX3=0.017); In addition, the avera-ge survival time in patients having ALDH1A3(+) and GPX3(-) exp-ression was significantly lower than in the ALDH1A3(-) and GPX3(+)(p=0.006). Cox multivariate analysis showed that ALDH1A3(+)(p=0.005) and GPX3(-)(p=0.010) expression had negative correlation with postoperative survival and positive correlation with mortality, suggesting that ALDH1A3and GPX3are independent risk factor on prognosis.ConclusionsThe expressions of ALDH1A3and/or GPX3were different in benign and malignant gallbladder lesions, and they are independent markers for reflecting the clinical biological behavior and prognosis of GBC. Part IV Identifying the function of ALDH1A3gene in GBC-SD cells in vitro by RNA interferenceObjectiveObserve the alterations of proliferation and invasive ability of GBC-SD cells by transfecting recombinant pRNA-siALDHlA3vector in vitro.MethodsGBC-SD cells without transfection and GBC-SD cells that transfected with pRNA-siNC (unrelated sequence) were set as contr-ol group, and three vectors (pRNA-siALDH1A3-1,2and3) were transfected into GBC-SD cells by using Liposome2000. The pRNA-siALDH1A3-1was verified to be the most effective recombinant vector by detecting fluorescence intensity of GFP. The proliferation activity was measured by MTT assay, and the changes of invasion and migration abilities were tested by scratch test and transwell assay.Results1. Visible green fluorescence protein could be found in GBC-SD cells that transfected with pRNA-siNC, pRNA-siALDH1A3-1,2and3through the inverted fluorescence microscope, confirming successful transfection. The most effective recombinant vector was pRNA-siALDH1A3-1, and green fluorescence protein could not be found in the GBC-SD cells without transfection. Thus pRNA-siALDH1A3-1was selected in following assays.2. MTT assay indicated that the ability of proliferation of GBC-SD cells that transfected with pRNA-siALDH1A3-1was depressed than that of pRNA-siNC group and non-transfected group cells from3rd day and continued to6th day (P<0.05). The proliferation ability of pRNA-siNC group and non-transfected GBC-SD cells had no statistical difference (P>0.05).3. GBC-SD cells transfected with pRNA-siALDH1A3-1, pRNA-siNC and non-transfected were observed at0hour and48hour for the scratch tests. The blank areas of the scratch in three groups were the same at the0hour. After48hours, a small num-ber of cells that transfected with pRNA-siALDH1A3-1migrated to the blank area, while the cells transfected with pRNA-siNC and non-transfected cells both migrated obviously.4. Transwell assay was carried out in GBC-SD cells that transfected with pRNA-siALDH1A3-1, pRNA-siNC and non-transfected. Cells that traveled across the artificial basement membrane of the well were27.5±2.38,55.4±5.43and59.8±6.23, respectively. The migratory ability of GBC-SD cells that transfected with pRNA-siALDHlA3-1was lower than that of pRNA-siNC and non-transfected cells (P<0.05), while no difference emerged between the cells transfected with pRNA-siNC and non-transfected cells.ConclusionsThe expression of ALDH1A3gene in GBC-SD cells was signif-icantly refrained after transfected with interference vector pRNA-siALDH1A3. Down regulation of ALDH1A3, which was a cancer-stem-cell-related gene, can reduce the proliferation activity and weaken the invasive ability in vitro of GBC-SD cell. |
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