| Part I Differential analysis of the genes in gallbladder cancer with TGF-β1induced epithelial-mesenchymal transitionObjectiveTo explore the mechanism of invasion and metastasis of gallbladder cancer, gene chips were applied to investigate the differential expression of genes between gallbladder cancer cells with TGF-β1induced epithelial mesenchymal transition (EMT) and gallbladder cancer cells without TGF-β1induced EMT, which might provide potential therapeutic intervention of gallbladder cancer.MethodsThe preliminary research in our lab had optimazied5ng/ml as the concentration of TGF-β1for EMT induction. GBC-SD cells with and without TGF-β1induced EMT were cultured, then the total RNA were extracted and labeled with fluorescent probe. The hybridazition was performed by using the human genome oligonucleotide microarray containing21,522transcripts. The image data were initially collected by LuxScan10KA dual Channel laser scanner. Final analysis was conducted by MAS2.0.Results264differentially expressed genes were definited between GBC-SD cells with and without TGF-β1induced EMT, among which166genes were up-regulated and98genes were down-regulated in gallbladder cancer cells after TGF-β1induced EMT. According to GO classification, the differentially expressed genes belong to the groups such as reduction, oxidation, protein binding, and cell adhesion. Most interestingly, revealed by the pathway analysis, these genes were associated with the cyclin dependent kinase pathway regulation, intrinsic prothrombin activation pathway, cell cycle regulation, phosphoinositide-mediated signaling, etc.ConclusionsThrough the anlysis and comparison of the gene expression profile, quite a few differentially expressed genes were newly identified. These genes were involved in a variety of signaling pathways. After reference review, we thought NT5E and FcGBP might play important roles in the invasion and metastasis of gallbladder cancer with TGF-β1induced EMT. So these two genes were choosed for further research. Part Ⅱ The significance of NT5E and FcGBP expression in gallbladder adenocarcinomaObjectiveThe in vivo study of gallbladder adenocarcinoma and chronic cholecystitis was performed to verify the results obtained in microarray study and further investigate the role of NT5E and FcGBP in invasion and metastasis of gallbladder adenocarcinoma.MethodsThe total RNA and protein were extracted from specimens both of15patients diagnosed as gallbladder adenocarcinoma and15patients diagnosed as chronic cholecystitis. The mRNA transcription and protein expression levels of NT5E and FcGBP were evaluated by RT-PCR and Western-blot respectively. Statistic analysis was performed afte data collectionResults1. NT5E mRNA was significantly lower in chronic cholecystitis than that in gallbladder adenocarcinoma,0.225±0.021vsl.012±0.019, P<0.01,while FcGBP mRNA was significantly higher in chronic cholecystitis than that in gallbladder adenocarcinoma (0.321±0.017vs0.078±0.020, P<0.01)2. Western blot demonstrated that NT5E expression was significantly higher in gallbladder adenocarcinoma than that in chronic cholecystitis (0.826±0.011vs0.412±0.017, P<0.01),while the expression of FcGBP protein was significantly lower in gallbladder adenocarcinoma than that in chronic cholecystitis (0.084±0.017vs0.187±0.025, P<0.01)ConclusionsNT5E expression was upregulated and FcGBP expression was downregulated in gallbladder adenocarcinoma compared to chronic cholecystitis in vitro that was constistent with in vitro study in part one.It was suggested that NT5E and FcGBP might play an important role in the process of gallbladder epithelial malignant transformationwhich is associated with gallbladder adenocarcinoma invasion and metastasis.Part Ⅲ Differential expression of NT5E and FcGBPand their clinicopathological significance in benign and malignant lesions of the gallbladderObjectiveThe differential expression of NT5E and FcGBP in benign and malignant lesions of gallbladder is investigated immunochemically and the clinicopathological significance between them is evaluated.MethodsSpecimens of gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenoma (n=30), polypus (n=15) and chronic cholecystitis (n=35) were paraffin-embedded to sections which detected by En VisionTM immunohistochemistry to evaluate the expression level of NT5E and FcGBP.Results(1) The NT5E expression was significantly up-regulated in gallbladder adenocarcinoma (54.6%) than that in peritumoral tissues (30.4%) and other benign lesions including adenoma (16.7%), polypus (13.3%) and chronic cholecystitis (11.4%)(P<0.01). The ratio of positive FcGBP expression was significantly down-regulated in gallbladder adenocarcinoma (48.1%) than that in peritumoral tissues (76.1%), adenoma (80.0%), polypus (86.7%) and chromic cholecystitis (85.7%)(P<0.05). Atypical hyperplasia in gallbladder epithelium was observed in the benign lesions with NT5E and/or FcGBP expression.(2) The NT5E expression in well-differentiated adenocarcinoma, small tumor size (diameter<2cm), no lymph node metastasis and no peripheral tissue invasion was significantly down-regulated than that in poor-differentiated adenocarcinoma, larger tumor size (diameter≥2cm), lymph node metastasis and peripheral tissue invasion (P<0.5or P<0.01). However, the expression of FcGBP showed a negative correlation in those cases (P<0.05or P<0.01).(3) The monovariable Kaplan-Meier survival analysis showed that the pathological type, tumor maxium diameter, lymph node metastasis, peripheral tissue invasion, NT5E and FcGBP expression were significantlyrelated to the average survival time of patients with the gallbladder adenocarcinoma (P<0.05or P<0.01). Furthermore, the survival time of patients with NT5E expression was significantly shorter than those without NT5E expression (P<0.05). And FcGBP showed a negative correlation in those cases (P<0.05). Interestingly, the overall survival time of cases withNT5E(-)FcGBP(+) was significantly longer than those with NT5E(+)FcGBP(-). Multivariable Cox analysis revealed that NT5E expression (P=0.019) or FcGBP expression (P=0.004) was negatively associated with survival period and positively associated with mortality after surgery.ConclusionsThe expression of NT5E and FcGBP in gallbladder adenocarcinoma is an independent marker for evaluation of the disease progression, clinical biological behaviors and prognosis. Patients with FcGBP expression had a better prognosis while patients with NT5E expression got a worse prognosis.
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