| Objective:To explore the expression and clinical significance of HCCR in pancreatic tumor and cell lines. To study the molecular mechanism of HCCR expression upregulation in PAN1 cell line via a PI3K/Akt/mT0R pathway treated with EGF.Methods:1. 178cases of pancreatic tumor, 47cases of paraneoplastic tissue and benign tumor were constructed on a tissue chip, and the expression and of HCCR and survivin were investigated by immunohistochemistry assay. The expression of HCCR protein in pancreatic tumor, paraneoplastic tissue and pancreatic cell line were determined by Western blot technique. The subcellular location of HCCR in PANC1, CFPAC1 and SW1990 pancreatic tumor cell lines were detected by immunofluorescence.2. Treated with increasing density(0,50,100,200ng/ml) of EGF for 24h, or with 100ng/ml EGF at different time(0,8,16,24,48,72h), the pancreatic tumor cells PANC1 were routine cultured. The expression of HCCR protein was detected by Western blot, and a properly EGF density and culture time was selected as the most suitable culture condition.3. PANC1 cells were cultured with EGF(100ng/ml) for different time(0,2,5,10,20,30min), the expression of phospho-Akt were detected by Western blot. Cells were pretreated with the specific inhibitor of PI3K and mTOR (LY294002 and rapamycin respectively) and then cultured with EGF(100ng/ml) for 24h, the expression of HCCR protein in these cells were measured by Western blot.4. PANC1 cells were transfected with constitutively active Akt kinase, dominant negative Akt kinase and HCCR1 by lipofectmine 2000, transfectants were selected and confirmed using Western blot technique. HCCR was measured on mRNA and protein level by RT-PCR, Western blot. The reproductive activity was detected by MTT assay.Results:1. The expression of HCCR in pancreatic cancer and cell lines.The HCCR protein was mainly located in the cell membrane and cytoplasmic in pancreatic cancer cells. The expression of HCCR and survivin were higher in pancreatic tumor, 77.5%(138/178) and 78.3%(137/175) respectively, than in paraneoplastic tissues and benign tumors(P<0.05). Western blot revealed HCCR expression was higher in pancreatic tumor than in paraneoplastic tissues. HCCR were overexpressed in pancreatic cell lines(PANC1,CFPAC1,SW1990).2. The relationship between HCCR expression and clinicopathologic features in pancreatic cancer.The HCCR expression was related to pathological stage (x~2=8.506 P<0.05 ), and it was higher in poorly differentiated pancreatic cancer. The expression of HCCR had no relation with lymphnode metastasis and nerve infiltration. The expression of HCCR and survivin coordinated in pancreatic cancer (r=0.236, P=0.002 ) .3. EGF treatment induced HCCR expression in PANC1 pancreatic cancer cell.In PANC1 cells, HCCR protein expression gradually increased in a time-dependent manner after treated with EGF at 100ng/ml for 16h to 72h. Cells were cultured with EGF at 100ng/ml or 200ng/ml for 24h, the HCCR protein were upregulated in a dose-dependent manner. EGF concentration at 100ng/ml and culture for 24h were employed for all subsequent experiments.4. EGF upregulated HCCR via a PI3K/Akt/mTOR signal transduction pathway in PANC1 cells.(1) EGF activated Akt in PANC1 cells. The phosphor-Akt was obviously increased with EGF treatment at 100ng/ml for 2min, and then gradually decreased. (2) The increased HCCR expression were depended on the activation of Akt in PANC1 cells. HCCR protein and mRNA were upregulated in constitutively active Akt transfectants, while downregulated in dominant negative active Akt. (3) Treatment of the cells with PI3K inhibitor (LY294002) or mTOR inhibitor (rapamycin) both significantly suppressed EGF induced HCCR expression. These results show that the PI3K/Akt/mTOR pathway is involved in the stimulation of HCCR production by EGF.5. HCCR overexpression promoted PANC1 cell proliferation.PANC1 cells were transfected with HCCR1. Increased reproductive activity was found in HCCR1 transfectants compared with vector transfected cells.Conclusion:1. The HCCR protein was mainly located in the cell membrane and cytoplasmic in pancreatic cancer cells, and it was overexpressed in pancreatic cancer and cell lines. The HCCR expression was related to pathological stage, HCCR may involve in the generation and development of pancreatic cancer. The expression of HCCR and surviving is coordinated in pancreatic cancer, which indicate they may inhibited cell apoptosis via involving in p53 network.2. EGF unregulated HCCR in pancreatic cancer cell PANC1 in a time-dependent and dose-dependent manner. EGF induced HCCR via a PI3K/Akt/mTOR signal transduction pathway in PANC1 cells. HCCR overexpression promoted PANC1 cell proliferation.
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