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Interferon-gamma Inhibited the Growth of Renal Cell Carcinoma throgh Hepacam via MAD1-dependent Manner

Author: ChenXiong
Tutor: WuXiaoHou
School: Chongqing Medical University
Course: Surgery
Keywords: hepaCAM Mitotic arrest deficiency-1 renal cell carcinomarenal renal carcinoma interferons hepaCAM protein Mitotic arrest deficiency-1protein cell proliferation
CLC: R737.11
Type: Master's thesis
Year: 2013
Downloads: 19
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PART ONE The expression of hepaCAM and Mitotic arrestdeficiency-1(MAD1) in renal cell carcinoma and adjacent tissues andthe correlation between hepaCAM and MAD1protein in RCC tissues.Objective: To examine the expression of hepaCAM and Mitotic arrestdeficiency-1(MAD1) in renal cell carcinoma and adjacent tissues.And toassay correlation between hepaCAM and MAD1protein and thecorrelations among each clinical index in RCC tissues.Methods: Immunohistochemical method was used to examine theexpression of hepaCAM and Mitotic arrest deficiency-1(MAD1) in renalcell carcinoma and adjacent tissues.Result: All the37cancer tissues was shown no expression ofhepaCAM or MAD1, while all adjacent tissues existed in various degree(hepaCAM, P<0.01and MAD1, P<0.01). HepaCAM expression mainly existed on the membrane, slightly on the cytoplasm, did not on the nucleus.Thirty-three adjacent tissues expressed MAD1mainly on the membraneand cytoplasm, while the other4expressed on the membrane, cytoplasmand nucleus (Figure1). There were no significant differences toward thedeficiency of hepaCAM and MAD1expression and various clinicalparameters (Table1and Table2). The results also revealed a positivecorrelation between hepaCAM and MAD1protein in same renal cellcarcinoma tissues (P<0.01, R2=0.881, Figure2).Conclusions: Both the protein expression of hepaCAM and MAD1were lost in renal cell carcinoma tissues. And the results also revealed apositive correlation between hepaCAM and MAD1protein in same renalcell carcinoma tissues. These offered a basis to investigate a mechanismthat how hepaCAM and MAD1to play a role in development and treatmentof renal cell carcinoma. PART TWO Effect and mechanism of Interferon-gamma on renalcancer786-0cells proliferationObjective: To investigate effect of proliferation of renal cancer786-0cellsafter being treated with γ-interferon (IFN-γ) and its molecular mechanism. Methods:786-0cells were treated with IFN-γ at five different finalconcentrations (1000U/mL,2000U/mL,3000U/mL,4000U/mL,5000U/mL), detecting the cells proliferation inhibition rate by CellCounting Kit-8method at three time points (24h,48h,72h). The finalconcentration (900U/mL) was then used to stimulate cells48h,and serve asthe experimental group, without IFN-γ povoked as a control group. Cellcycle distributions were analyzed by flow cytometry, expression ofhepaCAM gene assessed by RT-PCR and levels of MAD1protein weredetermined by Western blotting. The recombinant adenovirs vectorsplasmid containing hepaCAM gene or without were transfected into786-0cells, and then set up three groups: hepaCAM-adenovirus vectorgroup,empty-adenovirus vector group and control group. The hepaCAM andMAD1expression at protein level was measured by Western blotting.Results: The cell proliferation of786-0was suppressed in a time-dosedependent manner (n=5, P <0.05) and IC50of786-0cells affected by IFN-γat48h were900U/mL. Moreover,786-0were incubated with IFN-γ werearrested at G0/G1phase (n=3, P <0.01) and up-regulated the level ofhepaCAM mRNA (n=3, P <0.01), but no changes at the protein level (datanot show). Western blotting analysis showed that IFN-γ elevated thelevels of MAD1protein (n=3, P <0.05), hepaCAM adenovirus vectorsplasmid was successfully constructed and stably transfected into786-0 cells. Compared to the empty-adenovirus vector group and control group,the protein expression of MAD1is markedly increased (n=3, P<0.05).Conclusion: Taken together, our studies suggest that IFN-γ inhibits theproliferation of786-0maybe through re-expression of hepaCAM andarresting cancer cells at G0/G1phase via a MAD1-dependent manner.

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