Over-expression Of FBXW7 Promotes The Apoptosis Of Renal Carcinoma Cells

Objective:(1) To discuss the differences between the renal carcinoma tissues and para-carcinoma tissues in FBXW7 and the influences of FBXW7 on patient's prognosis through immunohistochemical detection on the expressions of FBXW7 in renal carcinoma tissues and para-carcinoma tissues.(2) To detect the viability and proliferation of renal carcinoma cells with over-expression of FBXW7 through CCK8, colony formation experiment, and flow cytometry on cell cycle and apoptosis.(3) To explore the relationship between FBXW7, c-myc and c-Jun by q PCR and Western blot.Methods: A total of 105 cases of renal carcinoma and corresponding para-carcinoma pathological samples were collected from Guangdong Second People's Hospital between 2008 and 2012. Immunohistochemical technique was used to dye the samples, and the expressions of FBXW7 were judged according to the color of the samples. q PCR was used to detect the m RNA expressions of FBWX7 in human renal carcinoma ACHN cells and A704 cells of the control group and the experimental group. Western blot was used to detect the protein expressions of FBWX7 as well as protein expressions of c-myc and c-Jun in ACHN cells and A704 cells of the control group and the experimental group. The control group and the experimental group were compared in the number of cloned cells, OD values and the changes in cell apoptosis and cell cycle by functional experiments such as colony formation and CCK8 to judge the influences of high expressions of FBXW7 on proliferation ability of renal carcinoma cells.Results: Immunohistochemical detection on protein expressions of the pathologic samples showed most of the carcinoma tissues were light dyed, while most of the para-carcinoma tissues were deeply dyed. Comparison on immunohistochemical integral(P<0.0001) indicated that FBXW7 expression in the carcinoma tissues was greatly lower than that of the para-carcinoma normal tissues. Postoperative survival of the high FBXW7 expression group(n=59) was obviously higher than that of the low FBXW7 expression group(n=22, log-rank test, P<0.001). Detection on cell line with western and Real-Time q PCR found that m RNA and protein expressions of FBXW7 in ACNH and A704 cells that were transfected by FBXW7 plasmid were obviously increased, compared with the control group, while c-myc and c-Jun proteins were significantly decreased. The absorbance of cells with FBXW7 plasmid at the wavelength of 450 nm was obviously lower than the control group that was transfected with blank plasmid, and the cell proliferation was significantly weakened(Vector vs. Plasmid A704: 1.327 ± 0.036vs1.035 ± 0.042 P<0.05; ACHN: 1.403 ± 0.024 vs. 1.166 ± 0.029 P<0.05). Cells with less than 50 clones in the experimental group were obviously less than that of the control group, and the proliferation ability of cells with FBXW7 plasmid was significantly inhibited(Vector vs. Plasmid ACHN: 567±19 vs. 412±21 P<0.05; A704: 279 ±13 vs. 144±7 P<0.05). Cell cycle was detected with flow cytometry instrument by PI method. In ACHN and A704 cells with high FBXW7 expression, the proportion of G0/G1 stage in the experimental group was higher than that of the control group(Vector vs. Plasmid ACHN 51.93% vs. 63.25% P<0.05; A704 60.31% vs. 68.32% P<0.05). ACHN and A704 cells with high FBXW7 expression expressed more early and late apoptosis than that of the control group(Vector vs. Plasmid ACHN 5.1% vs. 8.5% P<0.05; A704 14.1% vs. 20.4% P<0.05).Conclusion:(1) FBXW7 expression level in cancerous tissue is much higher than that of para-carcinoma tissues, and the vitality and proliferation ability of renal carcinoma cells with high FBXW7 expression are obviously lower than those of the control group. 59 cases are survived in the high FBXW7 expression group while 22 cases survive in the low FBXW7 expression group. The survival rate of high FBXW7 expression group is obviously higher than that of the low FBXW7 expression group(log-rank test, P<0.001). Results show that FBXW7 can play a tumor suppressor role.(2) Western blot finds that the expressions of c-myc and c-Jun are decreased with the increase of FBXW7, suggesting that FBXW7 may play a tumor suppressor role by reducing c-myc and c-Jun.