Upregulation Of Far Upstream Element- Binding Protein 1(FUBP1) Induces Tumor Proliferation And Tumorigenesis Of Clear Cell Renal Cell Carcinoma

Objective: 1. The clear cell renal cell carcinoma(ccRCC)is the second leading cause of death in adults among all types of urological cancers, which is due to lack of promising prognosis or predictors and effective target therapy.2.Using gene microarray technology to screen out genes that have significant difference between tumor tissues and normal tissues. Finally we focus on The far upstream element (FUSE)-binding protein 1 (FUBP1).3.FUBP1 is a transactivator of human c-myc proto-oncogene transcription, with important roles in carcinogenesis. However, the expression pattern and potential biological function of FUBP1 in ccRCC is yet to be established.Materials and methods:Message RNA and protein levels of FUBP1 were detected in ccRCC tissue samples and matched adjacent noncancerous tissue samples of 56 patients,as well as cell lines including the human renal proximal tubule epithelial cell line HKC and the human ccRCC cell lines( 786-O and caki-1) .Then,correlation analyses were utilized between mRNA expressions of FUBP1 and clinical-pathological parameters.Cell lines and culture: The human renal proximal tubule epithelial cell line HKC and the human ccRCC cell lines, including 786-0 and caki-1, were maintained at 37 ? in a humidified incubator with 5% CO2.FUBP1 was over-expressed and knockdown in the experimental group,and plasmid with the empty vector as control.All the cells were stable transfected and had been detected the efficiency of transfection. The biological function of FUBP1 during tumor cell proliferation was studied by MTS, colony formation, and soft-agar colony formation.Immunohistochemistry:A total of 49 pairs of 4 ?m-thick tissue sections of ccRCC patients sliced from paraffin-embedded blocks were deparaffinized twice with xylene and rehydrated in graded alcohol. The final staining index was determined by multiplying the intensity value with the percentage value, which ranged from 0 to 12. We defined 0 index as negative and 1-12 as positive.Results: 1.The levels of FUBP1 mRNA expression were markedly higher in ccRCC tissues than in the matched adjacent normal renal tissues. Similarly, Western blot analysis of 8 pairs of tissues revealed that the levels of FUBP1 protein expression were significantly increased in ccRCC tissues compared with the corresponding adjacent normal renal tissues(both P<0.05). These results suggested that the high expression of FUBP1 contributed to ccRCC cell proliferation.2. FUBP1 is involved in proliferation of ccRCC cells.Knockdown of FUBP1 protein inhibited the proliferation of 786-O and caki-1 cells, as assessed by MTS assays and a colony formation assay. In addition, we also observed a remarkable decrease in colonies in siRNA-treated 786-0 and caki-1 cells at soft agar colony formation assays.3.FUBP1 knockdown arrests 786-0 and caki-1 cell proliferations at the G1-S phase transition.It showed that FUBP1 knockdown increased the percentage of cells in the G0/G1 peak and decreased the percentage of cells in the S peak as compared with those in the matched controls.4. FUBP1 inhibited cell apoptosis of 786-0 and caki-1.It showed that FUBP1 knockdown of 786-0 and caki-1 cells had a remarkably higher percentage of annexin V-FITC-positive cells than that of the control cells. Consistent with the flow cytometry data,the expression levels of well-defined apoptosis protein markers, such as cleaved caspase 3,increased in the 786-0 and caki-1 cells with the knockdown of FUBP1 expression.5. c-myc and p21 mRNA expression levels are correlated with FUBP1 in ccRCC tissues.Conclusion:This study revealed that FUBP1 was upregulated in ccRCC patients. FUBP1 might play an important role in the initiation and development of ccRCC.