A Study Of The Function And Regulatory Mechanism Of Human Sprouty2 Protein In Renal Cell Carcinoma

Part 1 A study of the expression of Sprouty 2(Sprouty2) protein in renal cell carcinoma(RCC)Objectives: To investigate the expression of Sprouty 2(Sprouty2) protein in renal cell carcinoma(RCC) and try to clarify its association with the Clinicopathologic features and clinical outcome.Methods: RCC samples were obtained from 103 patients treated with radical nephrectomy(92 samples were clear-cell carcinoma and the another 11 samples were papillary carcinoma). The mean (SD, range) follow-up period was 51.2 months (15.5, 8-79). Immunohistochemical staining and Western blot analysis were performed. The degree of expression of Sprouty2 was compared with the clinicopathologic features. Results: Sprouty2 expression was down-regulated significantly in RCC compared with tumor-free kidney, associated with tumor size (P = 0.002),presence of metastasis(P<0.001), high pT stage (P <0.001),high-grade(P<0.001) and low survival(P=0.001). Univariate (P=0.001)and multivariate (P=0.035) Cox regression analysisi revealed thatSprouty2 expression is an independent prognostic parameter in renal cellcarcinoma.Conclusions: The results of this study suggest that inhibition of Sprouty2is associated with progression and recurrence of renal cell carcinoma.Thus, Sprouty2 is a useful independent prognostic marker for thiscondition.tPart 2 A study of the function of human Sprouty2 protein in renal cell carcinomaObjectives: To investigate the contribution of Sprouty2 protein with proliferation and invasion of renal cell carcinoma.Methods: The technique of RNAi was used to construct the plasmid transfected renal cell carcinoma cell line—786-O. MTT and Trans-well method were used to detect the expression level of Sprouty2 protein in 786-O cells and investigated it's influence with proliferation and invasion of renal cell carcinoma.Results: Sprouty2 expression was down-regulated in RCC and theproliferation and invasion of RCC cells were enhanced significantly.Conclusions: The expression lever of Sprouty2 was associated with thefunction of RCC. When Sprouty2 expression was down-regulated, theproliferation and invasion of renal cell carcinoma were enhanced. Thesesupported results of the relationship between the expression lever ofSprouty2 and patients' prognosis in Part 1.Part 3 A study of the regulatory mechanism of human Sprouty2 protein in renal cell carcinomaObjectives: To identify a possible mechanism which can account for the down-regulation of Sprouty2 in RCC.Methods: The 786-O cell was treated respectively with 5-aza-deoxycytidine(5-Aza-CdR) and trichostatin(TSA), MTT method was used to detect the proliferation of 786-O cell. Western blot was used to detect the expression level of Sprouty2 protein. The results were compared with the group which was not treated with the agents. Chromatin immunoprecipitation(CHIP) was performed to investigate the possibility in the regulatory function of Nuclear factor-κB(NF-κB) for the down-regulation of Sprouty2. TNF-α/SN50 were used to activate/inhibiting the expression level of NF-κB, and Western blot was used to detect the change of the level of Sprouty2 protein.Results: The expression of 786-O cell Sprouty2 protein after treated with 5-Aza-CdR and TSA remains a low level, the agents could not recover the Sprouty2 gene activation. The result of CHIP suggested that NF-κB could combine with the promotor of Sprouty2 gene to regulate its transcription. The expression of Sprouty2 and biological behaviour of RCC cells could be changed by activating or inhibiting NF-κB.Conclusions: NF-κB may play a important role as a transcription factor in the silence of Sprouty2 in RCC. The exact regulatory mechanism of human Sprouty2 protein in renal cell carcinoma could be focused on transcriptional level.