| Background and Objective: The vast majority of renal cell carcinoma(RCC)cases are of the clear cell type.In sporadic cc RCC tumors,about 75%-80% of them harbor biallelic inactivation of VHL through mutation,deletion,or hypermethylation of promoter that leads to the loss of its expression.Many studies have already confirmed that inactivation of VHL tumor suppressor gene plays an important role in the pathogenesis of cc RCC.The protein product of VHL tumor suppressor protein,p VHL,is the substrate recognition unit of an E3 ubiquitin ligase complex which targets the alpha subunits of the heterodimeric transcription factor HIF(Hypoxia-Inducible Factor)for ubiquitylation and destruction.Under normal oxygen tension,p VHL combines with HIF? then leads to quick proteasomal degradation.Increased HIF activity as a result of VHL inactivation further regulates transcription of HIF-responsive genes(HRGs).The effect of HIF-2? on 786-O cell lines was confirmed by the experiment in nude mice.However,some H R G s e n h a n c e d t u m o r g r o w t h,s o m e d i d n o t h i n g,w h i l e s o m e w e r e tumor-suppressive.The contributions to tumor growth by HRGs involved were not fully explored.We find DBC1 and STC1 are HRGs and regulated by HIF-2? and are essential for growth of some human malignant tumors.In order to improve the prognosis of patients with metastatic renal cell carcinoma,we examine the contribution of DBC1 and STC1 in VHL-/-RCC cells in this study.Methods:1.Renal cell carcinoma cell 786-O cells lines(VHL-/-?VHL+/+?VHL-/-HIF-2?sh566?VHL+/+ HIF-2?-d PA)was constructed and preserved in the lab.Real-time PCR detected the expression of DBC1 and STC1 in four RCC 786-O cells.2.Established the 786-O(VHL-/-)cells lines with and without DBC1/STC1 knocked down by lentiviral transfection carring sh RNA DBC1/STC1 and SCR respectively,the expression of DBC1 and STC1 protein was detected by Western Blot.3.In vitro cell proliferation assays in such lines were compared with the VHL-/-786-O cells expressing sh RNA and SCR useing MTT.4.VHL-/-786-O cells lines infected with sh RNA DBC1/STC1 were injected subcutaneously to one side of a nude mouse,and the same parental cells with SCR respectively were injected subcutaneously to another side of the same nude mouse,then we compared the tumor growth.The growth rates of the tumors evaluated by T test.Results:1.The expression of DBC1 and STC1 in 786-O(VHL+/+?VHL-/-HIF-2?sh566)cells was lower than that in 786-O(VHL-/-?VHL+/+ HIF-2?-d PA)cells,showing that DBC1 and STC1 are HIF-responsive genes and tightly regulated by HIF's transcriptional activity.2.MTT showed that suppression of DBC1 had no effect on the proliferation of 786-O VHL-/-cells in vitro,while suppression of STC1 imparied the proliferation of 786-O VHL-/-cells in vitro.3.Suppression of DBC1 and STC1 impaired tumor growth and the difference between the two groups were statistically significant(P<0.01).Conclusion:1.DBC1 and STC1 both are HRGs and regulated by HIF-2?.2.Suppression of DBC1 had no effect on the proliferation in vitro,while suppression of STC1 imparied the proliferation of 786-O VHL-/-cells in vitro.But both DBC1 and STC1 significantly impaired tumor growth of nude mice,suggesting that they are tumor-promoting genes in RCC and they could be potential drug targets for patients with m RCC.3.VHL-HIF-HRGs regulatory pathway is the key to the pathogenesis of RCC,there might be other HRGs that mediate the critical oncogenic activity of HIF in RCC.In oreder to find more genes can be targeted for efficient therapeutic intervention in RCC,further study is needed to understand the role of HRGs in VHL-HIF-HRGs-associated tumorigenesis. |
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