Pokemon/miR-137 Auto-regulatory Circuit Promotes The Progression Of Renal Carcinoma

Kidney cancer is a type of malignant tumor with higher morbidity and mortality in the past decade.Despite the advance in the therapeutic modalities,renal cell carcinoma patients benefit little from the current treatments,such as surgery,chemotherapy and radiotherapy.One of the major obstacles hampering kidney cancer treatment is that the underlying molecular mechanism has not been completely elucidated yet.Therefore,the identification of new genes and regulators involved with the initiation and progression of renal cancer is always of interest in the field of cancer research.Pokemon is a master oncogene identified to drive other genes to promote the progression of human tumors.Unlike its established role in many other types of cancers,the implication of Pokemon in renal cancer has not been studied yet.Furthermore,the molecular mechanism of Pokemon overexpression in cancers is still unknown.miRNAs have been well recognized as a key modulator for cancer initiation and progression.These non-coding RNAs can both act as oncogene and tumor suppressor,depending on their targets or the context.For renal cancer,miRNAs have been also shown to be associated with gastric cancer.miR-137,a tumor suppressor miRNA known for glioma and head neck cancer,is shown to be downregulated in renal cancer specimen and cell lines.The restoration of miR-137 was identified to suppress the growth of kidney cancer both in vitro and in vivo.However,mRNA molecules targeted by miR-137 is still not identified in renal cancer.Part One The expression of Pokemon in RCC tissue and RCC celllinesMethods:1.Tissue collection:Human RCC tissues and normal renal tissues of adjacent of cancer were harvested from patients with RCC who underwent tumor resection at the Department of Urology,2.Cell culture:Human RCC cell lines786-0,ACHN,Caki-1,Caki-2 and the nomal renal cell line HEK-293 cells were grown in 10%FBS-containing media recommended by ATCC in a 37℃atmosphere with 5%C02.3.The expression of Pokemon mRNA between RCC tissues and normal renal tissues of adjacent of cancer,and between RCC cell lines 786-0,ACHN,Caki-1 and Caki-2 and normal renal cell line HEK-293 were detected by qRT-PCR.4.The expression of Pokemon protein between RCC tissues and normal renal tissues of adjacent of cancer,and between RCC cell lines and normal renal cell line HEK-293 were detected by Western blotting.5.Statistical analyses were done using SPSS.The statistical significance of the difference was analyzed by ANOVA or post hoc Dunnett’s test.P-values<0.05 were considered to be statistically significant.Results:1.Pokemon was up-regulated in RCC tissues,as compared with that in the normal renal tissues of adjacent of cancer.2.Pokemon was up-regulated in RCC cell lines 786-0,ACHN,Caki-1 and Caki-2,as compared with that in HEK-293.Conclusion:Pokemon was up-regulated in RCC tissues and RCC cell lines 786-0,ACHN,Caki-1 and Caki-2.Part Two The effect of Pokemon inhibition or expression on the RCC cells or nomal kidney cells in vitro or in vivoMethods:1.Cell transfection was used to detect the effect of different vectors on cells.2.The proliferation of cells was analyzed by MTT assay.3.Transwell assays were used to detect the migration of cells.4.Establish a RCC xenograft in nude mice by inhibiting Pokemon gene,and the size and weight of tumor were monitored.Results:1.The proliferation and migration of RCC were inhibited when interfere the Pokemon gene.2.The proliferation and migration of nomal kidney cells were enhanced when up-expression the Pokemon gene.3.The tumor growth was inhibited when interfere the Pokemon gene in vivo.Conclusion:The proliferation,migration of RCC cells were regulated when interfere or over-expression of the Pokemon gene in vitro and in vivo.Part Three The mechanism research of miR-137 and Pokemon in renal cell carcinomaMethods:1.We instruct miR-137 mimics and a series of plasmids included psiCheck2,psiCheck2-MRE-wt and psiCheck2-MRE-mut.2.Luciferase assays were subjected to the quantification of luciferase expression level with Dual-Luciferase(?)Reporter Assay System/Dual-Luciferase(?)(Progema)according to the manufacturer’s instructions.Results:1.Pokemon is an authentic target of miR-13 7 in renal cancer cells,miR-137 can inhibit the expression of Pokemon.2.Pokemon suppresses the transcription of miR-137 by binding to its promoter.Conclusion:miR-137/Pokemon autoregulatory loop contributes to the proliferation and invasion of renal cancer cells.