Study On The Role Of SnoRNAs As New Biomarkers And N The Regulation Of Biological Behavior For CcRCC

Background:In recent years,renal cell carcinoma has been increasing at an annual rate of 2%-3%and has become the tenth most common cancer in urban areas of China,and the sixth most common cancer for male in the world.Due to the hidden location of kidney and lack of effective biomarkers for screening early renal cancer patients,about 30%of patients are diagnosed clearly in the progression of the tumor and lose the chance of surgical treatment.Therefore,it is urgent need to study the molecular regulation mechanism in the process of renal cancer development,which would provide sensitive and specific biomarkers for early diagnosis of renal cancer and assist decision-making for clinician.Small nuclear RNA(snoRNA)is widely distributed in the nucleolus of eukaryotic cells and is a kind of small molecule non-coding RNA of medium size.It mainly participates in the regulation of RNA maturation and post transcription chemical modification.The increasing evidence has indicated that snoRNAs are closely related to the occurrence and development of tumor.In addition,snoRNAs are not degraded by RNase and can be stably expressed and detected in body fluids,which has potential as tumor markers.However,at present,the role and mechanism of snoRNAs in the development of renal cancer are not clear.Therefore,we will verify the ability of snoRNAs in plasma and urine sediment to be novel biomarkers for the diagnosis of renal cancer.Moreover,we take SNORD4A as the research object to explore the molecular regulation mechanism of SNORD4A in the development of renal cell carcinoma.Methods:Based on the TCGA data of clear cell renal cell carcinoma(ccRCC),the differentially expressed snoRNAs in ccRCC were screened.RT-PCR was used to test snoRNAs expression in paraffin tissue,plasma,urine sediment for ccRCC patients.Then,the receiver operator characteristic(ROC)curves were applied to calculate the specificity and sensitivity.Moreover,area under curve(AUC)was used to evaluate diagnostic efficiency;Next,we took SNORD4A as the research object to explore its regulation on the biological behavior of renal cell carcinoma.The subcellular localization of SNORD4A was determined by RNA FISH and RT-PCR.Using a series of experimental methods,such as cell proliferation assay,colony formation assay and apoptosis assay,we explored the effects of SNORD4A on the biological behavior of RCC in vitro.Moreover,Xenograft tumor experiment in nude mice was used to study the effect of SNORD4A in vivo.Protein mass spectrometry was used to screen the differentially expressed proteins.Finally,using western blot we analyzed the differentially expressed and related molecular pathway proteins.Results:Firstly,four kinds of differentially expressed snoRNAs,including SNORD4A,SNORD63,SNORD96A and SNORD93,between RCC and healthy volunteers were screened according to TCGA database.The expression of these snoRNAs in ccRCC FFPE tissue and urinary sediment was increased(P<0.05).Moreover,the expression of SNORD96A in plasma of ccRCC patients was significantly increased(P<0.0001),while the expression of SNORD4A,SNORD63 and SNORD93 in plasma had no significant difference(P>0.05).Urinary sediment SNORD4A,SNORD63,SNORD96A and SNORD93 could be used as biomarkers for diagnosis of ccRCC.Interestingly,plasma SNORD96A had a good diagnostic value for ccRCC and even early ccRCC patients.Next,we took SNORD4A as an example to explore its regulatory ability to the biological behavior of RCC.The results revealed that interference with the expression of SNORD4A enhanced the proliferation and clone formation ability of RCC cells but decreased the apoptosis ability.After overexpression of SNORD4A,the proliferation and clone formation of RCC cells decreased.Over expression of SNORD4A promoted the proliferation and clone formation of tumor cells,and inhibited apoptosis.The results also have been confirmed in vivo,that was,interfering with the expression of SNORD4A,and the growth of tumor was slow.Protein function mass spectrometry analysis showed that SNORD4A may regulate the biological behavior of renal cancer through Wnt signaling pathway.Western blot revealed that SNORD4A played a role in Dvl2 protein,and silence or overexpression of SNORD4A caused Dvl2 mRNA expression level to change(P<0.05).After adding Wnt pathway activators or inhibitors to RCC cells,the effect of interference or over expression of SNORD4A on cell proliferation was rescued,which further indicated that SNORD4A played a role in the development of RCC through Wnt signaling pathway.Conclusion:SNORD4A,SNORD63,SNORD96A and SNORD93 in ccRCC FFPE tissues and urinary sediment were up-regulated.Moreover,the expression level of SNORD96A in the plasma of ccRCC patients was significantly higher than that of healthy volunteers.Urinary sediment snoRNAs can be used as novel diagnostic biomarkers for ccRCC and plasma SNORD96A has a good diagnostic value for ccRCC.Furthermore,SNORD4A could regulate the malignant phenotype of RCC,which played the role through Wnt signal.