Exploration And Validation Of The Prognostic Value Of RNA Methylation Factors In Clear Cell Renal Cell Carcinoma

Objective(s): Clear cell Renal Cell Carcinoma(ccRCC)is a highly prevalent malignancy with high incidence and mortality rates,therefore,it is necessary to investigate the pathogenesis of ccRCC and find new molecular therapeutic targets.RNA methylation is an important post-transcriptional modification,and previous studies have identified several RNA methylation regulators that are closely related to malignancy,but relatively few reports have comprehensively explored the clinicopathological effects of RNA methylation factors on ccRCC.Therefore,in this study,we applied bioinformatics analysis to investigate the correlation between RNA methylation-related factors and ccRCC,and then validated the biochemical results with cellular and tissue experiments.Methods: 1.The expression cohort of 48 RNA methylation regulators was extracted from the ccRCC full gene expression profile of The Cancer Genome Atlas(TCGA)database,from which differentially expressed genes were screened;functional annotation of differentially expressed genes was performed;and differentially expressed genes associated with ccRCC prognosis were screened using univariate cox analysis.2.The R package "Consensus Cluster Plus" was used to perform unsupervised cluster typing of prognosis-related differentially expressed genes,analyze the prognosis of different typing ccRCC,perform enrichment analysis in the typing,and compare the differences of immune microenvironment in different typing.3.The Lasso cox method in the "glmnet" package was used to construct a risk score model for ccRCC based on RNA methylation factors with prognostic value,and a column line graph was created to quantify the prognostic ability of the risk score.4.A combination of multifactorial cox analysis and literature search was used to screen a key factor for further analysis.5.RNA quantitative reverse transcription PCR(RT-qPCR)assay and Western Blot(protein blotting)were used to detect the mRNA and protein expression of key factors in ccRCC cell lines.6.RT-qPCR assay was used to analyze the mRNA expression of target genes in fresh ccRCC tissues and paraneoplastic kidney tissues.7.Paraffin tissues were randomly selected for immunofluorescence experiments to clarify the expression patterns of target proteins in cancer and normal tissues.Eighty paraffin specimens of ccRCC diagnosed by the Department of Pathology in our hospital from 2014-2018 were selected for immunohistochemical experiments of paraffin tissues of key factors to compare the differences in expression of target proteins in cancerous and normal kidney tissues.8.The immunohistochemical results of target proteins in paraffin tissues of 80 cases of ccRCC were combined with clinicopathological parameters such as tumor site,tumor diameter,gender,age,presence of intravascular cancer thrombus,tumor ISUP grade,tumor stage,Ki-67 index for correlation analysis.And the effect of target protein on the survival prognosis of ccRCC was explored by combining clinicopathological parameters for univariate and multifactorial cox analysis,and finally the prognostic column line graph model was constructed by combining clinical parameters.Results: 1.Thirty-nine of the 48 included RNA methylation regulators showed differential expression in ccRCC,of which 24 genes were up-regulated and 15 genes were down-regulated in expression.The functional annotation results of the differentially expressed genes showed that they were associated with methylation modification and substance metabolism of various types of RNAs.2.Univariate cox analysis of 39 differentially expressed genes showed that 12 factors were associated with the prognosis of ccRCC(P<0.05),and based on the expression data of these factors,it was possible to cluster ccRCC patients in the TCGA database into two molecular types,1 and 2.Kaplan-Meier(K-M)survival analysis showed that type 2 patients had a significantly worse survival prognosis than type 1patients;enrichment analysis showed that type 2 molecular typing was mainly related to substance metabolism,and type 2 patients had significantly higher immune scores than type 1(P<0.05),and in type 2 CD4+ T cells,CD8+ T cells,macrophages,myeloid derived suppressor cells(myeloid derived suppressor cells(MDSC),monocytes and other immune cells were significantly more infiltrated than type 1(P<0.01).3.A 7-gene risk score model was obtained by including 12 prognostic factors in the Lasso cox for analysis: Risk Score =0.571* NOP2+ 0.393* NSUN6-0.313*TRMT61B-0.049*ALKBH1+0.185* NSUN5-0.343*TET2 + 0.02 6 *DNMT3B.Of the 7 genes,4 were risk factors(NOP2,NSUN6,NSUN5,DNMT3B),which were associated with poor prognosis in ccRCC,and 3 were protective factors(TET2,TRMT61 B,ALKBH1),which were associated with relatively good prognosis.Using risk scores for grouping,followed by K-M analysis,patients in the low-scoring group survived significantly longer than those in the high-scoring group(P<0.001);univariate and multifactorial cox analyses showed that risk score,age,stage and grading were independent prognostic factors for ccRCC(P<0.05).Using these independent prognostic factors,a line graph predicting 1-,3-,and 5-year survival in ccRCC was constructed.4.This study identified NOP2,a constitutive factor in the risk score model,as a key gene for further experimental studies based on multifactorial cox analysis and literature search.5.The relative expression of NOP2 mRNA(P=0.0271)and protein(P=0.0129)were higher in ccRCC cell lines than in normal renal tubular epithelial cell lines.6.NOP2 mRNA was significantly upregulated in ccRCC fresh tissue(P=0.0082)compared to normal kidney tissue.Immunofluorescence experiments showed that the expression pattern of NOP2 protein in ccRCC is mainly nuclear positive staining,while in normal renal tissue,the expression pattern is mainly cytoplasm or cell membrane positive staining,with rare nuclear positive expression.The expression pattern and localization of NOP2 protein in immunohistochemistry are the same as those of immunofluorescence.Based on literature review,the nuclear positivity rate was used as an evaluation indicator for NOP2 protein expression.The nuclear positivity rate of NOP2 in ccRCC tissue was 42.9 ± 15.7%,and the nuclear positivity rate in renal tissue adjacent to cancer was 14.1 ± 13.5%.The statistical results showed that the expression level of NOP2 protein in ccRCC cancer tissue was significantly higher than that in normal renal tissue(P<0.001).7.Patients were divided into NOP2 protein high expression group and NOP2 protein low expression group using the median nuclear positivity rate of 43.67% as the cutoff value.The statistical results showed that the high NOP2 protein expression group was associated with high tumor grade(grade 3 and 4,P<0.001)and high Ki-67 index(P=0.0035)in ccRCC,but there was no statistical difference in parameters such as age,tumor site,tumor diameter,tumor stage,presence or absence of tumor thrombi,and gender among different NOP2 protein expression groups.8.NOP2 protein expression was associated with overall survival(Overall Survival,OS,P=0.0015)and progression-free survival(Progression-Free Survival,PFS,P<0.001)in ccRCC patients,and OS and PFS were worse in the group with high NOP2 protein expression than in the group with low expression.Univariate and multivariate cox analyses were performed for NOP2 protein expression and other clinicopathological parameters.NOP2 protein expression(P=0.0072)and tumor grade(P=0.015)were correlated with patients’ OS in univariate analysis;NOP2 protein expression(P=0.041)was correlated with patients’ OS in multivariate cox analysis and was an independent prognostic factor for ccRCC.Finally,based on NOP2 protein expression and other parameters,a column line graph model for predicting survival of ccRCC patients could be constructed.Conclusion(s): 1.Multiple differentially expressed RNA methylation regulators are present in ccRCC and are associated with the prognosis of the disease.2.Patients with ccRCC can be classified into type 1 and type 2 based on prognosisrelated RNA methylation-regulated differential expression factors,with type 2 patients having a worse prognosis than type 1.The reason for this difference may be related to immunosuppression caused by metabolic dysfunction in type 2 patients.3.The 7-gene risk score model constructed by machine learning model has strong prognostic assessment ability for ccRCC,and risk score,age,staging,and grading are independent prognostic factors for ccRCC,and the column line graph built by the combination of these factors can achieve the quantification of prognostic ability.4.Both mRNA and protein expression of NOP2,a key factor in RNA methylation regulation,were significantly upregulated in ccRCC tissues and cell lines,and high expression of both NOP2 mRNA and protein was associated with poor prognosis in ccRCC patients.5.NOP2 protein expression is an independent prognostic factor in ccRCC and may be involved in the disease progression of this tumor,playing a pro-cancer role in ccRCC.