Study On The Correlation Between The Expression Of HnRNPU And The Risk And Prognosis Of Gastric Cancer

Background: The occurrence of gastric cancer(GC)is a complex process of multi-factor participation,multi-gene abnormality,and multi-step development.Early detection of malignant transformation phenotypic characteristics and specific molecular events of gastric epithelial cells will help to identify individuals at high-risk of GC,screen potential therapeutic targets,and assess prognosis.Heterogeneous nuclear ribonucleoproteins(hnRNPs)are a large family of the RNA-binding proteins(RBPs),They play important roles in DNA repair,telomere biogenesis,cell signal transduction,alternative splicing,messenger RNA(m RNA)stabilization,and the regulation of transcription and translation.Abnormal expression of hnRNPs is one of the characteristics of a variety of malignant tumors,directly or indirectly involved in the regulation of tumorigenesis and development.In our previous study,we analyzed the correlation between hnRNPs family gene expression and GC risk using TCGA-STAD and Oncomine database,and found that hnRNPU was the only gene significantly differentially expressed in GC.hnRNPU is a bifunctional protein which participates in pre-m RNA packaging,alternative splicing regulation,and chromatin tissue remodeling.This study intends to investigate the correlation between hnRNPU expression and the occurrence,development,and prognosis of GC by integrating tissue detection and bioinformatics analysis,and to preliminary explore its possible mechanism.Materials and Methods: The subjects were from the First Hospital of China Medical University from 2012 to 2019,including 40 cases of cancerous tissues and non-cancer control tissues from GC patients,and 377 cases of paraffin-embedded tissues from different gastric diseases.Real-time quantitative PCR(Real-time PCR)and Immunohistochemistry(IHC)were used to detect the expression of hnRNPU m RNA and protein,respectively.The non-parametric test was used to analyze the differences in hnRNPU expression between groups and the correlation with clinicopathological parameters.The receiver operating curve(ROC)was used for diagnostic efficacy evaluation.Univariate and multivariate COX proportional hazard regression models were used to evaluate independent risk factors that affect the survival and prognosis.TCGA-STAD and Oncomine were used to jointly analyze the expression characteristics of hnRNPU in GC.hnRNPU interaction proteins was obtained by Bio GRID,and performed KEGG pathway enrichment analysis.Based on the median expression of hnRNPU,GC samples in TCGA were divided into high and low expression groups for GSEA analysis.Download the alternative splicing(AS)events of GC from the TCGA Splice Seq database,and screen for differentially expressed AS(DEAS)in GC.The cor.test function of R was used to calculate the correlation between hnRNPU m RNA expression and the percent-splice-in(PSI)of DEAS.Subsequently,Cytoscape was used to construct an alternative splicing regulatory network for hnRNPU.Results:(1)Correlation analysis of GC risk and prognosis: TCGA-STAD showed that,the expression of hnRNPU m RNA in GC tissues was significantly higher than that in control(P<0.001).From GES-1,AGS,to HGC27,the expression of hnRNPU m RNA gradually increased significantly in the gastric cell line(both P<0.05).The expression of hnRNPU m RNA and protein in GC tissues were significantly higher than those in non-cancer control tissues(both P<0.05).From superficial gastritis,atrophic gastritis,dysplasia,to GC,the expression of hnRNPU protein gradually increased significantly.The AUC of hnRNPU proteins for the diagnosis of diseased stomach and GC were 0.911(95% CI 0.877-0.945,P<0.001)and 0.847(95% CI 0.809-0.886,P<0.001),respectively.The expression of hnRNPU protein was significantly increased in GC patients with male(P=0.008)and the tumor size <5cm(P=0.035).Among GC with female(P<0.001),age≥60 years(P=0.048),HP positive(P=0.002),multifocal tumor(P=0.009),tumor size ≥5cm(P=0.001),Lauren diffuse type(P=0.034),diffuse infiltrating type(P=0.014),T3-T4(P=0.020),positive lymph node metastasis(P=0.020),and TNM II-IV stage(P=0.028),those with high expression of hnRNPU have a significantly poor prognosis.COX regression analysis showed that hnRNPU protein expression(P=0.016)and lymph node metastasis(P=0.002)were independent risk factors affecting the prognosis of GC.(2)Analysis of GC-related mechanism: 177 hnRNPU GC differentially expressed interacting proteins were screened from Bio GRID.KEGG and GSEA jointly analysis showed that hnRNPU was mainly enriched in the spliceosome pathway.Among the 10504 DEAS in GC,the differentially interacting protein of hnRNPU and AS events significantly related to hnRNPU expression were belonged to ECT2 and NKRF genes.Conclusion: The expression of hnRNPU increases significantly with the progress of gastric disease,which has a good performance in diagnosing diseased stomach and GC,and is an independent risk factor that affects the survival and prognosis of GC patients.hnRNPU may be involved in the occurrence and development of GC by regulating the alternative splicing of ECT2 and NKRF.