| Background: Hepatocellular carcinoma（HCC）is one of the malignant tumors with high morbidity and mortality among digestive cancers.It is characterized by insidious onset,not timely diagnosis,rapid progression,strong invasiveness and poor prognosis.At present,the prevention,early diagnosis and treatment of HCC are still urgent issues,so it is crucial to identify novel molecular markers that can be used for the diagnosis,treatment and prognosis evaluation of HCC.Progestin and adipo Q receptor4（PAQR4）,as one member of the PAQR family,has diverse functions,not only participating in multiple biological processes in the body,but also regulating multiple oncogenic pathways,However,it has been less studied in HCC.Objective: In this study,we clarified the correlation between expression levels of PAQR4 in HCC and clinicopathological characteristics of patients,evaluated its diagnostic and prognostic value in HCC.We analyzed the regulatory mechanisms and potential signaling pathways involved in its expression,explored its possible role in immunoregulation of HCC and its association with drug treatment sensitivity,and constructed a risk model for HCC clinical prognosis based on the PAQR family.Methods: Relevant data such as HCC RNA-seq and clinicopathological data were downloaded from TCGA,ICGC and GEO databases.The expression levels of PAQR4 in various human normal tissues and various types of tumors were analyzed using HPA and TIMER databases,the expression differences of PAQR4 in HCC tissues and adjacent noncancerous tissues were analyzed using TCGA,ICGC,and GEO databases,and the m RNA and protein expression levels of PAQR4 in HCC samples were verified by RT-PCR,WB,and immunohistochemistry experiments.Kaplan-Meier survival analysis and Cox regression analysis were used to evaluate the diagnostic prognostic value of PAQR4 in HCC.Genetic variations of PAQR4 in HCC were analyzed through c Bio Portal and COSMIC databases,utilizing mi RWalk and Targetscanv7.2 databases predicted potential regulators of PAQR4,and the signaling pathways enriched by PAQR4 were predicted with the help of GSEA.The CIBERSORT immune algorithm was used to analyze the relationship between infiltrating immune cells and PAQR4 in HCC and IC50 was used to determine the sensitivity to drug treatment.To analyze the expression differences of PAQR family genes in HCC,a Cox proportional hazards model was used to construct HCC prognostic risk model for PAQR family.Kaplan-Meier survival curves,ROC curves,Calibration curves,DCA curves and C-index were used to evaluate the predictive value of the nomogram model for HCC using the TCGA dataset as the training set and the ICGC dataset as the external validation set.Results:1.TIMER database analysis showed that PAQR4 was highly expressed in gastric cancer,lung cancer and HCC（p<0.001）.Analysis of TCGA,ICGC and GEO databases using Wilcoxon rank sum test showed that PAQR4 is upregulated in HCC（p<0.001）.2.Validation of 4 HCC samples by RT-PCR and WB experiments showed that both PAQR4 m RNA and protein expression were higher in cancer tissues than in adjacent tissues（p<0.05）.Meanwhile,immunohistochemistry of 25 HCC samples revealed that PAQR4 expression was significantly higher in cancer tissues than in adjacent tissues（p<0.05）.3.PAQR4 was associated with age,AFP,histological grade,T stage,clinical stage and survival status using Wilcoxon rank-sum test,Kruskal-Wallis test and Logistic regression test（p<0.05）.Fisher’s exact test analysis of 25 HCC samples verified by the experiment showed that high PAQR4 expression was correlated with tumor diameter（p<0.05）.4.The ROC curves indicated that PAQR4 had a better diagnostic value for HCC patients with different stages.5.The overall survival rate of patients with high PAQR4 expression was worse than that of patients with low PAQR4 expression by Kaplan-meier survival analysis（p<0.05）.Univariate Cox regression analysis showed that PAQR4 was associated with prognosis of HCC（HR=1.083,95%CI =1.035-1.135,p<0.001）and multivariate Cox regression analysis showed that PAQR4 was an independent prognostic factor（HR=1.062,95% CI=1.010-1.117,p=0.018）.6.PAQR4 overexpression in HCC is associated with increased DNA copy number（p<0.05）,and Pearson analysis showed that there was a negative correlation between PAQR4 m RNA level and PAQR4 DNA methylation level（p<0.05）.In addition,mi RWalk and Target Scanv7.2 database predictions showed that mi R-125b-5p is a potential regulator of PAQR4.7.GSEA enrichment analysis showed that the high expression of PAQR4 was significantly enriched in cell cycle,Notch signaling pathway,m TOR signaling pathway,apoptosis,p53 signaling pathway,etc.（FDR<0.05）.8.COSMIC database analysis showed that PAQR4 in HCC often missense mutation,synonymous mutation and nucleotide base G>T mutations.Analysis of mutations in TCGA-LIHC samples with high PAQR4 expression showed that TP53 mutation frequency was the highest（43%）,and PAQR4 overexpression was associated with TP53 mutation（p<0.05）.9.CIBERSORT analysis showed that PAQR4 expression was negatively correlated with monocyte and γδT cell infiltration（p<0.05）,and was positively correlated with the infiltration levels of helper T cells,regulatory T cells,myeloid dendritic cells and plasma cells（p<0.05）.In addition,PAQR4 was positively correlated with common immune checkpoint molecules such as PD1,TIM-3,CTLA-4,LAG3 and PD-L1（p<0.05）.HCC patients with low PAQR4 expression had better sensitivity to sorafenib,cisplatin and erlotinib,while patients with high PAQR4 expression had better sensitivity to paclitaxel,doxorubicin and mitomycin.10.PAQR family genes（except PAQR2）were up-regulated in HCC using Mann-whitney U test（p<0.05）.11.Cox regression analysis was used to identify PAQR4,PAQR8 and PAQR9 genes in the PAQR family as independent prognostic factors of HCC,and the HCC prognostic models of TCGA cohort and ICGC validation cohort were constructed.12.Patients with high risk-score had a worse prognosis in the prognostic model using Kaplan-meier survival analysis（p<0.05）.The performance of the prognostic model was evaluated by ROC curve,and its AUC in TCGA cohort was 0.712 and that in ICGC validation cohort was 0.698.13.Cox regression analysis showed that risk score was an independent prognostic factor（HR=1.527,95%CI =1.234-1.891,p<0.001）,and the AUC values of1-,3-and 5-year overall survival predicted by the nomogram model based on risk score were 0.726,0.709 and 0.687,respectively.In addition,the prediction model has a C-index of 0.65.Conclusion: PAQR4 is expected to be a novel biomarker for diagnosis,treatment and prognosis of HCC,and the clinical HCC prognosis model constructed based on the PAQR family can predict patient individualized survival expectation and plan individualized treatment with short-term follow-up. |
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