Immune prognostic implications of PSMD14 and its associated genes signatures in hepatocellular carcinoma

Background PSMD14 played a vital roles initiation and progression of hepatocellular carcinoma (HCC). However, PSMD14 and its-related genes for the immune prognostic implications of HCC patients have rarely been analyzed. Therefore, we aimed to explore gene signatures and immune prognostic values of PSMD14 and its-related genes in HCC. Method Analyzed the expression of PSMD14 in multiple databases, and clinicopathologic characteristics associated with PSMD14 overall survival using Wilcoxon signed-ranktest, logistic and Cox regression, Kaplan-Meier method. An immune prognostic signature (including RBM45, PSMD1, OLA1, CCT6A, LCAT and IVD) was constructed and validated using the co-expression and cox regression analyses in TCGA, ICGC and TIMER datasets. Gene Set Enrichment Analysis (GSEA) was performed using TCGA data set. Results Increased PSMD14 expression in HCC was signi�cantly associated with poor prognosis and clinicopathologic characteristics (grade, histologic stage, surgical approach and T stage, all p-values < 0.05). A total of six PSMD14-related genes were detected, which markedly related to overall survival and immune in�ltrating levels in HCC patients. Using cox regression analysis, the PSMD14 and its-related genes were found to be an independent prognostic factor for HCC survival. Calibration curves con�rmed good consistency between clinical nomogram prediction and actual observation. Immune prognostic model suggests that patients in the high ‐ risk group shown signi�cantly poorer survival than patients in the low ‐ risk group. Conclusion We screened potential immune prognostic genes and constructed and veri�ed a novel PSMD14-based prognostic model of HCC, which provides new potential prognostic biomarkers and therapeutic targets and lays a theoretical foundation for immunotherapy of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancy in the world and high rate of metastas is a vital biological feature that leads to unfavorable prognosis [1][2].Although surgery, radiofrequency ablation and chemoembolization have been widely applied for HCC therapy, the survival rate of HCC patients is still low, partly due to high heterogeneity of HCC [3].Furthermore, due to the fact that biological processes involved in the occurrence and progression of HCC are very complicated, there have not been effective prognostic biomarkers until now [4].Therefore, it is necessary to explore new HCCrelated molecules for the diagnosis, prognosis and treatment of HCC.
PSMD14 acts as an oncogene that promotes tumor progression by deubiquitinating of different protein substrates.For example, Deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signalling pathway, resulting in increased stability of the ALK2, either PSMD14 or ALK2 depletion signi cantly decreases colorectal cancer growth and chemoresistance [6].Similarly, PSMD14 inhibits degradation of GRB2 by deubiquitinating this oncoprotein and stabilizing GRB2, which it enhances hepatocellular carcinoma growth and metastasis [5].POH1 deubiquitinates the TGF-β receptors and CAV1, contributes to hyperactivation of TGF-β signaling and facilitates hepatocellular carcinoma metastasis, which it negatively regulates lysosome pathway-mediated turnover of TGF-β receptors [8].However, other mechanisms of the contribution of PSMD14 in the progression of HCC remained to be further explored.
Here, we evaluated the PSMD14 mRNA and protein levels and the prognostic value in Multiple databases, increased PSMD14 expression associated with poor survival in HCC.Moreover, we screened prognostic genes signature closely related to PSMD14 through bioinformatics analysis and constructed the PSMD14-related prognostic model, and validated in The Cancer Genome Atlas (TCGA) and independent International Cancer Genome Consortium (ICGC) database.In addition, con rmed the correlation PSMD14 expression with clinicopathological characteristics, signature (RBM45, PSMD1, OLA1, CCT6A, LCAT and IVD) and immune in ltration of hepatocellular carcinoma patients.In conclusion, our study con rms the important role of PSMD14 and its associated genes in Liver cancer as well as correlated with prognosis and immune in ltrating levels in HCC patients, providing new insights relevant to individualized treatment.

PSMD14 mRNA expression
Oncomine database analysis demonstrated that signi cantly upregulation in 2 Liver cancer data set compared to the normal liver tissues (Fig. 1A).Complementary, PSMD14 mRNA expression was higher in most tumors, such as bladder cancer, brain and CNS, breast cancer, cervical, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, myeloma, pancreatic cancer and sarcoma.To further assess PSMD14 expression of liver cancer, we veri ed PSMD14 mRNA expression by TIMER data set (Fig. 1B).PSMD14 mRNA expression was signi cantly higher in Liver Hepatocellular Carcinoma (LIHC) compared with adjacent normal tissues.

PSMD14 expression and its clinical signi cance in HCC
To further assess the role of PSMD14 in HCC, we rst analyzed four independent microarray datasets from Oncomine database.The median rank of PSMD14 in up-regulated genes of HCC was 1197.5 based on a meta analysis across the four datasets, including 4 analyses using the Oncomine algorithms (760 samples, P = 2.96E-5, Fig. 2A) [14][15][16].In the meantime, we examined the protein expression of PSMD14 using the Human Protein Atlas (HPA).Imàges revealed a markedly high expression of PSMD14 could be observed compared to the normal Liver tissues using the HPA002114 antibody in Liver cancer (Fig. 2B).
Next, we evaluated PSMD14 mRNA expression compared with normal tissues in multiple HCC studies from TCGA and ICGC databases, and found that the mRNA level of PMSD14 was signi cantly higher in HCC patients than normal liver tissues (Fig. 2C-D).Besides, the results of TCGA database, which were mainly analyzed by the web-based tools, UALCAN, GEPIA and LinkedOmics database, showed that PSMD14 expression was signi cantly overexpressed in human HCC tissues compared with normal tissues (Fig. 2E-F), and elevated PSMD14 expression level corresponds to a shorter overall survival in HCC (Fig. 2G).These ndings suggested that PSMD14 is highly expressed in HCC and elevated PSMD14 may predict a poor outcome for HCC patients.
We analyzed 377 HCC samples with PSMD14 expression and clinical characteristics from the TCGA database.As shown in Fig. 3 (A-D).Signi cant increase in PSMD14 expression correlated with tumor histological grade (p = 0.046), histological stage (p < 0.001), surgical approach (p = 0.001) and T stage (p = 0.013).Categorical dependent variable using logistic regression, univariate analysis showed that PSMD14 expression was signi cantly associated with poor prognostic clinicopathologic characteristics (Table 2).Increased PSMD14 expression was signi cantly associated with grade (OR = 1.9 for well vs. moderate), stage (OR = 2.5 for I vs. II, III), surgical approach (OR = 0.26 for HL vs. SSLR), tumor size (OR = 1.65 for ≤ 5 cm vs. > 5 cm) in HCC (all p-values < 0.05).The results suggested that HCC with high PSMD14 expression was progressed to a more advanced stage than those with low PSMD14 expression.

Survival and independent prognostic analysis
Kaplan-Meier survival analysis indicated that HCC with PSMD14-high had a poor prognosis than the PSMD14-low in TCGA and ICGC database in Fig. 3 (E-F) (p < 0.001).The univariate analysis revealed that PSMD14-high correlated signi cantly with a poor OS (hazard ratio [HR]: 2.17; 95% con dence interval [CI]: 1.32-3.57;p = 0.002).Other clinicopathologic variables related to poor survival include status, stage, T stage and distant metastasis.Multivariate analysis with HR of 1.9 shown that the PSMD14 remained independently associated with overall survival (CI: 1.08-3.33,p = 0.026), along with status in TCGA (Table 3).Therefore, we rst revealed that PSMD14 was associated with poor prognosis, as an independent prognostic factor for HCC survival.

Construction and validation of signature
We selected the genes signatures associated with PSMD14 in the TCGA-LIHC database.A total of 2478 PSMD14-associated genes (Pearson |R| > 0.4, p < 0.001) were chosen to generate prognosis gene signatures and the top 10 positively and negatively genes were selected for further analysis (Fig. 4A).All genes were analyzed by univariate Cox regression.A total of 17 genes were signi cantly related to the OS in TCGA-LIHC database (Fig. 4B).Then, the LASSO COX regression analysis and the regression coe cient was computed, the model achieved the best performance at 6 genes (Fig. 4C).Finally, constructed a risk signature for HCC using multivariate COX regression (Table 4).All patients were divided into high-and low-risk sets based on median risk score in the TCGA and ICGC database.Status, survival time and gene expression levels of patients were shown in TCGA (Fig. 4D) and ICGC (Fig. 4E).The survival analysis indicated that the OS of the low-risk set was better than that of high-risk set in the TCGA database (P < 0.001) (Fig. 5A).The results were consistent in the ICGC database (P < 0.001) (Fig. 5B).The area under the ROC curve (AUC) for 1-, 3-, and 5-year OS were 0.723, 0.653, 0.645, 0.657, 0.705, 0.683 in the TCGA (Fig. 5C) and ICGC (Fig. 5D) cohorts, respectively.Together, Results revealed that the signature showed excellent performance for OS prediction.6D) was signi cant independent risk factors in the ICGC database.These data demonstrated that this signature was an independent risk factor of HCC.

Nomogram construction
Based on the prognostic signature and clinical factors, such as age, gender, vascular invasion, tumor status and stage, a nomogram was constructed (Fig. 7A).The calibration curve was used to describe the prediction value of the nomogram and the 45-degree line indicates the actual survival outcomes.The results showed that the nomogram-predicted survival closely matched with the best prediction performance for predicting 1-, 3-and 5-year OS (Fig. 7B).The 1-year AUC was 0.765 for nomogram, and 0.481 for age, 0.490 for grade, 0.425 for status, 0.711 for stage.The 3-year AUC was 0.697 for nomogram, and 0.508 for age, 0.525 for grade, 0.567 for status, 0.706 for stage.Moreover, the 5-year AUC was 0.715 for nomogram, and 0.594 for age, 0.508 for grade, 0.607 for status, 0.667 for stage (Fig. 7C-E).These showed that compared with a single clinical factor, the nomogram had great predictive accuracy combined the signature and clinical factors.

Model gene veri cation
Consistent with our results, IVD and LCAT were found to be signi cantly downexpressed, while CCT6A and OLA1 were signi cantly overexpressed for liver cancer in the Oncomine (Fig. 8A), TIMER (Fig. 8B), TCGA (Fig. 8C) and ICGC (Fig. 8D) database.Though lack in the Oncomine database, the mRNA expression of PSMD1 and RBM45 were also found to be signi cantly overexpressed for liver cancer in the TIMER, TCGA and ICGC database.Taking together, we further veri ed aberrant expression of six prognostic genes and found that IVD and LCAT genes were signi cantly decreased, while CCT6A,OLA1,PSMD1 and RBM45 genes were increased in HCC tissues.

Gene set enrichment analyses
We carried on the Gene Set Enrichment Analysis (GSEA) between PSMD14 (high/low) expression and high/low risk model data sets in TCGA-LIHC.GSEA reveal signi cant difference (FDR < 0.05, NOM p-val < 0.05) in enrichment of MSigDB Collection (c2.cp.kegg.v7.0.symbols.gmts).We selected the most signi cantly enriched signaling pathways based on their normalized enrichment score (NES).A great majority of the enriched pathways were metabolism related, such as the purine metabolism, pyrimidine metabolism and ubiquitin mediated proteolysis are differentially enriched in PSMD14 high expression and high-risk group phenotype (Fig. 9A).Besides, the glycine serine and threonine metabolism, primary bile acid biosynthesis and retinol metabolism were enriched in PSMD14 low expression and low-risk group phenotype (Fig. 9B).

Discussion
HCC remains a common malignant tumor in the world and the important reasons for the low survival rate.Thus, in view of the low survival rate for HCC, investigation of novel biomarkers and models is necessary.Recently, PSMD14-based signatures have revealed excellent potential in prognosis prediction of multiple cancers.the prognosis value of PSMD14 in cancers have been reported, including HCC, esophageal cancer, breast cancer [5,[12][13].Our results are in accordance with these studies, implying that PSMD14 could be a potential prognostic gene for HCC [5].To our knowledge, the expression of PSMD14 and its potential immune prognostic impact on HCC has not yet been explored, the potential immune prognostic role of PSMD14 and its associated genes in HCC was the focus on the present study.
In the present study, we veri ed the high expression of PSMD14 in liver cancer through Multiple databases, including ONCOMINE, TIMER, HPA, TCGA, ICGC, UALCAN and GEPIA.Bioinformatic analysis from TCGA and ICGC database demonstrated that high expression of PSMD14 in HCC was associated with clinical pathologic characteristics (high grade, histologic stage, surgical approach, T stage), survival time and poor prognosis.Univariate and multivariate COX regression shows PSMD14 as an independent prognostic factor (Table 3).In addsion, we identi ed the PSMD14 associated novel biomarkers base on TCGA data set, and con rmed PSMD14 associated genes model signi cantly correlated with prognosis using COX regression and LASSO analysis.Finally, six genes (RBM45, PSMD1, OLA1, CCT6A, LCAT and IVD) were selected to fabricate a prognostic signature for HCC, validated its e ciency in TCGA and ICGC data set.A robust nomogram consisted of the 6-genes signature, age, gender, vascular invasion, tumor status and the stage was constructed for prognostic prediction of HCC patients.At the same time, the AUC value of the signature-based nomogram was nearly better than the AUC values of age, grade status and stage in 1-, 3, 5-years.Next, we further veri ed the mRNA expression of 6-genes (RBM45, PSMD1, OLA1, CCT6A, LCAT and IVD) for HCC patients in the Oncomine, TIMER, TCGA and ICGC database, which is consistent with the results of previous studies [17][18][19][20].What's interesting, no reports of RBM45 and IVD genes in liver cancer.They may become new targets for the treatment of liver cancer, which need to be further studied.
A total of 7 key prognostic genes of HCC patients were selected in this paper.However, no review had been studied about intriguing mechanisms of these key genes in HCC except PSMD14 and CCT6A.POH1 was rst found as a human pad1 homologue in 1997, a previously unidenti ed component of the human 26S proteasome, that degrades proteins targeted for destruction by the ubiquitin pathway [21].Lv [5] reveal that PSMD14, as a novel posttranslational regulator of GRB2, inhibits degradation of GRB2 via deubiquitinating this oncoprotein in HCC cells.Overexpressing CCT6A Contributes To Cancer Cell Growth By Affecting The G1-To-S Phase Transition And Predicts A Negative Prognosis In Hepatocellular Carcinoma.CCT6A may contribute to HCC cell proliferation by accelerating the G1-to-S transition and predicts a negative prognosis in Hepatocellular Carcinoma [19].Consistent with the previously published papers, the high expression of PSMD14 and CCT6A were found positively related to the poor prognostic of HCC patients in our research.
Enrichment analysis revealed many signi cantly enriched pathways for the signature, of which most were metabolism related.To further investigate the functions of PSMD14 and its associated genes in HCC, we performed GSEA using TCGA data, GSEA showed that purine metabolism, pyrimidine metabolism and ubiquitin mediated proteolysis are differentially enriched between PSMD14 gene high expression and PSMD14 associated genes high risk phenotype.Besides, glycine serine and threonine metabolism, primary bile acid biosynthesis and retinol metabolism are differentially enriched between PSMD14 gene low expression and PSMD14 associated genes low risk phenotype.
Another important aspect of this study, seven genes mRNA expression was correlated with diverse immune in ltration levels in LIHC.PSMD14, RBM45, PSMD1, OLA1 and CCT6A mRNA expression level was signi cantly positively correlated with in ltrating levels of B Cell, CD8 + T cell, CD4 + T cell, Macrophage, Neutrophils and Dendritic cells.In addition, LCAT mRNA expression had a signi cantly negative correlation with in ltrating levels of tumor purity, B Cell, CD8 + T cell, CD4 + T cell, Macrophage, Neutrophils and Dendritic cells.IVD mRNA expression was signi cantly positive related to tumor purity and had a signi cantly negative correlation with in ltrating levels of B Cell, CD8 + T cell, CD4 + T cell, Macrophage and Dendritic cells and no signi cant correlations with Neutrophils.Moreover, the correlation between seven immune genes expression and the signature genes of immune cells imply the role of its in regulating tumor immunology.Of note, among the 7 genes, only the PSMD14 and CCT6A genes, have been reported that correlated with diverse immune cells levels.For instance, POH1 de ciency in macrophages resulting in deubiquitination of pro-IL-1β that restrains in ammatory responses for the maintenance of immune homeostasis [22].CCT6A research shown that CD8(+) T cells provide functional in cytokine secretion and lytic activity upon of their cognate antigens, it was used in personalised adoptive T-cell therapy of melanoma [23].These correlations could be indicative of a potential mechanism that 7 immune-related genes regulated immune cells in HCC.The above suggested that PSMD14 and signature genes could play a signi cant role in recruitment and regulation of immune in ltrating cells in HCC.
In summary, here a systemic analysis was performed on PSMD14 expression and prognosis, which provided a further understanding of the biomolecular properties of HCC.Next, we identi ed and veri ed that immune-related genes ( PSMD14, RBM45, PSMD1, OLA1, CCT6A, LCAT and IVD) as prognostic signatures for HCC.Therefore, it deepens our understanding of liver cancer immune-related genes and provides new potential biomarkers for prognosis and treatment.

Materials And Methods
Oncomine database PSMD14 expression levels were identi ed in the Oncomine database (https://www.oncomine.org/resource/login.html) in various common of cancers.The data is de ned as a P-value of 0.01, a fold change of 1.5, a top 10% gene ranking, and the data has to be from mRNA.

PSMD14 mRNA expression and survival in public databases
To investigate the expression level and prognostic role of PSMD14 mRNA in HCC, the Gene Expression

TIMER database
We analyzed the correlation between signature gene expression and 6 types of immune in ltrating cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages and dendritic cells) in HCC patients via The Tumor IMmune Estimation Resource (TIMER) algorithm database (https://cistrome.shinyapps.io/timer/).Tumor purity is a vital factor that in uences the analysis of tumor immune in ltration by genomic approaches.

Data collection
RNA-sequencing and clinical information for hepatic carcinoma were acquired from the Cancer Genome Atlas (TCGA) database and The International Cancer Genome Consortium (ICGC) database.The clinical features of patients with hepatic carcinoma in the TCGA cohorts are presented in Table 1.

Figures
Figures

Figure 5 Association
Figure 5

Figure 13 Construction
Figure 13

Figure 15 Expression
Figure 15

Table 3
Univariate and multivariate analyses of overall survival in hepatocellular carcinoma patients of TCGA

Table 4
The HR and p values of 6 Genes