Expression and clinical significance of PRKCD in liver hepatocellular carcinoma analyzed based on bioinformatics

Lian-Tao Hu, Wen-Jun Deng, Luo Sun, Shi-Zhen Lu, Xue-Bing Li, Chu-Hao Li,Yi-Bo Wang, Xin-Ran Wang, Yue Li, Wei-Qun Wang✉

1. Basic Medical College, Jiamusi University, Jiamusi 154007, China

2. Key Laboratory of Microecology‑immune Regulatory Network and Related Diseases, Jiamusi 154007, China

3. First Affiliated Hospital, Jiamusi University, Jiamusi 154007, China

Keywords:Protein kinase C delta (PKCδ,PRKCD)Liver hepatocellular carcinoma Bioinformatics analysis Prognosis

ABSTRACT

1. Introduction

Hepatocellular carcinoma (LIVER hepatocellular carcinoma,LIHC) is a common digestive malignant tumor, accounting for more than 90% of all liver cancer cases, with the sixth and fourth highest morbidity and mortality rates in the world, respectively [1].Although clinical and experimental treatment of LIHC has made great progress with the improvement of surgical techniques, the overall prognosis of patients is still poor due to the high recurrence and metastasis rate of postoperative tumors [2, 3]. At the same time,it is worth noting that many biomolecules can play a key role in the occurrence and development of LIHC [4]. Therefore, finding reliable LIHC biodiagnostic markers and developing new treatment strategies are still necessary prerequisites for effective treatment of LIHC patients.

Protein kinase C (PKC) is a multi-gene family composed of silk/sulphine protein kinases, including PKC alpha, PKC beta, PKC gamma, PKC, and PKC, etc., PKC can regulate a variety of cell functions, such as proliferation, differentiation and apoptosis,by triggering its downstream cascade reactions [5]. Protease C(PRKCD, PKC) is one of the new isoenzymes in the protein kinase C family, and like other PKCs, PRKCD is also composed of the kinase domain, the amino end regulation domain and the hinge area. However, the PRKCD regulated domain contains only diamyl glycerin (DAG) or other phospholipid messenger activation sites, and is therefore insensitive to Ca2+[6]. PRKCD is widely found in tissues and organs of the body and plays a key role in a variety of diseases, including inflammation and tumors. Studies have shown that functional genomic changes to PRKCD are rare and not directly linked to cancer, supporting the idea that PRKCD is a cancer "passenger gene" rather than a cancer "driven gene".Thus, the activation, expression, positioning and/or carcinogenic environment of PRKCD may largely determine the function of this kinase in cancer [7]. This study analyzes the expression and clinical significance of PRKCD in LIHC by bioinformatics method, with a view to providing new ideas for the establishment of prognostic molecular markers and targets of LIHC diagnosis and treatment.

2. Materials and methods

2.1 Mining of clinical data

The original data of mRNA-seq of normal liver tissue and LIHC tissue were downloaded from the Cancer Genome Atlas database(https://portal.gdc.cancer.gov/ repository) using Rstudio software,while the preliminary clinical data came from the UCSC Xena platform. The two raw data were then sorted and summarized, and no TNM staged data and other incompletely matched data were filtered, resulting in 419 sample data, including 369 LIHC and 50 cancer-side tissue (normal tissue, NT) samples. The normalization of the original count data and the identification of the expression gene between the cancer side and THEC tissue are done by the R-pack"edgeR".

2.2 Study of differences in the expression of tissues next to LIHC and cancer

Using R software to read the organized TCGA-LIHC database gene expression data, the "limma" package and "beeswarm" package statistical analysis of the difference expression of THECC mRNA in LIHC and cancer-side tissue, and the drawing of the difference map of the expression scatter difference of the LIHC, from the Human Protein Atlas (HPA) database to retrieve the immunohistochemical(IHC) staining data (including stain strength and quantity), to clarify differences in PRKCD protein expression between LIHC and normal tissues.

2.3 Study on the relationship between PRKCD expression and LIHC clinical pathological state

The R software reads the gene expression data of the TCGA-LIHC database and then calculates the area below the curve (AUC) using the receiver operating characteristics (ROC) curve to determine the diagnostic value of THEC in LIHC, and the correlation between the expression of PRKCD in pearson Chi-square test LIHC and its pathological parameters is calculated.

2.4 Study on the relationship between PRKCD expression and LIHC prognosis

Using the Rstudio software to read TCGA-LIHC gene expression data and clinical data respectively, the "survival" package was used to use the median expression of PRKCD mRNA in the sample as the demarcation point, which divided the patient into high and low expression groups and analyzed the survival of the two groups. At the same time, the "survival" and "survminer" packages were applied to analyze PRKCD expression, patient age, gender, family cancer history, TNM staging, histological rating, Ishak score, Child-Pugh grading, vascular invasion, and other single and multifactor Cox regressions associated with total lifetime.

2.5 Study on the relationship between PRKCD expression and LIHC biological processes and related signal pathways

To analyze cancer pathways associated with PRKCD expression in LIHC, GSEA analysis was performed using the TCGA-LIHC data set. The reference gene set is c2.cp.kegg.v7.symbols.symbols.gmt,which is used to calculate the normalized enrichment score (NES).When both the p-value and the false discovery rate (FDR) q values are less than 0.05, the gene set is considered significantly rich.

2.6 Study on the relationship between PRKCD expression and LIHC genetics and epigenetics

In order to analyze the causes of changes in the expression of the PRKCD gene, the relationship between PRKCD DNA methylation and copy number mutation (CNA) and its expression was detected by R software from the perspective of genetics and epigenetics.The original data came from the organized TCGA-LIHC dataset,and because there was too little data on the depth loss and deep amplification of the number of copies in the CNA data, only the difference between the mild loss of the number of copies and the light number of copies amplification (gain) and the normal diploid sample was taken into account.

2.7 Statistical analysis

The R software and SPSS Statistics 19.0 software are used for analysis. The Expression Differences of LIHC and PRKCD in Cancer-Side Tissue were analyzed by the Mann-Whitney U test, the correlation between PRKCD expression and clinical pathological parameters was analyzed by Pearson Chi-square, the relationship between Kaplan-Meierr's expression and LIHC patient survivability was analyzed, and the prognostic significance of PRKCD in LIHC patients was analyzed by single-factor and multi-factor Cox regression model. The difference of P≤0.05 is statistically significant.

3. Results

3.1 PRKCD expressed in the LIHC organization

The results of the analysis of non-paired LIHC and cancer side samples in TCGA-IHC data showed that the expression level of PRKCD mRNA in LIHC tissue was significantly higher than that of cancer-side tissue (P＜0.01, Figure 1A). Further analysis using the HPA database showed that the PRKCD protein levels in most LIHC samples were also higher than in cancer-side tissue (Figure 1B).

3.2 PRKCD expression is correlated with clinical pathological characteristics in LIHC patients

Using TCGA-LIHC data, the correlation between PRKCD expression and clinical pathological parameters in LIHC patients showed that the level of gene expression of LCDs was related to patient TNM staging (Figure 2A), histological levels (Figure 2B), AFP content (Figure 2C) and survival status (Figure 2D).And with the increase of TNM stage, the decrease of histological differentiation, and the increase of the level of A-fetoprotein,the level of gene expression of PRKCD has an upward trend. In addition, the ROC curve is calculated to show that the AUC is 0.744(p＜0.0001), indicating that the level of expression of PRKCD has some diagnostic accuracy for clinical diagnosis (Figure 2E).

3.3 PRKCD expression increase is associated with poor prognosis in LIHC patients

Kaplan-Meier's differential analysis of the overall survival rate of the PRKCD gene in the TCGA-LIHC data and found that the overall survival rate of patients with low expression PRKCD was significantly higher than that of the high expression group,indicating that the level of PRKCD expression was correlated with the prognosis of LIHC patients (Figure 3A). A single-factor and multi-factor Cox regression analysis of TCGA-LIHC data showed that PRKCD could be used as an independent prognostic factor for LIHC patients (Table 1). In addition, because Cox regression analysis shows that TNM staging can affect the survival ability of patients, Kaplan-Meier further applied Kaplan-Meier in the TNM I-IV period to analyze the relationship between THECCD expression and the overall survival rate, and found that the risk ratio of III and IV survival curve (HR) is significantly greater than that of Phase I and II, indicating that the risk effect of TNM staging on patients increases (Figure 3B, C).

3.4 PRKCD enriches in tumor processes and associated signaling pathways

To clarify the function of PRKCD in LIHC, we performed GSEA analysis in the gene onto body. The results showed that PRKCD was mainly enriched in processes such as cell cycle and DNA replication and was positively correlated with both (Figure 4A, B). In addition,further analysis of the relevant signal pathways shows that PRKCD can mainly participate in chemokine (Figure 4C), NOD-like receptors (Figure 4D), PPAR (Figure 4E), and adipocytokine (Figure 4F) and T cell receptor signaling pathway (Figure 4G).

3.5 PRKCD CNA and methylation levels affect their expression

Finally, in order to understand the reasons for the change of PRKCD expression, this study used R software to detect the relationship between the expression and copy number variation(CNA) and methylation of PRKCD in TCGA-LIHC from the perspective of genetics and epigenetics. The results showed that the increase in the number of copies of the PRKCD gene increased its expression level (p＜0.05, Figure 5A), while the expression decreased gradually with the increase of PRKCD methylation level (Figure 5B).

4. Discussion

Previous studies have shown that PRKCD inhibits the proliferation of colon cancer cells [8]. Other studies, however, have shown that PRKCD can promote tumor development as a cancer gene for breast and lung adenocarcinoma [9, 10]. This shows that in the occurrence and development of tumors, the regulatory effect of PRKCD is extremely complex and contradictory, and it may have the effect of suppressing or promoting cancer in different tumors. In the area of anti-cancer, Gonzalez-Guerrico et al. have found in prostate cancer studies that foposter can induce tumor apoptosis, a process that is achieved by PRKCD's involvement in the secretion of death receptor ligands and the transduction of the death receptor's downstream apoptosis signal [11]. Similarly, studies such as Liu's have found that PRKCD can regulate the expression of death receptors to influence endotrophic apoptosis, which is mediated by endoplasmic mesh stress [12]. The above studies show that the activation of PRKCD is very important for apoptosis induced by death receptors such as tumor necrosis factor-related apoptosis ligands (TRAIL) and tumor necrosis factors α (TNF alpha). In addition, studies have shown that the depletion of PRKCD has a protective effect in degenerative diseases and tissue damage caused by cell death, and by inducing apoptosis with various toxins and simultaneously raising the PRKCD, PRKCD may be the global regulator of apoptosis [13, 14, 15].In the area of cancer promotion, Yuan and other studies have found that PRKCD inhibitors can regulate the activity of tumor stem cells and inhibit the metastasis of stomach cancer cells [16]. Similarly,studies such as Berardi and Chen have reported that PRKCD promotes the survival of tumor stem cells in a variety of tumors [17,18]. For example, in a study on tumor malignant phenypes, it was found that when the expression of PRKCD was reduced, the selfrenewal potential and carcinogenic potential of stem cells were inhibited, whereas the activation of PRKCD could promote the above potential of tumor stem cells, and further mechanism research confirmed that a persistent self-secretion feedback mechanism driven by the PRKCD/STAT 3/IL-23/JAK signal axis played an important regulatory role in the activation of PRKCD [19]. Similarly, similar studies have shown that PRKCD depletion inhibits the formation of breast, pancreatic and lung cancer tumors in mouse models [20, 21,22].

Although a great deal of research has been carried out on the correlation between PRKCD and many tumors, its role in the occurrence and development of LIHC is rarely reported. This study first looked at data from TCGA-LIHC and HPA data sets and found that CD was significantly higher in LIHC than cancerside or normal liver tissue in both mRNA and protein expression levels. Subsequently, we also confirmed that PRKCD expression and a variety of LIHC clinical pathological characteristics have a significant correlation, as shown that PRKCD level increase and high TNM staging, high AFP content, low differentiation, and ROC curve also shows that PRKCD in clinical diagnosis has a certain degree of accuracy. In addition, survival curve and COX survival analysis showed that the survival ability of patients with low expression PRKCD was significantly improved, and with the increase of TNM stage, the risk effect of PRKCD expression on the survival of LIHC patients also increased, so PRKCD can be used as an independent prognosis factor in LIHC patients. At the same time,GSEA analysis found that PRKCD may be involved in cell cycle and DNA replication processes through cheetification factors, NODlike receptors, PPARs, fat cytokines, and T-cell receptor signaling pathways, which in turn can promote the proliferation of LIHC cells.Final genetic and epigenetic analysis found that an increase in the number of copies of the PRKCD gene increased its expression level,while the level of PRKCD methylation was negatively correlated with its expression level.

To sum up, this study mainly analyzes the expression and clinical significance of PRKCD in LIHC by bioinformatics method,and concludes that PRKCD gene may become a new target for molecular target therapy of LIHC clinical prognosis evaluation,and discusses the reasons for the signaling pathway and expression change of PRKCD's possible participation in LIHC. Because the above research results are derived from big data analysis, so there are certain limitations. Based on this, our follow-up studies need to further validate and elaborate the expression, function and related molecular mechanisms of PRKCD in LIHC from clinical practical samples and basic experiments.

Author’s contribution Wang Wei-qun teachers participate in the guidance and revision of the paper, Hu Lian-tao participates in the writing of the paper and the production of charts, Deng Wen-jun and others are responsible for some of the data.

Acknowledgement

This experiment thanks Heilongjiang Province North Medicine and functional food specialty disciplines of technical support and financial assistance.