Gene Regulation Network and prognosis analyses of PCK1 in Hepatocellular Carcinoma

Background: The cytoplasmic isoform of phosphoenolpyruvate carboxykinase (PCK1) is a rate-limiting enzyme in gluconeogenesis. Mounting studies have shown that PCK1 plays a key role in human cancers including hepatocellular carcinoma (HCC). In this study, the mRNA expression, the prognostic value and the gene networks of PCK1 in HCC were investigated. Methods: Expression analysis of PCK1 in HCC was obtained from Oncomine, UALCAN, Human Protein Atlas (HPA) and HCC samples. Prognostic value related to PCK1 expression in HCC patients were evaluated by Kaplan–Meier Plotter. The function effects were analyzed by constructing xenograft tumor mice. Gene functional enrichment was performed via LinkedOmics and GeneMANIA. Results: PCK1 expression at mRNA level and protein level was both downregulated in liver cancer tissues compared with normal liver tissues. The decreased expression of PCK1 at protein level was also validated in collected HCC samples. What’s more, PCK1 expression was negatively associated with cancer stages, tumor grades and metastasis nodal of HCC. Meanwhile, decreased mRNA expression of PCK1 was associated with poor overall survival of HCC patients. Furthermore, PCK1 overexpression in xenograft tumor of nude mice repressed hepatoma tumor growth and made hepatoma mice survive longer. Next, enrichment and functional interaction analysis of PCK1 in HCC by using LinkedOmics and GeneMANIA suggested that PCK1 and neighbor genes were involved in the spliceosome, cell cycle, DNA replication pathways. Conclusions: In sum, our results reveal the PCK1 expression patterns, the potential prognosis value and predicted functional network in HCC, which illuminates the key role of PCK1 in HCC development.


Background
Hepatocellular carcinoma (HCC), the most common primary malignancy of the liver, is the fth leading cancer that ranked second mortality rate in the world [1,2]. Nowadays, the prognosis and recurrence of HCC patients have been signi cantly improved for the common treatment including surgical resection, radiotherapy, chemotherapy, targeted therapy and so on [3,4]. Nevertheless, it is not optimistic about the 5-year survival rate of HCC. Therefore, it is urgently needed to further investigate the underlying pathogenesis and etiology of HCC, which will contribute to the discovery of the advanced treatment and diagnostic markers.
Metabolic disorders contributes to tumorigenesis [5,6]. It is the major way to obtain energy in normal cells by mitochondrial oxidative phosphorylation. However, cancer cells rewire speci c metabolic patterns such as aerobic glycolysis, which is also known as the "Warburg effect" [7]. Phosphoenolpyruvate carboxykinase (PCK) acts as the rst rate-limiting enzyme of gluconeogenesis, which catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP). It has been reported that the dysfunction of PCK is related to a series of metabolic diseases such as diabetes, fatty liver, obesity and so on. PCK1 [8]. There are two isoforms of PCK: a cytoplasmic isoform (PCK1) and a mitochondrial isoform (PCK2) [9,10]. Studies have shown that PCK was involved in tumorigenesis in certain cancers.
PCK2 was found upregulated in human colon cancers and might be a useful predictor of a response to chemoradiotherapy in patients with rectal cancer [11]. In melanoma cells, upregulation of PCK1 promoted melanoma cells tumor growth in vitro and in vivo [12]. The expression level of PCK1 was negatively correlated with tumor progression in clear cell renal cell carcinoma (ccRCC), of which PCK1 suppressed ccRCC cell growth and metastasis in vitro and inhibited tumorigenesis in nude mice by blocking the aerobic glycolysis pathway [13]. However, studies showed that PCK1 was downregulated in HCC. Up to now, little is known about the expression pattern, functional role, and prognostic role of PCK1 in HCC.
As far as we know, the bioinformatics analysis has yet been applied to explore the role of the PCK1 in HCC. Based on the analyses of PCK1 gene expression patterns with pathological characteristics in HCC from online database, we demonstrated the expression patterns with clinicopathological features, prognosis, potential pathways and functions of PCK1 in HCC.

Ethics statement
This study was approved by the Academic Committee of Tongji University. All of the datasets were retrieved from the online databases and published literature, so it was con rmed that all written informed consent had been obtained.

ONCOMINE database
The mRNA expression of PCK1 in HCC was analyzed within the Oncomine database (www.oncomine.org), an integrated online cancer microarray database [22][23][24]. This analysis included these studies of Chen Liver [14], Roessler Liver 2, Roessler Liver [15] and Wurmbach Liver [16]using a Student's t test. The cutoff of p value and fold change were de ned as 0.05 and 2, respectively.

UALCAN
The relative expression of PCK1 between HCC tumors and normal liver tissues, with individual cancer stages, tumor grade or other clinicopathological features was analyzed by UALCAN [25] (http://ualcan.path.uab.edu), which was an interactive web-portal covering level 3 RNA-seq and clinical data from 31 cancer types via TCGA.

Human Protein Atlas
The expression of PCK1 at protein level between human HCC tumors and normal tissues was shown as immunohistochemistry image, which derived from the Human Protein Atlas (https://www.proteinatlas.org). The Human Protein Atlas was a website focusing on a particular aspect of the genome-wide analysis of the human proteins covering common kinds of cancers and normal tissues [26].

Western blot
Tissues were lysed with lysis buffer and harvested. The loading samples were quanti ed with BCA protein quanti cation kit (Yeasen). The equal tissue lysates were prepared for sodiumdodecyl sulfatepolyacrylamide gel (SDS-PAGE) electrophoresis and then transferred to nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). The NC membranes were blocked with 5% non-fat milk then incubated with the primary antibodies at 4°C overnight. Next, the membranes incubated with horseradish peroxidaselabelled secondary antibody (Abcam). The protein bands were detected with the enhanced chemiluminescence kit (Beyotime).

Kaplan-Meier Plotter
The prognostic value of PCK1 expression at mRNA level in HCC was analyzed by using Kaplan Meier plotter (http://kmplot.com/analysis/), which is capable to assess the effects of 54k genes on survival in 21 cancer types [27][28][29][30]. The largest datasets include breast cancer, ovarian cancer, lung cancer, and gastric cancer. The Information including the number-at-risk cases, median values of mRNA expression levels, HRs, 95% CIs and p-values was shown on the K-M plotter chart. Statically signi cant difference was considered when a p value < 0.05.

Xenograft tumor mice
The male nude mice (5~6 weeks old) were used for the subcutaneous xenograft model. Hepatoma cells were injected subcutaneously into the anks of nude mice (1×10 5 cells/inoculation). For tumor weight analysis, mice were sacri ced and tumors tissues were obtained for assessment. Tumor volumes were measured every seven days after inoculation. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Tongji University School of Medicine.
cBioPortal The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access website including data sets of multidimensional cancer genomics [31]. We used c-BioPortal to analyze PCK1 alterations in the TCGA liver hepatocellular carcinoma (LIHC) samples.

LinkedOmics
LinkedOmics ((http://www.linkedomics.orglogin.php) is publicly available portal that includes multiomics data from all 32 TCGA Cancer types [32]. Our study used the LinkInterpreter module to perform enrichment analysis based on Gene Ontology, biological pathways, network modules, among other functional categories.

GeneMANIA
GeneMANIA is a website for constructing protein protein interaction (PPI) network by using a large amount of functional association data [33]. These association data include protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity. In our study we analyzed PCK1 associated genes networks and the predicted potential functions by using GeneMANIA.

PCK1 expression in HCC
It has been reported that PCK1 functions as an oncogenic gene during certain tumors development [10,13]. To evaluate the prognostic and potential clinical value of PCK1 in liver cancer, we analyzed PCK1 expression at mRNA and protein levels using ONCOMINE database (www.oncomine. org) and Human Protein Atlas (https://www.proteinatlas.org). As shown in Figure 1, PCK1 was mostly downregulated at mRNA level among 20 types of cancers including the liver cancer compared to normal tissues by using ONCOMINE database. PCK1 expression was signi cantly decreased in HCC tissues in four datasets ( Figure 2 and Table 1). In Chen Liver dataset, PCK1 down-regulation was found in HCC tissues compared with normal tissues with a fold change of 5.155 (p=1.15E-17) (Figure 2A) [14]. The result from Roessler Liver 2 dataset showed that there was 7.603-fold (p=7.05E-48) decrease in PCK1 mRNA expression in HCC tissues ( Figure 2B) [15]. Roessler found 10.352-fold decrease in PCK1 mRNA expression in HCC samples (p=6.58E-6) and Roessler observed 4.351-fold decrease in PCK1 mRNA expression in HCC tissues (p=3.24E-6) ( Figure 2C and 2D) [16]. Next, we analyzed the protein expression pattern of PCK1 in HCC using the Human Protein Atlas. We observed that PCK1 was not detected in HCC tissues while low expressed in normal liver tissues ( Figure 3A). Moreover, we detected PCK1 expression in 6 pairs of HCC tissue samples. PCK1 expression was also downregulated in HCC tissues compared to adjacent normal liver tissues ( Figure 3B). Taken together, our results showed that PCK1 was downregulated in HCC at transcription and protein levels.
Relationship between PCK1 expression at mRNA level and clinicopathological features of HCC patients Furthermore, we assessed the clinical pathological relevance of PCK1expression, TCGA data of 371 hepatocellular carcinoma (HCC) patients and 50 controls were downloaded from UALCAN (http://ualcan.path.uab.edu/analysis.html). Consistently, PCK1 was also remarkably decreased in primary HCC tissues compared to normal samples ( Figure 4A, p<0.001). What's more, PCK1 was expressed at mRNA level less and less with more advanced individual cancer stages and tumor grades ( Figure 4B and 4C). The lowest expression of PCK1 was found in stage 3 but not stage 4 perhaps for the reason that there were only 6 samples in HCC patients at stage 4. Similarly, the expression of PCK1 at mRNA level was signi cantly decreased associated with tumor grades. The expression of PCK1 tended to be lower as the tumor grade increased ( Figure 4C). In addition, it seemed that PCK1 was also related to the nodal metastasis status of HCC patients, lower expression of PCK1 associated with increased nodal metastasis except for N1 (The sample size was too small only including 4 samples shown in Figure 4D). Above all, the results suggested that PCK1 expression was closely related to the clinicopathological characteristics in HCC patients.
We also detected PCK1 expression in HCC cell lines by using the Cancer Cell Line Encyclopedia (CCLE) (https://www.broadinstitute.org/ccle) and the European Bioinformatics Institute (EMBL-EBI) (https://www.ebi.ac.uk/gxa/home) as shown in Supplementary Figure 1. We discovered that PCK1 expression was almost decreased in cell lines of liver (Supplementary Figure 1A) via CCLE and the EMBL-EBI result showed that PCK1 was downregulated in most cell lines of liver (Supplementary Figure 1B).

The Prognostic Value of PCK1 in HCC patients
Moreover, we investigated the prognostic values of PCK1 in HCC patients via the Kaplan-Meier plotter (http://kmplot.com/analysis/). Results showed that the low expression of PCK1 at mRNA level was associated with better overall survival (OS) (HR=0.61 (0.42-0.86), p=0.005, Figure 5). The analysis suggested that the transcription expression of PCK1 might be considered as an independent prognostic factor for OS of HCC patients.

Antitumor effects of PCK1 in vivo
Next, we investigated the in vivo effects of PCK1 on hepatoma tumorigenesis, PCK1 overexpressing cells and control cells were subcutaneously inoculated into nude mice. We observed that the mean tumor volume of PCK1-overexpressing group was signi cantly smaller than the control group as shown in Figure 2E. The mice were sacri ced and the tumors were weighted after 28-day inoculation. The mean tumor weight of PCK1-overexpressing group was lower than the control group ( Figure 6B). Consistently, the mean tumor volumn of PCK1-overexpressing group was also smaller than the control group ( Figure  6C). As above analyzed, high expression of PCK1 was associated with poor overall survival of HCC patients, we wondered the overall survival of PCK1-overexpressing of hepatoma mice. We observed that the hepatoma mice with high expression of PCK1 survived longer than the control group ( Figure 6D). The in vivo results con rmed that high PCK1 expression of HCC patients tend to be a better prognosis.
Predicted functions and pathways of PCK1 in HCC In order to further explore the functional network of PCK1 on HCC development, we performed the enrichment and functional interaction analysis of PCK1 in HCC by using LinkedOmics. The result of the volcano plot showed that there were 2987 genes (dark dots by the red line) positively correlated with PCK1 while 6599 genes (dark dots by the green line) negatively correlated with PCK1 in statistical signi cance (false discovery rate (FDR) < 0.01, Figure 7A). The 50 representative genes sets signi cantly negative and positive related with PCK1 were presented as the heat maps ( Figure 7B and 7C). These results revealed the co-expression genes associated with PCK1 in HCC at transcription level. Functions of PCK1 were analyzed by gene set enrichment analysis (GSEA). Results showed that genes differentially expressed in correlation with PCK1 participated in the main biological processes such as biological regulation, metabolic process, response to stimulus and multicellular organismal process, which were mainly distributed on the membrane and nucleus. Moreover, the molecular functions such as protein binding and ion binding were predominantly affected by PCK1 and its neighboring genes ( Figure 8A-8C). KEGG pathway analysis showed enrichment in the spliceosome, cell cycle, DNA replication pathways ( Figure 8D).
Moreover, the protein-protein interaction network analyzed by GeneMANIA revealed that the correlated genes with PCK1 was responsible mainly for cellular response to xenobiotic stimulus, xenobiotic metabolic process, drug metabolic process and uronic acid metabolic process (Figure 9).

Discussions
Previous studies have showed that abnormal expressions and dysfunctions of PCK1 advance certain cancers [10][11][12][13]. Lin et.al found that downregulation of PCK1 promoted TXNRD1 expression and hepatoma cell growth via the Nrf2/Keap1 pathway [17]. To further investigate the detailed potential functional and regulatory networks of PCK1 in HCC, we conducted comprehensive bioinformatics analysis of PCK1 in HCC based on the public literature and online databases. Herein, the expression, the prognostic value, the enrichment of functional networks and the mutation of PCK1 in HCC were analyzed and summarized. we hope our ndings could contribute to the treatment and prognosis for patients with HCC.
Our study from a variety of perspectives demonstrated the importance of PCK1 in HCC development. Our results revealed that expressions of mRNA and protein of PCK1 was downregulated in HCC samples. Besides, PCK1 expression was also downregulated in liver cancer cell lines. Next, PCK1 mRNA level was negatively associated with HCC patients' cancer stages, tumor grades and nodal metastasis. What's more, decreased mRNA expression of PCK1 was closely related to poor overall survival of liver cancer patients. Finally, enrichment and functional interactive analysis showed that PCK1 and neighboring genes were mainly involved in the biological processes such as biological regulation, metabolic process, response to stimulus and multicellular organismal process, which were mainly distributed on the membrane and nucleus. Moreover, the molecular functions such as protein binding and ion binding were predominantly affected by PCK1 and its neighboring genes. PCK1 and neighboring genes were responsible for the regulation of spliceosome, cell cycle, DNA replication pathways.
Downregulation of PCK1 was found in HCC tissues and was associated with poor prognosis of HCC patients [18]. Functional study revealed that forced expression of PCK1 promoted liver cancer cells apoptosis and suppressed liver tumorigenesis in mice. Similarly, in our study, PCK1 expression at mRNA and protein levels were also found in HCC tissues, mRNA expression of PCK1 was signi cantly associated with HCC patients' individual cancer stages, tumor grades and nodal metastasis. And that, decreased mRNA expression of PCK1 was also signi cantly associated with poor survival of HCC patients. Taken together, our ndings discussed above indicated that PCK1 acted as a tumor suppressor role in HCC.
Studies have shown that mutagenesis could aggravate human cancers [19][20][21]. We wondered whether PCK1 gene alterations had effects on HCC tumorigenesis. The data of the types and frequency of PCK1 alterations in HCC patients were obtained from cBioPortal (www.cBioPortal.org) (Supplementary Figure  2). PCK1 was altered in 40 of 363 (11%) HCC cases (Supplementary Figure 2). These alterations were mRNA upregulation in 25 cases (6.89%), mutation in 7 cases (1.93%), ampli cation in 4 cases (1.1%), and multiple alterations in 2 cases (0.55%) ( Table 2). On the whole, ampli cation is the most common type of PCK1 in HCC. These results suggested that mutagenesis of PCK1 might not occurred frequently in HCC.
However, there are some limitations in our research. Firstly, we did not explore the detailed molecular mechanism of PCK1 in HCC tumorigenesis, which could be included in the follow-up plan. Secondly, we did not make any analysis of the diagnostic role of PCK1 in HCC.it is worth to be investigated in the future research.
To sum up, we comprehensively study the expression and prognostic value of PCK1 in HCC and have important implications for the potential molecular mechanisms of PCK1-involved HCC tumor growth and development. Our results indicated that PCK1 was down-regulated in HCC and was closely related to the clinicopathological features such as the tumor stage, the tumor grade and the nodal metastasis status of HCC patients, suggesting that PCK1 played an important role in HCC development. Moreover, low PCK1 expression was associated with poor survival which could be considered as an independent prognostic factor for high risk HCC patient. Our study indicated that PCK1 might be a potential prognostic markers in HCC patients.

Conclusions
In summary, we comprehensively study the expression and prognostic value of PCK1 in HCC and have important implications for the potential molecular mechanisms of PCK1-involved HCC tumor growth and development.

Declarations
Ethics approval and consent to participate The research was approved by the Academic Committee of Tongji University. All written informed consent had been obtained.

Consent for publication
All presentations have consent for publication.

Availability of data and material
All data generated or analysed during this study are included in this published article [and its supplementary information les].

Competing interests
All authors declared no con icts of interests with this paper.