A novel correlation between ATP5A1 gene expression and progression of human clear cell renal cell carcinoma identified by co‑expression analysis
- Lushun Yuan
- Liang Chen
- Kaiyu Qian
- Gang Wang
- Mengxin Lu
- Guofeng Qian
- Xinyue Cao
- Wei Jiang
- Yu Xiao
- Xinghuan Wang
- Published online on: December 4, 2017 https://doi.org/10.3892/or.2017.6132
- Pages: 525-536
Copyright: © Yuan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Renal cancer is one of the ten most common cancers, with an annual incidence of 2–4%. Approximately 90% of renal cancers are renal cell carcinoma (RCC), most of which (70–85%) are clear cells subtype (ccRCC) (1).
Localized renal cell carcinoma can be cured by surgery. However, the survival rate of patients sharply declines once the disease become metastatic. ccRCC is usually resistant to chemotherapy, targeted therapies have been exploited for their target specificity and low toxicity, so they can be the best choice of non-surgical treatments (2). Many of them have been approved for clinical use such as multi-kinase inhibitors, anti-VEGF antibodies and mTOR (3).
Survival of patients indeed have been improved by the new therapies, however, median progression-free and overall survival are nearly 2 years, most patients eventually become resistance and surrender (2). Therefore, more effective biomarkers and therapeutic targets are urgently needed.
At present, with the development of high-throughput microarray technology, gene expression profiles have been used to identify genes associated with progression of renal cancer (4–6). However, most studies focused on the screening of differentially expressed genes and ignored the high degree of interconnection between genes, although genes with similar expression patterns may be functionally related (7).
We attempted to construct a co-expression network of relationships between genes through a systematic biology method based on a weighted genome expression network (WGCNA) and to identify network-centric genes associated with different stages of disease progression of renal cancer (8–10).
Materials and methods
Ethical statement for human kidney tissues
The Ethics Committee at Zhongnan Hospital of Wuhan University approved the experiments using human ccRCC and paracancerous tissues for RNA isolation and qRT-PCR (approval no. 2015029). All methods used for human ccRCC tissue samples were performed in accordance with the approved guidelines and regulations. Informed consent was obtained from all individual participants included in the study.
Study design and data collection
In order to clarify our study, we designed a flow diagram to demonstrate the data preparation, preprocessing, analysis and validation (Fig. 1). Firstly, expression profiles of mRNA of clear cell renal cell carcinoma were downloaded from Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). Dataset GSE68417 performed on Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] (Affymetrix, Santa Clara, CA, USA) was used to construct co-expression networks and identify hub genes in this study. This dataset included 14 normal kidney tissues (controls), 6 kidney samples from patients with benign, 13 samples from patients with low grade ccRCC (Fuhrman grades 1 and 2), and 16 samples from patients with high grade (Fuhrman grades 3 and 4). Another independent dataset of GSE40435 was downloaded from GEO and used as a test set to verify our results. This dataset included clear cell renal carcinoma patients from Czech patients (including ccRCC of Fuhrman grades 1, 2, 3 and 4).
For the analyses, the raw expression data were firstly performed RMA background correction, and the processed signals were log2 transformed and normalized by quantile normalization. Then median-polish probesets were summarized by using the ‘affy’ R package. Probes were annotated by the Affymetrix annotation files. Microarray quality was assessed by sample clustering according to the distance between different samples in Pearson's correlation matrices and average linkage, and no samples were removed from subsequent analysis in GSE68417 (Fig. 2).
Samples clustering to detect outliers (GSE68417). (A) Cluster dendrogram. (B) Sample dendrogram and trait indicator. The color intensity was proportional to ccRCC grade.
Screening of differentially expressed genes (DEGs)
The ‘limma’ R package was used to screen the DEGs between normal kidney and ccRCC tissues in the expression data. The SAM (significance analysis of microarrays) with FDR (false discovery rate) <0.05 and |log2 fold change (FC)| >0.585 were applied to select genes further considered in the network construction.
Co-expression network construction
Firstly, expression data profile of DEGs was tested to check if they were good samples and good genes. Then, we used the ‘WGCNA’ package in R to construct co-expression network for the DEGs (11,12). First, the Pearson's correlation matrices were both performed for all pair-wise genes. A weighted adjacency matrix was constructed using a power function amn=|cmn|β (cmn=Pearson's correlation between gene m and gene n; amn=adjacency between gene m and gene n). β was a soft-thresholding parameter that could emphasize strong correlations between genes and penalize weak correlations. Here, the power of β = 10 (scale free R2 = 0.86) was selected to ensure a scale-free network (Fig. 3). Next, the adjacency was transformed into topological overlap matrix (TOM), which could measure the network connectivity of a gene defined as the sum of its adjacency with all other genes for network generation (13). To classify genes with similar expression profiles into gene modules, average linkage hierarchical clustering was conducted according to the TOM-based dissimilarity measure with a minimum size (gene group) of 50 for the gene dendrogram (14). To further analyze the module, we calculated the dissimilarity of module eigengenes, chose a cut line for module dendrogram and merged some modules.
Determination of soft-thresholding power in the weighted gene co-expression network analysis (WGCNA). (A) Analysis of the scale-free fit index for various soft-thresholding powers (β). (B) Analysis of the mean connectivity for various soft-thresholding powers. (C) Histogram of connectivity distribution when β = 10. (D) Checking the scale free topology when β = 10.
Identification of clinical significant modules
Two approaches were used to identify modules related with the progression of ccRCC. First, gene significance (GS) was defined as the log10 transformation of the P-value (GS = lgP) in the linear regression between gene expression and Furhman grade. In addition, module significance (MS) was defined as the average GS for all the genes in a module. In general, the module with the absolute MS ranked first or second among all the selected modules was considered as the one related with clinical trait. Module eigengenes (MEs) were considered as the major component in the principal component analysis for each gene module and the expression patterns of all genes could be summarized into a single characteristic expression profile within a given module. In addition, we calculated the correlation between MEs and clinical trait to identify the relevant module.
Hub gene analysis and validation
Hub genes, highly interconnected with nodes in a module, have been shown to be functionally significant. In our study, we chose an interesting module, and hub genes were defined by module connectivity, measured by absolute value of the Pearson's correlation (cor.geneModuleMembership >0.8) and clinical trait relationship, measured by absolute value of the Pearson's correlation (cor.geneTraitSignificance >0.2) (Fig. 4). In order to screen a key candidate among the hub genes, a linear regression analysis was performed to calculate the relationship between the hub gene expressions and the Furhman grades of ccRCC and R2 was defined as the relationship between them. Furthermore, we uploaded all genes in the hub module to the STRING (Search Tool for the Retrieval of Interacting Genes) database (http://www.string-db.org/) to construct protein-protein interaction (PPI), choosing confidence score >0.40 as the cut-off to screen hub nodes in PPI network (15,16).
Detection of hub genes and protein-protein network (PPI). (A) Scatter plot of module eigengenes in turquoise moudule. (B) Heatmap of the expression of hub genes in different stages of ccRCC. (C) Protein–protein interaction network of genes in the turquoise module. The color intensity in each node was proportional to the degree of connectivity in the weighted gene co-expression network (positive correlation in red and negative correlation in green). The nodes with bold circle represented network hub genes identified by WGCNA. The edge width was proportional to the score of protein-protein interaction based on the STRING database.
In the test set of GSE40435, downloaded before background correcting, normalizing and expression calculating, the expression values of the candidate hub gene in normal kidney and 4 grades ccRCC were collected to perform t-test, and P<0.05 were considered statistically significant. Moreover, we used additional 3 databases: Oncomine (http://www.oncomine.org), The Human Protein Atlas (http://www.proteinatlas.org) and Gene Expression Profiling Interactive Analysis (GEPIA) database (http://www.gepia.cancer-pku.cn) to perform validation of expression, immunohistochemistry (IHC) and prognosis of the candidate hub gene (17). Oncomine is a database consisting of microarray data of various tumors; in our study, we used the data of the expression of the candidate hub gene in 5 subtypes of renal carcinoma. Human Protein Atlas is a database providing immunohistochemistry staining of common cancers, normal tissues and cell lines; in our study, we used the IHC of hub gene in normal and tumor tissues. GEPIA database is based on TCGA data; in our study, we used it to perform survival analysis and assessment of the hub gene expression levels in different pathological stages.
Functional and pathway enrichment analysis
The Database for Annotation, Visualization and Integrated Discovery (DAVID) database (http://david.abcc.ncifcrf.gov/) is an online program providing a comprehensive set of functional annotation tools for investigators to understand biological meaning behind large list of genes (18). Enriched biological themes of DEGs in hub module, particularly GO terms and visualization of those on KEGG pathway maps were performed using DAVID database. P<0.05 was set as the cut-off criterion.
Gene set enrichment analysis (GSEA)
In the test set of GSE40435, 101 samples of ccRCC were divided into two groups according to the expression level of valid hub gene. To identify potential function of the hub gene, GSEA was conducted to detect whether a series of a priori defined biological processes were enriched in the gene rank derived from DEGs between the two groups. P-value <0.05 was chosen as the cut-off criteria.
Preparation for human ccRCC samples
The ccRCC and paracancerous tissues samples were collected from patients after surgery at Zhongnan Hospital of Wuhan University. The histology diagnosis was confirmed by two pathologists independently. The ccRCC and paracancerous tissues were immediately frozen and stored in liquid nitrogen or fixed in 4% PFA after collection. The study using ccRCC and paracancerous tissue samples for total RNA isolation and qRT-PCR analysis was approved by the Ethics Committee at Zhongnan Hospital of Wuhan University (approval no. 2015029). Informed consent was obtained from all subjects.
Total RNA isolation
Total RNA from ccRcc tissues were isolated using RNeasy Mini kit (cat. no. 74101, Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNase I digestion (cat. no. 79254) was used in each RNA preparation to remove genomic DNA. After that, total RNA quantity was measured using NanoPhotometer (cat. no. N60, Implen, München, Germany).
Quantitative real-time PCR (qRT-PCR)
The cDNA was synthesized using 1 µg of total RNA isolated from PCa cells by ReverTra Ace qPCR RT kit (Toyobo, Shanghai, China) and qRT-PCR was performed using 400 ng cDNA per 25 µl reaction. Each reaction was conducted with iQ™ SYBR® Green Supermix (Bio-Rad, China) using 400 or 500 ng of cDNA in a final volume of 25 µl. Primers used for ATP5A1: 5′-ATGACGACTTATCCAAACAGGC-3′ (forward), 5′-CGGGAGTGTAGGTAGAACACAT-3′ (reverse), annealing temperature was 60°C. Primers used for GAPDH (loading control): 5′-TGCACCACCAACTGCTTAG-3′ (forward), 5′-GATGCAGGGATGATGTTC-3′ (reverse), annealing temperature was 60°C.
After data preprocessing and quality assessment, the expression matrices were obtained from the 49 samples in training set GSE68417. Under the threshold of FDR <0.05 and |log2FC| >0.585, a total of 3,495 DEGs (1,549 upregulated or 1,946 downregulated) were selected for subsequent analysis.
Sample cluster and quality assessment
In Fig. 2, sample cluster of GSE68417 was performed, using average linkage method and Pearson's correlation method to compare sample cluster in order to screen outlier samples. Moreover, no samples were deleted. The color intensity was proportional to stage of ccRCC. In Fig. 3, the quality assessment for expression data matrix was performed. In addition, when we chose the correct β = 10, the expression data matrix could construct scale-free network to perform further analysis.
Weighted co-expression network construction and identification of key modules
We used ‘WGCNA’ package in R to put the DEGs with similar expression patterns into modules by average linkage clustering, and a total of 9 modules were identified (Fig. 5A). Two methods were used to test the relevance between each module and the ccRCC progression. Firstly, modules with greater MS were considered to have more connection with the disease progression, and we found that the MS of turquoise module and blue module were higher than those of any other MS (Fig. 5B). Afterwards, the ME in the turquoise module and brown module showed a higher correlation with disease progression than the other modules (Fig. 5C). Based on the two methods, we identified the turquoise module was the module most relevant to the disease progression of ccRCC.
Identification of modules associated with the progression of ccRCC. (A) Dendrogram of all differentially expressed genes clustered based on a dissimilarity measure (1-TOM). (B) Distribution of average gene significance and errors in the modules associated with the progression of ccRCC. (C) Heatmap of the correlation between module eigengenes and the disease progression of ccRCC.
Hub gene identification
Defined by module connectivity, measured by absolute value of the Pearson's correlation (cor.geneModuleMembership >0.8) and clinical trait relationship, measured by absolute value of the Pearson's correlation (cor.geneTraitSignificance >0.2), 13 genes with the high connectivity in turquoise module were taken as hub genes (DHRS11, NDUFV1, ATP5A1, PDHA1, PTRH1, ACAA1, LINC00467, MCCC2, MARVELD2, GOT2, COX11, MRPL41, IMMT) (Fig. 4A and B). Moreover, we also constructed a network of protein-protein interaction (PPI) for all genes in turquoise module by Cytoscape according to the STRING database, and genes connected with >15 nodes were identified as hub nodes in the PPI network (ATP5A1, CS, CYC1, DLST, NDUFA5, NDUFS1, NDUFS2, NDUFV1, SUCLG1, SUCLG2, UQCRFS1) (Fig. 4C) (19).
Hub gene validation
Among all genes in two networks, ATP5A1 and NDUFV1 were genes in both networks. Here, concerning genes with the most relevance to ccRCC stage, we chose ATP5A1 which had the top 1 relevance to the clinical feature in the hub module. Moreover, linear regression analyses were conducted to validate hub genes in the training set. Most genes showed a moderate correlation with the disease progression, and only ATP5A1 had a higher correlation than other genes (P=0.001219) (Fig. 6A). Therefore, ATP5A1 was chosen as the candidate gene for further validation. In the test set, ATP5A1 expression was significantly higher in normal kidney tissues than that in ccRCC tissue of any grade (Fig. 4B). In the dataset of GSE40435, ATP5A1 also showed its high expression in normal kidney tissues and low expression in ccRCC tissues of any grade (Fig. 6C). Based on Oncomine database, interestingly, we found that the expression of ATP5A1 was not only highly-expressed in normal kidney, but also had a strong relation with malignancy with pathological grade and differentiation (Fig. 6B). In GEPIA database, we found that the expression of ATP5A1 was decreased with the progression of ccRCC (Fig. 6D). More convincingly, the result of qRT-PCR using 11 ccRCC tissues and matched paracancerous tissues exhibited a significant downregulation in ccRCC compared to paracancerous tissues (P<0.001) (Fig. 7A). In addition, immunohistochemistry staining obtained from The Human Protein Atlas database, revealed strong decrease of ATP5A1 protein in ccRCC tissues, compared with normal kidneys (Fig. 7B). In addition, we discovered that patients with lower expression of ATP5A1 had a significantly shorter overall survival and disease-free survival time (Fig. 7C and D).
Bioinformatical analysis suggested ATP5A1 was induced in ccRcc tissues. (A) ATP5A1 expression was correlated with the disease progression of ccRCC (GSE68417). (B) Oncomine database indicated that ATP5A1 was downregulated in ccRCC, compared with other subtypes of renal cancer. (C) ATP5A1 expression in different stages of ccRCC was significantly lower than normal kidney (GSE40435). (D) ATP5A1 expression was significantly decreased with the progression of ccRCC.
ATP5A1 was negatively correlated with tumorigenesis of ccRcc. (A) ATP5A1 mRNA was validated using 11 ccRCC tissues and matched paracancerous tissues by qRT-PCR. (B) The Human Protein Atlas database suggested that ATP5A1 protein was strongly downregulated in ccRCC tissues compared with normal kidneys. Normal kidney tissue (patient id. 2887; male, age 2); renal carcinoma tissue (patient id. 2545; female, age 72). (C and D) Kaplan-Meier survival curve obtained GEPIA database revealed that ccRCC patients with lower expression of ATP5A1 had a significantly shorter overall survival time and disease-free survival time.
Functional and pathway enrichment analysis
To obtain further insight into the function of DEGs in hub module, they were uploaded to the DAVID database. GO analysis results showed that ATP5A1 was significantly enriched in biological process (BP), including ATP hydrolysis coupled proton transport, lipid metabolic process, mitochondrial ATP synthesis coupled proton transport and ATP biosynthetic process. Moreover, ATP5A1 was overrepresented in five KEGG pathways, including metabolic pathways, oxidative phosphorylation, Parkinson's disease, Alzheimer's disease and Huntington's disease (Fig. 8).
Bioinformatical analysis of differentially expressed genes (DEGs). (A) GO analysis and (B) KEGG pathway enrichment of ATP5A1.
Gene set enrichment analysis
The pathway enrichment analysis of DAVID just used differentially expressed genes, whereas, GSEA analysis used all genes or probes in the chips regardless the genes were differentially expressed or not, which could supplement other evidence in pathway enrichment. Therefore, GSEA was performed using a test set. To identify the potential function of ATP5A1 in ccRCC, GSEA was conducted to search biological processes enriched in ATP5A1 highly-expressed samples (Table I). Twelve gene sets were enriched, including ‘PROPANOATE_METABOLISM’, ‘FATTY_ACID_METABOLISM’, ‘PROXIMAL_TUBULE_BICARBONATE_RECLAMATION’, ‘CITRATE_CYCLE_TCA_CYCLE’, ‘PYRUVATE_METABOLISM’, ‘OXIDATIVE_PHOSPHORYLATION’ in Fig. 9, and ‘BUTANOATE_METABOLISM’, ‘VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION’, ‘BETA_ALANINE_METABOLISM’, ‘RETINOL_METABOLISM’, ‘LYSINE_DEGRADATION’, ‘PARKINSONS_DISEASE’ in Fig. 10.
Gene set enrichment analysis (GSEA). The gene sets of (A) ‘PROPANOATE_METABOLISM’, (B) ‘FATTY_ACID_METABOLISM’, (C) ‘PROXIMAL_TUBULE_BICARBONATE_RECLAMATION’, (D) ‘CITRATE_CYCLE_TCA_CYCLE’, (E) ‘PYRUVATE_METABOLISM’ and (F) ‘OXIDATIVE_PHOSPHORYLATION’ were significantly enriched in ATP5A1 highly-expressed human ccRcc samples (GSE40435).
GSEA analysis for biological processes related with ATP5A1 expression. The gene sets of (A) ‘BUTANOATE_METABOLISM’, (B) ‘VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION’, (C) ‘BETA_ALANINE_METABOLISM’, (D) ‘RETINOL_METABOLISM’, (E) ‘LYSINE_DEGRADATION’ and (F) ‘PARKINSONS_DISEASE’ were significantly enriched in human ccRCC samples with induced ATP5A1 (GSE40435).
ATP5A1 (ATP synthase, H+ transporting, mitochondrial F1 complex, α subunit 1, cardiac muscle) encoding a subunit of mitochondrial ATP synthase plays a critical role in catalyzing ATP synthesis. Only a few studies have reported the function of ATP5A1. Xu and Li reported that ATP5A1 and ATP5B, which plays an important role in pathogenesis of glioblastoma, are highly expressed in glioblastoma tumor cells and endothelial cells of microvascular proliferation (20). Seth et al supposed that higher levels of ATP5A1 were associated with certain SNPs and with TP53 mutation. Moreover, highly-expressed ATP5A1 occurs in chromosomal instability and may facilitate tumor development along this pathway. Conversely, low levels of ATP5A1 may facilitate development of tumors with microsatellite instability (21). As mitochondrial dysfunction often occurs in encephalopathy, Jonckheere et al discovered a complex V ATP5A1 which could cause fatal neonatal mitochondrial encephalopathy (22).
In this study, WGCNA was performed to identify gene co-expression modules related with the progression of ccRCC. The turquoise module was identified, and 13 hub genes were derived from the module. Furthermore, relating the results of PPI network, only ATP5A1 and NDUFV1 were hub nodes in both the co-expression module and PPI network, indicating that the two hub genes had high connection with clinical trait as well as vital biological processes. In validation, ATP5A1 was more highly-correlated with the clinical trait estimated by log rank test than any other genes in the hub module.
As a tumor suppressor, ATP5A1 was correlated with the pathological malignant of renal cell carcinoma (Fig. 6A and B). Ranking by pathological malignancy and differentiation, clear cell renal cell carcinoma and sacomatoid renal cell carcinoma were highly malignant, papillary renal cell carcinoma and granular renal cell carcinoma were moderately malignant and chromophobe renal cell carcinoma had low malignancy (23). Thus, we found a significant difference of the expression of ATP5A1 in different pathological type of renal cell carcinoma. Also, through the Oncomine database, we found a significant difference of the expression of ATP5A1 in renal cortex and renal tissues comparing with ccRCC tissues. Moreover, in the test set, we found a trend that the expression of ATP5A1 decreased with the increasing Furhman grade, but there is no statistic difference between the 4 grades of ccRCC. However, the expression of ATP5A1 of each grade was significantly upregulated compared with normal kidneys (P<0.001), which also illustrated the critical role of ATP5A1 in the progression of ccRCC. Interestingly, we found that based on TCGA data, the expression of ATP5A1 was significantly decreased with the progression of ccRCC. To verify the results of the expression of ATP5A1 at the transcriptional level, we used 12 pairs of ccRCC tissues and paracancerous tissues to perform real-time PCR, and the results showed that the expression of ccRCC tissues was significantly downregulated comparing with the paracancerous tissues (P<0.001). As shown in Figs. 6 and 7, the fold changes of ATP5A1 were significant, indicating the differential expression of ATP5A1 in transcriptional level. To obtain further insight of translational level of the expression of ATP5A1, we observed the immunohistochemistry staining of ATP5A1 in both normal kidney and renal carcinoma in the Human Protein Atlas database. We discovered that compared with renal carcinoma tissue, the expression of ATP5A1 was significantly upregulated in normal kidney tissue. Interestingly, we found that the expression of ATP5A1 in glomeruli was lower than renal tubules, representing that the function of ATP5A1 might correlate with transmembrane and transportation. As to the prognostic value, according to the GEPIA database, we found that lower expression of ATP5A1 causes lower survival rate and shorter overall survival time and disease-free survival time, on the contrary, higher expression of ATP5A1, as a protective tumor suppressor, causes higher survival rate and longer survival time.
Considering the functional and pathway enrichment analysis as well as GSEA, ATP5A1 was overrepresented in metabolic pathways and oxidative phosphorylation. Many studies had reported that mitochondrial DNA mutations leading to changes in enzymes, may affect the process of oxidative phosphorylation, and ultimately cause the occurrence of tumors (24–28). Combing the subcellular location that ATP5A1 was mostly in mitochondrion inner membrane and cell membrane and the gene function in biological process, we could speculate the potential role of ATP5A1 in the progression of ccRCC by regulating important proteins of signaling pathways regarding oxidative phosphorylation (29,30).
In conclusion, this study used systems of biology-based WGCNA to construct a gene co-expression network, to identify and validate network hub genes associated with the progression of ccRCC. ATP5A1 was identified and validated in association with the progression of human ccRCC probably by regulating tumor-related phosphorylation.
The excellent technical assistance of Yuan Zhu, Shanshan Zhang and Danni Shan is gratefully acknowledged. This study was supported in part by grants from Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (grant nos. cxpy20160049 and cxpy20160010) and Natural Sciences Foundation of Hubei Province (2014CFA006).
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