Bioinformatic analysis identifies potentially key differentially expressed genes in oncogenesis and progression of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein–protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers.


INTRODUCTION
Renal cell carcinoma (RCC) is a heterogeneous group of cancers, and is one of the 10 most common cancers in the world. Based on the histopathological and molecular characterization of RCC (Hes, 2014;Moch et al., 2016), clear cell RCC (ccRCC), papillary RCC and chromophobe RCC are the major subtypes with ≥5% incidence (Cancer Genome Atlas Research Network, 2013;Cancer Genome Atlas Research Network et al., 2016;Chen et al., 2016). Clear cell RCC is the most prevalent subtype and accounts for about 80% of all RCC (Hsieh et al., 2017;Shenoy & Pagliaro, 2016). Although somatic gene mutations, including VHL, BAP-1, PBRM-1, KDM5C, SETD2, and MTOR genes (Chow, Dong & Devesa, 2010;Ricketts et al., 2018) are involved in the pathogenesis of ccRCC, the molecular mechanism of ccRCC is still not fully elucidated. Moreover, up to one-third of RCC patients have already presented with primary metastases at the time of diagnosis (Fujioka et al., 2012). Therefore, it is important to explore the molecular mechanisms of RCC and find effective biomarkers for early diagnosis. In the last decade, the high-throughput analysis platform for gene expression, such as microarray technology, has been widely used to obtain genetic alteration during tumorigenesis. Bioinformatics is a study field that uses computation to extract knowledge from biological data. It includes the collection, retrieval, manipulation and modeling of data for analysis, visualization or prediction through the algorithms and software. Bioinformatics analysis can help us identify differentially expressed genes (DEGs) and functional pathways related to the carcinogenesis and progression of cancer.
In the present study, mRNA microarray dataset from Gene Expression Omnibus (GEO) was downloaded and analyzed to obtain DEGs between ccRCC and paracancerous tissue. Afterwards, protein-protein interaction (PPI) network, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were performed, which help us understand the molecular mechanism of carcinogenesis and progression. In summary, in total of eleven up-regulated and one down-regulated genes were identified as hub gene, which may be candidate biomarkers for ccRCC.

Microarray data
The gene expression profile of GSE53757 was downloaded from the Gene Expression Omnibus (GEO) database. GSE53757, which is based on Affymetrix GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array), was submitted by Copland et al. (Von Roemeling et al., 2014). The GSE53757 dataset contained 144 samples, including 72 ccRCC samples (Stage I 24, stage II 19, stage III 14 and stage IV 15 cases) and 72 matched normal kidney tissue.

Affymetrix microarray data processing
Raw data (CEL file) were read by an Affy package (http://bioconductor.org/packages/ release/bioc/html/affy.html) of R (version 3.4.4; http://r-project.org/). Chip data preprocessing includes background correction, data normalization, combining normal and tumor group data, ID transform gene symbol, and probe supplemental missing value. The data normalization was conducted using a robust multi-array average analysis method (Hochreiter, Clevert & Obermayer, 2006). Probe supplemental missing value was performed using k-nearest neighbor method (Sim, Kim & Lee, 2005). When multiple probes were mapped to the same gene ID, the mean expression of those probes was calculated. The linear models of microarray data (limma) package (http: //bioconductor.org/packages/release/bioc/html/limma.html) in R language was used to identify DEGs between ccRCC and normal samples. Only genes with an adjusted p < 0.05 and |log 2 FC|>1 were selected as DEGs (where FC = fold change).

KEGG and GO enrichment analyses of DEGs
The Database for annotation, visualization and integrated discovery (DAVID, https: //david.ncifcrf.gov/tools.jsp) is an online program that provides a comprehensive set of functional annotation tools for researchers to understand biological meaning behind plenty of genes (Sherman et al., 2007). Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed for identified DEGs using DAVID database. P < 0.05 was set as the cut-off criterion.

Construction of protein-protein interaction network and module analysis
The functional interactions between proteins may provide insights into the molecular mechanism of cellular processing. In this study, protein-protein interaction (PPI) networks were constructed for the DEGs using the STRING database (https://string-db.org/) (Szklarczyk et al., 2015), which provides a critical integration of PPIs, including known and predicted interactions. The interacting pairs with a combined score >0.7 (high confidence) were selected for the PPI network construction. Subsequently, PPI network was visualized using Cytoscape software (3.6.1) (Demchak et al., 2014). The molecular complex detection (MCODE, version 1.5.1) algorithm is a Cytoscape plugin (Bader & Hogue, 2003), which clusters a given network based on topology to find densely connected regions. The most significant module in the PPI networks was screened using MCODE with score >5, degree cut-off = 10, node score cut-off = 0.2, k-core = 2 and max depth = 100.
The functional enrichment analysis of genes in each module was performed by DAVID.

Hub genes selection
In this study, Cytoscape plugin cytoHubba is used for selected hub genes. CytoHubba is a novel Cytoscape plugin for exploring important nodes in an interactome network by several topological algorithms, including degree, edge percolated component (EPC), maximum neighborhood component (MNC) and maximal clique centrality (MCC) (Chin et al., 2014). The overlapped genes were selected as candidate hub gene in the four algorithms of cytoHubba.

Validation of the hub genes using MEXPRESS database and survival analysis
MEXPRESS (http://mexpress.be/) is a data tool designed for the easy visualization of the Cancer Genome Atlas (TCGA) expression and clinical data, as well as the relationships between them (Koch et al., 2015). To confirm the reliability of the hub genes, we used the MEXPRESS to validate the expression level of the candidate hub genes in ccRCC. The overall survival analyses of hub genes were performed using gene expression profiling interactive analysis (GEPIA, http://gepia.cancer-pku.cn/index.html) online platform, which based on TCGA datasets.

Validation of the hub genes by quantitative real-time PCR
Validation of selected hub gene was conducted using quantitative real-time PCR. Forty-four primary ccRCC and paired normal tissues were obtained from the operative specimens. The patients consisted of 6 females and 38 males, with age from 17 to 85 years (55.88 ± 13.36). All the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration, and were approved by the ethics committee on clinical new technologies and scientific research of Wuxi People's Hospital (Permit number: KS00025). The written informed consents were obtained before the specimens were collected. The total RNA was extracted using Trizol (Invitrogen GIBco) following the manufacturer's instructions. First-strand cDNA was synthesized using MMLV reverse transcriptase (Promega, America) and random primers according to the manufacturer's instructions. 20 µL PCR reaction system contains 2 µL 25 mM/L MgCl 2 , 5mM/ µL sense and antisense primers (1.0 µL each), 0.4 µL 10 mM/L dNTP, 1.0 µL Evagreen (Biotium), 2.0 µL 5*PCR buffer, 2.0 µL cDNA, 0.5 Unit Taq DNA polymerase (Promega). Quantitative real-time PCR analysis was conducted on Lightcycler 480 (Roche, Switzerland) with the following PCR profile: predenaturation at 95 • C for 5 min; 40 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 10 s and elongation at 72 • C for 15 s. The expression changes of candidate hub genes between malignant and adjacent normal tissues were calculated as 2 − CT using the comparative CT method.

Statistical analysis
The student's t -test was used to check whether real-time PCR expression data in malignant and adjacent normal tissues differ significantly. Gene with p < 0.05 and >2-fold change was considered to be significantly different.

KEGG and GO enrichment analyses of DEGs
To gain further insight into the identified DEGs, functional and pathway enrichment analyses were conducted using DAVID. Gene ontology (GO) enrichment analysis showed that up-regulated DEGs were mainly involved in biological processes (BP), including immune response, inflammatory response, and interferon-gamma-mediated signaling pathway, while down-regulated DEGs were significantly enriched in oxidation-reduction process, sodium ion transport and excretion process. KEGG pathway analysis showed the up-regulated DEGs were enriched in phagosome and allograft rejection, while the downregulated DEGs were enriched in metabolic pathways and biosynthesis of antibiotics. The top five over-represented GO terms under biological process (BP), cellular component (CC) and molecular function (MF), and the main enriched pathways are shown in Table 1.

PPI network construction and modules analysis
The PPI network of DEGs was constructed using Cytoscape. The PPI networks of up and down-regulated DEGs consisted of 543 nodes and 3,590 edges, 301 nodes and 507 edges, respectively (Figs. 2 and 3). Using the MCODE app, the most significant module with highest score in the PPI network for ccRCC was detected. The most significant module from up-regulated DEGs PPI network contains 31 nodes and 432 edges (Fig. 4A), and eight nodes and 28 edges form down-regulated DEGs PPI network (Fig. 4B). The enrichment analysis of DEGs in the modules was also carried out with DAVID. The results showed that these genes were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation.

Hub gene selection
In the present study, Cytoscape plugin cytoHubba is used for selected hub genes. Degree is equal to or greater than 10 as the threshold of the hub gene. In this PPI network, the degree of 267 up-regulated and 16 down-regulated genes is equal to or greater than 10. Twentyone genes appeared in the top 50 up-regulated genes list in terms of degree, EPC, MNC  and MCC, simultaneously. Similarly, four genes appeared in the top 10 down-regulated genes list. These genes were selected as candidate hub gene which overlapped in the four algorithms of cytoHubba.

Hub genes validation using MEXPRESS database
To confirm the reliability of the hub genes, we used MEXPRESS to validate the expression level of candidate hub genes in ccRCC. This TCGA dataset contains RNA-seq v2 (log2) from 343 clinical ccRCC specimens. The data showed that except for two down-regulated candidate hub genes, the expression of other candidate hub genes were consistent with the TCGA database (p < 0.05). Nevertheless, MEXPRESS visualization showed that for some genes with significant differences in expression, the distribution of tumor specimens was not highly concentrated. In order to ensure the reliability of hub gene selection, we screened 15 up-regulated and one down-regulated genes with p < 1.0E-10 for further verification (Fig. 5).

Validation of hub gene by quantitative real-time PCR
Quantitative real-time PCR was used to verify the expression profiles of 15 up-regulated genes and one down-regulated gene in 44 patients with ccRCC. Table 2 lists the primers for real-time PCR detection of 16 candidate hub genes. Table 3 shows mRNA expression of candidate hub genes between tumor and paracancerous tissue. The data showed that the expression of 11 up-regulated genes and one down-regulated gene had significant difference between tumors and adjacent tissues, but not for CCNA2, NUSAP1, PBK and RRM2 gene. Therefore, a total of 11 up-regulated genes and one down-regulated gene were identified as the hub genes. The names, abbreviations and functions of hub genes are shown in Table 4. Table 5 shows the average fold change of hub genes between malignant and normal tissue samples from chip dataset.

DISCUSSION
Clear cell renal cell carcinoma (ccRCC) is the most frequent form of urologic malignancy with numerous genetic alterations. The most common genetic changes associated with the development of ccRCC are the deletion of the short arm of chromosome 3. Other genetic alterations include gain of 5q, partial loss of 14q, 8p deletion, 9p loss and 7q gain (Li et al., 2017). Despite our understanding of the biology and pathophysiology of ccRCC has improved significantly over the past decade, the overall mortality of ccRCC has remained

Notes.
All results were expressed as the Means ± SD of cycle threshold (Cq). *p < 0.01. # p < 0.05. largely unchanged. The lethality of ccRCC is mainly due to the difficulty of early detection and the lack of effective treatment. Traditional chemotherapy and radiotherapy are almost ineffective in the treatment of ccRCC (Makhov et al., 2018). Therefore, potential markers for early diagnosis and effective treatment are urgently demanded. With the development of high-throughput technology nowadays, bioinformatics analysis enables us to explore the genetic alterations and identify new biomarkers in ccRCC (Batai et al., 2018), which have possible clinical applications for diagnosis, therapeutic, and prognosis. In the present study, we analyzed the mRNA microarray dataset to obtain DEGs between ccRCC and paracancerous tissues. A total of 896 up-regulated and 653 down-regulated genes were identified in four stages of ccRCC. We constructed the PPI network of DEGs and got the most significant module. Functional annotation showed that DEGs in the modules were mainly enriched in mitotic cell cycle regulation. High expression of these genes accelerates cell cycle progression and promotes the proliferation of cancer cells (Santo, Siu & Raje, 2015). Eleven up-regulated and one down-regulated gene were identified as hub genes by validation of TCGA database and clinical specimens. All the up-regulated hub genes are located in the most significant module.
Aberrations of the mitotic cell cycle play important roles in the carcinogenesis or progression of tumors. Dysregulation of the cell cycle is recognized as a hallmark of malignancy (Hanahan & Weinberg, 2011). In the present study, we found five high expression hub genes involved in the cell cycle, including DTL, MELK, NDC80, NUF2 and Microtubule Nucleation Factor Spindle assembly factor required for normal assembly of mitotic spindles.

UBE2C
Ubiquitin Conjugating Enzyme E2 C Acts as an essential factor of the anaphase promoting complex.
ACADM Acyl-CoA Dehydrogenase Medium Chain Catalyzes the initial step of fatty acid beta-oxidation.  UBE2C. DTL (Denticleless E3 Ubiquitin Protein Ligase Homolog) is a critical regulator of cell cycle progression and genome stability. DTL mediates the poly-ubiquitination and subsequent degradation of CDKN1A/p21 and CDT1 (Baraniskin et al., 2012;Ma et al., 2014). Overexpression of DTL is related to the poor outcome in gastric carcinoma (Kobayashi et al., 2015), breast and lung cancers (Perez-Pena et al., 2017). MELK (Maternal embryonic leucine zipper kinase) is involved in various processes such as cell cycle, apoptosis and splicing regulation. MELK is an oncogenic kinase essential for early recurrence of hepatocellular carcinoma (Xia et al., 2016). Studies have shown that MELK is a novel biomarker and a potential therapeutic target in cervical cancer (Lv et al., 2018), triplenegative breast cancer (Moreno, 2016) and gastric cancer (Zhang et al., 2016a). NDC80 (Nuclear division cycle 80) is also known as highly expressed in cancer 1 (HEC1). NDC80 is a mitotic protein that regulates cell cycle by interacting with SAC protein kinase (Ji, Gao & Yu, 2015), binding with phosphorylated spindle and kinetochore-associated protein 3 (Zhang et al., 2017). Studies have shown that NDC80 is overexpressed in colorectal cancer (Yan et al., 2018), pancreatic cancer (Meng et al., 2015) and gastric cancer (Qu et al., 2014), which may play a crucial role in carcinogenesis. NUF2 also called as CDCA1, is a member of the NDC80 complex, which plays an important role in regulating mitosis. NUF2 is a novel cancer biomarker overexpressed in lung cancer (Harao et al., 2008), colorectal cancer and pancreatic cancer (Hu et al., 2015;Kobayashi et al., 2014). Study suggests NUF2 as a novel prognostic biomarker and therapeutic target for cancers (Thang et al., 2016). UBE2C (Ubiquitin conjugating enzyme E2C) is a member of the E2 ubiquitin conjugating enzyme family. UBE2C is necessary for the destruction of mitotic cyclins and cell cycle progression, and may participate in the cancer progression (Zollner et al., 2017). UBE2C is considered to be a crucial factor upregulated in various malignancies, including breast cancer, melanoma, esophageal squamous cell carcinoma, colorectal cancer and gastric cancer (Kraft et al., 2017;Ma et al., 2018;Palumbo Jr et al., 2016;Pellino et al., 2016;Qin et al., 2017).
In recent years, kinesin motor proteins have become a potential target for cancer therapy (Huszar et al., 2009;Kaestner & Bastians, 2010). In the present study, kinesin motor protein KIF4A, KIF20A and CENPE are involved in the carcinogenesis and progression of ccRCC. KIF4A and KIF20A are members of the kinesin protein superfamily, which is microtubule-dependent molecular motor and mediates the transport of organelles, vesicles and chromosomes, as well as the movement of microtubules within the cell. In hepatocellular carcinoma (Huang et al., 2018), breast cancer (Xue et al., 2018 and prostate cancer (Huang & Gao, 2018), the up-regulation of KIF4A can predict poor prognosis. Increased expression of KIF4A is also associated with lymph node metastasis in colorectal cancer (Matsumoto et al., 2018). KIF20A expression is aberrant in various cancers, such as cervical squamous cell carcinoma (Zhang et al., 2016b), pancreatic cancer (Stangel et al., 2015 and glioma (Saito et al., 2017). Overexpression of KIF20A is correlated with poor overall survival of hepatocellular carcinoma (De Carcer et al., 2018), lung adenocarcinoma (Zhao et al., 2018) and ccRCC (Yuan et al., 2017). CENPE (Centrosome-associated protein E) is a kinesin-like motor protein that accumulates in the G2 phase of the cell cycle. CENPE plays an important role in chromosome congression, microtubule-kinetochore conjugation and spindle assembly checkpoint activation. CENPE is highly expressed in lung adenocarcinoma (Gao, Wang & Zhang, 2019), breast cancer (Yuan et al., 2018 and esophageal adenocarcinoma (Zhu et al., 2019). The study showed the overall survival rate of NSCLC patients with high expression of CENPE was poor (Hao & Qu, 2019).
Microtubule-associated proteins are involved in various cellular functions, such as motility, intracellular trafficking and mitotic spindle formation. Drugs that interfere with microtubule function can cause cell cycle arrest or apoptosis and prevent cells from mitosis. Owing to its critical role in mitotic exit and cytokinesis, microtubules have gained significant interest as important target of cancer therapy (Tangutur et al., 2017). In the present study, microtubule-associated protein TPX2 was highly expressed in ccRCC. TPX2 (Targeting protein for Xenopus kinesin-like protein 2) is required for microtubule formation in human cells. As a critical regulator of mitosis, TPX2 cooperates with Aurora-A kinase and Eg5 kinesin to control microtubule assembly and spindle stability. Several studies have demonstrated that TPX2 is overexpressed in esophageal squamous cell carcinoma, breast, colon and prostate cancer (Hsu et al., 2014;Wei et al., 2013;Yang et al., 2015;Zou et al., 2018). TPX2 was reported as a prognostic marker and potential therapeutic target in ccRCC (Glaser et al., 2017).
The multiple genomic alterations in most cancers may be linked to various DNA metabolic processes, including fidelity of the DNA synthesis and mismatch repair. In this study, hub gene TOP2A is involved in the DNA synthesis and highly expressed in ccRCC. TOP2A (Topoisomerase 2-alpha) is a critical enzyme in DNA replication, transcription and regulating the topologic states of DNA. Studies have confirmed that TOP2A is implicated in various types of tumors, such as gastric cancer (Terashima et al., 2017), colon cancer (Hou et al., 2018), pancreatic cancer (Pei, Yin & Liu, 2018) and papillary renal cell carcinoma (Ye et al., 2018). TOP2A is reported to be a novel prognostic marker in renal cell carcinoma (Lu et al., 2017).
In addition, hub gene NCAPG is highly expressed in ccRCC. NCAPG (Non-SMC condensin I complex subunit G) is a mitosis-related chromosome condensation protein, which reconstitutes long and thin chromatin strands into compact short chromosomes. The non-SMC subunits control the activity of ATP-dependent DNA supercoiling and chromosome segregation. Dysregulation of NCAPG may contribute to the progression of gastric cancer (Song et al., 2018). Overexpression of NCAPG is associated with recurrence and survival of tumor patients (Sun et al., 2018).
Fatty acid metabolic disorders are considered to be a component of malignant transformation in many cancers (Monaco, 2017). In the present study, low-expression of ACADM is involved in the ccRCC tumorigenesis. ACADM (Acyl-CoA Dehydrogenase Medium Chain) is specific for acyl chain of 4 to 16 lengths. The homotetramer ACADM catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Although the molecular pathological mechanisms of low expression of ACADM in cancer remain unclear, study has shown that HIF-1 mediated suppression of acyl-CoA dehydrogenases and fatty acid oxidation is critical for cancer progression (Huang Li et al., 2014). Another study also showed that in a breast cancer transgenic mouse model, attenuating medium-chain acyl-CoA dehydrogenase activity accelerated cancer progression (Niu et al., 2017).
In the present study, the survival analyses revealed that ten up-regulated and one down-regulated hub genes were significantly correlated with worse overall survival of ccRCC patients. These genes include CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2, UBE2C and ACADM. The survival analyses indicate that these hub genes may play important roles in the carcinogenesis, progression, invasion or recurrence of ccRCC.
The pathogenesis of ccRCC is a complex process driven by specific genetic and epigenetic alterations. The discovery of new potential markers will contribute to the early diagnosis and effective treatment of ccRCC. Literature retrieval results showed that some hub genes in present study (DTL, KIF20A, KIF4A, MELK, NCAPG, NUF2, TOP2A, TPX2 and UBE2C) were consistent with previous studies (Chen et al., 2018;Wang et al., 2019;Yuan et al., 2018). Meanwhile, we found three new hub genes (NDC80, CENPE and ACADM) related to the prognosis of ccRCC. These findings may advance the understanding of the pathogenesis of ccRCC and provide novel targets for diagnosis, clinical treatment and prognosis.
Some limitations of our study should be mentioned. First, relatively few samples carry a risk of failing to demonstrate the hub genes. This may explain four candidate hub genes have no statistically significant differences in 44 pairs of specimens. Similarly, two candidate hub genes identified from chip dataset with 72 pairs of samples can't be validated in TCGA database with 343 samples. Second, 44 pairs of samples used for hub genes validation originate from Chinese Han ethnicity, which is different from that of chip dataset samples (American population). This may be the reason why some hub genes cannot be verified. Therefore, these hub genes may need to be validated in different ethnic population.

CONCLUSIONS
In conclusion, the present study identified 12 hub genes that may be involved in the carcinogenesis or progression of ccRCC. Among them, 11 hub genes are closely related to the prognosis of ccRCC. These hub genes may be regarded as diagnostic and prognostic biomarkers, and could become potential targets for future ccRCC therapeutic strategies. However, the function of these genes in ccRCC needs further study to elucidate the biological characteristics.
• Ying Yin analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, approved the final draft.
• Bo Zhang, Yaling Hu and Jingjing Wangperformed the experiments, contributed reagents/materials/analysis tools, prepared figures and/or tables, approved the final draft.
• Huijun Mu conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Human Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The ethics committee of the Affiliated Wuxi People's Hospital of Nanjing Medical University approved to carry out the study (Permit number: KS00025).

Data Availability
The following information was supplied regarding data availability: