High Expression of JMJD4 Is a Potential Diagnostic and Prognostic Marker of Renal Cell Carcinoma

Histone demethylase JMJD4 is a burgeoning tumor marker, which has been proven to be associated with colon cancer, but the role it plays in kidney cancer has not yet been investigated. In the present study, we evaluated whether JMJD4 can be a prognostic marker of patients with clear cell renal cell carcinoma (ccRCC) using data from public platform and in vitro experiments. Our results revealed that the expression of JMJD4 is higher in cancerous tissue than in normal tissues (p < 0.001). High expression of JMJD4 is associated with a poor overall survival (OS) of ccRCC as compared with low expression of JMJD4 (p = 0.015). JMJD4 showed significant relevance with M stage (p = 0.016), gender (p = 0.003), OS (0.018), disease-specific survival (DSS) (0.007), and percussion free interval (PFI) (0.041). Univariate and multivariate Cox analyses demonstrated that high JMJD4 expression had independent predictive value for OS in ccRCC patients (hazard ratio (HR) = 1.563, 95%confidence interval (CI) = 1.055‐2.316, and p = 0.026). Besides, in vitro experiments confirmed that high expression of JMJD4 can significantly promote the invasion ability (p < 0.001), cloning ability (p < 0.001), and proliferation (p < 0.001) of renal cell carcinoma. In summary, high JMJD4 expression may be a prognostic marker in patients with kidney cancer.


Introduction
Renal cell carcinoma (RCC) is a common malignant tumor of the genitourinary system. It can be mainly divided into three subtypes, clear cell renal cell carcinoma (ccRCC), chromophobe, and papillary renal cell carcinoma, which comprise 65-70%, 15-20%, and 5-7% of all RCCs [1,2]. RCC accounts for nearly 3% of all adult malignant tumors, and its incidence and related mortality have been increasing globally in the past decades [3,4]. Usually, some patients have been prompted to have advanced RCC at the time of diagnosis due to the insidious onset of RCC. This tricky feature leads to poor prognosis and high mortality of patients with RCC. In advanced RCC, increased resistance to targeted therapies which may be mediated by gene disorders, angiogenesis, and decreased intake of tyrosine kinase inhibitors by cancer cells is common. In view of the hidden onset of RCC and the high resistance rate of targeted drug therapy for advanced RCC, it is urgent to explore effective biomolecular markers to contribute to the early diagnosis and prognostic information for RCC.
The modification of histones is one of the epigenetic mechanisms that regulate gene expression without changing the genotype [5,6]. Hitherto, histone modifications that have been widely studied are histone methylation and acetylation modifications, which are closely related to the occurrence of many cancers [7,8]. Histone demethylase is one of the most important modification enzymes involved in the regulation of chromatin function by catalyzing the removal of the methyl group on the N-terminal lysine residue of histone [9]. Many histone demethylases have been proven to promote the genesis and development of cancer, such as KDM3A, KDM4A, KDM4D, and JMJD6. Study has confirmed that KDM3A, which is highly expressed in colorectal cancer, is conducive to metastasis of colorectal cancer [10]. KDM3A has also been found to be increased in pancreatic tumors and promote cancer genesis by regulating expression of DCLK1 [11]. KDM4A could function as an oncogene in lung cancer by mediating expression of Myc through the Wnt/β-catenin signaling pathway [12]. In our previous study, we found that KDM4D inhibition can reduce the proliferation and development of kidney cancer. In addition, JMJD6 has been confirmed to be related to the progression of kidney cancer recently [13]. JMJD4 is a newly discovered histone demethylase which is homologous to JMJD6. However, the relationship between JMJD4 and tumor, especially kidney cancer, has not been reported yet.
This study is aimed at evaluating the prognostic value of JMJD4 in kidney cancer. We also conducted in vitro experiments to evaluate the effect of JMJD4 on ccRCC cells. Our results demonstrated that JMJD4 may be a cancerpromoting gene, and high expression of JMJD4 portends a poor prognosis in these patients.

Materials and Methods
2.1. Data Acquisition. The gene expression data and clinical data of ccRCC patients (Workflow Type: FPKM) were acquired from the online TCGA website (https://portal.gdc .cancer.gov/). Immunohistochemistry staining of ccRCC tissues was downloaded from The Human Protein Atlas (https://www.proteinatlas.org/). Patients with missing data, unknown staging status, and unknown survival status were excluded. Finally, a total of 539 patients with ccRCC with clinical characteristics information were obtained.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes
and Genomes Pathway (KEGG) Enrichment Analysis. Differentially expressed genes in the low and high JMJD4 data set were identified and categorized into specific signaling pathways by GO (https://www.geneontology.org) and the KEGG database (http://www.genome.ad.jp/kegg/). The significant GO and KEGG pathways were identified by Fisher's exact test. Adjusted p value < 0.05 and q value < 0.2 were considered as statistically significant.

Colony Formation Assay.
Caki-1 cells were digested and resuspended in medium and seeded on a 6-well plate at a density of 500 cells per well. The culture was maintained in McCoy's 5A medium and terminated when visible clones appeared. Next, the cells were harvested and fixed with 4% paraformaldehyde for 15 minutes. The cells were then stained with crystal violet and dried at room temperature. Finally, the number of clones which contains more than 50 cells was counted under the microscope.
2.6. Tumor Invasion Assays. Caki-1 cells were trypsinized, resuspended in serum-free culture medium, and counted. The upper chamber membranes of Transwell were evenly covered with Matrigel (BD science, New Jersey, USA) and incubated for 1 h before the cells were seeded. After 24 hours of conventional culture, cells in the upper chamber were gently wiped off by a cotton swab. Each chamber was fixed    3.2. Expression of JMJD4 in ccRCC Tissues. JMJD4 was significantly higher in ccRCC tissues (p < 0:001) than in normal tissues both in paired and unpaired box plot analysis (Figures 1(a) and 1(b)). The expression of JMJD4 was also higher in bladder cancer, breast cancer, cholangiocarcinoma, colon cancer, renal cancer, lung cancer, etc., than in normal tissue (p < 0:01 or p < 0:001, Figure 1(c)).

Correlation of High Expression of JMJD4 with Clinical
Characteristics. The relationships between JMJD4 and clinical characteristics of ccRCC patients are displayed in Table 2. JMJD4 showed significant relevance with M stage (p = 0:016), gender (p = 0:003), OS (0.018), DSS (0.007), and PFI (0.041). As shown in Table 3     3.6. Genes Enriched in Different Signaling Pathways. We analyzed the RNA-seq data from TCGA database. Volcano map of differentially expressed genes is shown in Figure 6(a), and more genes were downregulated. The result of KEGG pathway enrichment is shown in Figure 6(b). KEGG analysis has shown that three most enriched pathways were estrogen signaling pathway, Staphylococcus aureus infection, and neuroactive ligand-receptor pathway. There are 31 genes enriched in tumor-related pathways as shown in Figure 6(c), suggesting that JMJD4 is closely related to cancer development. GO analysis and KEGG pathway enrichment were performed to investigate genes correlated with JMJD4 associated with ccRCC. Pathways that meet p value < 0.05 and q value < 0.2 are considered to be statistically significant. In biological process (BP) of GO analysis, genes were mainly associated with cornification, keratinization, keratinocyte differentiation, epidermal cell differentiation, and skin development (Figure 7 Figure 8(a)). The efficiency of overexpression was verified by qRT-PCR and western blot (Figures 8(b) and 8(c)). As a result, western blot analysis and qRT-PCR showed that the expression of JMJD4 was significantly upregulated after transfection of pcDNA-3.1(+)-JMJD4. To investigate the effect that JMJD4 exerts on renal cell proliferation, we performed CCK-8 assays. The results showed that overexpression of JMJD4 in Caki-1 cells promoted the renal cell growth (p < 0:001, Figure 8(d)).

Discussion
JumonjiC (JmjC) domain-containing histone demethylase (JMJD) is a protein family that plays an important role in   Disease Markers cancer growth and development by regulating gene transcription and chromatin structure. Among the members of family, JMJD6 was reported to be tumorigenic factor which involves in various kinds of cancers such as prostate cancer [15], breast cancer [16,17], melanoma [18], colon cancer [19], and lung cancer [20]. JMJD4 is a paralog of JMJD6 which shares 34% sequence with JMJD6 [21]. Knockout of the JMJD6 gene in mice will significantly reduce the proliferation of NIH3T fibroblasts, while knocking out JMJD4 will cause the same effect by RNA interference [22]. Study has confirmed that JMJD4-knockout embryos are born at a normal Mendelian ratio which proved that JMJD4 is unessential in embryogenesis [23]. Currently, only one study has reported that JMJD4 expression could be a prognostic indicator for patients with colon cancer [24]. The interaction between JMJD4 and other tumors still needs more research to reveal. Therefore, we investigate the relationship between JMJD4 and renal cancer.  Figure 3: (a-e) Subgroup analysis of overall survival for patients in advanced Fuhrman grade, female patients, patients in T1 to T3 stage, patients age no greater than 60, and patients with low or normal hemoglobin. (f, g) Subgroup analysis of DSS for patients with ccRCC in female patients and patients age no greater than 60. (h) Subgroup analysis of PFI for patients with ccRCC in T1 to T3.

Disease Markers
In this study, we first use bioinformatics analysis to assess the role of JMJD4 in ccRCC from online public database such as TCGA and TIMER. The results of box plot analysis demonstrate that high expression of JMJD4 is a common feature of multiple human cancers, including ccRCC. And Logistic regression analysis indicated that high JMJD4 is closely related to M stage, gender, and prognosis in ccRCC patients. Then, to evaluate the prognostic value of JMJD4 in ccRCC patients, we performed Kaplan-Meier survival analysis, and results showed that patients with high JMJD4 expression had poor OS, DSS, and PFI. Moreover, Cox regression analysis confirmed that high JMJD4 expression has independent prognostic value for OS in ccRCC patients. Other clinicopathological characteristics are also related to the poor OS of ccRCC, including advanced Fuhrman grade, M stage, T stage, and age, as shown in Figure 4.

Disease Markers
Next, we performed ROC analysis and compute the AUC. The results suggest that high JMJD4 expression has certain diagnostic value in ccRCC patients ( Figure 5(a)). Besides, to help practitioners predict the risk of individual death and optimize treatment options, we also constructed a prognostic nomogram ( Figure 5(b)).
To further evaluate the mechanism of high expression of JMJD4 in promoting the progression of renal cancer, we performed GO and KEGG analysis. We found that the high JMJD4 expression was associated with collecting duct acid secretion, neuroactive ligand-receptor interaction, and fat digestion and absorption pathway in KEGG analysis. GO analysis shows that keratinization, keratinocyte differentiation, and epidermal cell differentiation were most enriched genes in BP. Skin development extracellular region, intermediate filament, and intermediate filament cytoskeleton were most enriched pathways in CC, while transmembrane trans-porter activity, channel activity, and passive transmembrane transporter activity were most enriched pathways in MF. The main genes enriched in these pathways include DSG3, KRT5, KRT6, and KRT14. DSG3 was reported to promote the growth and invasion of cancer cells [25]. KRT5 deficiency can prevent the migration of ovarian cancer cells, which prompts that it may act as an oncogene [26]. KRT6 is associated with notch1 signaling and contributes the progression of renal cancer [27]. KRT14 was proved to be a key regulator in metastasis of breast cancer [28]. The above data shows that the genes related to JMJD4 are mainly cancer-promoting genes, indicating that JMJD4 may promote the invasion and progression of cancer. Furthermore, to clarify the effect of JMJD4 on kidney cancer, we transfected the pcDNA-3.1(+)-JMJD4 plasmid into Caki-1 cells and verified the overexpression by qRT-PCR and western blot (Figures 8(a) and 8(b)). Then, we performed Transwell      16 Disease Markers invasion experiments and plate colony formation experiments. The results showed that JMJD4 overexpression significantly promoted the invasiveness and colony formation ability of Caki-1 cells (Figures 8(c), p < 0:001). We also conducted the CCK-8 experiment, and the results demonstrate that JMJD4 can significantly promote the proliferation of Caki-1 cells (Figures 8(d), p < 0:001). The above in vitro data prove that JMJD4 acts as a cancer-promoting gene in renal cancer cells. One limitation of our research is that we did not conduct in vivo experiments for verifica-tion. Subsequent experiments can be performed on animal experiments to verify the cancer-promoting effect of JMJD4 in vivo.
In summary, our study is the first research that focuses on the role of JMJD4 played in ccRCC. We validated the predictive value and diagnostic value of JMJD4 in ccRCC patients, as well as the effect of JMJD4 on promoting the invasion and proliferation of ccRCC cells. Our results showed that high expression of JMJD can be a potential prognostic marker for ccRCC patients.