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Pan-Cancer Analysis of NEDD4L and Its Tumor Suppressor Effects in Clear Cell Renal Cell Carcinoma Huiyue Dong , Ling Zhu , Jingj

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Pan-Cancer Analysis of NEDD4L and Its Tumor Suppressor Effects in Clear Cell Renal Cell Carcinoma Huiyue Dong , Ling Zhu , Jingj 1 Pan-cancer Analysis of NEDD4L and Its Tumor Suppressor Effects in Clear Cell 2 Renal Cell Carcinoma 3 Huiyue Dong#1,2, Ling Zhu#1,2, Jingjing Sun#2, Yi Zhang2, Qiang Cui4, Lin Wu2, 4 Shushang Chen3, Jun Lu1,2* 5 1. Fujian Provincial Key Laboratory of Transplant Biology, Fuzong Clinical College, 6 Fujian Medical University, Fuzhou 350025, China 7 2. Laboratory of Basic Medicine, Dongfang Hospital (900 Hospital of the Joint 8 Logistics Team), Xiamen University, Fuzhou 350025, China 9 3. Department of Urology, 900 Hospital of the Joint Logistics Team, Fuzhou 350025, 10 Fujian, China 11 4. Nephrology and Urology Department, The Second Affiliated Hospital of Wannan 12 Medical College, Wuhu, China. 13 # These authors have contributed equally to this work. 14 *Correspondence: Jun Lu, Xierhuan Road No. 156, Fuzhou General Clinical College / 15 Dongfang Hospital / 900th Hospital of the Joint Logistics Team, Fuzhou, 350025, China. 16 E-mail addresses: [email protected]. 1 17 Abstract 18 The expression level of NEDD4L, an E3 ubiquitin ligase, has changed 19 significantly in human cancers. In this study, we aimed to study the expression of 20 NEDD4L in pan-carcinoma and its function in malignant tumors. We analyzed the gene 21 expression level of NEDD4L in pan-cancer from The Cancer Genome Atlas (TCGA) 22 microarray data set, the correlation between gene expression and overall survival, 23 disease-specific survival, and tumor immune microenvironment changes. NEDD4L 24 expression changes in half of the cancer types. Low expression of NEDD4L gene 25 predicts poor overall survival and disease-specific survival (DSS) in renal clear cell 26 carcinoma (KIRC) and renal chromophobe cell carcinoma (KIRP). NEDD4L is 27 negatively related to interstitial cell infiltration and immune cell infiltration in most 28 common cancers. Furthermore, the low expression of NEDD4L was verified in our 29 clear cell renal cell carcinoma (ccRCC) clinical tissues. In ccRCC cells, NEDD4L 30 overexpression significantly reduced cell proliferation and migration. In the functional 31 analysis, we proved that NEDD4L could inhibit ERBB3 and MAPK signaling pathways. 32 When cells are deficient in nutrition, NEDD4L promoted the degradation of the 33 autophagy regulatory protein ULK1. Our study provides novel insights into the role of 34 NEDD4L in pan-cancer. NEDD4L may play a tumor suppressor effect in ccRCC, 35 through tumor immune regulation and ubiquitination of key intracellular kinases. 36 Keywords: NEDD4L; pan-cancer; TCGA; immune cell infiltration; ubiquitination 37 38 2 39 Introduction 40 Ubiquitination mediated by the ubiquitin-proteasome system is an important 41 process of post-translational modification of biological proteins, and plays a very 42 important role in protein localization, metabolism, regulation and degradation [1]. 43 Studies have found that the ubiquitin-proteasome system can regulate the biological 44 functions of tumor cell proliferation and metastasis by mediating the degradation of a 45 variety of tumor-related proteins [2]. The key enzymes involved in this system include 46 ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase 47 E3 [3]. Different ubiquitin ligase E3 mediates the transfer of activated ubiquitin from 48 the conjugate enzyme E2 to the substrate, and acts on different substrate proteins, which 49 determines the specificity of ubiquitination modification. The substrate specificity of 50 ubiquitin ligase E3 makes it considered a good target for cancer treatment. 51 NEDD4L (Neural Precursor Cell Expressed Developmentally Down-Regulated 4- 52 Like), also known as NEDD4-2, is a member of the neural precursor cell expression 53 down-regulated factor 4 (NEDD4) protein family. The expression level of NEDD4L 54 has also changed significantly in human cancers. In gallbladder cancer cells, the 55 expression level of NEDD4L was significantly up-regulated compared with normal 56 epithelial cells [4]. At the same time, NEDD4L is generally highly expressed in 57 melanoma cells [5]. However, in prostate cancer specimens, the expression of NEDD4L 58 was significantly reduced compared with neighboring normal tissues [6]. It is reported 59 that NEDD4L expression level is significantly down-regulated in non-small cell lung 60 cancer [7]. Down-regulation of NEDD4L expression enhances the aggressiveness of 3 61 NSCLC tumors [8]. At the same time, NEDD4L low expression group has a shorter 62 survival time than non-low expression group. Similar observations have also been 63 reported in colorectal cancer [9]. NEDD4L is related to the regulation of many central 64 pathways in cancer, including TGF-β, Wnt and EGFR signaling pathways [9, 10]. 65 The Cancer Genome Atlas (TCGA) contains data on 33 cancers from more than 66 10,000 patients [11]. The expression of NEDD4L in the data of TCGA pan-cancer 67 species, its correlation with patient survival, the main function of genes, and whether it 68 will affect the tumor immune microenvironment and other issues are in this paper 69 through TCGA data analysis to draw conclusions. Select a tumor type whose NEDD4L 70 gene expression significantly affects the prognosis of patients, and focus on how 71 NEDD4L affects the malignant phenotype of tumor cells. 72 Materials and Methods 73 Data obtained from UCSC Xena Databases 74 We downloaded gene expression RNASeq (HTSeq-Fragments Per Kilobase per 75 Million (FPKM)) data, somatic mutation data (varscan2_snv.tsv.gz), survival data and 76 an integrated TCGA pan-cancer clinical resource (TCGA-CDR) from UCSC Xena 77 Databases (http://xena.ucsc.edu/). 78 Pan-Cancer NEDD4L Expression Profile Analysis 79 We used a Perl script to generate a txt file of NEDD4L gene expression. Gene 80 expression summary was performed by a R package “ggpubr”. NEDD4L protein 81 expression summary among cancer types was obtain from The Human Protein Atlas 82 (www.proteinatlas.org). 4 83 Survival Analysis of NEDD4L Expression 84 According to the median expression value of NEDD4L, we divided the patients in 85 each cohort into "high" and "low" groups. The Kaplan–Meier method is used to 86 estimate the survival function, and the R software package is used for overall survival 87 analysis and the corresponding visualization "survival" and "survminer". Disease- 88 related survival analysis and forest plots of all tumors are visualized using R packages 89 "survival", "survminer", and "forestplot". 90 Gene Set Enrichment Analysis (GSEA) analysis of KIRC 91 The c2.cp.kegg.v7.1.symbols.gmt and c5.all.v7.1.symbols.gmt files are 92 downloaded from the molecular signature database (MSigDB, 93 http://software.broadinstitute.org/gsea/index.jsp). These two files were used for GSEA 94 analysis. According to the median expression of NEDD4L gene in renal clear cell 95 carcinoma samples, we divided them into high expression group and low expression 96 group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) 97 pathway analysis were completed by using the clusterprofiler R package. According to 98 the default weighted enrichment statistical method, this process is repeated 1,000 times 99 for each analysis. According to p value < 0.05, false discover rate (FDR) < 0.25 and p 100 value < 0.05, FDR < 0.05 were used to screen statistically significant pathways and 101 biological processes. 102 The relationship between NEDD4L gene expression and tumor microenvironment 103 The content of stromal cells and total immune cells in tumor tissues is realized by 104 Pearson correlation analysis, which is realized by R package "estimate" analysis [12]. 5 105 Only the tumor sample data in TCGA-kidney renal clear cell carcinoma (KIRC) is 106 retained. CIBERSORT's (cell type identification by estimating relative subsets of 107 known RNA transcripts) algorithm has been used to evaluate the composition of 108 immune cells in many cancer types [13]. The content of different types of immune cells 109 in KIRC tumor tissues is achieved by "CIBERSORT.R", p Value < 0.001. Relevant 110 pictures are visualized by R package "ggplot2" "ggpubr" "ggExtra". 111 Clinical specimen 112 From November 2013 to November 2015, 50 cases of renal clear cell carcinoma 113 and matched adjacent tissues (> 2 cm from the edge of the cancerous tissue) were 114 selected from the renal cancer patients in Fuzhou General Hospital. The patients were 115 28-77 years old, with an average age of 55.5 years. All cancer tissue specimens were 116 confirmed to be clear cell renal cell carcinoma (ccRCC) by pathology. The isolation 117 and use of human tissues were approved by the Human Research Ethics Review 118 Committee of Fuzhou General Hospital (approval number 2013-017). All patients 119 provided written informed consent. The pathological classification was verified 120 according to Furhman classification. The details were described in our previous study 121 [14, 15]. 122 Cell culture 123 ACHN and 786-O cells were provided by Shanghai Jikai Gene Technology Co., 124 Ltd. (Shanghai, China). Cells are routinely checked for mycoplasma contamination 125 every 2-3 months. ACHN and 786-O cells were cultured in RPMI-1640 medium 126 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% (v/v) fetal bovine 6 127 serum (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Thermo Fisher 128 Scientific, Inc.). All the cells were cultured at 37 °C in a humidified incubator under 5% 129 CO2. 130 Quantitative polymerase chain reaction 131 Total RNA was extracted from tissues using Total RNA Kit II (Omega Bio-Tek, 132 Inc., Norcross, GA, USA). After RNA quantification, 3 μg of total RNA was reverse 133 transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit. PowerUp 134 SYBR Green mix (Applied Biosystems, Thermo Fisher Scientific, Inc.) is used for 135 quantitative polymerase chain reaction (qPCR). Use NEDD4L and β-actin primers to 136 perform qPCR on cDNA: NEDD4L forward primer, 5'- 137 CAGGGTCCAGAAGCAGATGA-3' and reverse primer, 5'- 138 GAGTAGCACAGCCTTCCAGA-3'; α-actin forward primer 5'- 139 TGACGTGGACATCCGCAAAG-3' and reverse primer 5'- 140 CTGGAAGGTGGACAGCGAGG-3'.
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