521 Original Article The prognosis analysis of RFWD2 inhibiting the expression of ETV1 in colorectal cancer Wei Huang1#, Xiumei Tian2,3#, Xiaoying Guan2,3 1Department of Pathology, The Affiliated Shunde Hospital of Guangzhou Medical University, Foshan 528315, China; 2Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Affiliated Stomatology Hospital of Guangzhou Medical University, Guangzhou 510500, China; 3Department of Biomedical Engineering, Basic Medicine School, Guangzhou Medical University, Guangzhou 511436, China Contributions: (I) Conception and design: W Huang, X Guan; (II) Administrative support: Guangzhou Medical University; (III) Provision of study materials or patients: W Huang; (IV) Collection and assembly of data: X Tian; (V) Data analysis and interpretation: W Huang, X Guan; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. #These authors contributed equally to this work. Correspondence to: Xiaoying Guan. Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Affiliated Stomatology Hospital of Guangzhou Medical University, Huangsha Avenue 39#, Liwan District, Guangzhou 510500, China; Department of Biomedical Engineering, Basic Medicine School, Guangzhou Medical University, Xinzao, Panyu District, Guangzhou 511436, China. Email: [email protected]. Background: The poor prognosis is partly due to the lack of efficient methods for early diagnosis on colorectal cancer (CRC). Methods: Bioinformatic analysis and Immunohistochemical analysis were used to evaluate E3 ubiquitin ligase Ring finger and WD domain 2 RFWD2( ) and ETS variant 1 (ETV1) mRNA and protein expression levels. Results: The abundance of RFWD2 and ETV1 proteins from 76 CRC patients were examined. The relationship between their expression levels and clinic pathological parameters including prognostic significances were also detected. The expression of RFWD2 and ETV1 and the relative genes functions in CRC through bioinformatics methods were further analyzed. Conclusions: In conclusion, RFWD2 functioning as a tumor suppressor by negative regulating ETV1, which might play an important role in the development and progression of CRC. RFWD2 and ETV1 have the potential to serve as a pair of molecular biomarkers for the early diagnosis of CRC. Keywords: E3 ubiquitin ligase Ring finger and WD domain 2 RFWD2( ); ETS variant 1 (ETV1); colorectal cancer (CRC); bioinformatics; prognosis Submitted Apr 05, 2019. Accepted for publication Nov 01, 2019. doi: 10.21037/tcr.2019.11.35 View this article at: http://dx.doi.org/10.21037/tcr.2019.11.35 Introduction in China gradually is increasing (5). With improving diagnostic techniques, and by surgery and chemotherapy Colorectal cancer (CRC) is one of the common for CRC had better effect on early treatment (6). However, gastrointestinal malignancies, its incidence ranks third in for advanced CRC patients, due to the chemotherapy- the world, the tumor-related lethality ranks fourth in the insensitive and easy to relapse lead to the five year survival world. CRC is a heterogeneous disease that caused by the rate is only 50% (7). Tumor metastasis and recurrence is the interaction of genetic and environmental factors (1,2). CRC most important factor affecting the survival rate of CRC is one of the most prevalent and deadly incident cancers patients. Therefore, studying the molecular mechanism of worldwide (3,4). In recent years, with the improvement CRC metastasis and looking for new targets for prevention of living standards, the incidence and mortality of CRC and treatment are still the hot topics in the field of cancer © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(2):508-521 | http://dx.doi.org/10.21037/tcr.2019.11.35 Translational Cancer Research, Vol 9, No 2 February 2020 509 research. in a moist box with antibodies of ETV1 (1:100; ABCAM, RFWD2 also known as COP1 (constitutive USA) or RFWD2 (1:200; ABCAM, USA) in PBS at photomorphogenic 1) protein, comprising RING finger, 4 . Poly peroxidase anti-mouse/rabbit IgG (GSGB-BIO, coiled-coil and WD40 domains, is conserved in both higher China) was subjected to the sections for 30 minutes at room ℃ plants and vertebrates (8). Scientists have pointed at possible temperature after washing with PBS. Diaminobenzidine roles for mammalian COP1 in tumorigenesis and the stress was used for colorimetric detection and the sections were response through regulating the activities of p53 (9) and counterstained with haematoxylin and mounted with c-Jun (10). Recent genetic studies have shown that RFWD2 distyrene plasticizer xylene (DPX). Negative controls deficiency leads to spontaneous tumor formation in mice, were performed by replacing the primary antibody with and have identified mutations in RFWD2 and its substrates preimmune rabbit serum. Positive controls were conducted in various human cancers (11). RFWD2 was identified with according to the manufacture’s suggestion. For each run relevant roles in tumorigenesis process. of immunohistochemistry, negative and positive controls The oncoproteins ETV family is implicated in were performed. Immunostained tissue array sections were melanomas, breast and other types of cancer. Complex reviewed under a microscope by two pathologists, who were post-translational modifications govern the activity of PEA3 blinded regarding the clinicopathological characteristics factors, which promote cell proliferation, motility and and outcome of the patients, while visually scoring each invasion (12). The ETS family is one of the largest families individual tissue core. For positively stained cells: 0 of signal-dependent transcriptional regulators (13). It is (negative) was denoted for <10% positive cells, 1 (weak) involved in tumorigenesis and development of a variety of for <25% positive cells, 2 (moderate) for <50% positive tumors (14). The ETS family is overexpressed in breast cells, and 3 (strong) for >50% positive cells. The staining cancer (15), prostate cancer (16), melanoma (17), Ewing’s intensity was defined as: 0 for no stain, 1 for weak-positive tumor (18) and gastrointestinal stromal tumor (19) and also (faint yellow), 2 for moderate-positive (yellowish-brown), involved in the development of tumors. and 3 for strong-positive (brown). Scores of the proportion Herein, we determined the expression of RFWD2 and and intensity of positively stained tumor cells were added ETV1 in CRC through bioinformatic analysis, aiming to and stratified as having negative (−) expression (0–3 score) ascertain whether they are potential molecular biomarkers and positive (+) expression (4–6 score). for the early diagnosis of CRC and to obtain clues for the pathogenesis of CRC. Bioinformatic analysis of RFWD2 and ETV1 Methods The human patients’ samples from The Cancer Genome Atlas (TCGA; https://cancergenome.nih.gov/) were Ethics statement analyzed to investigate the clinical significance of RFWD2 The research was approved by the Ethics Committee of and ETV1 expression in CRC patients. Cancer Cell Guangzhou Medical University (No. 2018-KY-106C), and Line Encyclopedia databases (CCLE, https://portals. written informed consent was obtained from each patient broadinstitute.org/ccle) was used to investigate the involved in the study. expression levels of RFWD2 and ETV1. In Linkedomics (http://www.linkedomics.org/) we dug out the positively and negatively associated genes regulated by RFWD2 and Immunohistochemical analysis ETV1. To explore the functional annotation and pathway Archived and paraffin-embedded samples were obtained enrichment of RFWD2 and ETV1 genes, the Gene from 76 CRC patients who underwent surgical resection Ontology (GO; http://www.geneontology.org/) and Kyoto between January 2013 and March 2019 in the Second Encyclopedia of Genes and Genomes (KEGG; http:// Affiliated Hospital of Guangzhou University and the www.kegg.jp/kegg/kegg2.html) database analyses were Affiliated Shunde Hospital of Guangzhou Medical conducted using a Database for Annotation, Visualization University. Immunohistochemical staining of ETV1 or and Integrated Discovery (DAVID) v6.7 online analysis tool RFWD2 was carried out according to the manufacture’s with P<0.05 as the significant threshold to obtain significant protocol. In briefly, the sections were incubated overnight gene sets. © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(2):508-521 | http://dx.doi.org/10.21037/tcr.2019.11.35 510 Huang et al. RFWD2 inhibiting the expression of ETV1 in CRC Statistical analysis databases was consistent with that of TCGA, demonstrating the mRNA expression level of RFWD2 was more than 1.8 Data were expressed as the mean ± SD from at least three times of ETV1 (Figure 3A,B), the spearman correlative independent experiments for each group. The differences analysis showed that their expression was negative relevant between RFWD2 or ETV1 expression levels with the in colorectal tumor tissue (r2=0.2015, F=14.13, P<0.001, clinicopathological features groups were analyzed using a Figure 3C). factorial model one-way analysis of variance. All statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and P less than 0.05 was considered Immunohistochemical and prognosis Analysis of RFWD2 statistically significant. and ETV1 in CRC patients Among the 76 cases of CRC 25 cases (32.9%) were Results RFWD2-positive and 51 (67.1%) were RFWD2-negative; whereas 47 cases (61.8%) were ETV1-positive and 29 Association of RFWD2 and ETV1 expression with (38.2%) were