Journal of Cancer 2020, Vol. 11 6025 Ivyspring International Publisher Journal of Cancer 2020; 11(20): 6025-6037. doi: 10.7150/jca.47538 Research Paper TRIM67 inhibits tumor proliferation and metastasis by mediating MAPK11 in Colorectal Cancer Ying Liu1*, Guiqi Wang1*, Xia Jiang1*, Wei Li1, Congjie Zhai1, Fangjian Shang1, Shihao Chen1, Zengren Zhao1 and Weifang Yu2 1. Department of General Surgery, Hebei Key Laboratory of Colorectal Cancer Precision Diagnosis and Treatment, The First Hospital of Hebei Medical University, Donggang Road No.89, Shijiazhuang, Hebei 050031, P.R. China. 2. Department of Endoscopy Center, The First Hospital of Hebei Medical University, Donggang Road No.89, Shijiazhuang, Hebei 050031, P.R. China. *These authors contributed equally to this work. Corresponding authors: Prof. Zengren Zhao or Weifang Yu, The First Hospital of Hebei Medical University, Donggang Road No.89, Shijiazhuang, Hebei 050031, P.R. China; Tel: +86 0311 85917217; E-mail: [email protected] or [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.04.28; Accepted: 2020.08.04; Published: 2020.08.18 Abstract Purpose: We recently reported that tripartite motif-containing 67 (TRIM67) activates p53 to suppress colorectal cancer (CRC). However, the function and mechanism of TRIM67 in the inhibition of CRC cell proliferation and metastasis remains to be further elucidated. Methods: We detected the expression of TRIM67 in CRC tissues compared with normal tissues and confirmed its relationship with clinicopathological features. DNA methylation of TRIM67 was analyzed to determine its significantly hypermethylated sites in CRC tissues. CCK-8, colony formation, transwell migration, and Matrigel invasion assays were performed to evaluate the effects of TRIM67 on cell proliferation and metastasis in CRC cells. RNA sequencing of TRIM67 and TRIM67 rescue experiments were performed to reveal its mechanisms in CRC cell proliferation and metastasis. Results: TRIM67 expression was significantly downregulated in CRC tissues and its expression was associated with clinical stage, invasive depth, tumor size, lymph node metastasis, and Dukes’ stage. Three methylation sites were significantly hypermethylated and negatively correlated with TRIM67 expression in CRC tissues. TRIM67 suppressed proliferation, migration, and invasion in CRC cells. RNA sequencing revealed that protein mitogen-activated protein kinase 11 (MAPK11) was a potential downstream negative regulatory gene of TRIM67. Reversing MAPK11 expression could rescue the effects of TRIM67 on the proliferation and metastasis of CRC cells. Conclusion: TRIM67 inhibited cell proliferation and metastasis by mediating MAPK11 in CRC, and may be a potential target to inhibit CRC metastasis. Key words: TRIM67, colorectal cancer, MAPK11, cell proliferation, metastasis Introduction Colorectal cancer (CRC) is a main cause of death of RBCC proteins of which nearly 70 members have worldwide, and its morbidity and mortality have been identified in humans, while fewer than 20 increased in recent years [1]. Multiple genes and members have been identified in invertebrates and signaling pathways have been identified but the none in single-celled organisms. The high number of precise molecular mechanism of CRC is not fully TRIM members in humans indicates that it is a rapidly understood. Therefore, clarifying its pathogenesis and evolving family [3]. Members of the TRIM protein identifying novel molecular biomarkers to improve family are involved in many biological processes the diagnosis and prognosis of patients with CRC are including proliferation, differentiation, development, urgently required [2]. autophagy, and apoptosis of cancer cells [4-8]. In The tripartite motif (TRIM) family is a large class recent years, various studies have associated several http://www.jcancer.org Journal of Cancer 2020, Vol. 11 6026 TRIM family members, TRIM15, TRIM27 and pairs of human CRC tissues and adjacent normal TRIM29, with CRC through AKT or JAK2/STAT3 tissues were collected from the First Hospital of Hebei signaling to regulate CRC cell migration and Medical University (Shijiazhuang, Hebei, China). All epithelial–mesenchymal transition [9-11]. patients were without any therapeutic invention and TRIM67, a TRIM family member, is a had signed informed consent prior to surgery. All protein-coding gene located at 1q42.2 consisting of cancer tissues were histologically confirmed as approximately 59 kb DNA bases that encodes TRIM67 colorectal adenocarcinoma or mucinous carcinoma (84 kDa) [12]. To our knowledge, TRIM67 has been and were immediately preserved in liquid nitrogen reported to be involved in neuroprotective effects and within 5 min following resection and then placed at tumor processes. Nicholas P. Boyer et al. [13] found −80°C for long-term preservation. that TRIM67 is necessary for appropriate brain development and behavior. TRIM67 is also associated DNA methylation assay with ubiquitination and interacts with PRG-1 and Genomic DNA (gDNA) was extracted from 10 80K-H to regulate the RAS signaling pathway to pairs of human CRC tissues and adjacent normal inhibit cell proliferation and enhance synapse tissues using the DNeasy Blood and Tissue Kit production in neuroblastoma [14]. Le Duy Do et al. (Qiagen, Valencia, CA, USA) according to the [15] found that autoantibodies against TRIM67 manufacturer's protocol. DNA quality assessment and appeared to be specific biomarkers of paraneoplastic quantification were performed using the NanoDrop cerebellar degeneration and associated with lung 1000 (Thermo Scientific). Bisulphite conversion was carcinoma. A recent study implicated that TRIM67 performed according to the manufacturer’s activates the NF-κB pathway and promotes cell recommendations for the Illumina Infinium Assay apoptosis in GA-13315-treated lung cancer cells [16]. (EZ DNA methylation kit, ZYMO, Orange, CA). The Our previous study found that in The Cancer Genome converted and amplified gDNA fragment was used Atlas (TCGA), TRIM67 is frequently methylated in for hybridization with the 15-mers specific capture CRC compared with adjacent normal tissues, and probe (Table 2) according to the Illumina Infinium HD prevents intestinal tumorigenesis in mice and methylation protocol for genomic facilities improves chemotherapy efficacy by activating the (Novogene, Beijing, China). The nucleotide substrates TRIM67–p53 axis [17]. In the present study, TRIM67 (A/T and C/G) were labeled by different fluorescent expression levels and clinicopathological features dyes and only the probe with complementary binding based on TCGA dataset and our cohort was further of gDNA could be single base extended. Finally, iScan examined. We also investigated the direct functions of software was used to read and output the methylation TRIM67 in CRC cells and studied its potential level results according to the fluorescence. mechanisms as a tumor suppressor in CRC. Cell culture and transfection Materials and Methods Human CRC cell lines HCT116, SW480, LoVo, SW620, SW1116, Caco2, SW1463, and HT29 cell lines TCGA data download and analysis were obtained from Prof. Jun Yu (The Chinese mRNA sequencing data of 647 CRC tissues and University of Hong Kong, Hong Kong, China). 51 normal tissues was downloaded from TCGA HCT116 and HT29 were cultured in McCoy’s 5A (https://tcga-data.nci.nih.gov/), and data for the medium (Gibco, Grand Island, NY, USA) and the mRNA expression of 19,640 genes were obtained. other cells lines were cultured in DMEM medium Immunohistochemistry (IHC) data based on TCGA (Gibco), supplemented with 10% fetal bovine serum was downloaded from The Human Protein Atlas (FBS; Gibco) and 1% penicillin-streptomycin (https://www.proteinatlas.org/). The staining (Invitrogen, Carlsbad, CA, USA) in a 37°C humidified intensity was scored as negative (0), weak (1), incubator with 95% air and 5% CO2. moderate (2), or strong (3). The staining quantity was PLV-TRIM67-shRNA and negative control scored as 1 (≤10%), 2 (11%–50%), 3 (51%–75%), or 4 plasmids were obtained from Prof. Jun Yu. (>75%). The total immunostaining score was M98-TRIM67, M98-MAPK11, siMAPK11, and negative calculated by multiplying the staining intensity score control plasmids were purchased from GeneCopoeia with the quantity and ranged from 0 to 12 [18]. (Guangzhou, China). The plasmids were transfected into CRC cells using Lipofectamine 2000 (Invitrogen) Patients and tissue specimens according to the manufacturer’s protocol. The present study was approved by the Ethics Committee of The First Hospital of Hebei Medical University (No. 2016004) and was in accordance with the principles of Declaration of Helsinki. A total of 60 http://www.jcancer.org Journal of Cancer 2020, Vol. 11 6027 Reverse transcription-quantitative polymerase colony formation assay, transfected cells were seeded chain reaction (RT-qPCR analysis) into 10-cm plates. The visible colonies were fixed with Total RNA was extracted from frozen tissues and 4% formaldehyde and stained with crystal violet after cells using TRIzol® reagent (Invitrogen) according to 12 days. Colonies containing more than 50 cells were the manufacturer’s protocol. cDNA was produced counted and each condition was performed in from the RNA by performing reverse transcription triplicate. using a PrimeScript™ RT Reagent Kit (Perfect Real