High mical l2 expression and its role in the prognosis of colon adenocarcinoma

(2022) 22:487 Yang et al BMC Cancer https://doi.org/10.1186/s12885-022-09614-0 Open Access RESEARCH High MICAL‑L2 expression and its role in the prognosis of colon adenocarcinoma Yixing Yang1†, Fengwen Ye2†, Tianxiang Xia2, Qianwen Wang2, Yujie Zhang2* and Jun Du2*  Abstract  Background:  MICAL-like protein (MICAL-L2), a member of the molecules interacting with CasL (MICAL) family of proteins, is strongly associated with the malignancy of multiple types of cancer However, the role of MICAL-L2 in colon adenocarcinoma (COAD) has not been well characterized Methods:  In this study, we analyzed the role of MICAL-L2 in COAD using datasets available from public databases The mRNA and protein expression of MICAL-L2 was investigated using TCGA, UALCAN, and independent immunohistochemical assays Overall survival (OS) and disease-specific survival (DSS) of COAD patients were assessed based on the MICAL-L2 expression level using the Kaplan–Meier method Univariate and multivariate analysis was employed to determine whether MICAL-L2 could serve as an independent prognostic indicator of OS Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were further utilized to explore the possible cellular mechanism underlying the role of MICAL-L2 in COAD In addition, the correlation between MICAL-L2 expression and immune cell infiltration levels was investigated via single-sample gene set enrichment analysis (ssGSEA) Results:  Data from TCGA, HPA, and UALCAN datasets indicated that MICAL-L2 expression was significantly higher in COAD tissue than in adjacent normal tissues, and this was confirmed by immunohistochemical assays Kaplan–Meier survival analysis revealed that patients with MICAL-L2 had shorter OS and DSS Furthermore, multivariate Cox analysis indicated that MICAL-L2 was an independent risk factor for OS in COAD patients ROC analysis confirmed the diagnostic value of MICAL-L2, and a prognostic nomogram involving age, M stage, and MICAL-L2 expression was constructed for OS Functional enrichment analyses revealed that transport-related activity was closely associated with the role of MICAL-L2 in COAD Regarding immune infiltration levels, MICAL-L2 was found to be positively associated with ­CD56bright NK cells Conclusions:  Our results suggested that MICAL-L2 is a promising biomarker for determining prognosis and correlated with immune infiltration levels in COAD Keywords:  MICAL-L2, Overall survival, Prognosis, COAD *Correspondence: zeater87@126.com; dujun@njmu.edu.cn † Yixing Yang and Fengwen Ye contributed equally to this work Department of Physiology, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing 211166, China Full list of author information is available at the end of the article Background Colon cancer is a commonly diagnosed malignant tumor of the digestive tract and a leading cause of cancerrelated death worldwide [1] It usually affects adults at 40–50 years of age and occurs more often in males than females The etiology of colon cancer is mainly associated with a high-fat diet, colonic polyps, genetic make-up, and chronic inflammation [2] Patients in the early stage of colon cancer may not present obvious clinical symptoms © The Author(s) 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/ The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Yang et al BMC Cancer (2022) 22:487 As the tumor volume increases, patients may display abdominal distension and dyspepsia, and may even be able to feel a lump/mass in the abdomen Although the 5-year survival rate of patients in the early stages of colon cancer can be higher than 90%, that of patients diagnosed at an advanced stage is lower than 20% [3–5] These observations underline the need to further unravel the mechanisms underlying colon cancer progression and identify novel therapeutic targets for the treatment of this disease Molecules interacting with CasL (MICALs) represent an evolutionarily conserved family of proteins with roles in the regulation of cytoske leton dynamics [6] MICALlike protein (MICAL-L2), a member of the MICAL family, has three conserved domains, namely, a calponin homology (CH) domain; a Lin11, Isl-1, and Mec-3 (LIM) domain; and a C-terminal coiled-coil (CC) domain [7] The CH and LIM domains link MICAL-L2 to the actin cytoskeleton, while the CC domain is required for interaction with Rab GTPases MICAL-L2 exerts its multiple biological functions primarily via processes involving cargo transportation For example, by binding to Rab13, MICAL-L2 triggers the transportation of glucose transporter-4 (GLUT4) and mediates GLUT4-containing vesicle localization and fusion with the muscle cell membrane [8] Rab13 and MICAL-L2 also act together in the transfer of actinin-4 from the cell body to the tips of neurites [9] It has been well documented that MICAL-L2 is highly expressed and promotes cell migration and invasion in multiple types of cancer, including gastric cancer, ovarian cancer, and breast cancer [10–12] In ovarian cancer cells, the silencing of MICAL-L2 was shown to inhibit canonical Wnt/β-catenin signaling and induce mesenchymal–epithelial transition [11] We have previously shown that MICAL-L2 facilitates the proliferation of lung cancer cells via the de-ubiquitination of c-Myc, which blocks its degradation [13] Recently, another MICAL family member, MICAL1, which shares sequence similarity with MICAL-L2 [14], was found to play a key role in the migration and growth of colorectal cancer cells by suppressing the ERG1/β-catenin signaling pathway [15] However, the role of MICAL-L2 in the prognosis and possible pathogenesis of colon cancer has not been fully elucidated Colon adenocarcinoma (COAD) is one the most common type of colon cancer In this study, several informatics tools were used to evaluate the expression profile and the prognostic significance of MICAL-L2 in COAD Moreover, the correlation between MICAL-L2 expression and immune infiltration, and the putative mechanisms underlying the role of MICAL-L2 in COAD, were also investigated This is the first comprehensive study of the association between MICAL-L2 expression and its Page of 12 clinical characteristics in COAD and our findings may contribute to our understanding of MICAL-L2-related processes in this cancer Methods Ethics statement All immunohistochemical assays with human tumor specimens were conducted according to the institutional guidelines of Jiangsu Province MICAL‑L2 mRNA expression and analysis of prognosis The mRNA expression of MICAL-L2 in COAD and the corresponding clinical information data were downloaded from The Cancer Genome Atlas (TCGA) database (https://​tcga-​data.​nci.​nih.​gov/​tcga/) [16] MICAL-L2 mRNA expression and its association with overall survival (OS) and disease-specific survival (DSS) of patients with COAD were also analyzed using the TCGA–COAD dataset The expression of MICAL-L2 was assessed in 456 COAD and 41 adjacent normal tissue samples from the TCGA database According to the median values of mRNA expression, patients with COAD were divided into high and low expression groups Data were collected and analyzed using R3.6.3 software [17] MICAL‑L2 protein expression analysis The Human Protein Atlas (HPA) database (https://​www.​ prote​inatl​as.​org) and the University of Alabama Cancer Database (UALCAN) (http://​ualcan.​path.​uab.​edu/​index.​ html) were used to compare MICAL-L2 protein expression between normal and COAD tissues Immunohistochemistry Immunohistochemistry was performed as previously described [18] COAD tissue microarrays were purchased from Outdo Biotech (Shanghai, China) Thirty paired COAD and paracancerous tissue samples were used for MICAL-L2 immunohistochemical assays After dewaxing and hydration, the microarray was incubated with 3% ­H2O2 for 30 min, subjected to antigen retrieval with citric acid at 95 °C for 20 min, blocked for 2 h at room temperature, incubated with primary antibody against MICAL-L2 at 4 °C overnight, and then with a species-matched secondary antibody for 2 h at room temperature DAB staining was employed to detect the expression of MICAL-L2, with hematoxylin serving as the counterstain Images were captured using an Olympus BX51 microscope The immunoreactivity score (IRS) was obtained by multiplying the percentage of stained cells by the staining intensity scores of MICAL-L2, as previously described [19, 20] Yang et al BMC Cancer (2022) 22:487 Enrichment analysis for MICAL‑L2 function An ordered list of genes was generated based on the correlation between all genes and MICAL-L2 expression Enriched pathways were determined using Gene Ontology (GO) [21, 22], KEGG [23–25], and GSEA [26, 27] In the KEGG analysis, genes were determined to be differentially expressed based on a log2 fold-change of > 1.0 and an adjusted P-value