Meng et al. Cancer Cell Int (2021) 21:332 https://doi.org/10.1186/s12935-021-02039-y Cancer Cell International PRIMARY RESEARCH Open Access Down-regulation of BCL2L13 renders poor prognosis in clear cell and papillary renal cell carcinoma Fei Meng1,2,3 , Luojin Zhang4, Mingjun Zhang5, Kaiqin Ye1,3, Wei Guo1,2, Yu Liu1,2,3, Wulin Yang1,3, Zhimin Zhai4, Hongzhi Wang1,3, Jun Xiao6* and Haiming Dai1,3* Abstract Background: BCL2L13 belongs to the BCL2 super family, with its protein product exhibits capacity of apoptosis-medi- ating in diversifed cell lines. Previous studies have shown that BCL2L13 has functional consequence in several tumor types, including ALL and GBM, however, its function in kidney cancer remains as yet unclearly. Methods: Multiple web-based portals were employed to analyze the efect of BCL2L13 in kidney cancer using the data from TCGA database. Functional enrichment analysis and hubs of BCL2L13 co-expressed genes in clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) were carried out on Cytoscape. Evaluation of BCL2L13 protein level was accomplished through immunohistochemistry on parafn embedded renal cancer tissue sections. Western blotting and fow cytometry were implemented to further analyze the pro-apoptotic function of BCL2L13 in ccRCC cell line 786-0. Results: BCL2L13 expression is signifcantly decreased in ccRCC and pRCC patients, however, mutations and copy number alterations are rarely observed. The poor prognosis of ccRCC that derived from down-regulated BCL2L13 is independent of patients’ gender or tumor grade. Furthermore, BCL2L13 only weakly correlates with the genes that mutated in kidney cancer or the genes that associated with inherited kidney cancer predisposing syndrome, while actively correlates with SLC25A4. As a downstream efector of BCL2L13 in its pro-apoptotic pathway, SLC25A4 is found as one of the hub genes that involved in the physiological function of BCL2L13 in kidney cancer tissues. Conclusions: Down-regulation of BCL2L13 renders poor prognosis in ccRCC and pRCC. This disadvantageous factor is independent of any well-known kidney cancer related genes, so BCL2L13 can be used as an efective indicator for prognostic evaluation of renal cell carcinoma. Keywords: BCL-rambo, Cell death, Renal cancer, Prognosis, ANT Introduction Kidney cancer remains one of the malignant tumors with *Correspondence: [email protected]; [email protected] the incidence increased notably in recent years [1]. It is 1 Anhui Province Key Laboratory of Medical Physics and Technology, composed of heterogeneous subtypes with histological Institute of Health and Medical Technology, Hefei Institutes of Physical and molecular abnormalities. Clear cell renal cell carci- Science, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031, Anhui, China noma (ccRCC) accounts for about 75% of all the cases, 6 Department of Urology, The First Afliated Hospital of USTC, Division which is mainly characterized as constitutional chro- of Life Sciences and Medicine, University of Science and Technology mosome 3p deletion, dysfunctional von Hippel-Lindau of China, Hefei 230001, China Full list of author information is available at the end of the article (VHL) gene and uncontrolled stabilization of hypoxia © The Author(s) 2021. 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. Meng et al. Cancer Cell Int (2021) 21:332 Page 2 of 12 inducible factors (HIFs) by molecular features. Papillary Materials and methods renal cell carcinoma (pRCC), which occupies about 15% University of California Santa Cruz (UCSC) genome browser of the incidence, usually displays loss of chromosome Y, and UCSC Xena gain of chromosome 7 and/or chromosome 17. Moreo- Protein–protein interaction (PPI) network analysis of ver. diferent molecular characteristics are assigned to BCL2L13 was performed using UCSC genome browser each pathological subtype, including both Type I and (https:// genome. ucsc. edu), which ofers visualized inter- Type II pRCC [2, 3]. Chromophobe renal cell carcinoma connection of the concerned genes [18, 19]. BCL2L13 (chRCC) and some other rare types make up the rest. mRNA and exon expression were completed on UCSC Mutations of VHL have been proved to be one of the Xena platform (http:// xena. ucsc. edu), complied with data typical genetic causes of kidney cancer, which often lead from TCGA [20]. to stabilization of HIF1α and HIF2α, creating an illusion of pseudohypoxia in stricken renal tissues [4]. Te accu- mulated HIF1α-HIF1β and HIF2α-HIF1β dimers will Catalogue of Somatic Mutations in Cancer (COSMIC) cause an elevation in growth factors, including platelet- and Open Targets Platform derived growth factors (PDGFs) and vascular endothe- Te most frequent somatic mutations in ccRCC and lial growth factors (VEGFs), which will prompt tumor pRCC tissues were queried by COSMIC cancer browser angiogenesis [5, 6]. Te VHL-HIF-VEGF axis repre- (http:// cancer. sanger. ac. uk), showing the top 20 can- sents one of the canonical pathways for renal carcino- didate genes curated from published research for each genesis, while some other gene mutations or epigenetic cancer type [21]. Heritable kidney cancer-predisposing changes have also been reported [7, 8], including muta- syndrome related genes are searched from Open Targets tions of polybromo-1 (PBRM1) [9], SET domain-con- Platform (https:// www. targe tvali dation. org). Te over- taining 2 (SETD2) [10] and tuberous sclerosis complex all target-disease association score is the harmonic sum 1/2 (TSC1/2) [11, 12]. Nevertheless, nearly 40% of the aggregated from genetics, genomics, drugs, animal mod- resected kidney cancer patients are confronted with risk els and text mining data [22]. of recurrence, the efective predictive markers are absent so far [13]. BCL-2-like protein 13 (BCL2L13), also termed BCL- University of Alabama Cancer Database (UALCAN) rambo, is encoded by the BCL2L13 gene [14]. It has and cBioPortal for cancer genomics manifested that BCL2L13 participates in drug resist- Te BCL2L13 mRNA expression and corresponding ance in several tumors. For example, BCL2L13 was efect on tumor prognosis were analyzed on UALCAN found elevated in tumors like glioblastoma (GBM) and (http:// ualcan. path. uab. edu/) [23–25]. Lymph node childhood acute lymphoblastic leukemia (ALL) [15, 16]. metastasis of renal cancer: N0, No regional lymph node In GBM, BCL2L13 interacts with ceramide synthases 2 metastasis; N1, Metastases in 1 to 3 axillary lymph nodes; and 6 (CerS2/6), inhibiting the leakage of cytochrome c N2, Metastases in 4 to 9 axillary lymph nodes. Mutations into cytoplasm [15]. Augmented BCL2L13, which takes and copy number alterations (CNAs) of BCL2L13, the part in L-asparaginase resistance, executes as an inde- impact of these alterations on patients’ overall survival pendent adverse prognostic factor in ALL [16]. On the (OS), and BCL2L13 co-expressed genes in kidney cancer other hand, BCL2L13 has been demonstrated to inter- were achieved by cBioPortal for cancer genomics stud- act with adenine nucleotide translocator (ANT, encoded ies (http:// www. cbiop ortal. org/) [26]. |Spearman’s r|> 0.4 by SLC25A4), a component of mitochondrial perme- was set to sort out the BCL2L13 co-expressed genes in ability transition pore (MPTP), therefore promoting ccRCC and pRCC. cytochrome c release from mitochondrial intermem- brane space to cytoplasm, resulting in activation of the apoptotic caspase cascade [17]. TargetScan and the encyclopedia of RNA interactomes Te prognostic value of BCL2L13 in kidney cancer is (ENCORI) still unclearly. In this study, we profled BCL2L13 across TargetScan (http:// www. targe tscan. org) was engaged 33 cancer types in the Cancer Genome Atlas (TCGA), to analyze the potential target microRNA (miRNA) in and found that its mRNA expression is signifcantly homo sapiens, conformed to the canonical transcript reduced in ccRCC and pRCC. Moreover, low BCL2L13 of BCL2L13, which supported by 736 3P-seq tags [27]. expression correlates with lessened survival probability Correlation between BCL2L13 and the target miRNA of kidney cancer. It poses an attractive hypothesis that in kidney cancer were analyzed on ENCORI pan-cancer BCL2L13 may be a promising prognosis marker for kid- analysis platform (http:// starb ase. sysu. edu. cn/ panCa ncer. ney cancer. php), upon the expression data from TCGA [28]. Meng et al. Cancer Cell Int (2021) 21:332 Page 3 of 12 Cytoscape and Search Tool for the Retrieval of Interacting 5.1) were used to capture the morphological changes Genes (STRING) of 786-0 cells after treatments. For quantitative assess- Kyoto Encyclopedia of Genes and Genomes (KEGG) ment, 786-0 cells were harvested after treatments pathway enrichment analysis and hubs of BCL2L13 co- and stained with both FITC Annexin V and PI (BD expressed genes were accomplished within Cytoscape, Pharmingen, 556547), then subjected to fow cytom- with the plug-in ClueGO, CluePedia, MCODE and etry (Beckman Coulter, CytoFLEX). Annexin V + / CytoHubba, which realized by the grid topology algo- PI − , Annexin V + /PI + and Annexin V − /PI + cells rithm density of maximum neighborhood component were used to represent the types of early apoptotic, late (DMNC) [29–31].