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CIRP Promotes the Progression of Non-Small Cell Lung Cancer Through

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CIRP Promotes the Progression of Non-Small Cell Lung Cancer Through Liao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:275 https://doi.org/10.1186/s13046-021-02080-9 RESEARCH Open Access CIRP promotes the progression of non- small cell lung cancer through activation of Wnt/β-catenin signaling via CTNNB1 Yi Liao1,2†, Jianguo Feng3†, Weichao Sun1, Chao Wu2, Jingyao Li1, Tao Jing4, Yuteng Liang5, Yonghui Qian5, Wenlan Liu1,5* and Haidong Wang2* Abstract Background: Cold-inducible RNA binding protein (CIRP) is a newly discovered proto-oncogene. In this study, we investigated the role of CIRP in the progression of non-small cell lung cancer (NSCLC) using patient tissue samples, cultured cell lines and animal lung cancer models. Methods: Tissue arrays, IHC and HE staining, immunoblotting, and qRT-PCR were used to detect the indicated gene expression; plasmid and siRNA transfections as well as viral infection were used to manipulate gene expression; cell proliferation assay, cell cycle analysis, cell migration and invasion analysis, soft agar colony formation assay, tail intravenous injection and subcutaneous inoculation of animal models were performed to study the role of CIRP in NSCLC cells; Gene expression microarray was used to select the underlying pathways; and RNA immunoprecipitation assay, biotin pull-down assay, immunopurification assay, mRNA decay analyses and luciferase reporter assay were performed to elucidate the mechanisms. The log-rank (Mantel-Cox) test, independent sample T- test, nonparametric Mann-Whitney test, Spearman rank test and two-tailed independent sample T-test were used accordingly in our study. Results: Our data showed that CIRP was highly expressed in NSCLC tissue, and its level was negatively correlated with the prognosis of NSCLC patients. By manipulating CIRP expression in A549, H460, H1299, and H1650 cell lines, we demonstrated that CIRP overexpression promoted the transition of G1/G0 phase to S phase and the formation of an enhanced malignant phenotype of NSCLC, reflected by increased proliferation, enhanced invasion/metastasis and greater tumorigenic capabilities both in vitro and in vivo. Transcriptome sequencing further demonstrated that CIRP acted on the cell cycle, DNA replication and Wnt signaling pathway to exert its pro-oncogenic action. Mechanistically, CIRP directly bound to the 3′- and 5′-UTRs of CTNNB1 mRNA, leading to enhanced stability and translation of CTNNB1 mRNA and promoting IRES-mediated protein synthesis, respectively. Eventually, the increased CTNNB1 protein levels mediated excessive activation of the Wnt/β-catenin signaling pathway and its downstream targets C-myc, COX-2, CCND1, MMP7, VEGFA and CD44. * Correspondence: [email protected]; [email protected] †Yi Liao and Jianguo Feng contributed equally to this work. 1The Central Laboratory, Shenzhen Second People’s Hospital/First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, Guangdong 518035, P. R. China 2Department of Thoracic Surgery, Southwest Hospital, Army Medical University, Chongqing 400038, P. R. China Full list of author information is available at the end of the article © The Author(s). 2021 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:275 Page 2 of 19 Conclusion: Our results support CIRP as a candidate oncogene in NSCLC and a potential target for NSCLC therapy. Keywords: Non-small cell lung cancer, CIRP, CTNNB1, Wnt/β-catenin Background senescence [12–16]. In addition, CIRP could post- Non-small cell lung cancer (NSCLC) is one of the most transcriptionally regulate the expression of tumoral bio- malignant tumors, with an increasing incidence world- markers RPA2 and TRX [17, 18], mediating the expres- wide [1]. Although the diagnostic and treatment algo- sion of poor prognosis-related genes such as hypoxia rithms for NSCLC are constantly improving, it is inducible transcription factor-1 (HIF-1) and vascular difficult to obtain satisfactory curative effects, and the endothelial growth factor (VEGF) [19–21]. Moreover, long-term survival rate is still low [2]. A relatively poor studies have also reported that CIRP upregulation in- understanding of the molecular mechanisms underlying creases the expression of cell cycle-related proteins, such the pathogenesis of NSCLC is one of the reasons. Al- as cyclin D1 and c-Myc [22, 23]. In another study, CIRP though, many studies have focused on the identification was shown to negatively regulate p53 levels, thereby of NSCLC associated genes [3–5], the molecular patho- downregulating proapoptotic genes and upregulating genesis of NSCLC is far from clear. Therefore, searching antiapoptotic genes [24]. These findings all support for new genes that are involved in the development of CIRP as a new proto-oncogene to promote the progres- NSCLC is critical for improving the molecular typing sion of various cancers through multiple cellular signal- and completing the regulation network of NSCLC, and ing pathways, such as brain cancer, breast cancer, oral may provide novel targets for precision therapy [6]. cancer, colon cancer, prostate cancer, and liver cancer Cold-inducible RNA binding protein (CIRP) is consti- [25]. However, the mechanisms underlying the role(s) of tutively expressed in the nucleus at low levels in various CIRP in the regulation of NSCLC progression remain tissues, and its expression can be induced by mild largely unknown. hypothermia, UV irradiation, hypoxia, osmotic pressure, In this study, we investigated CIRP expression and its and ischemic disorders [7]. CIRP contains an RNA rec- functions in NSCLC. Our data showed that CIRP was ognition motif (RRM) domain with RNA binding func- overexpressed in NSCLC cell lines and human NSCLC tion and a glycine-rich C-terminal domain involved in tissue, and CIRP overexpression was correlated with a cell localization [8]. Study have shown that sepsis and poor prognosis in NSCLC patients. Mechanistically, hemorrhagic shock can also induce CIRP to be released CIRP directly bound to the 3′-and5′-UTRs of into extracellular (eCIRP) through the lysosomal secre- CTNNB1 mRNA to enhance its stability and translation, tion, and act as a damaged-associated molecular pattern and the resultant accumulation of CTNNB1 protein in- (DAMP )[9]. Hitherto, several signaling pathways of in- duced excessive activation of Wnt/β-catenin signaling flammation induced by eCIRP have been uncovered. and promoted the progression of NSCLC. Wang et al. found that TREM-1 and TLR4-MD2 com- plexes act as eCIRP receptors to activate the inflamma- tory cascade and amplify the inflammation of Materials and methods macrophages [9, 10]; while the combination of eCIRP Tissue arrays and IL-6R induces activation of the STAT3 pathway to Eighty-six tumor tissues and their paired noncancerous make macrophages tend to the M2 phenotype, which tissues were randomly collected from NSCLC patients may prevent the amplification of inflammation providing who had undergone surgical resection in the Depart- a negative feedback loop [11]. Whether eCIRP has bio- ment of Thoracic Surgery, Southwest Hospital, Army logical effects other than inflammation regulation has Medical University (Chongqing, China). No antitumor not been studied yet. treatment was performed before the surgery. All samples Intracellular, it is believed that CIRP is a stress- were frozen in liquid nitrogen within 10 min following induced protein and participates in multiple cellular sig- surgical resection and stored at − 80 °C until analyses. naling pathways by post-transcriptional regulation of the Tissue array blocks containing NSCLC tissues and adja- translation of specific mRNAs. Studies have shown that cent noncancerous tissues were established using a tis- tumor necrosis factor-alpha (TNF-α), cycloheximide, or sue microarrayer (Leica, Germany). Procedures for the hypothermia treatment could upregulate the expression collection of human samples and their usage for tissue of CIRP and thus increase the expression of phosphory- arrays were approved by the Ethical Committee of the lated extracellular signal-regulated kinase 1/2 (ERK1/2), Army Medical University (Chongqing, China). Informed leading to the inhibition of the apoptosis signal acquisi- consent forms were obtained from the patients before tion factor caspase-3 and the bypassing of replicative participating in this study. Liao et al. Journal of Experimental & Clinical Cancer Research (2021) 40:275 Page 3 of 19 Immunohistochemistry (IHC) and hematoxylin-eosin (HE) performed in accordance with
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