Abstract
BackgroundCold-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.MethodsTissue 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.ResultsOur 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.ConclusionOur results support CIRP as a candidate oncogene in NSCLC and a potential target for NSCLC therapy.
Highlights
Cold-inducible RNA binding protein (CIRP) is a newly discovered proto-oncogene
Wang et al found that TREM-1 and TLR4-MD2 complexes act as Extracellular CIRP (eCIRP) receptors to activate the inflammatory cascade and amplify the inflammation of macrophages [9, 10]; while the combination of eCIRP and IL-6R induces activation of the STAT3 pathway to make macrophages tend to the M2 phenotype, which may prevent the amplification of inflammation providing a negative feedback loop [11]
CIRP directly bound to the 3′- and 5′-Untranslated region (UTR) of CTNNB1 mRNA to enhance its stability and translation, and the resultant accumulation of CTNNB1 protein induced excessive activation of Wnt/β-catenin signaling and promoted the progression of non-small cell lung cancer (NSCLC)
Summary
Cold-inducible RNA binding protein (CIRP) is a newly discovered proto-oncogene. Many studies have focused on the identification of NSCLC associated genes [3,4,5], the molecular pathogenesis of NSCLC is far from clear. Searching for new genes that are involved in the development of NSCLC is critical for improving the molecular typing and completing the regulation network of NSCLC, and may provide novel targets for precision therapy [6]. Cold-inducible RNA binding protein (CIRP) is constitutively expressed in the nucleus at low levels in various tissues, and its expression can be induced by mild hypothermia, UV irradiation, hypoxia, osmotic pressure, and ischemic disorders [7]. Study have shown that sepsis and hemorrhagic shock can induce CIRP to be released into extracellular (eCIRP) through the lysosomal secretion, and act as a damaged-associated molecular pattern (DAMP )[9]. Whether eCIRP has biological effects other than inflammation regulation has not been studied yet
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