CIRP Promotes the Progression of Non-small Cell Lung Cancer Through Activation of Wnt/β-catenin Signaling via CTNNB1

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 clinic 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 injecting and subcutaneously inoculating of animal models were performed to study the role of CIRP in NSCLC cells; Gene expression microarray was used to select the underlying pathways; RNA immunoprecipitation assay, biotin pull-down assay, immuno-purification assay, mRNA decay analyses and luciferase reporter assay were performed to elucidate the mechanisms. The log-rank (Mantel-Cox) test, independent sample T test, the 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 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 cell cycle, DNA replication and Wnt signaling pathway to exert its pro-oncogenic action. Mechanistically, CIRP directly bound to the 3’- and 5'-UTR of CTNNB1 mRNA, leading to enhanced stability and translation of CTNNB1 mRNA and promote IRES-mediated protein synthesis, respectively. Eventually, the increased CTNNB1 protein levels mediated excessive activation of the Wnt/β-Catenin signaling pathway and its downstream C-myc, COX-2, CCND1, MMP7, VEGFA and CD44. Conclusion: Our results support CIRP as a candidate oncogene in NSCLC and a potential target for NSCLC therapy.