Circular RNA circ-CMPK1 contributes to cell proliferation of non-small cell lung cancer by elevating cyclin D1 via sponging miR-302e

Abstract

Background: It is well recognized that competing endogenous RNA (ceRNA) regulatory network is linked to the development and progression of cancer, including non-small cell lung cancer (NSCLC). Herein, we aimed to explore the functional role of circ-CMPK1/miR-302e/cyclin D1 ceRNA signaling in NSCLC.

Methods: GEO database (GSE102287) was utilized to screen differentially expressed miRNAs in NSCLC. Quantitative reverse transcription PCR (qRT-PCR) and western blotting assays were used to determine gene expression. Cell proliferation analysis was performed with Cell Counting Kit-8 (CCK-8) and cell cycle assays. Luciferase reporter and RNA pull-down assays were conducted to identify the interaction among circ-CMPK1, miR-302e, and cyclin D1. Xenograft tumor model was established to evaluate the role of circ-CMPK1/miR-302e/cyclin D1 axis in vivo.

Results: miR-302e expression was significantly down-regulated in NSCLC cell lines and tissues and its decrease was closely associated with aggressive clinicopathological features and unfavorable outcome. Overexpression and knockdown of miR-302e obviously retarded and enhanced the growth of NSCLC, respectively. Furthermore, we found that miR-302 was sponged by circular RNA CMPK1 (circ-CMPK1, hsa_circ_0012384), which was remarkably up-regulated in NSCLC and predicted poor prognosis. Circ-CMPK1 was capable to promote NSCLC cells proliferation by increasing the expression of cyclin D1 via inhibiting miR-302 activity. Moreover the miR-302e-mediated tumor inhibition could be effectively counteracted by ectopic expression of circ-CMPK1 or cyclin D1 both in vitro and in vivo.

Conclusion: Our data demonstrate for the first time that circ-CMPK1/miR-302e/cyclin D1 signaling plays an essential regulatory role in NSCLC and targeting this axis may be an efficacious avenue for treatment of NSCLC patients.

Keywords: NSCLC; biomarker; ceRNA; circular RNA; microRNA; proliferation.

Figure 1

miR‐302e is frequently down‐regulated in NSCLC cells and tissues. (a) The top five up‐ and down‐regulated miRNAs in normal and NSCLC tissues (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102287). FC, Fold change. (b, c) qRT‐PCR analysis for miR‐302e expression in NSCLC cell lines (b) and tissues (c). (d, e) The survival curves of NSCLC patients with low and high miR‐302e expression in our (d) or KM‐plotter (e) survival data (http://kmplot.com/analysis/). ** p < .01, *** p < .001

Figure 2

miR‐302e is a proliferation inhibiting factor in NSCLC cells. (a) qRT‐PCR analysis for miR‐302e expression in A549 and H460 cells transfected with control or miR‐302e mimics. (b) CCK‐8 assay at the indicated time in control or miR‐302e‐overexpressing A549 and H460 cells. The absorbance was recorded at 450nM. (c) Cell cycle analysis with PI staining in A549 and H460 cells. (d) qRT‐PCR analysis for miR‐302e expression in 95‐D and H1299 cells transfected with control or miR‐302e inhibitors. (e, f) CCK‐8 (e) and cell cycle (f) assays were used to test the proliferative abilities of control or miR‐302e‐silencing 95‐D and H1299 cells. * p < .05, ** p < .01, *** p < .001

Figure 3

Circ‐CMPK1 acts as a sponge of miR‐302e in NSCLC cells. (a) The wild‐type and mutant sequence of circ‐CMPK1 and miR‐302e binding site. (b) Luciferase reporter assay for wild‐type and mutant circ‐CMPK1 luciferase vectors in A549 and H460 cells transfected with control or miR‐302e mimics. (c) RNA pull‐down assay was carried out in A549 and H460 cells transfected with biotinylated control or miR‐302e probe, followed by qRT‐PCR analysis for circ‐CMPK1 expression. (d, e) qRT‐PCR analysis for circ‐CMPK1 expression in NSCLC tissues (d) and cell lines (e). (f) The survival curves of NSCLC patients with low and high circ‐CMPK1 expression. (g) qRT‐PCR analysis for miR‐302e expression in 95‐D and H1299 cells with or without circ‐CMPK1 overexpression. (h) The correlation between circ‐CMPK1 and miR‐302e expression in NSCLC tissues. (i) Cell cycle analysis with PI staining in circ‐CMPK1‐overexpressing 95‐D and H1299 cells with or without miR‐302e overexpression. ** p < .01, *** p < .001

Figure 4

Novel circ‐CMPK1/miR‐302e/cyclin D1 ceRNA regulatory network functions in vivo and in vitro. (a) The wild‐type and mutant sequence of miR‐302e and cyclin D1 binding site. (b) Luciferase reporter assay for wild‐type and mutant cyclin D1 3′‐UTR luciferase vectors in A549 and H460 cells transfected with control or miR‐302e mimics. (c, d) qRT‐PCR (c) and western blot (d) assays for cyclin D1 expression in miR‐302e‐overexpressing A549 and H460 cells with or without circ‐CMPK1 overexpression. (e) qRT‐PCR analysis for cyclin D1 expression in adjacent normal and NSCLC tissues. (f) The correlation between circ‐CMPK1 and cyclin D1 expression in NSCLC tissues. (g) Cell cycle assays with PI staining in miR‐302e‐overexpressing A549 and H460 cells with or without circ‐CMPK1 or cyclin D1 overexpression. (h, i) The volume and weight of tumors in nude mice bearing miR‐302e‐overexpressing A549 cells with or without circ‐CMPK1 or cyclin D1 overexpression (n = 5 in each group). * p < .05, ** p < .01, *** p < .001