CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27(Kip1) mRNA translation

Abstract

The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.

Figure 1. CPEB1 is downregulated in glioblastoma and affects proliferation of glioma cell lines.

(A) Microarray data for CPEB1 gene expression from TCGA dataset consisting of normal (n = 10) and GBM (n = 542) samples; or from “Sun brain” dataset consisting of normal (n = 23) and GBM (n = 81) samples, are shown as a box-plots. A non-parametric t test was performed to calculate p values as indicated in the text. The horizontal line in each plot represents the mean value. (B) Total RNA was extracted from A172, LN18, U87 and T98G glioblastoma cell lines, or from cultured human astrocytes and endogenous CPEB1 mRNA expression was analysed by RT-qPCR. CPEB1 mRNA expression in all samples is reported as compared to that in “normal human brain” (Ambion-Thermofisher #AM7962), set as = 1. ANOVA test in combination with Dunnett’s test were performed to calculate p values. (C) Cells were transfected with the empty plasmid (C) or pCPEB1 plasmid and, after 48 h, viable cells were counted using a hemocytometer. Student’s t-test was performed. D, T98G cells transfected with siRNAs against CPEB1 or a non-targeting control siRNA (siC) were analysed as in (C). In this and all the subsequent figures, *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were performed three times (biologic replicates). All histograms in all figures refer to mean ± S.D.

Figure 2. CPEB1 directly interacts with the 3′UTR of p27^kip1.

(A) T98G and A172 cells were transfected with the empty vector (C) or pCPEB1. After 48 h, the whole-cell lysates were run on the same gel, blotted onto nitrocellulose and the membrane cut to allow probing with antibodies against p27 or Gapdh. Relative signal intensities (right panel) for p27 in the Western blots normalized to the Gapdh loading control, are included for comparison of p27 levels. Bars represent average +/− SD of three independent experiments. Student’s t-test was utilized to calculate p values. (B) Cell lysates from T98G cells trasfected with the empty vector (C), pCPEB1 or pEZH2, were subjected to RIP assay with Flag antibody. P27 and GAPDH mRNA levels were detected using RT-PCR as shown in the representative cropped gel. (C) Proliferation assay of HeLa cells co-transfected with pCPEB1, siRNAs against p27 and relative controls was performed as in Fig. 1C. ANOVA test in combination with Dunnett’s test were performed to calculate p values, comparing the mean of each column with the mean of the control column. (D) pUTRp27 luciferase and a renilla control luciferase constructs were co-transfected with the empty vector (C) or pCPEB1. After 48 h from transfection, T98G and HeLa cells were harvested and assayed with Dual Luciferase Assay (Promega) according to the manufacturer’s instructions. Relative luciferase activity is the ratio between firefly luciferase and renilla control luciferase. Student’s t-test was utilized to calculate p values. (E) pUTRp27 luciferase constructs were co-transfected with siRNA negative control (siC) or siRNA against CPEB1, and cells were treated as in (D). (F) HeLa cells were transfected with the indicated luciferase constructs in the presence of empty vector (C) or pCPEB1 and processed as in (D). (G) Protein complexes extracted from HeLa cells, transfected with the indicated luciferase constructs, were immunoprecipitated and treated as indicated in (B). The cDNA was amplified by specific oligos corresponding to the Firefly luciferase mRNA.

Figure 3. CPEB1 competes with miR221/222 binding at an overlapping site in the 3′UTR of p27^kip1.

(A) p27 mRNA 3′UTR putative sites targeted by CPEB (orange) and by miR-221/222 seed sequence (green). (B) pUTRp27 or pUTRp27m1 were co-transfected with a plasmid expressing miR221/222 (p222/221) and pCPEB1 (last bar). After 48 h, HeLa cells were subjected to luciferase assay as in Fig. 2D. (C) pUTRp27 was co-transfected with pCPEB1 and/or LNA antisense oligonucleotides directed against endogenous miR-221 and miR-222 (antimiR). After 48 h, cells were treated as in (B). (D) HeLa cells were transfected with either pUTRp27 or pSeedMut together with pCPEB1 or an empty vector. After 48 h, cells were subjected to luciferase assay as in Fig. 2D. ANOVA test in combination with Dunnett’s test were performed to calculate p values. The mean of each column was compared with the mean of the control column.

Figure 4. CPEB stimulates p27 expression by promoting the elongation of polyA tail and translation efficiency.

(A) HeLa cells transfected with either empty vector, or pCPEB1 or siRNAs against CPEB1 were treated with Actinomycin D (10 μg/ml). RNA was isolated at various time points after the treatment and analyzed by RT-qPCR. (B) HeLa cells were transfected as indicated in the panels and after 48 h, total RNA was isolated and subjected to PAT assays. (C) Cells transfected with control vector or pCPEB1 were lysed and cytoplasmic extracts were subjected to polysome gradient. RNA extracted from gradients collected in fraction HP (Heavy Polysomes), fraction LP (Light Polysomes) and fraction NP (Non-Polysomes) was reverse transcribed into cDNA and analysed by qPCR with primers specific for p27 (upper panel) or GAPDH (lower panel). The final result was reported as the percentage of RNA in each fraction. (D) Cells co-transfected with pCPEB1 and either pUTRp27 or pUTRp27m1 were treated as in (C). The RT-qPCR was performed with primers specific for the Firefly luciferase mRNA. ANOVA test in combination with Tukey’s multiple comparisons test were performed to calculate p values in (C,D).