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. 2015 Oct 6;6(30):29413-27.
doi: 10.18632/oncotarget.5003.

The PTTG1-targeting miRNAs miR-329, miR-300, miR-381, and miR-655 inhibit pituitary tumor cell tumorigenesis and are involved in a p53/PTTG1 regulation feedback loop

Hai-qian Liang  1 , Ren-jie Wang  1 , Cai-feng Diao  2 , Jian-wei Li  1 , Jing-liang Su  1 , Sai Zhang  1
Affiliations

The PTTG1-targeting miRNAs miR-329, miR-300, miR-381, and miR-655 inhibit pituitary tumor cell tumorigenesis and are involved in a p53/PTTG1 regulation feedback loop

Hai-qian Liang et al. Oncotarget. .

Abstract

Deregulation of the pituitary tumor transforming gene (PTTG1), a newly discovered oncogene, is a hallmark of various malignancies, including pituitary tumors. However, the mechanisms regulating PTTG1 expression are still needed to be explored. MicroRNAs (miRNAs) are a novel class of small RNA molecules that act as posttranscriptional regulators of gene expression and can play a significant role in tumor development. Here, we identified a series of miRNAs, namely, miR-329, miR-300, miR-381 and miR-655, which could target PTTG1 messenger RNA and inhibit its expression. Interestingly, all four miRNAs significantly that are downregulated in pituitary tumors were mapped to the 14q32.31 locus, which acts as a tumor suppressor in several cancers. Functional studies show that the PTTG1-targeting miRNAs inhibit proliferation, migration and invasion but induce apoptosis in GH3 and MMQ cells. Furthermore, overexpression of a PTTG1 expression vector lacking the 3'UTR partially reverses the tumor suppressive effects of these miRNAs. Next, we identified the promoter region of PTTG1-targeting miRNAs with binding sites for p53. In our hands, p53 transcriptionally activated the expression of these miRNAs in pituitary tumor cells. Finally, we found that PTTG1 could inhibit p53 transcriptional activity to the four miRNAs. These data indicate the existence of a feedback loop between PTTG1 targeting miRNAs, PTTG1 and p53 that promotes pituitary tumorigenesis. Together, these findings suggest that these PTTG1-targeting miRNAs are important players in the regulation of pituitary tumorigenesis and that these miRNAs may serve as valuable therapeutic targets for cancer treatment.

Keywords: PTTG1; miRNAs; p53; pituitary tumor.

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Conflict of interest statement

CONFLICTS OF INTEREST

None declare.

Figures

Figure 1
Figure 1. Identification of PTTG1-targeting miRNAs
A. Potential miRNAs predicted to down-regulate the expression of PTTG1 (Targetscan6.2 and Miranda). B. MicroRNAs that target PTTG1 3′UTR, both predicted and previously described. C. A screenshot (http://genome.ucsc.edu; GRCh37/hg19 assembly) of the region of interest at the chromosomal level (red horizontal bar, top image) and in detail (bottom image), showing the 51 miRNAs located within 10 kb of each other. D. Real-time PCR analysis indicated that PTTG1 expression was significantly decreased in GH3 cells transfected with miRNA mimics (hsa-miR-NC, hsa-miR-374c, hsa-miR-3941, hsa-miR-603, hsa-miR-4477a, hsa-miR-4714-5p, hsa-miR-329, hsa-miR-300, hsa-miR-381, hsa-miR-655) (*P < 0.05). E. Measurement of PTTG1 expression levels by western blot analysis. Protein was extracted from GH3 cells transfected with miRNA mimics. The endogenous expression levels of the GAPDH protein were used for normalization, and the relative PTTG1 protein expression levels are shown. F, G, H. and I. Real-time PCR analysis indicated that miR-329, miR-300, miR-381, and miR-655 expression was significantly decreased in pituitary tumors relative to normal controls.
Figure 2
Figure 2. MiR-329, miR-300, miR-381 and miR-655 suppress cell proliferation activity and cell viability of GH3 and MMQ cells
A. and B. Measurement of miR-329, miR-300, miR-381 and miR-655 expression levels by real-time RT-PCR. U6 snRNA served as an endogenous normalizer of expression. The relative miRNA expression levels (mean ± SEM) are shown (*P < 0.05). C. and D. Cell viability was detected using the MTT assay. The relative cell growth activity was normalized to the growth activity of GH3 and MMQ cells in the control groups (*P < 0.05). E, F, G. and H. MiR-329, miR-300, miR-381 and miR-655 inhibit GH3 and MMQ colony formation. GH3 and MMQ cells transfected with miR-329, miR-300, miR-381, and miR-655 mimics or scrambled were seeded in 12-well plates. On the 14th day after seeding, the number of colonies was counted. The quantitative results are shown as the mean ± SEM. The experiments were repeated three times (*P < 0.05). I. and J. The GH3 pituitary tumor growth in vivo was determined based on the tumor weight after euthanasia. A representative image of an in vivo tumor in each group is shown. Group 1–6 is the group of mice injected with Scramble, miR-329, miR-300, miR-381, miR-655 or mixed miRNAs. Each group contained 5 nude mice. The error bars represent the SEM. (*P < 0.05). K. The tumors in each group were collected and combined, and then digested for western blot analysis to detect PTTG1 expression.
Figure 3
Figure 3. MiR-329, miR-300, miR-381 and miR-655 decrease cell motility in vitro and induce cell apoptosis of GH3 and MMQ cells
A–F. Transwell assays were used to evaluate the migration and invasion of GH3 (A, B and C) and MMQ (D, E and F) cells transfected with miR-329, miR-300, miR-381, miR-655, miR-329+miR-300+miR-381+miR-655 or scrambled. Representative fields of invasive cells on the membrane. The data were drawn from three independent experiments (*P < 0.05). G. and H. Apoptosis of GH3 and MMQ cells after transfection with miR-329, miR-300, miR-381, miR-655, miR-329+miR-300+miR-381+miR-655 or scrambled were determined by flow cytometry. The incidence of apoptotic cells is shown. The cells were stained with annexin V-fluorescein isothiocyanate and counterstained with 7-ADD (*P < 0.05).
Figure 4
Figure 4. MiR-329, miR-300, miR-381 or miR-655 target PTTG1
A. The construction and sequencing map of PTTG1–3′UTR. B. The predicted miR-300, miR-381, miR-329 or miR-655 binding site on the PTTG1 mRNA 3′UTR and the mutation at the miRNA “seed region” binding site are shown. C, D, E. and F. GH3 and MMQ cells were transfected with the wild type version of the luciferase-PTTG1 3′-UTR reporter vector as well as the miR-300, miR-381, miR-329, or miR-655 mimics or scrambled. The miR-300, miR-381, miR-329, and miR-655 mimics reduced the intensity of the luciferase-PTTG1 3′-UTR reporter vector relative to the scrambled group, while the miR-300, miR-381, miR-329, and miR-655 mutants failed to alter the luciferase intensity. (*P < 0.05).
Figure 5
Figure 5. PTTG1 Overexpression Counteracts miR-329, miR-300, miR-381 and miR-655 induced pituitary tumor cell malignant inhibition
A. Transfection with pcDNA3.1/PTTG1 increased PTTG1 protein levels in GH3 cells compared with the control group (*P < 0.05). B. and C. GH3 and MMQ cells were transfected with plasmids expressing PTTG1 or control vector, together with miR-329, miR-300, miR-381 and miR-655. Cell viability was detected by the MTT assay (*P < 0.05). D. and E. PTTG1 overexpression reversed the cell growth suppression caused by miR-300, miR-381, miR-655 and miR-329 in the colony formation assay. GH3 cells and MMQ cells were transfected with pcDNA3.1/PTTG1(PTTG1) or pcDNA3.1 with miR-300, miR-381, miR-655 and miR-329, and then seeded in 12-well plates. The colony formation rate is shown (*P < 0.05). F. and G. In GH3 and MMQ cells, PTTG1 ectopic expression reduced the cell apoptosis caused by miR-300, miR-381, miR-655 and miR-329. The cells were stained with annexin V-fluorescein isothiocyanate and counterstained with 7-ADD (*P < 0.05). H–J. Cell migration and invasion were increased by ectopic expression of PTTG1 in GH3 and MMQ cells (*P < 0.05).
Figure 6
Figure 6. p53 binds the promoter of PTTG1-targeting miRNAs, activating miR-300, miR-381 and miR-655 transcription
A. Schematic representation of potential p53 binding sites on the miRNA cluster promoter. Twelve predicted binding sites are shown. p53 is shown as a symbolic tetramer occupying a p53 binding motif containing two palindromic DNA sequences. P1-P12 represents the DNA-binding domain of p53 with PTTG1-targeting miRNAs. B. p53 binds the PTTG1-targeting miRNA promoter. Normalized inputs of HEK293 chromatin DNA were pulled down by p53 or negative IgG antibodies, and the DNA template was amplified by real-time PCR with specific PTTG1-targeting miRNA promoter primers (mentioned in Materials and Methods). A negative control primer was designed to assess the contribution of nonspecific binding. p53 binding sites in the p21 promoter were used as a positive control for the ChIP assay. Each negative IgG control was normalized to unit 1, each real-time PCR was performed in triplicate, and the ChIP experiments were repeated twice (*P < 0.05). C. p53 was reduced in GH3 cells treated with pSilencer2.1/p53-shRNA. Under this condition, PTTG1-targeting miRNAs were decreased in the GH3 cells. D. p53 was induced in GH3 cells treated with 1.0 mg/ml doxo for 16 h. Under this condition, PTTG1-targeting miRNAs were increased in the GH3 cells. Western blot analysis showed that p53 was reduced in GH3 cells treated with siRNA-p53 and induced in GH3 cells treated with 1.0 mg/ml doxo for 16 h.
Figure 7
Figure 7. PTTG1, PTTG1-targeting miRNAs and p53 form a feedback loop
A. Activity of the promoter constructs in Dox- and DOx+ HEK293 cells. The construct R1 indicates the full length from the position of the transcription start site (−1) to the upstream 4900 bp (−4900) regions containing the P2 p53 binding sites. Error bars represent standard deviations from three independent transfections each measured in triplicate. B. PTTG1 reduced p53 protein expression. GH3 cells were transfected with pcDNA3.1/PTTG1 and pcDNA3.1/p53. The p53 and PTTG1 protein was measured by western blotting. Western blots were quantified by Image-J and normalized to GAPDH. The experiments were repeated three times (*P < 0.05). C, D. and E. PTTG1 overexpression decreases the expression activity of miR-381, miR-300 and miR-655. GH3 cells transfected with pcDNA3.1/p53 together with increasing amounts of pcDNA3.1/PTTG1 (*P < 0.05). F. A schematic illustration of the proposed model depicting a feedback loop between PTTG1, PTTG1-targeting miRNAs and p53 that promotes pituitary tumor progression.
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