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. 2012;7(9):e45133.
doi: 10.1371/journal.pone.0045133. Epub 2012 Sep 19.

TSA suppresses miR-106b-93-25 cluster expression through downregulation of MYC and inhibits proliferation and induces apoptosis in human EMC

Zhi-Ning Zhao  1 , Jiu-Xu Bai, Qiang Zhou, Bo Yan, Wei-Wei Qin, Lin-Tao Jia, Yan-Ling Meng, Bo-Quan Jin, Li-Bo Yao, Tao Wang, An-Gang Yang
Affiliations

TSA suppresses miR-106b-93-25 cluster expression through downregulation of MYC and inhibits proliferation and induces apoptosis in human EMC

Zhi-Ning Zhao et al. PLoS One. 2012.

Abstract

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TSA inhibited EMC cells proliferation and downregulated miR-106b-93-25 cluster and its host gene MCM7.
(a) Growth inhibitory effects of TSA on ECC-1 cells and HEC-1A cells (left) and Cytometric detection of apoptosis in cells treated with TSA (right) by Annexin V-FITC staining of EMC cells, ECC-1 cells (top) and HEC-1A cells (bottom). (b)Cell cycle analysis of ECC-1 (top) and HEC-1A cells (bottom) by flow cytometry. Control cells were treated with vehicle alone. Results represent the mean ± SD of three independent experiments. (c)Microarray results showing decreased expression levels of miR-106b, miR-93 and miR-25 in the ECC-1 cells treated with TAM (5 nM) combined with TSA (100 ng/mL) while they were unchanged when treated with TAM alone compared with control. (d) qRT-PCR analysis of miR-106b, miR-93, miR-25 and their host gene MCM7 expression in ECC-1 and HEC-1A cells treated with TSA (100 ng/mL) for 24 h. Relative expression levels were normalized using the human housekeeping GAPDH gene. Error bars represent the S.D. of mean values (expressed as percent of control) from three independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05, paired t-test.
Figure 2
Figure 2. The miR-106b-93-25 cluster promoted EMC cell growth.
(a)At indicated times post-transfection, MTT assays were used to compare the effects of miR-106b, miR-93, miR-25 and their inhibitors. miR-106b, miR-93, miR-25 promoted the growth of ECC-1 cells, while their inhibitors significantly decreased proliferation in ECC-1 cells. Values represent the means ± SD from triplicate determinations. (b) Effects of miR-106b, miR-93 or miR-25 on the cell cycle. FACS analysis was performed on cells transfected with either miRNA or miRNA inhibitors. miR-106b and miR-93 gain of function led to an increase in cells in S-phase, while the G1-phase population was increased in the cells transfected with their inhibitors. miR-25 gain of function had no effects on the cell cycle. (c) Effects of miR-106b or miR-93 or miR-25 on apoptosis. FACS analysis was performed on cells transfected with either miRNA or miRNA inhibitors. miR-25 gain of function decreased apoptosis of cells. Apoptosis increased significantly in cells transfected with the miR-25 inhibitor. miR-106b and miR-93 had no effect on the apoptosis levels of the cells.
Figure 3
Figure 3. Regulation of P21 and BIM by the miR-106b-93-25 cluster.
(a) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3′UTR target sequence of miR-106b or miR-25 in the P21 or BIM gene were co-transfected into ECC-1 cells with either miRNA-negative control, miRNA mimics or empty pGL3 control vector (each n = 3). Luciferase activity was determined in the cell extracts after 24 h. In the presence of the wild-type P21 3′UTR, the miR-106b mimics significantly inhibited the luciferase activity compared with vector control. This inhibition was not observed with the mutant 3′UTR reporter construct (left). In the presence of the wild-type BCL2L11 3′UTR, the miR-25 significantly inhibited the luciferase activity compared with vector control. This inhibition was attenuated with the mutant 3′UTR reporter construct (right). (b) qRT-PCR analysis of P21 and BIM mRNA levels after the transfection of ECC-1 cells with miR-106b or miR-25 mimics. P21 mRNA was significantly decreased by miR-106b mimics while BIM mRNA was not significantly changed by miR-25 mimics. (c) Western blot analysis showing downregulated P21 and BIM proteins in ECC-1 and HEC-1A cells transfected with miR-106b and miR-25 mimics. (d) qRT-PCR and Western blot analysis showing upregulated P21 and BIM mRNA and protein level in ECC-1 and HEC-1A cells treated with TSA for 24 h. **, P<0.01; *, P<0.05, paired t-test.
Figure 4
Figure 4. TSA downregulated the miR-106b-93-25 cluster by downregulating MYC.
(a) The mRNA and protein levels of MYC was downregulated in ECC-1 and HEC-1A cells treated with TSA for 24 h. (b) Depletion of MYC inhibited the expression of miR-106-93-25, as well as its host gene MCM7, and promoted the expression of P21 and BIM mRNA and protein expressions in ECC-1 and HEC-1A cells. (c) Overexpression of MYC upregulated expressions of miR-106b-93-25 and MCM7, and downregulated P21 and BIM mRNA expressions. (d) P21 mRNA and protein levels were upregulated in ECC-1 cells treated with TSA for 24 h, while it was partially neutralized by miR-106b (left). BIM mRNA was upregulated in ECC-1 cells treated with TSA for 24 h, while it was not neutralized by miR-25, but its protein level was neutralized in miR-25 overexpressing ECC-1 cells. **, P<0.01; *, P<0.05; ns, not significant; paired t-test.
Figure 5
Figure 5. MYC activation of human MCM7 promoter activity was dependent on the E-box sequence.
ECC-1 cells were transfected to detect the human MCM7 promoter activity. (a) Schematic of the core promoter region of the MCM7 promoter. Location of the mutated sequence for the E box (AGTTTA) is indicated by the black box. (b) Luciferase activity of constructs of the MCM7 promoter and its truncations. ECC-1 cells transfected with luciferase reporter constructs containing different 5′flanking segments of hMCM7 alone or co-transfected with pBABE-MYC. The numbers on the left side of the panel represent the relative position of each deletion. The luciferase activities were normalized to that of ECC-1 cells transfected with the pGL3(-) vector (control) was set to 1.0. Results are means ± S.D. All assays were repeated for at least 3 independent experiments. In each experiment, the individual data points were calculated as the means of triplicates. (c) Effect of mutated E-box site on activity of the MCM7 promoter. The MCM7 (−185/+44)/luciferase reporter construct was co-transfection with its E-box site mutant and with or without the MYC expression plasmid. (d) ChIP assays were performed in ECC-1 cells using antibodies directed against MYC protein or an IgG control. The sequences of all primers for ChIP-PCR were based on the MYC-binding sites (−756 to −50 relative to the TSS) identified from TSS analysis. After immunoprecipitation, DNA was eluted and amplified by PCR using primers designed to amplify the minimal promoter region of MCM7. *, P<0.05, paired t-test.
Figure 6
Figure 6. qRT-PCR analysis of miRNAs and related transcripts in clinical EMC tissues.
(a) Twenty clinical EMC samples and the normal adjacent tissues (NAT) were analyzed. The average expression values of adjacent normal tissues is set at 1. Horizontal bars represent average levels. The expression of miR-106b, miR-93, miR-25 and their host gene MCM7 were upregulated in EMC tissues compared to the normal adjacent tissues. (b) MYC expression was upregulated in EMC tissues. (c) P21 and BIM expression in EMC tissues. ***, P<0.001, paired t-test.
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References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71–96. - PubMed
    1. Di Cristofano A, Ellenson LH (2007) Endometrial carcinoma. Annu Rev Pathol 2: 57–85. - PubMed
    1. Smith LT, Otterson GA, Plass C (2007) Unraveling the epigenetic code of cancer for therapy. Trends Genet 23: 449–456. - PubMed
    1. Hake SB, Xiao A, Allis CD (2004) Linking the epigenetic ‘language’ of covalent histone modifications to cancer. Br J Cancer 90: 761–769. - PMC - PubMed
    1. Szyf M (2005) DNA methylation and demethylation as targets for anticancer therapy. Biochemistry (Mosc) 70: 533–549. - PubMed

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Grants and funding

This study was funded by National High Technology Research and Development Program of China grant 2007AA021104 (A.G. Yang), National Basic Research Program of China 2010CB529905 (L.T. Jia), National Major Science and Technology Specialized Project for New Drugs Formulation 2009ZX09103-664 (L.T. Jia), Natural Science Foundation of China 81172289 (T. Wang). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.