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Histone deacetylases inhibitor trichostatin A increases the expression of Dleu2/miR-15a/16-1 via HDAC3 in non-small cell lung cancer

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Abstract

Histone deacetylases (HDACs) inhibitor is a promising new approach to the treatment of lung cancer therapy via inhibiting cell growth and inducing apoptosis. miR-15a and miR-16-1 are important tumor suppressors through modulating B cell lymphoma 2 (Bcl-2), Cyclin D1, D2, and others. However, whether HDACs inhibitor modulates the expression of miR-15a/16-1 in lung cancer is still unknown. The purpose of our study was to identify a new miRNA-mediated mechanism which plays an important role in the anti-cancer effects of HDACs inhibitor. We found HDACs inhibitors trichostatin A (TSA) and sodium butyrate upregulated the expression of miR-15a/16-1, residing in the host tumor suppressor Dleu2 gene, through increasing the histone acetylation in the region of Dleu2/miR-15a/16-1 promoter in lung cancer cells. Moreover, among class Ι HDACs subtypes, only knockdown of HDAC3 by specific siRNA increased the hyperacetylation of Dleu2/miR-15a/16-1 promoter region and finally resulted in the upregulation of miR-15a/16-1. Furthermore, overexpression of miR-15a/16-1, which were always deleted or downregulated in lung cancer cells, effectively suppressed cell growth and reduced colony formation. Finally, TSA reduced the expression of Bcl-2, an important survival protein in lung cancer cells, partly through upregulation of miR-15a/16-1. Therefore, this offers a therapeutic strategy that lung cancer patients who exhibit low level of miR-15a/16-1 or high activity of HDACs may benefit from HDACs inhibitor-based therapy.

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Acknowledgments

The Project is supported by National Natural Science Foundation of China (81270131, 81200350), Zhejiang Provincial Natural Science Foundation of China (LY12H08003, 2010C14011, Z2080988), and Foundation of Wenzhou Municipal Science and Technology Bureau (H20080058).

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Authors and Affiliations

  1. Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, 2, FuXue Road, Wenzhou, 325000, China

    Chi-qi Chen, Jian-bo Wu & Shen-meng Gao

  2. Department of Respiration, The First Affiliated Hospital of Wenzhou Medical College, 2, FuXue Road, Wenzhou, 325000, China

    Cheng-shui Chen, Jun-jie Chen, Lian-ping Zhou & Hong-lei Xu

  3. Department of Obstetrics and Gynecology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, 75, Jinxiu Road, Wenzhou, 325000, China

    Wei-wei Jin

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  1. Chi-qi Chen
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Correspondence to Shen-meng Gao.

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Chi-qi Chen and Cheng-shui Chen have contributed equally to this work.

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11010_2013_1762_MOESM3_ESM.tif

Supplemental Fig. 1: Representative of cell cycle analysis in A549 (A-D) and H1299 (E–H) cells treated with different concentrations of TSA for 24 h

11010_2013_1762_MOESM4_ESM.tif

Supplemental Fig. 2: Effects of SB on anti-proliferation, colony formation and apoptosis in lung cancer cells. (A and B) Cell proliferation was detected by CCK-8 assay in A549 and H1299 cells treated with indicated concentrations of SB for 24, 48 and 72 h. (C and D) Colony formation assay in A549 (C) and H1299 (D) treated with 1.0 and 2.0 mM SB. (E and F) Apoptosis was detected by annexin V/PI assay in A549 and H1299 cells treated with 1.0 and 2.0 mM SB for 48 h. *P < 0.01 versus untreated cells

11010_2013_1762_MOESM5_ESM.tif

Supplemental Fig. 3: Knockdown of HDAC3 by siRNA increases the expression of Dleu2/miR-15a/16-1 in A549 and H1299 cells. A549 and H1299 cells were transfected with Scramble or si-2-HDAC3 for 48 h. (A and B) The transcript and protein levels of HDAC3 was detected by quantitative real-time PCR (A) and Western blotting (B). (C and D) The relative expression of Dleu2, miR-15a and miR-16-1 was measured by quantitative real-time PCR. *P < 0.01 versus Scramble

11010_2013_1762_MOESM6_ESM.tif

Supplemental Fig. 4: 5-AZA fails to modulate the expression of miR-15a/16-1. (A) A representative of CpG dinucleotides at miR-15a/16-1 promoter region. No obvious CpG islands were found at miR-15a/16-1 promoter region when the length is longer than 200 nucleotides. (B) The relative expression of miR-15a/16-1 was detected in A549 and H1299 cells treated with 1.0 μM 5-AZA for 2, 4 and 6 days

11010_2013_1762_MOESM7_ESM.tif

Supplemental Fig. 5: TSA inhibits cyclin E1 but not cyclin D1 and D2 expression. (A) A549 and H1299 cells were treated with 0.5 μM TSA for 48 h. The protein levels of cyclin D1, D2 and E1 were measured by Western blotting, respectively

11010_2013_1762_MOESM8_ESM.tif

Supplemental Fig. 6: The expression of HDAC2 and HDAC3 are elevated in lung cancer tissues compared with adjacent normal tissues. A quantitative real time PCR analysis of HDAC2 (A) and HDAC3 (B) expression in 27 cases of lung cancer tissues and corresponding adjacent normal tissues

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Chen, Cq., Chen, Cs., Chen, Jj. et al. Histone deacetylases inhibitor trichostatin A increases the expression of Dleu2/miR-15a/16-1 via HDAC3 in non-small cell lung cancer. Mol Cell Biochem 383, 137–148 (2013). https://doi.org/10.1007/s11010-013-1762-z

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