Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis
Sichuan Xi, Hong Xu, Jigui Shan, Yongguang Tao, Julie A. Hong, Suzanne Inchauste, Mary Zhang, Tricia F. Kunst, Leandro Mercedes, David S. Schrump
Sichuan Xi, Hong Xu, Jigui Shan, Yongguang Tao, Julie A. Hong, Suzanne Inchauste, Mary Zhang, Tricia F. Kunst, Leandro Mercedes, David S. Schrump
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Research Article Oncology

Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis

Abstract

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

Authors

Sichuan Xi, Hong Xu, Jigui Shan, Yongguang Tao, Julie A. Hong, Suzanne Inchauste, Mary Zhang, Tricia F. Kunst, Leandro Mercedes, David S. Schrump

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Figure 8

miR-487b inhibits cellular proliferation via cell-cycle arrest and senescence but not apoptosis in normal respiratory epithelia and lung cancer cells.

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miR-487b inhibits cellular proliferation via cell-cycle arrest and senes...
(A) Flow cytometry analysis demonstrating that miR-487b overexpression significantly impairs progression of SAECs, HBECs, Calu-6, H841, and H358 cells from G0/G1 into S and G2/M phases. Endogenous depletion of miR-487b enhanced cell-cycle progression in these cells. Data are represented as percentage of gated cells ± SD. (B) In vitro apoptosis assay demonstrating no change in apoptosis index in lung cancer cells exhibiting overexpression or knockdown of miR-487b. (C) Representative microscopic images (senescence staining) of SAECs, Calu-6, H841, and H358 cells exhibiting overexpression or depletion of miR-487b. Scale bars: 50 mm. Original magnification, ×100. (D) Quantitation of miR-487b–induced cellular senescence in normal respiratory epithelia and lung cancer cells. Ectopic expression of miR-487b significantly increased percentage of senescence in these cells. However, depletion of miR-487b did not affect senescence in these cells. Data are represented as percentage of cells ± SD. **P < 0.01.