SABiosciences Human miRNA PCR array assay of normal respiratory epithelia and lung cancer cells with or without treatment of CSC

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 in these cells. Subsequent experiments demonstrated that miR-487b directly targets SUZ12, BMI1, Wnt5a, c-Myc and K-ras. Repression of miR-487b correlated with over-expression 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. Deoxyazacytidine de-repressed miR-487b, and attenuated CSC-mediated silencing of miR-487b. TGF-β1 recapitulated CSC-mediated repression 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 over-expression 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 novel 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. MicroRNA PCR array analysis. Human RT2 miRNA PCR Arrays (MAH-3100E-12) were obtained from SA Biosciences. Two hundred ng of isolated miRNA was used for reverse transcription and the entire first strand cDNA was diluted and distributed amongst the 384 wells of the super-array plate. The reactions were performed with RT² SYBR Green / ROX PCR Master Mix (SABiosciences). Results were analyzed using software provided by the vendor (