Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on microRNA expression in the NCI-H460 human lung large cell carcinoma cell line

To understand the effect of nicotine on sensitivity of cancer cells to radiation or anti-cancer drugs, NCI-H460 human lung large cell carcinoma cells were treated with 6 Gy ionizing radiation or 1 uM cisplatin after exposure to 0.5 uM nicotine or cigarette smoke extract. MicroRNA expression in total RNA extracted from the treated cells was quantified using the 7th generation miRCURY™ locked nucleic acid microarray platform from Exiqon® (Vedbaek, Denmark). Cells were subjected to one of nine types of treatment in triplicate and harvested for RNA extraction after 2 or 24 hours. Thus, 54 RNA samples representing biological triplicates for nine types of treatments and two time-points were obtained and analyzed using microarrays. Three RNA samples were assayed in duplicate (thus, 3 sets of technical duplicates: samples 5 & 55, 7 & 56, and 28 & 57). Reference RNA created by pooling the 54 unique RNA samples (at equal concentrations) was used in the reference channel (channel 2) of all microarrays. The reference RNA was also assessed in the test channel (channel 1) of one microarray (i.e., both test and reference channels had the pooled RNA).