Expression profiling by small RNA-seq for identifying miRNA associated with epithelial-mesenchymal transition and acquired resistance to ALK inhibitors

Treatment with ALK tyrosine kinase inhibitors often elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. In this study, we aimed to identify molecular events that limit the response to ALK inhibition using genetic and epigenetic approaches. To identify novel mechanisms of acquired resistance to ALK inhibitors, we established in vitro models of acquired resistance to ceritinib using H3122 cell. For in vitro model, H3122 parental cells, ceritinib-treated resistant cells, and non-resistant cells that combinely treated with certinib and panobinostat were used for small RNA-seq based miRNA expression profiling. Overall design: miRNA-seq data of 6 samples, two H3122 parental cells, two ceritinib-treated resistant cells, and two non-resistant cells that combinely treated with certinib and panobinostat, were generated. Total RNA was isolated using the mirVana miRNA isolation kit (Ambion). Small RNAs (20 - 30 nt) were purified from 15% Novex TBE-Urea gel (Invitrogen) and ligated first with the 5' RNA adaptor and then with the 3' RNA adaptor provided by Illumina TruSeq small RNA sample preparation protocol. In each step, the ligated product was PAGE-gel purified. After first-strand synthesis and 11 cycles of PCR amplification, the product was PAGE-gel purified. Sequencing was performed in single end reads (75 bp) using NextSeq 500 platform (Illumina).