Transcriptomic analysis of imatinib-resistant K562 cells with knocked down Pleomorphic Adenoma Gene 1 (PLAG1) to identify the targets
Genomic profiling of K562 cells resistant to imatinib as well as imatinib resistant CML patients harboured a recurrent chromosomal amplification in 8q11.2-12.1. Gene encoding PLAG1, a transcription factor associated with cancers and chemo resistance, resides on this locus and was found to be amplified in resistant cells. Upon PLAG1 knockdown we observed a decrease in imatinib resistance, and to understand the molecular events underlying PLAG1-mediated imatinib resistance, RNA sequencing was carried out.  Imatinib-resistant K562 cells were transfected with lentiviral particles containing shRNA against PLAG1 and lentiviral particles containing pLKO.1-empty vectors. Monoclonal populations were established. Total RNA sequencing was performed. For QC, RNA samples were quantified using the Qubit RNA BR Assay kit (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert, and RNA integrity was assessed on TapeStation using High Sensitivity RNA ScreenTape® (Agilent, Cat# 5067-5579). QC passed RNA samples were subjected to the library preparation, and NEB Ultra RNA-Seq Library Prep kit protocol (Cat# E7530L) was used and sequenced on Illumina NovaSeq 6000 instrument.  Transcriptomic analysis identified several genes down-regulated upon PLAG1 knockdown which were further investigated.