Multiple gene silencing in STAT pathway in K562 cells

Abstract

Context: Chronic myeloid leukemia (CML) is characterized by the presence of a fusion oncoprotein BCR-ABL. This mutation imparts a constitutive phosphorylation activity of tyrosine residues in the cellular proteins. One of the targets of BCR-ABL is the STAT5 protein, which when phosphorylated induces gene expression of antiapoptotic proteins such as BCL-XL. The STAT pathway has been targeted in the past by disrupting any one protein only. A multiple gene silencing has never been done in this pathway. Aim: The aim of this study was to compare the effects of downregulation of BCR-ABL, STAT5A, STAT5B, and BCL-XL, individually and simultaneously, in human CML cell line (K562 cells) through RNA interference (RNAi). Further, gene expression, inhibition of proliferation, and apoptosis induction were assessed in K562 cells. Materials and Methods: K562 cells were transfected with various combinations of small iRNA (siRNA) and the expressions of aforesaid genes were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. K562 cell proliferation and apoptosis were analyzed using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry, respectively. The results were compared through one-way analysis of variance. Results: qPCR and western blotting results post-siRNA transfection confirmed the targeted gene suppression and protein reduction in K562 cells. The cell proliferation assay and apoptosis assay revealed that simultaneous gene silencing of BCR-ABL, STAT5A, STAT5B, and BCL-XL had the highest killing effect on K562 cells as compared to knocking down these genes individually or in any other combinations. Conclusions: This was the first time it was shown that multiple gene silencing in STAT pathway in CML cell line K562 was better as compared to individual gene silencing.