Revista de Bioanálise e Biomedicina

Validating the Efficacy of Shrna Vs Sirna In Silencing Hsp90α In Gliomas

Abstract

Chinmay Munje, Leroy Shervington, Adi Mehta, Zarine Khan and Amal Shervington

The stress dependent tumour cells found to harbour Hsp90 and its inducible component Hsp90á in activated superchaperone complexes are highly sensitive to pharmacological Hsp90 inhibitors compared to normal cells where Hsp90 is present in an uncomplexed state. Downregulating Hsp90á can be achieved using chemical inhibitors and RNAi such as siRNA’s or shRNA’s. This study aimed at assessing the efficacy of siRNA (targeting hsp90á exon 5) and shRNA (targeting hsp90á exon 4 and 5 border) in three glioma cell lines (1321N1, GOS-3 and U87-MG). hsp90á expression at mRNA and protein levels was monitored using qRT-PCR and immunofluorescence, respectively. The downstream effect of silencing hsp90á was determined by measuring the Akt/PKB kinase activity level. While siRNA treatment decreased hsp90α mRNA copy numbers by ~35%, shRNA decreased it by ~63% (three glioma cell lines). Furthermore, hsp90α inhibition by siRNA resulted in downregulating Akt/PKB kinase by ~29%, whereas shRNA downregulated it to ~3% (three glioma cell lines). Considering the vital role of Akt kinase in cell signalling, anti-apoptotic and drug resistant pathways in tumours, the treatment induced sensitivity of Akt to degradation results in a novel therapeutic strategy emphasising a greater potential of shRNA as opposed to siRNA in silencing hsp90á and subsequently Akt in glioma cells.