Knock-down of human Beclin1 by small interfering RNA targeting untranslated coding region
HUANG Zhen-jun
LIU Shao-jun
Abstract：Objective To establish and evaluate an approach of small interfering RNA ( siRNA) targeting human Beclin1 mRNA non-coding region.Methods The siRNAs targeting the 5′-UTR (siRNA-#1), the 3′-UTR (siRNA-3#and siRNA-4#), or the coding region (siRNA-2#, positive control) of human Beclin1 mRNA were designed, respec-tively.HEK293 cells were transfected with these siRNAs .After 48 h transfection, the cells were harvested and subjected to Western blot analysis with anti -Beclin 1 and anti-LC3 antibodies;or co-transfected with plasmids encoding mRFP -GFP tandem fluorescent -tagged LC3(tfLC3)for an additional 24 h and observed by fluorescence microscopy .Results The siRNA-#3 and the siRNA-#4, targeting human Beclin13′-UTR, similar to positive control siRNA-2#that targe-ting Beclin1 coding region, significantly reduced the protein expression of Beclin1 and LC3.And siRNA-#3 and siRNA-#4 apparently reduced the LC3 autophagosome (yellow puncta) and autolysosome (red puncta) under fluorescence microsco-py.Conclusion The 3′-UTR specific siRNA effectively knock down Beclin 1 protein expression and reduce the cellular autophagy activity , providing a helpful way to study the role of mutation Beclin 1 in autophagy regulation .
Keywords：Beclin 15′-UTR3′-UTRsiRNAautophagy
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 3776-3779 )