Abstract #387: Regulation of autophagy by the beclin 1-targeted microRNA in cancer cells

Beclin 1, the mammalian homologue of the yeast Atg6, is a key autophagy gene whose product is part of class III Phosphoinositide 3-kinase complex that participates in the formation of autophagosome. It is now widely acknowledged that beclin1- mediated autophagy plays an essential role in the regulation of cell survival and death, and implicates in many physiologic and pathophysiologic processes such as aging, cancers, and neurodegenerative diseases. Nevertheless, expression of beclin 1, both protein and mRNA, was found to be aberrant in several types of cancers. The mechanism underlying the altered expression of beclin 1, however, is largely unknown. The objective of this study was to explore the role of microRNAs (miRNAs) in the regulation of beclin 1 expression and autophagy. MiRNAs are a class of endogenous, 22 - 24 nucleotides non-coding RNA molecules able to affect stability and/or translation of mRNA, thereby modulating the expression of genes involved in many biological processes. In the current study, we found that miRNA miR-30a could negatively regulate beclin 1 expression resulting in decreased autophagic activity. We observed that following nutrient depletion or rapamycin treatment, while autophagy was increased, endogenous miR-30a expression was down-regulated in T98G glioma and MDA-MB-468 breast cancer cells, as analyzed by the miRNA microarray expression profiling and qRT-PCR. An in silico search for miRNA binding sites using the PicTar algorithm (http://pictar.bio.nyu.edu) revealed that the 3\#8217; - UTR of beclin 1 contains consensus sequences for miR-30a. To confirm that the miR-30a binding sequences in the 3\#8217; - UTR of Beclin 1 contribute to the modulation of beclin 1 expression, we co - transfected T98G, MDA-MB-468 and H1299 cells with a reporter gene vector (psiCHECK2) containing the wild-type or mutated binding sequences and a mimic of miR-30a, then measured the luciferase activity. We showed that the miR-30a mimic significantly decreased the luciferase activity in cells transfected with the wild-type reporter plasmid, but had no effect on the luciferase activity in cells transfected with the reporter plasmid containing the mutated miR-30a binding sequences. Treatment of cells with the miR-30a mimic also significantly decreased the beclin 1 mRNA and protein, as measured by qRT-PCR and Western blot, respectively. Furthermore, inhibition of beclin 1 expression by the miR-30a mimic blunted activation of autophagy induced by rapamycin, as evidenced by the decreases in LC3-II turnover, formation of GFP-LC3 aggregation and double membrane vacuoles in the cytoplasm. These results demonstrate that beclin 1 is a target for miR-30a, and that miRNA can control autophagy though modulating the expression of beclin 1. Our study of the role of miR-30a in regulating autophagy reveals a novel function for miRNA in a cellular event with significant impacts in cancer development and progression.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 387.