Interplay of microRNA-20a and HuR in the regulation of beclin1 during Withaferin-A mediated impaired autophagy in Breast Cancer cell-line, MCF-7

Dysfunctional autophagy is connected to multiple diseases. Withaferin-A, a biologically active withanolide, has been shown to impair autophagy in the breast cancer cell line, MCF-7; however, the underlying mechanism remains unclear. Here while uncovering the mechanism, it was found that treatment of MCF-7 cells with WA declines Beclin1 protein synthesis by restricting association of its mRNA to polysomes with no significant reduction in its mRNA level. Reporter assay established that WA-treatment enhanced the expression level of mature miR-20a which directly interacts with the 3UTR of beclin1 mRNA. RNA affinity chromatography revealed association of mRNA stabilizing protein, HuR with the 3UTR of beclin1 mRNA. Additionally, co-immunoprecipitation assay determined the interactions of GW182, an essential component of GW-body and Ago2, a crucial member of miRNA related silencing complex (RISC) with the 3UTR of beclin1 mRNA. In parallel, it was found that knocking down of HuR eliminates the interaction of GW182 with beclin1 3UTR, while the association of miRNA (Ago2) remains unaffected. These results suggested simultaneous association of HuR, GW182 and microRNA with beclin1 mRNA which could be pivotal for sequestration of beclin1 mRNA in GW-bodies. Thus, our findings culminated in identifying an unconventional mechanism of regulation of Beclin1 expression by the dual interplay between hsa-miR-20a and HuR during WA-induced impaired autophagy in MCF-7 cells.