EphrinA 1 mediated anti-oncogenic effects are regulated by microRNA-122 in NSCLC cells

EphrinA1, a ligand for receptor EphA2 exerts an anti-oncogenic effect through the activation and down regulation of the EphA2 receptor in Non-Small Cell Lung Cancer (NSCLC). The underlying mechanisms of ephrinA1 mediated anti-oncogenic signals in NSCLC remains largely undefined. MicroRNAs (miR) are small non-coding RNAs that play a key role in tumorogenesis. The current study focuses on determining the mechanisms by which ephrinA1 induces anti-oncogenic effects in NSCLC cells. A-549 and H-23 NSCLC cell lines were treated with ephrinA1 and miR-122 expression was determined by quantitative-PCR and In-situ hybridization. In addition, transcription factor Bach-1 was determined. In order to determine the direct effect of miR-122 on NSCLC cells, A-549 and H-23 were transfected with pre-miR-122 and tumorsphere growth and migration were determined by matrigel and Wound healing assays. Additionally, miR-122 induced apoptosis was evaluated by Annexin-V expression. The Bcl-w, Bak, Bad-1 and Caspase-3 expression levels were also determined. EphrinA1 enhanced miR-122 and Bach-1 expression in a time dependent manner in NSCLC cells. Silencing Bach-1 gene expression using silencing interference RNA (siRNA) significantly down-regulated ephrinA1 induced miR-122 expression, suggesting that ephrinA1 mediated miR-122 induction was regulated by the transcription factor Bach-1 in NSCLC. Pre-miR-122 transfection significantly attenuated tumorsphere growth and cell migration in NSCLC. Moreover, miR-122 directly targets Bcl-w which harbours a putative miR-122 binding site in its 3’-UTR region. NSCLC cells, when transfected with miR-122, showed significant down-regulation of Bcl-w expression and enhanced the expression of pro-apoptotic genes Bad, Bak and Caspase-3. Taken together our study indicates that ephrinA1 mediated miR-122 expression is regulated by Bach-1. MiR-122 targets Bcl-w, and triggers apoptosis in NSCLC cells. Thus, we provide new insight into the complex ephrinA1/EphA2 receptor signalling process in understanding the direct role of miR-122 in ephrinA1 mediated signalling which may lead to new therapeutic strategies for NSCLC.

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