Non-Small Cell Lung Cancer

AEG-1[Coding-mRNA]

miR-30a[miRNA]

SNAIL[mRNA]

A549

Homo sapiens (human)

Eur J Cell Biol 2015 Jan 94, 22-31 doi:10.1016/j.ejcb.2014.10.006 PMID:25484183

AEG-1 3'-untranslated region functions as a ceRNA in inducing epithelial-mesenchymal transition of human non-small cell lung cancer by regulating miR-30a activity.

qRT-PCR

Astrocyte Elevated Gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC/3D3. RT-PCR analyses were performed to examine the expres- sion of AEG-1 and hallmark EMT genes by using human-specific primers. Cell analysis of miR-30a mimics, inhibitor and negative control transfected cells were subjected to PCR analysis, Western blot and immunoblot analysis. AEG-1 protein expression in the same cells was inhibited by miR-30a. For ceRNA verification, HEK 293T cells were co-transfected with pMiR-Report/Snail, pMiR-Report/Mut-Snail, pMiR-Report/Vimentin, or pMiR-Report/Mut-Vimentin and AEG-1 mRNA 3' UTR vector (PAU)or control vector (PSI). The cells were lysed and luciferase activity was measured by luminometer 48 h afterwards. Overexpression of AEG-1 3' UTR increased the 3' UTR activity of luc-Snail and luc-Vimentin as assumed, due to less available common miRNAs. The protein level increased by western blot analysis in the AEG-1 3'UTR overexpression lung cancer A549 cells. Therefore, AEG-1 3'UTR as a ceRNA regulated the expression of Snail and Vimentin post-transcriptly based on the activity of miR-30a. we also used AEG-1 CDS overexpression plasmid (PA) to increase the protein expression of AEG-1, and determine the expression of Vimentin and Snail by western blot analysis. It was suggested the regulation effect of AEG-1 on the expression of Snail was not only at RNA level but also protein level.