EVADR ceRNA transcript variants upregulate WNT and PI3K signaling pathways in SW480 and HCT116 cells by sponging miR-7 and miR-29b

Abstract

Long noncoding RNAs are cancer regulators and EVADR-lncRNA is highly upregulated in colorectal cancer (CRC). Accordingly, we aimed to functionally characterize the EVADR in CRC-originated cells. Firstly, during the amplification of EVADR full-length cDNA (named EVADR-v1), a novel/shorter variant (EVADR-v2) was discovered. Then, RT-qPCR analysis confirmed that EVADR is upregulated in tumors, consistent with RNA-seq analysis. Interestingly, bioinformatics analysis and dual-luciferase assay verified that EVADR sponges miR-7 and miR-29b. When both EVADR-v1/-v2 variants were overexpressed in SW480/HCT116 cells, miR-7 and miR-29b target genes (involved in the WNT/PI3K signaling) were upregulated. Furthermore, EVADR-v1/-v2 overexpression resulted in elevated PI3K activity (verified by western blotting and RT-qPCR) and upregulation of WNT signaling (confirmed by western blotting, TopFlash assay, and RT-qPCR). Consistently, overexpression of EVADR-v1/-v2 variants was followed by increased cell cycle progression, viability and migration as well as reduced early/late apoptotic rate, and Bax/Bcl2 ratio of the CRC cells, detected by the cell cycle analysis, MTT, wound-healing, Annexin-V/PI, and RT-qPCR methods, respectively. Overall, we introduced two oncogenic transcript variants for EVADR that by sponging miR-7/miR-29b, upregulate WNT and PI3K signaling. Given the crucial role of these pathways in CRC, EVADR may present potential therapy use.

Keywords: EVADR; LncRNA; WNT/PI3K signaling; colorectal cancer; miRNAs.