Abstract 1959: Identification of microRNAs that target Id-1 in esophageal cancer.

Abstract

Abstract Inhibitor of DNA binding-1 (Id-1) belongs to a family of helix-loop-helix (HLH) proteins that lack the fundamental domain for DNA binding. Since Id-1 is overexpressed in esophageal squamous cell carcinoma (ESCC), and its expression level is associated with cancer invasion and metastasis, suppression of Id-1 may have therapeutic significance. MicroRNAs (miRNAs), which constitute non-coding small RNAs of around 22-nucleotides and negatively regulate gene expression as post-transcriptional regulator, were reported to be involved in carcinogenesis. However, there is limited information about the miRNAs that regulate Id-1. This project aims to identify miRNAs that can target Id-1 and exert tumor suppressive functions. In this study, several bioinformatics software programs, including TargetScan, RegRNA, MicroRNA and EMBL-EBI, were used to predict miRNAs that can directly bind to the 3’UTR of Id-1 mRNA. The miRNAs which have high scores and are predicted to target Id-1 by at least 3 different target prediction algorithms were chosen as candidates for this study. These miRNA candidates are: miR-29 family (miR-29a, miR-29b and miR-39c), miR-330, miR-300, miR-593, miR-654, miR-338, miR-346, miR-632. Plasmids which can over-express these miRNAs were designed and constructed using BLOCK-iT™ Pol II miR RNAi Expression system (Invitrogen®). Successful over-expression of the miRNAs in ESCC cell lines were verified by qRT-PCR. The transient and stable miRNA over-expressing ESCC cells were harvested and subjected to western blot analysis for Id-1 protein expression level. Direct binding of miRNAs to the 3’UTR of Id-1 was analyzed by 3’UTR luciferase assay. The pGL3-Id-1 3’UTR luciferase reporter vector was constructed by cloning the Id-1 3’ UTR downstream of the firefly luciferase open reading frame and the luciferase activities were measured using the Dual Luciferase Assay System. The cell invasion ability of miRNA over-expressing ESCC cells was analyzed by using BD BioCoat™ Matrigel™ Invasion Chamber. Cell proliferation and colony formation assays were performed to investigate the anti-proliferation effect of these miRNAs. In this study, ten miRNA over-expressing plasmids were constructed and over-expression of several miRNAs was achieved. We found that miR-29c could directly target the 3’UTR of Id-1 and down-regulate Id-1 protein level. miR-29c exerts tumor suppressing effects by reducing the ESCC cell invasion ability. Further study using miR-29c inhibitors in western blot assay, Id-1 3’UTR luciferase assay and cell invasion assay will be conducted. In summary, this study shows that miR-29c can suppress Id-1 and inhibit cell invasion in ESCC cells. This study is supported by General Research Funds from Research Grants Council of the Hong Kong SAR (Project Nos. HKU762610M and HKU763111M) Citation Format: L Han, B Li, SW Tsao, ALM Cheung. Identification of microRNAs that target Id-1 in esophageal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2013-1959