Abstract 1157: Identification of miRNAs that target the Epithelial-Mesenchymal Transition regulator, Zeb2, using transcriptome PCR arrays

Abstract

Abstract Epithelial-mesenchymal transition (EMT) is the process of molecular reprogramming from polarized immotile epithelial cells to motile mesenchymal cells and is activated during cancer cell invasion of tissues and metastasis. Zeb2 is a transcription factor that has been implicated in EMT, in part, through decreasing levels of E-cadherin. Loss of E-cadherin leads to cell motility which is required for tumor invasion and metastasis. The miRNA family of miR-200 has been shown to inhibit EMT through direct targeting of Zeb2. Currently, only members of the miR-200 family (miR-200bc/429, miR-200a/141) have been identified to target and decrease the expression of Zeb2. Therefore, to identify additional potential miRNAs that target Zeb2, a reverse genetics approach was used. The SureFind Transcriptome PCR Array is an array of 90 cDNA preparations obtained from HeLa cells that were transfected with various miRNA mimics to simulate overexpression of the mature miRNAs. Using a cancer-focused array, qPCR was performed to quantify the expression of Zeb2 wherein changes in mRNA expression identified specific miRNAs that alter Zeb2 expression. In addition to confirming miR-200c (miR-200ab/141/429 were not on the array) as a regulator of Zeb2, five new miRNAs, not previously predicted to target Zeb2 using bioinformatics or reported in the literature, were identified as targeting Zeb2 mRNA. Four of the miRNAs, Let-7g, miR-122, miR-142-5p, and miR-29b, caused at least a two-fold decrease in Zeb2 expression compared to the negative control, which was from cells transfected with a negative mimic control. In addition one miRNA, miR-34c-5p caused a 2.4 fold increase in Zeb2 expression, identifying it as a potential positive regulator of Zeb2 expression. Next to determine if the identified miRNAs inhibit translation of Zeb2, a reporter consisting of firefly luciferase coupled to the 3’UTR of Zeb2 was used to measure the activity of these miRNAs toward the 3’UTR of Zeb2. Let-7g, miR-142-5p, miR-200c, and miR-29b inhibited luciferase activity by at least 1.5 fold compared to negative mimic control. Interestingly, miR-34c-5p and miR-122 exhibited no statistical difference in luciferase activity compared to the negative control. An explanation for this is that miR-34c-5p and miR-122 do not target the 3’UTR of Zeb2 or that they require contextual cues from full-length RNA for their activity. In conclusion, five novel miRNA/Zeb2 interactions were identified in addition to the previously reported miR-200 family. To date only miR-200 has been implicated in EMT though it is possible that let-7g, miR-122, miR-142-5p and miR-29b maybe involved in inhibition of EMT through downregulation of Zeb2 expression. Additionally, these experiments demonstrated that the SureFIND Transcriptome PCR Arrays identify novel miRNA/gene interactions. This application is for research use only, not diagnostic/clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1157. doi:10.1158/1538-7445.AM2011-1157