Deciphering the role of the microRNA-200c in breast cancer metastasis

ITQB/IBET Seminar

Title: Deciphering the role of the microRNA-200c in breast cancer metastasis

Speaker: Ricardo Perdigão Henriques

From: Engineering Cellular Applications Laboratory

Abstract:

MicroRNAs are short, non-coding RNA molecules that post-transcriptionally regulate gene expression. Our laboratory showed that the microRNA-200 (miR-200), which controls the mesenchymal to epithelial transition, regulates the last step of breast cancer metastasis formation, the colonization of distant sites. miR-200 family members are poorly expressed in mouse 4TO7 breast cancer cells, which are invasive but incapable of forming macrometastasis, but highly expressed in metastatic isogenic 4T1 cells. Ectopic expression of miR-200 in 4TO7 cells conferred metastatic capability to these otherwise non-metastatic cancer cells after orthotopic transplantation. To understand the role of miR-200 in metastasis, we sought to identify the genes regulated by miR-200c. Using a novel microRNA pull-down technique developed in our laboratory, we identified 517 putative miR-200c target genes. These genes are highly enriched for miR-200c recognition elements and cancer-related functions. We selected 17 genes for further validation on the basis of their high connectivity to the remaining genes in the list. To validate these putative targets, we transfected 4TO7 mouse cells with a miR-200c mimic to determine whether miR-200c over-expression suppresses their mRNA and protein expression. Transcripts of 15 of the 17 genes tested were downregulated after miR-200c overexpression, and two (Ywhag and Smad5) of the genes tested so far also showed substantially reduced protein. We then investigated whether these genes are direct targets of miR-200c by cloning their 3’UTR into a luciferase reporter vector. The 3’UTRs of eleven genes (Xbp1, Map3k1, Smad2, Mapk12, Snail, Tob1, Zfp36, Ywhag, Fhod1, Scmh1, Crtap) were confirmed to be directly regulated by miR-200c. We also identified the miR-200c recognition elements in the 3’UTRs of nine of the genes tested so far by showing that mutation of miR-200c seed region in the full 3’UTR rescues reporter expression. We are currently studying the effect of miR-200c overexpression on the protein expression of the remaining genes. We are also investigating the effect of miR-200 regulation of these novel target genes on the expression of epithelial genes involved in cellular adhesion. Together, these results will define novel biological pathways regulated by miR-200c and help us to better understand the role of miR-200 in breast cancer metastasis.