Suppression of breast cancer metastatic behavior by microRNAs targeting EMT transcription factors. A relevant participation of miR-196a-5p and miR-22-3p in ZEB1 expression.

Purpose: Metastasis, the leading cause of cancer-associated deaths, is promoted by transcription factors SNAIL, SLUG, ZEB1 and TWIST through the activation of epithelial-mesenchymal transition (EMT). MicroRNAs can suppress EMT, emerging as candidate molecular biomarkers and novel therapeutic targets. Herein, we evaluated microRNAs downregulated in breast cancer (BC) tissues expressing EMT transcription factors, to find new potential regulators of EMT.

Methods: Candidate microRNAs were selected from microarray data by their inversely correlated expression with SNAIL, SLUG, ZEB1 and TWIST, evaluated in BC tissues through immunohistochemistry. We selected eight microRNAs predicted in silico as probable modulators of SNAIL, SLUG, ZEB1 and TWIST, and validate their interaction through the 3'UTR region in luciferase reporter gene assays. MDA-MB-231 cells were transfected with selected microRNAs to perform migration, invasion and cell proliferation assays, and western blot was used to evaluate protein levels.

Results: MiR-30a-5p, miR-1271-5p, miR-196a-5p, miR-202-3p, miR-210-3p, miR-22-3p and miR-331-3p decreased luciferase activity through SNAIL, SLUG, ZEB1 and/or TWIST 3'UTR. These microRNAs, including miR-34b-3p, decreased migration, invasion and cell proliferation in MDA-MB-231 cells. MiR-30a-5p, miR-202-3p and miR-22-3p decreased vimentin expression, whereas miR-196a-5p and miR-22-3p decreased endogenous ZEB1 levels. MiR-196a-5p, miR-202-3p and miR-30a-5p also decreased CCR7 expression, a chemokine receptor involved in lymph node metastasis.

Conclusion: microRNAs selected in this work can regulate gene expression trough 3'UTR region of EMT-transcription factors. In BC cells, miR-196a-5p and miR-22-3p decrease ZEB1 levels, being novel modulators of EMT. Also, the eight evaluated microRNAs, reduced the metastatic hallmarks in BC cells.