MiR-3194-3p Inhibits Breast Cancer Progression by Targeting Aquaporin1

Abstract

Increasing evidence indicates that the Aquaporin1 (AQP1) aberrant expression may be related to a wide variety of human cancers, including breast cancer (BC). In the present study, we explore the effects and possible mechanism of miR-3194-3p on the biological behaviors of BC. At first, miR-3194-3p is found to modulate AQP1 expression targeting the 3'-UTR using miRNA target prediction algorithms. MiR-3194-3p expression is markedly downregulated, and AQP1 expression is upregulated in BC tissues compared with adjacent normal breast tissues. Moreover, the differential expression of miR-3194-3p and AQP1 are observed in four BC cells with different malignancy degree. Meanwhile, a significant negative correlation between AQP1 and miR-3194-3p expressions in tumor tissues from 30 BC patients is revealed. miR-3194-3p mimic remarkably inhibits cell proliferation, migration, and invasion as well as promotes apoptosis in MDA-MB-231 cells while miR-3194-3p inhibitors exert an opposite role in MCF-7 cells. Dual-luciferase reporter system demonstrates that AQP1 is a direct target gene of miR-3194-3p. Overexpression of AQP1 by pBABE-puro-AQP1 vector partially abrogates the effect of miR-3194-3p mimic in MDA-MB-231 cells. In short, our results suggest that miR-3194-3p suppresses BC cell proliferation, migration, and invasion by targeting AQP1, providing a novel insight into BC tumorigenesis and treatment.

Keywords: AQP1; apoptosis; breast cancer; invasion; miR-3194-3p; migration; proliferation.

FIGURE 1

The miR-3194-3p and AQP1 expression levels in BC tissues and cell lines. Comparing differences in the expression levels of miR-3194-3p between (A) BC and adjacent non-tumor tissues (n = 30); (B) BC cell lines and the control breast epithelial cells (MCF-10A); differences in AQP1 mRNA level between (C) BC and adjacent non-tumor tissue; (D) BC cell lines and MCF-10A; differences in AQP1 protein level between (E) BC cell lines and MCF-10A. (F) A significant inverse correlation between miR-3194-3p and AQP1 expression was observed in BC tissues. All PCR reactions were performed independently in triplicate (*P < 0.05, **P < 0.01, and ***P < 0.001).

FIGURE 2

miR-3194-3p expression regulates cell proliferation, migration, invasion, and apoptosis in BC. (A) miR-3194-3p expression was measured by qRT-PCR in MDA-MB-231 and MCF-7 cells transfected with miR-3194-3p mimic/inhibitor and their negative controls. Cell proliferation was assessed with (B) CCK-8 assay and (C) Edu assay. (D) Cell migration was measured by wound-healing assay. (E) Cell invasion was measured by transwell assay. (F) Cell apoptosis was measured by Flow cytometry analysis (*P < 0.05 and **P < 0.01).

FIGURE 3

miR-3194-3p negatively regulates the expresssion of AQP1 by binding to the 3′-UTR of its mRNA. (A) The predicted miR-3194-3p-binding site in the AQP1 3′-UTR. (B) Mutations were generated and sequenced at the putative miR-3194-3p-binding site located in the AQP1 3′-UTR. (C) Either the WT or MUT reporter plasmid was cotransfected with either miR-3194-3p or NC oligo into HEK293T cells, and the luciferase activity was determined. (D) qRT-PCR analysis and (E) Western blot analysis of AQP1 expression in MDA-MB-231 and MCF-7 cells transfected with miR-3194-3p mimic/inhibitor and their negative controls. (F) Western blot analysis of AQP1 expression in MDA-MB-231 cells cotransfected with pBABE-puro-AQP1 and miR-3194-3p mimic. (G) Cell migration, (H) cell invasion, and (I) cell apoptosis was measured in MDA-MB-231 cells cotransfected with pBABE-puro-AQP1 and miR-3194-3p mimic (**P < 0.01).