Re-expression of miR-200s in claudin-low mammary tumor cells alters cell shape and reduces proliferation and invasion potentially through modulating other miRNAs and SUZ12 regulated genes

Abstract

Background: MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. One family of microRNAs known as the miR-200 family is an important regulator of epithelial identity. The miR-200 family consists of five members expressed in two distinct clusters; the miR-200c/141 cluster and the miR-200b/200a/429 cluster. We have found that murine and human mammary tumor cells with claudin-low characteristics are associated with very low levels of all five miR-200s.

Methods: To determine the impact of miR-200s on claudin-low mammary tumor cells, the miR-200c/141 cluster and the miR-200b/200a/429 cluster were stably re-expressed in murine (RJ423) and human (MDA-MB-231) claudin-low mammary tumor cells. Cell proliferation and migration were assessed using BrdU incorporation and transwell migration across Matrigel coated inserts, respectively. miRNA sequencing and RNA sequencing were performed to explore miRNAs and mRNAs regulated by miR-200 re-expression while Enrichr-based pathway analysis was utilized to identify cellular functions modified by miR-200s.

Results: Re-expression of the miR-200s in murine and human claudin-low mammary tumor cells partially restored an epithelial cell morphology and significantly inhibited proliferation and cell invasion in vitro. miRNA sequencing and mRNA sequencing revealed that re-expression of miR-200s altered the expression of other microRNAs and genes regulated by SUZ12 providing insight into the complexity of miR-200 function. SUZ12 is a member of the polycomb repressor complex 2 that suppresses gene expression through methylating histone H3 at lysine 27. Flow cytometry confirmed that re-expression of miR-200s increased histone H3 methylation at lysine 27.

Conclusions: Re-expression of miR-200s in claudin-low mammary tumor cells alters cell morphology and reduces proliferation and invasion, an effect potentially mediated by SUZ12-regulated genes and other microRNAs.

Keywords: Claudin‐low breast cancer; H3K27me3; Migration; RNA sequencing; SUZ12; miR-200; miRNA sequencing.

Fig. 1

MicroRNA and gene expression. Expression of the miR-200 family (a, c) and epithelial and mesenchymal genes (b, d) in RJ345, RJ423, RJ423EV, RJ423c141 and RJ423ba429 cells (a, b) and MCF-7, MDA-MB-231, MDA-231EV, MDA-231c141 and MDA-231ba429 cells (c, d). miR-200 expression was determined using Taqman qRT-PCR while gene expression was determined using qRT-PCR. The graphs represent the mean ± SEM (n = 3) and the * indicates as significant difference (p < 0.05) from RJ423EV cells (a, b) or MDA-231EV cells (c, d)

Fig. 2

Cell morphology. Crystal violet staining of murine (a–e) and human (f–j) mammary tumor cells. Mammary tumor cells with a luminal phenotype (a, f) display a more rounded/cuboidal morphology while mammary tumor cells with a claudin-low phenotype (b, g) display a more spindle-like morphology. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells (e) and the miR-200c/141 cluster in MDA-MB-231 cells (i) reverted these cells to a more luminal morphology. Scale bars are 25 µm

Fig. 3

Cell proliferation and invasion. Proliferation (a, b) and transwell migration across Matrigel coated inserts (c–l) in murine (a, c–g) and human (b, h–l) mammary tumor cells. Proliferation was determined using BrdU incorporation while transwell migration was determined by calculating the percentage of the lower transwell membrane covered with crystal violet stained cells. Representative images of the crystal violet stained cells on the bottom of the transwell insert have been provided for murine (c–f) and human (h–k) mammary tumor cells. Graphs (a, b, g, l) represent mean ± SEM with n = 3 for the BrdU assay and n = 4 for the transwell assay. * Indicate values significantly different (p < 0.05) from either RJ423EV or MDA-231EV cells

Fig. 4

Hierarchical clustering of RNA sequencing data (n = 4). Hierarchical clustering (a, c) and Venn diagram (b, d) analysis of mRNAs differentially expressed in RJ423c141 and RJ423ba429 cells relative to RJ423EV cells (a, b) as well as in, MDA-231c141 and MDA-231ba429 cells relative to MDA-231EV cells (c, d). The Venn diagrams also indicate the percentage of differentially expressed genes with predicted miR-200 binding sites as determined by the miRDB database

Fig. 5

Pathway analysis of genes regulated by miR-200s. KEGG Pathways (a), Gene Ontology (b), and ENCODE/ChEA analysis (c) of genes differentially expressed in RJ423ba429 cells (white bars) or MDA-231c141 cells (grey bars)

Fig. 6

Venn diagrams and pathway analysis of shared genes. Venn diagram (a), KEGG Pathways (b), Gene Ontology (c) and ENCODE/ChEA analysis (d) of genes differentially expressed in both RJ423ba429 and MDA-231c141 cells

Fig. 7

Quantification of H3K27me3 levels using flow cytometry. The levels of H3K27me3 in RJ345, RJ423c141, and RJ423ba429 cells were expressed relative to the levels of H3K27me3 in RJ423EV cells (a) while the levels of H3K27me3 in MCF-7, MDA-231c141 and MDA-231ba429 cells were expressed relative to the levels of H3K27me3 in MDA-231EV cells (b). The graphs represent the mean ± SEM (n = 5) and the * indicates p < 0.05 relative to RJ423EV cells (a) and MDA-231EV cells (b)