Abstract
BackgroundMicroRNAs 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.MethodsTo 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.ResultsRe-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.ConclusionsRe-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.
Highlights
MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation
Results miR‐200 expression and the impact on mesenchymal gene expression and cell shape As shown in Fig. 1, the murine (RJ345, Fig. 1a) and human (MCF-7, Fig. 1c) mammary tumor cell lines with epithelial characteristics expressed high levels of all 5 miR-200 family members compared to the murine (RJ423/RJ423 cells stably expressing an empty vector (RJ423EV)) and human (MDA-MB-231/MDA231EV) claudin-low mammary tumor cell lines, respectively
Since the miR-200 family has been implicated in regulating epithelial to mesenchymal transition (EMT) [19, 21,22,23,24,25], the expression of the epithelial gene Cdh1, and several mesenchymal genes were evaluated
Summary
MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. The interaction between miRNA and mRNA is primarily regulated by nucleotides 1–8 of the miRNA. More recent studies have shown that non-canonical sites can influence miRNA-mRNA interaction [13, 14] and miRNAs have been found to directly regulate gene transcription, activating Toll-like receptors and upregulating protein expression [15, 16], further increasing their complexity. MiR-200 family members negatively regulate mesenchymal transcription factors such as Zeb1/2, Twist1/2, Snai1/2, [19, 21,22,23] while inducing the expression of epithelial genes [24, 25]. Loss of miR-200s facilitates epithelial to mesenchymal transition (EMT)
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