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.

The miR-200 family in normal mammary gland development

“…The miR-200 family has been extensively studied in mammary tumorigenesis, particularly the claudin-low breast cancer subtype by our lab [ 41 , 45 , 46 ] and others [ 47 – 60 ]. From this research, it has been postulated that miR-200s may serve as therapeutic targets for the treatment or prevention of breast cancer.…”

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

“…The function of miR-200s in mammary tumor growth and metastasis have been extensively studied using human and murine mammary tumor cell lines. These studies from our lab [23] , [24] , [25] and others [62] , [63] , [64] generally show that miR-200s can inhibit the growth of primary mammary tumors and suppress metastatic spread. However, the ability of miR-200s to prevent mammary tumor initiation, has not been explored.…”

“…SUZ12 is a component of the polycomb repressor complex 2 (PRC2) that mono-, di-, and trimethylates histone H3 on lysine 27 (H3K27) leading to chromatin compaction and suppression of transcription [98] , [99] , [100] . Saa1 [77] and Spp1 [101] have been shown to be regulated by H3K27 methylation and our previous study found that increased expression of miR-200s in murine and human breast cancer cells resulted in an elevation of H3K27me3 [24] . Therefore, miR-200s may directly target genes critical for tumor initiation and/or influence histone methylation to regulate gene expression.…”

“…RNA extraction was completed as previously described [23] , and RNA sequencing and analysis was performed by Arraystar (Arraystar Inc, Rockville, MD). Fastq files were analyzed using Genialis software (Genialis Inc, Houston, TX) following the standard RNA-seq pipeline as previously described [30] . Hierarchical clustering was performed using Genialis software (Genialis Inc, Houston, TX) with all genes and Pearson distance measure and average linkage clustering.…”

“…The most studied function of the miR-200 family in breast cancer is the suppression of epithelial to mesenchymal transition (EMT) and thus tumor migration and metastasis. Work from our lab and others showed that increased expression of miR-200s in murine and human mammary tumor cells inhibited migration and invasion in vitro and metastasis in vivo [23] , [24] , [25] , [26] , [27] , [28] , [29] . miR-200s have also been reported to influence proliferation, stem/progenitor cell number and the expression of immune regulatory molecules [ 23 , 25 , 27 , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] ].…”