Regulatory interplay between miR-21, JAG1 and 17beta-estradiol (E2) in breast cancer cells

Abstract

Overexpression of the oncomir miR-21 is associated with many cancers, including breast cancer. Elevated levels of Jagged-1 (JAG1), a predicted miR-21 target, are implicated in estrogen receptor negative (ER−) breast cancer. We demonstrate (by ablation of the miR-21 binding site in the JAG1 3′UTR) that miR-21 directly targets and represses JAG1 levels in MCF-7 (ER+) breast cancer cells. MiR-21 targeting of JAG1 in MDA-MB-231 (ER−) breast cancer cells is dependent on miR-21 dosage (levels). In both cell lines, miR-21 and JAG1 expression levels were negatively correlated due to their regulatory relationship. In addition, 17beta-estradiol (E2) increases JAG1 levels by limiting (via downregulating miR-21 levels) the repressive effects of miR-21 on the JAG1 3′UTR. Our results reveal a regulatory interplay between miR-21, JAG1 and E2 that is important for advancing understanding of how the oncogenic potential of miR-21 and JAG1 manifests in different sub-types of breast cancer.

Introduction

MicroRNAs (miRs) are small non-coding RNAs regulating gene expression post-transcriptionally [1]. Numerous miRs are dysregulated in cancer [2], [3] and can have tumor-suppressor [4], [5] or oncogenic activity (oncomirs) [6]. For instance, miR-21 promotes cancer cell growth via regulating several genes through their 3′UTR complementary target sites [7].

The miR:mRNA interaction can depend on the cellular context, including whether miR/target are co-expressed, tissue type and cellular stage [8]. For instance, endogenous levels of JAG1 protein are reduced by miR-34a and miR-21 in human monocyte-derived dendritic cells whereas mRNA levels of JAG1 were upregulated during differentiation [9]. A similar lack of association between JAG1 mRNA and protein levels has been previously described [10].

Previous studies have observed differential expression levels of JAG1 expression in cancer cells. High levels of JAG1 have been observed in oral squamous cell [11] and adrenocortical [12] carcinomas. Higher JAG1 levels were associated with poor prognosis [13], [14], [15], high tumor grade, and ER/PR negativity [10] in breast cancer patients.

JAG1 encodes a ligand important for the Notch intercellular signaling pathway and for angiogenesis [16]. Notch signaling is important for development and tissue homeostasis and is activated in many human cancers [17]. Triple negative (ER/PR/Her−) breast cancer cell lines display higher expression levels of JAG1 compared to ER+ cell lines, indicating that in the absence of ER/PR/Her stimulation, activation of Notch signaling by JAG1 promotes cell growth [18].

In a cDNA microarray study, MCF-7 cells stimulated with 17beta-estradiol (E2) showed upregulation of JAG1 [19]. In contrast to JAG1, miR-21 is downregulated by E2 via promoter binding in MCF-7 [20] and also other breast cancer cell lines [20]. These studies indicate that JAG1 and miR-21 are both downstream targets of estrogen signaling (via E2 stimulation). In addition, miR-21 [7] and JAG1 [13], [14] are independently associated with cancer, yet the possible regulatory interplay between miR-21, JAG1 and estrogen has not been investigated.

In this study, we investigated the miRNA-mediated regulation of JAG1 expression levels by miR-21 in MCF-7 and MDA-MB-231 (as ER+ and ER−) breast cancer cell lines and have elucidated the effects of 17beta-estradiol (E2) on miR-21:JAG1 interaction. We demonstrate that JAG1 is targeted and its expression levels suppressed by miR-21 in MCF-7 cells, but not in MDA-MB-231 cells. However, by changing the levels of miR-21 dosage ectopically we could control the JAG1 suppression by miR-21 in both cell lines. We also demonstrate that miR-21 and JAG1 levels display an inverse correlation in both breast cancer cell lines and that E2 interferes with the miR-21:JAG1 interaction by decreasing the expression of miR-21 in estrogen responsive MCF-7 breast cancer cells.