Abstract
Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development, and sexual maturation. Accumulated studies have reported that autocrine/paracrine hGH is an orthotopically expressed oncoprotein that promotes normal mammary epithelial cell oncogenic transformation. Autocrine/paracrine hGH has also been reported to promote mammary epithelial cell epithelial-mesenchymal transition (EMT) and invasion. However, the underlying mechanism remains largely obscure. MicroRNAs (miRNAs) are reported to be involved in regulation of multiple cellular functions of cancer. To determine whether autocrine/paracrine hGH promotes EMT and invasion through modulation of miRNA expression, we performed microarray profiling using MCF-7 cells stably expressing wild type or a translation-deficient hGH gene and identified miR-96-182-183 as an autocrine/paracrine hGH-regulated miRNA cluster. Forced expression of miR-96-182-183 conferred on epithelioid MCF-7 cells a mesenchymal phenotype and promoted invasive behavior in vitro and dissemination in vivo. Moreover, we observed that miR-96-182-183 promoted EMT and invasion by directly and simultaneously suppressing BRMS1L (breast cancer metastasis suppressor 1-like) gene expression. miR-96 and miR-182 also targeted GHR, providing a potential negative feedback loop in the hGH-GHR signaling pathway. We further demonstrated that autocrine/paracrine hGH stimulated miR-96-182-183 expression and facilitated EMT and invasion via STAT3 and STAT5 signaling. Consistent with elevated expression of autocrine/paracrine hGH in metastatic breast cancer tissue, miR-96-182-183 expression was also remarkably enhanced. Hence, we delineate the roles of the miRNA-96-182-183 cluster and elucidate a novel hGH-GHR-STAT3/STAT5-miR-96-182-183-BRMS1L-ZEB1/E47-EMT/invasion axis, which provides further understanding of the mechanism of autocrine/paracrine hGH-stimulated EMT and invasion in breast cancer.
Highlights
HGH is an orthotopically expressed oncoprotein associated with mammary epithelial cell tumorigenesis
Identification of miR-96-182-183 Cluster as Autocrine hGHstimulated miRNAs—To determine specific miRNA expression patterns regulated by autocrine Human growth hormone (hGH) in breast cancer cells, we utilized a well characterized cellular model of autocrine hGH expression [18]: MCF-7-hGH cells, which stably synthesize and secrete hGH, and MCF-7-MUT cells, which stably express the hGH gene but with the start codon disabled
We performed microarray profiling by use of this cell model and identified a set of autocrine hGH-regulated miRNAs (Fig. 1, A and B). qRTPCR of the relative expression levels of four miRNAs in these two cell lines was performed to verify the microarray data (Fig. 1C). Among these autocrine hGH-regulated miRNAs, we focused on the miR-96-182-183 cluster, which resides within the intergenic region on human chromosome 7q32.2 (Fig. 1C) and has been reported to be processed from a common primary transcript in mice [19]
Summary
HGH is an orthotopically expressed oncoprotein associated with mammary epithelial cell tumorigenesis. We have further reported that autocrine hGH promotes epithelial-mesenchymal transition (EMT) in epithelioid breast cancer cells, resulting in an invasive phenotype with increased MMP activity through repression of plakoglobin expression and relocalization of E-cadherin to the cytoplasm [6].
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