Abstract
The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.
Highlights
MicroRNAs are a class of small highly conserved non-coding RNAs that provide widespread expressional control through repression of mRNA translation
We found that mean miR-21 expression was fourfold higher in the laser microdissection (LMD) stroma of Colorectal cancer (CRC) specimens compared with paired normal tissue (P 1⁄4 0.005, Figure 1c)
Targeted therapeutic strategies based on an improved understanding of the molecular biology of malignancy have been applied to CRC with little success;[4,5] recent insights linking deregulated miRNA expression to malignant change[16,27] and to the acquisition of metastatic capabilities[17,28,29,30,31] have brought renewed optimism
Summary
MicroRNAs (miRNAs) are a class of small highly conserved non-coding RNAs that provide widespread expressional control through repression of mRNA translation. They are strongly implicated in the pathogenesis of numerous malignancies,[12] and hold promise as therapeutic targets as they regulate fundamental intracellular processes and are deregulated, often in a tissue- and tumour-specific manner, in all cancer types examined to date.[13]. MiR-21 suppression with antisense inhibitors was shown to prevent the fibroblast-to-myofibroblast transdifferentiation in response to transforming growth factor beta (TGF-b).[19] Together, these data suggest that the pro-metastatic influence of miR-21 may be mediated through the tumour microenvironment; biological and functional evidence to support this model has not been deeply investigated. The aims of the current study are to provide multimodal validation of miR-21 expression patterns in CRC and to gain improved understanding of the pathological relevance of deregulated stromal miR-21 by dissecting out molecular interactions within the tumour microenvironment
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