In the present report, we derived an invasive subline of U87 cells, U87-2M1. Enhanced malignancy of U87-2M1 cells as opposed to parental U87 cells could be in part owing to increased expression of miR-10b. A baculoviral vector encoding decoy miR-10b-binding sites was found to sufficiently silence miR-10b activity, resulting in the detection of broad changes in U87-2M1 cells from cell growth, invasion to angiogenicity.
miRNA-10b's pleiotropic action in this study stood in direct contrast to earlier reports on miR-10b's exclusive regulation of either glioma cell growth3
or glioma invasion.5
Although miR-10b's impact on cell cycle and apoptosis may appear to confound our findings on miR-10b's regulation of glioma invasion, miR-10b gain-of-function studies showed that miR-10b is able to positively regulate cell invasion. In addition, invasive proteins such as MMP13, MMP2, CTNNB1 and HGF were downregulated after miR-10b silencing, supporting miR-10b's regulation of glioma invasion. Several laboratories have undertaken efforts using unsupervised hierarchical clustering to delineate gene expression signatures that are preferentially activated in different subtypes of GBM,2, 33
suggesting that the biological behavior and malignancy of each GBM subtype is likely different. We noted that the lack of correlation between miR-10b and glioma invasion was made in the context of all glioblastoma cases under the TCGA study,3
where only <30% of all glioblastoma cases were categorized as having a mesenchymal signature.2, 34
Although miR-10b may universally regulate glioma cell growth and death regardless of the GBM subtype being studied, it seems plausible that additional roles for miR-10b may arise depending on the repertoire of mRNA expression. In our study, we have shown that HOXD10, PAX6, TP53 and NOTCH1 may coordinately regulate invasion via suppression of proteins such as RHOC, PLAUR, MMP2 and CTNNB1.
Other than governing invasiveness of U87-2M1 glioma cells, we have also shown that miR-10b displays the same propensity toward regulation of U87-2M1 cell death and growth. This challenges the notion that miR-10b exclusively performs a singular role of either promoting cell invasion4
or cell growth.3
Of particular interest, Ma et al.4
observed a significant increase in the weight of SUM149 mammary tumors stably expressing miR-10b as opposed to control tumors from the ninth week of SUM149 xenograft development. Although the authors suggested that the invasiveness of the cells provided better access to vasculature that aid tumor development, we reckon the development of tumors in vivo
provided microenvironmental signals, for example hypoxia, which may modulate the repertoire of mRNA expression and allow miR-10b to simultaneously promote cancer cell growth, angiogenesis and invasion while suppressing apoptosis.
We have demonstrated a previously unappreciated role for miR-10b in regulating glioma angiogenesis. This is not improbable as many proteins are pleiotropic suppressors of tumorigenesis. Furthermore, Ma et al.4
reported the finding of highly proliferative and angiogenic SUM149 mammary cancer cells at the invasive fronts of an in vivo
SUM149 mammary tumor stably expressing miR-10b, suggesting that it is possible for miR-10b to act in a pleiotropic manner that was previously unknown. Microarray analyses revealed a panel of 13 angiogenic molecules that was downregulated after miR-10b silencing. Out of these 13 proteins, 11 of them have been implicated in glioma malignancy and angiogenesis. ANGPT1,35
COL1A2, JAG1, ITGA4, LAMA4, THBS1 and TGFB2,36
CTGF, ANXA2 and LOX,37
have been shown to underlie the angiogenic, invasive or mesenchymal gene expression of glioma cells while LEPR is a key receptor for leptin-mediated angiogenesis in acute myeloid leukemia.39
RECK is a well-characterized MMP-dependent anti-angiogenic molecule whose expression in U87-2M1 cells seems paradoxical. Interestingly, RECK has been shown to possess an MMP-independent function. Osteoarthritic chondrocytes proliferating in response to TGF-β
required the presence of RECK and silencing of RECK-reduced proliferation of TGF-β
This finding suggests that RECK and TGF-β
coordinately and positively regulates cell proliferation in an inflammatory setting. As the mesenchymal subtype of GBM is characterized by a high expression of genes from the NF-κ
B pathway, RECK likely has a pro-proliferative, rather than anti-angiogenic, role in U87-2M1 cells. Moreover, RECK overexpression is associated with poor patient survival rate in the REMBRANDT dataset (P
=0.002), suggesting that RECK is a glioma oncogene (Supplementary Figure S4
). Similarly, THBS1 has been widely regarded as a tumor suppressor via its inhibition of angiogenesis. However, patient survival data from REMBRANDT database reveals a highly significant link between THBS1 overexpression and poor survival rates ().32
We speculate that THBS1 acts as an oncogene via activation of latent TGF-β
in the extracellular environment.40
Downregulation of pro-angiogenic Notch ligand, JAG1, enables NOTCH1 receptor to act as a tumor suppressor via its sequestration of CTNNB1, hence avoiding conflict with its potential angiogenic contribution via JAG1 activation. Our results collectively indicate a reduced angiogenicity of U87-2M1 glioma cells after miR-10b silencing.
Our findings further confirmed the utility of miR-10b silencing for glioma therapy. Although miR-10b has been established to regulate cell growth and apoptosis across all gliomas, miR-10b's role should be expanded to include regulation of angiogenesis and invasion, at least, in the mesenchymal subtype of gliomas. Whether miR-10b possesses such a pleiotropic role in other subtypes of glioma requires development of in vitro models that accurately mimic other GBM subtypes.