miR-30e* upregulation correlates with progression of human gliomas.
To determine which miRNA species may be involved in promoting the aggressiveness of gliomas mediated through NF-κB signaling, we first compared miRNA expression profiles in cells obtained from matched pairs of glioma and adjacent non-tumor brain tissues from 4 patients. Microarray analysis identified 15 upregulated and 7 downregulated miRNAs from 960 analyzed miRNAs in glioma cells (Figure A), including upregulated miR-21, miR-10b, and miR-9/9*, which had been previously found to be associated with glioma invasiveness (37
). Further real-time PCR analysis of 8 paired glioma and adjacent tissues demonstrated a previously unidentified alteration, namely, the consistent overexpression of miR-30e*
, which is produced from the same miRNA precursor and imperfectly complementary to miR-30e
was found to be overexpressed in 10 samples microdissected from regions with glioma cells compared with those derived from the adjacent glial brain tissues and in all 17 tested glioma cell lines compared with primary normal human astrocytes (12
) (Supplemental Figure 1, A–C; supplemental material available online with this article; doi:
Upregulation of miR-30e* enhances invasiveness of glioma cells.
For evaluation of the clinical relevance of the upregulated miR-30e*, paraffin-embedded specimens of 5 normal brain tissues and 127 archived clinical gliomas classified as grades I–IV according to the WHO definition were microdissected and comparatively examined for miR-30e* expression using real-time PCR and in situ hybridization (Supplemental Table 1). miR-30e* levels remained low in tumors of grades I and II but became markedly higher in those at grade III and further elevated in grade IV tumors (Figure B). Moreover, miR-30e* levels also significantly correlated with patient survival (P < 0.001) (Figure C, Supplemental Figure 1, G and H, and Supplemental Table 2). High miR-30e* expression was closely associated with shorter overall survival time (P < 0.001) (Supplemental Table 3), which suggests a possible link between high-level miR-30e* expression and progression of human gliomas and highlights the potential value of the molecule as a predictive biomarker for disease outcome.
miR-30e* enhances the invasiveness of glioma cells.
To understand the biological function of miR-30e* in glioma cells, we then transfected glioma cell lines U87MG, LN444, and SNB19 with hsa-miR-30e* mimic oligonucleotides and examined the effect of upregulated miR-30e* on the expression of genes related to cell invasion. Northern blot analysis confirmed that a high level of miR-30e* expression was achieved in transfected cells compared with the negative control–transfected cells. Furthermore, the miR-30e* expression in transfected cells was higher than that in one, but lower than those in three, WHO grade IV glioma samples, indicating that the expression levels of miR-30e* in the experimentally modified glioma cells were within the range of those endogenously expressed in human gliomas (Figure D). Subsequent microarray and gene ontology (GO) enrichment analysis showed that genes upregulated in miR-30e*–transfected cells with GO biological process terms “extracellular matrix organization,” “response to wounding,” “immune response,” “inflammatory response,” and “chemotaxis” were enriched; meanwhile, the downregulated genes with GO terms “cell adhesion” and “biological adhesion” were enriched in these cells (at enrichment cutoff P ≤ 0.001; Supplemental Figure 2, A and B), predicting that miR-30e* may be involved in the development of the migration/invasion phenotype of glioma cells.
We next tested the effect of miR-30e* on the migration and invasiveness of all three glioma cell lines. Wound healing and Transwell (without Matrigel) assays demonstrated that ectopic expression of miR-30e* accelerated migration of glioma cells, while inhibiting endogenous miR-30e* using complementary oligonucleotides dramatically slowed down the migration (Supplemental Figure 2, C and D). Strikingly, in a 3D spheroid invasion assay, miR-30e*–transfected glioma cells displayed morphologies typical of highly aggressive/invasive cells, presenting more outward projections compared with the control cells; conversely, suppression of miR-30e* caused the cells to be immotile and to display spheroid morphologies (Figure E). The Transwell matrix penetration assay (TMPA) with Matrigel showed that overexpression of miR-30e* increased, while inhibition of miR-30e* reduced, the number of invaded glioma cells (Figure F).
MMP-9 mediates miR-30e*–induced glioma invasion.
GO enrichment analysis of miR-30e*–regulated genes found several transcripts with known functions related to regulation of the ECM (Supplemental Figure 2, A and B). Among these, the MMP family members, including MMP-1, -3, -9, -10, -12, and -13, were of particular interest (Figure A). The transcripts of these MMPs were upregulated in miR-30e*–transfected and downregulated in miR-30e*–inhibited glioma cells (Supplemental Figure 3A). Moreover, higher MMP9 production and proteolytic activities were found in miR-30e*–transfected glioma cells (Figure B and Supplemental Figure 3B). When activity of the MMPs was restrained by an inhibitor, however, the enhancing effect of miR-30e* on glioma cell invasiveness was blocked (Figure C and Supplemental Figure 3C), suggesting the importance of MMPs in mediating miR-30e*–induced glioma invasion.
miR-30e* induces invasion of glioma cells through MMP activation.
miR-30e* activates NF-κB.
Gene set enrichment analysis (GSEA) showed an apparent overlap between NF-κB–dependent and miR-30e*–upregulated gene sets, suggesting that miR-30e* may activate NF-κB signaling. Indeed, ectopic expression of miR-30e* significantly increased, and miR-30e* downregulation attenuated, the NF-κB–driven luciferase reporter activity and expression of 9 classically recognized NF-κB target genes (Figure , A and B). Furthermore, the abundance of nuclear p65 was significantly increased in miR-30e*–overexpressing cells and decreased when miR-30e* was suppressed (Figure C). Moreover, luciferase activity driven by the MYC, TNFA or MMP9 promoter was enhanced in miR-30e*–overexpressing cells and reduced in miR-30e*–suppressed cells (Supplemental Figure 4A). EMSA showed that the endogenous NF-κB activity in miR-30e*–overexpressing glioma cells was dramatically increased as compared with that in control cells (Supplemental Figure 4B). In agreement with results obtained from the glioma cell lines, inhibition of miR-30e* in two collections of primary glioma cells (PGCs), which exhibited normal levels of genomic IκBα and high levels of miR-30e* expression, significantly inhibited the NF-κB luciferase activity, decreased expression of 9 classical NF-κB target genes and reduced the invasiveness of PGCs (Supplemental Figure 5, A–D). Collectively, our results suggest that miR-30e* plays important roles in activation of the NF-κB pathway.
NF-κB activation mediates miR-30e*–induced invasiveness.
The invasiveness of miR-30e*–overexpressing glioma cells was dramatically reversed upon treatment with an inhibitor, JSH-23 or SN-50, that could block NF-κB nuclear translocation (Figure D), accompanied by a robust reduction of the general transcriptional activity of NF-κB (Supplemental Figure 4C), suggesting that functional NF-κB activation is key to the development of the invasive phenotype of glioma cells induced by miR-30e*. Meanwhile, the transcriptional activity of AP-1, a factor also known to activate MMP transcription, was not enhanced by miR-30e* (Supplemental Figure 4D), indicating a specific role of NF-κB activation in mediating miR-30e*–induced upregulation of MMPs.
Consistent with the results obtained from the glioma cell lines tested above, ectopic expression of miR-30e* in PGCs significantly decreased the expression of IκBα and increased NF-κB–driven luciferase activity and expression of 9 classically established NF-κB target genes (Supplemental Figure 5, A–C). Furthermore, TMPA showed that the invasive ability of PGCs dramatically increased upon transfection of the miR-30e* mimic but decreased in response to transfection of miR-30e* inhibitor (Supplemental Figure 5D). The invasive ability of PGCs induced with miR-30e* could be abrogated by treatment with an MMP inhibitor, further confirming the role of MMPs in the enhancing effect of miR-30e* on glioma invasion (Supplemental Figure 5D).
miR-30e* directly targets the IκBα 3′-UTR.
Interestingly, while IκBα mRNA was upregulated in miR-30e*–overexpressing cells and downregulated in miR-30e*–suppressed cells (Supplemental Figure 6A), its protein level displayed the reverse pattern relative to miR-30e* expression (Figure A). Together with the lack of alteration in expression of phosphorylated IKKs and IKK activity upon miR-30e* overexpression or downregulation (data not shown), these results indicate that miR-30e* may directly modulate IκBα expression at the translational level. Indeed, the 3′-UTR of IκBα contains conserved critical nucleotides that may serve as a legitimate target of miR-30e* (Figure B). When tested using the pEGFP-C3 and pGL3 dual luciferase reporter vectors containing a complete wild-type IκBα 3′-UTR, miR-30e* robustly inhibited the expression of GFP, but not the GFP–γ-tubulin control, in glioma as well as 293FT cells (Figure C). A consistent and dose-dependent reduction in luciferase activity upon miR-30e* transfection was observed, which could be reversed by transfection with the miR-30e* inhibitor (Figure D). Point mutations in the tentative miR-30e*–binding seed region in the IκBα 3′-UTR abrogated the aforementioned repressive effect of miR-30e*, demonstrating that IκBα is a bona fide target of miR-30e* (Figure , B and E).
miR-30e* directly targets the 3′-UTR of IκBα.
IκBα repression is essential for miR-30e*–induced invasiveness.
In order to understand the role of IκBα repression in miR-30e*–induced invasiveness, we studied the effect of knockdown of IκBα by two specific IκBα siRNAs and found that it enhanced the NF-κB activity and transcription of NF-κB–regulated genes, including MMPs, IL8, and VEGFC (Supplemental Figure 6, B–F). Concomitant overexpression of the IκBα ORF (without 3′-UTR) and miR-30e* in glioma cells robustly abrogated the enhancement of NF-κB activation and cell invasiveness by miR-30e*; meanwhile, when the IκBα ORF was replaced with the same amount of IκBα cDNA (with 3′-UTR) in the co-transfection experiment, the enhancing effects of miR-30e* were only partially compromised (Figure , B and C, and Supplemental Figure 7A). Notably, the invasiveness of miR-30e*–suppressed cells could be rescued by silencing IκBα (Figure D and Supplemental Figure 7B), but further ectopically expressing miR-30e* in IκBα-downregulated cells did not increase invasion (Figure A), suggesting that IκBα repression is essential for miR-30e*–induced invasiveness.
miR-30e* induces glioma invasiveness through direct targeting of IκBα.
Overexpression of miR-30e* induces invasiveness of glioma cells in vivo.
To test whether miR-30e* induces in vivo progression of glioma in an orthotopic tumor model, we engineered U87MG, LN444, and SNB19 glioma cells to stably overexpress miR-30e*/30e. As expected, the modified cells displayed upregulated miR-30e*, reduced IκBα, and increased MMP9 expression and NF-κB activity (Figure , A–D). Additionally, a highly invasive phenotype was shown by the TMPA and 3D spheroid invasion assay (Figure , E and F). Of note, oligonucleotides mimicking miR-30e had no effect on miR-30e* expression or the invasive/migratory ability of glioma cells (Supplemental Figure 8, A–D), nor on the expression of IκBα and MMP9 or NF-κB activity (data not shown). Furthermore, suppression of miR-30e* drastically reduced the number of invaded glioma cells and decreased the invasive ability of miR-30e/30e*–overexpressing glioma cells as compared with control gliomas cells (Supplemental Figure 8, E–G). Consistent with the observed effects of the miR-30e* inhibitor, co-transfection with mixed miR-30e and miR-30e* inhibitors also reduced the invasiveness of miR-30e/30e*–overexpressing glioma cells. However, inhibition of miR-30e alone had no effect on the invasion of miR-30e/30e*–overexpressing glioma cells (Supplemental Figure 8, E and F). Taken together, our results suggest that the observed molecular and phenotypic changes in the glioma cells stably transduced with the miR-30e/30e* expression cassette was caused by miR-30e* rather than miR-30e.
miR-30e/30e* induces invasiveness of glioma in vitro.
The above engineered and control glioma cells were implanted in the brains of nude mice. As shown in Figure A, the control cells generally formed oval-shaped intracranial tumors and exhibited sharp edges when expanding as spheroids. By contrast, tumors formed by the miR-30e/30e*–transduced glioma cells exhibited highly invasive morphology, with the borders displaying a palisading pattern of tumor cell distribution and forming spike-like structures invading into the surrounding regions. Notably, the pattern of MMP9 staining was highly diffusive at the invasion fronts as well as in the disseminated tumor clusters; meanwhile, MMP9 expression in the control tumors was low and mostly localized in the primary loci of the implanted tumors (Figure A).
miR-30e* induces invasiveness and angiogenesis of glioma in vivo.
When miR-30e* expression in the above engineered miR-30e/30e*–overexpressing glioma cells was inhibited by a retrovirus-mediated miR-30e* inhibitor, the IκBα levels recovered, accompanied by robustly decreased expression of MMP9, NF-κB activity, and invasiveness in vitro (Supplemental Figure 9, A–F). Strikingly, suppressing miR-30e* in these cells that had been initially engineered to overexpress miR-30e*/30e dramatically reduced the invasiveness, MMP9 expression, and nuclear NF-κB p65 in the tumors they formed (Figure A). It is particularly worth noting that retrovirally reintroducing IκBα cDNA into two miR-30e/30e*–overexpressing glioma cell lines substantially reversed tumor invasiveness (Figure A), strongly supporting IκBα as a key mediator for miR-30e*–induced invasion.
Overexpression of miR-30e* promotes glioma angiogenesis.
In parallel with the miR-30e*–induced invasive phenotype demonstrated in vivo by the above animal experiments, we also observed drastically enhanced microvascular outgrowth and increased MVD in miR-30e/30e*–transduced tumors (P < 0.01, Figure B), accompanied by an upregulation of VEGF-C, which was downregulated in tumors transduced with miR-30e* inhibitor or IκBα cDNA (Figure A). Secretion of VEGF-C was increased by miR-30e* overexpression, and decreased by miR-30e* suppression, in glioma cells (Figure C). When the clinical glioma samples were analyzed, MVD was found to correlate with not only the WHO grading and patient survival (P < 0.001, Supplemental Figure 10, A–C), but also with the miR-30e* levels (P < 0.01, Supplemental Figure 10D). Collectively, our data support a role for miR-30e* as an angiogenesis inducer in gliomas.
Ectopic expression of miR-30e* strongly provoked, while miR-30e* inhibition abrogated, the ability of glioma cells to induce tube formation and migration of HUVECs and the formation of second- and third-order vessels in chicken chorioallantoic membranes (CAMs) (Figure , A and B). Restoration of IκBα by co-transfecting the IκBα ORF (without 3′-UTR) into miR-30e*–overexpressing glioma cells hampered their ability to induce HUVEC tube formation to a level similar to that of the negative control cells. By contrast, IκBα cDNA with the 3′-UTR only mildly attenuated the tube formation ability induced by miR-30e* (Figure A). In addition, HUVEC tube formation and migration and CAM neovascularization induced by conditioned medium of miR-30e*–overexpressing glioma cells were markedly reduced when the cells were treated with an NF-κB inhibitor (Figure , A and B, and Supplemental Figure 11A). Notably, miR-30e* induction of VEGF-C in glioma cells could be prevented by re-introduction of the IκBα ORF (without 3′-UTR) (Supplemental Figure 11, B and C), indicating that VEGF-C is regulated by miR-30e* through modulation of IκBα. Moreover, the same phenomenon was also observed in the PGCs, in which overexpression of miR-30e* enhanced, but suppression of miR-30e* reduced, HUVEC tube formation (Supplemental Figure 5E), and the HUVEC tube formation ability induced by miR-30e* could be significantly reduced by treatment with a VEGF-C inhibitor (Supplemental Figure 5E). Taken together, our results suggest that miR-30e* exerts proangiogenic effects through suppression of IκBα expression, which in turn activates NF-κB signaling.
miR-30e* promotes glioma angiogenesis in vitro.
Clinical relevance of miR-30e*–triggered NF-κB activation in human gliomas.
Finally, we examined whether the NF-κB–activating functions of miR-30e* in glioma cells identified in the in vitro experiments as well as animal studies are clinically relevant. As shown in Figure , A and B, correlation studies in 127 glioma specimens using IHC analysis showed that miR-30e* expression inversely correlated with IκBα expression (P < 0.001) but strongly positively correlated with MMP9 (P < 0.001) and VEGF-C levels (P < 0.001). Furthermore, 52 of 79 (65.8%) specimens with high miR-30e* expression exhibited increased nuclear localization of NF-κB p65, whereas cytoplasmic NF-κB p65 was found in 37 of 48 low-miR-30e* specimens (77.1%) in which nuclear NF-κB was absent or only marginally detectable. Thus, a statistically significant correlation between miR-30e* expression and NF-κB nuclear localization was found (Figure B, r = 0.541, P < 0.001). Moreover, in 10 freshly collected clinical glioma samples, miR-30e* expression was shown to be strongly correlated with MMP9 and VEGF-C (r = 0.732, P < 0.001; r = 0.773, P < 0.001) and inversely with IκBα levels (r = –0.722, P < 0.001; Figure C). In the cohort study, the IκBα levels were found to correlate with patient survival time (P = 0.004), which was also inversely associated with high MMP9 (P < 0.001) and VEGF-C (P = 0.0232) levels (Figure , A, C, and D). In addition, patients with cytoplasmic NF-κB had higher cumulative 3-year survival rates than those with NF-κB mainly present in the nucleus (P = 0.001) (Figure B). Interestingly, while genomic PCR analysis showed that 1 of the 10 glioma cases tested (tumor 3) may be IκBα monoallelic (data not shown), real-time PCR analysis demonstrated that the mRNA levels of IκBα in all 10 gliomas samples were upregulated compared with those in 2 normal brain tissues (Supplemental Figure 12), further supporting the notion that the upregulation of miR-30e* post-transcriptionally represses IκBα expression, activates NF-κB signaling, promotes MMP9 and VEGF-C production, and ultimately leads to poor clinical outcomes for human gliomas (Figure ).
Clinical relevance of miR-30e* expression in human gliomas.
The prognostic significance of the NF-κB signaling pathway in gliomas.
Model of miR-30e*–mediated constitutive activation of the NF-κB pathway via epigenetic disruption of IκBα negative feedback, leading to an aggressive glioma phenotype.