MiR-218 is frequently downregulated in gliomas
To investigate the functional role of miR-218 in glioma tumorigenesis, we analyzed the expression of miR-218-1 and miR-218-2 in gliomas and normal brain tissues (control subjects) using The Cancer Genome Atlas (TCGA) dataset. As shown in Fig. 1a, compared to control subjects, the expression of both miR-218-1 and miR-218-2 was significantly downregulated in gliomas. Moreover, we found that the expression levels of miR-218-2 were significantly higher than those of miR-218-1 in gliomas (4.99 ± 1.95 vs. 0.25 ± 0.43, P < 0.001), indicating that mature miR-218 in gliomas was mostly constituted by miR-218-2, which was consistent with a previous study in thyroid cancers [22]. We further analyzed the expression of miR-218-1 and miR-218-2 in gliomas with different histologic grades. The results showed that expression levels of miR-218-1 were not significantly different between gliomas with histologic grade 2 (G2) and grade 3 (G3) (P = 0.71) (Fig. 1b, left panel). However, the gliomas with histologic G3 had a significant lower miR-218-2 expression than those with histologic G2 (P = 0.002) (Fig. 1b, right panel).
Next, Kaplan-Meier analysis of survival was performed according to their expression levels in a large cohort of gliomas using TCGA dataset. The results showed that the expression of miR-218-1 or miR-218-2 almost did not affect the survival of glioma patients when their survival time were less than 2000 days (Fig. 1c). However, decreased expression of miR-218-2 but not miR-218-1 was significantly associated with poor patient survival when their survival time more than 2000 days (Fig. 1d). The above findings suggest that miR-218-2 may be a potential biomarker for predicting long-term survival of glioma patients.
MiR-218 inhibits glioma cell growth
To determine biological role of miR-218 in glioma, a series of in vitro experiments were performed with gain-of-function of miR-218 in glioma cells. First, we validated ectopic expression of miR-218 mimics in U251 and SHG44 cells by qRT-PCR assay (Fig. 2a). Next, we evaluated the effect of miR-218 mimics on malignant phenotypes of glioma cells. The results showed that miR-218 mimics significantly inhibited the proliferation of U251 and SHG44 cells compared to the control (Fig. 2b). The inhibitory effect of miR-218 mimics on cell growth was further confirmed by soft agar colony formation assay. As shown in Fig. 2c, the colonies formed in miR-218 mimics-transfected cells were fewer than those formed in control cells. We also evaluated in vivo tumor-inhibitory effect of miR-218 in nude mice. As shown in Fig. 2d, the tumors induced by U251 cells stably expressing miR-218 showed significantly longer latency and smaller mean tumor volume than those induced by control cells. At the end of the experiments, the xenograft tumors were isolated and weighed. The mean weight of the tumors stably expressing miR-218 was significantly less relative to control tumors (P = 0.0009) (Fig. 2e). As expected, our data showed that the percentage of Ki-67 positive cells was significantly decreased in the tumors stably expressing miR-218 relative to control tumors (Fig. 2f).
Next, we tested the effect of miR-218 mimics on cell cycle contributions and apoptosis in U251 and SHG44 cells. As shown in Fig. 3a, compared to control cells, cell cycle was arrested at the G0/G1 phase in miR-218 mimics-transfected cells. The percentage of G0/G1 phase was increased from 51.7 ± 2.4% to 62.3 ± 2.0% in U251 cells (P = 0.004) and from 52.3 ± 2.7% to 66.6 ± 3.7% in SHG44 cells (P = 0.005), respectively. In addition, we found that miR-218 mimics transfection showed an increase in both early and late apoptosis in comparison with the control (20.5 ± 1.1% vs. 28.9 ± 1.8% in U251 cells, P < 0.002; 7.2 ± 1.3% vs. 16.0 ± 2.1% in SHG44 cells, P = 0.003) (Fig. 3b). Collectively, our results further support tumor suppressor role of miR-218 in glioma cells.
MiR-218 inhibits glioma cell migration and invasion
We attempted to assess the effect of miR-218 on migration and invasion potential in U251 and SHG44 cells. As shown in Fig. 4, there were a significantly lower number of migrated cells in the miR-218 mimics-transfected cells than that in control cells. In addition, by in vitro invasion assay, we found that miR-218 mimics clearly decreased the ability of cells to pass through the matrigel-coated membrane compared to the control (Fig. 4). These data indicate that there is a close correlation between miR-218 expression and metastatic phenotypes of glioma cells.
TNC is identified as a new target of miR-218
Using target prediction tools such as miRDB, miRanda and TargetScan, we identified a panel of candidate genes that are potentially targeted by miR-218. Among them, genes involved in vital signal pathways were selected to accept further detection, including IKBKB, TNC and WNT2B. Next, we assessed the effect of miR-218 on their expression in U251and SHG44 cells. The results showed that, among these three genes, only TNC was dramatically downregulated by miR-218 mimics in these two cell lines at both mRNA and protein levels (Fig. 5a, b; Supplementary Fig. 1). To determine whether TNC is a target of miR-218, we constructed a luciferase reporter plasmid, where the 3’ UTR (containing putative miR-218 binding sites: 5’-AAGCACA-3’) of TNC was attached to the coding region of luciferase (Fig. 5c). Meanwhile, to further determine that the modulation of TNC by miR-218 is caused by direct interaction, we also constructed a miR-218 binding sites of TNC mutated (5’-ACGAATA-3’) luciferase reporter plasmid (Fig. 5c). As shown in Fig. 5d, relative to the control, miR-218 mimics significantly suppressed luciferase activity in U251 and SHG44 cells transfected with wild-type (WT) luciferase reporter plasmid, while almost did not affect luciferase activity in these cells transfected with mutated (MUT) luciferase reporter plasmid. These data support TNC as a direct target of miR-218.
Next, we analyzed TNC expression in normal brain tissues and gliomas using TCGA dataset. As shown in Fig. 5e, compared to normal brain tissues, TNC was significantly elevated in gliomas, which was consistent with a previous study [18]. In addition, we also investigated the relationship between the expression of miR-218-1/miR-218-2 and TNC in gliomas. The results showed that TNC expression was not significantly correlated with miR-218-1 expression (P = 0.08, r = 0.08; Pearson’s correlation coefficient) (Fig. 5F, left panel), while was strongly correlated with miR-218-2 expression (P < 0.0001, r = 0.18; Pearson’s correlation coefficient) (Fig. 5f, right panel).
MiR-218 functions as a tumor suppressor in gliomas cells by inhibiting TNC/AKT/AP-1/TGFβ1 positive feedback loop
We next attempted to explore the mechanism of miR-218 inhibiting malignant phenotypes of glioma cells. There is evidence revealing that TNC can increase phosphorylation of AKT at Ser 473 by interacting with intergrins, thereby activating the PI3K/AKT signaling pathway [23–25]. Thus, we speculated that miR-218 plays its tumor suppressor role in glioma cells by inhibition of the PI3K/AKT signaling via targeting TNC. As shown in Fig. 6a, miR-218 mimics expectedly decreased TNC expression, and clearly inhibited phosphorylation of AKT at Ser 473, while almost did not affect phosphorylation of AKT at Thr 308 in U251 and SHG44 cells.
Evidently, transcription factor AP-1 as a target of the PI3K/AKT signaling pathway plays an important role in cell proliferation and is constitutively activated in glioma [26–29]. AP-1 is composed primarily of heterodimers of various proteins of the FOS and JUN families, that bind to a common DNA binding sequence. AP-1 activation contains complex process, such as increased expression or phosphorylation of FOS and JUN [30]. As shown in Fig. 6a, we found that miR-218 mimics strongly inhibited JNK phosphorylation, while almost did not affect the expression of FOS and JUN in U251 and SHG44 cells. Considering that TGFβ1 is a well-known target of AP-1 [31–33], thus we speculated that miR-218 could downregulate TGFβ1 expression by suppressing AP-1 activity, thereby inhibiting malignant phenotypes of glioma cells. The results showed that miR-218 mimics expectedly decreased TGFβ1 expression in U251 and SHG44 cells compared to the control (Fig. 6a). These results suggest that transcriptional activity of AP-1 can be inhibited by miR-218, as supported by the AP-1 luciferase reporter assay (Fig. 6b).
Next, to confirm the above observations in vivo, we performed western blot analysis in the xenograft tumors using the indicated antibodies. As shown in Fig. 6c, TNC expression was significantly downregulated in miR-218-overexpression tumors relative to control tumors. As expected, phosphorylation of Akt at Ser 473 and JNK and TGFβ1 expression were dramatically downregulated in miR-218-overexpression tumors relative to control tumors, but not phosphorylation of Akt at Thr 308 and the expression of FOS and JUN, further supporting the in vitro results.
It should be noted that TGFβ1 has been reported to, in turn, induce TNC expression involving Smad3/4, Sp1, Ets1 and CBP300 [34]. Thus, we suppose that TGFβ1 is able to activate AKT/AP-1 signaling axis by increasing TNC expression, thereby forming a positive feedback loop. To prove this, we treated U251 and SHG44 cells with recombinant human TGFβ1 proteins. The results showed that TGFβ1 treatment markedly induced TNC expression and subsequently increased phosphorylation of Akt at Ser 473 and JNK compared to the control, while this effect could be reversed by miR-218 mimics (Fig. 6d). This was also supported by the AP-1 luciferase reporter assay (Fig. 6e). Altogether, our data indicate that miR-218 exerts its tumor suppressor function in glioma cells by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop.
Give the above, we propose a model to explore the mechanism of miR-218 inhibiting malignant progression of glioma (Fig. 6f). Briefly, miR-218 binds to the 3’ UTR of its target TNC, and represses its expression. This will reduce AKT phosphorylation and subsequently suppress transcriptional activity of AP-1 by decreasing JNK phosphorylation, thereby downregulating the expression of TGFβ1, which be able to, in turn, activate the TNC/AKT/AP-1 signaling axis. Thus, miR-218 acts as a potent tumor suppressor in glioma by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop.