miR-4792 and EGFR expression in BV2 cells induced by C. neoformans (WM148)
It has been shown that miR-4792 is downregulated in nasopharyngeal carcinoma, uterine leiomyoma, during submucosal fibrosis and in glioblastoma-infiltrating CD14 + cells. In contrast, miR-4792 is upregulated in glioma microvesicles [13–17]. Our previous studies have shown that the expression of miR-4792 increases in THP-1 cells infected with C.neoformans (WM148) (Fig. 1a) . To further study the expression of miR-4792 in microglia, BV2 cells were treated with WM148 for 0, 3, 6 and 9 and 12 h and qRT-PCR was performed to detect miR-4792 expression. We found that the expression of miR-4792 decreased over time, reaching its lowest after 6 h (Fig. 1b), while the expression of EGFR gradually increased, peaking at 6 h (Fig. 1c). ELISA assays showed that the inflammatory factors TNF-α and IL-6 secreted by microglia gradually increased over time, while IL-1β levels showed no obvious changes (Fig. 1d).
EGFR is targeted by miR-4792.
To further define the molecular mechanisms governing the regulatory effects of miR-4792, we performed targeted bioinformatics to investigate novel miR-4792 targets using MIRADA and target scan. These analyses revealed EGFR as miRNA target, the sequence of which was assumed to be in the 3’UTR (Fig. 2a). We additionally performed western blot analysis to assess the expression of the Phosphorylation of EGFR (pEGFR) in BV2 cells. We found that miR-4792 overexpression significantly reduced pEGFR levels, and dual luciferase assays confirmed EGFR as a direct target of miR-4792 (Fig. 2b). The exogenous overexpression of miR-4792 significantly inhibited WT EGFR 3’-UTR activity, but had minimal effect on mutant EGFR 3’UTR sequence (Fig. 2c). These data strongly implicate EGFR as a direct target for miR-4792.
EGFR blockade inhibits WM148 induced microglia activation
Microglia regulate the innate immune responses of the CNS [18, 19]. Given that microglia are responsible for pro-inflammatory cytokine production , we investigated the effects of WM148 on microglia induction. Western blot analysis was performed to assess the expression of the cell surface molecule CD11b, considered a phenotypic marker of microglial activation . The data showed that, after 6 h of WM148 treatment, higher levels of CD11b expression and EGFR activation (evident through the enhanced levels of pEGFR) occurred in treated BV2 cells (Fig. 3a). In contrast, 24 h treatment with EGFR inhibitor (AG1478) led to a loss of CD11b and pEGFR expression (Fig. 3b) confirmed by western blot analysis. Interestingly miR-4792 expression inversely correlated with EGFR levels, further highlighting EGFR as a miR-4792 target (Fig. 3c).
Inhibiting EGFR prevents MAPK kinase mediated cytokine production in BV2 cells
Activated microglia stimulate neuronal inflammation and enhance the levels of proinflammatory cytokines including IL-1β and TNF-α in the CNS . Extracellular signal-regulated kinases (Erk1/2), c-Jun terminal kinase (JNK) and p38 MAPK are key cellular signaling cascades MAPKs are known to enhance the production and secretion of cytokines [23–25]. For example, MAPK signaling promotes the LPS-induced synthesis of IL-1β and TNF-α . MAPK is also stimulated following EGFR activation [27, 28]. Consistent with previous findings, we found that AG1478 treatment inhibited MAPK phosphorylation leading to a loss of ERK1/2, p38MAPK and JNK activation in WM148 treated BV2 cells, while no significant differences in p-JNK levels were observed (Fig. 4a). Similarly, AG1478 treatment inhibited both IL-1β and TNF-α production in WM148 infected BV2 cells (Fig. 4b). We simultaneously assessed the secretion of other inflammatory factors in WM148 infected BV2 cells. The results showed that IL-1α, IL-12, Eotaxin, GM-CSF, MCP-1/CCL2 levels decreased after AG1478 treatment compared to the control group (Fig. 4c), but the differences were not significant.
miR-4792 regulates the EGFR/MAPK axis in BV2 cells
We next investigated the effects of the miR-4792-mediated regulation of EGFR on ERK1/2, JNK and P38MAPK signaling in BV2 cells infected with WM148. As shown in Fig. 2b and Fig. 5a, the levels of active pEGFR and p-ERK1/2, p-p38 and p-JNK decreased in the presence of miR-4792 mimics compared to the WM148 group, and the levels of the inflammatory cytokines IL-1β and TNF-α also decreased (Fig. 5b). While there is no significant different when BV2 cells were treated with miR-4792 inhibitors. All findings were confirmed by qRT-PCR analysis in which the expression of miR-4792 significantly increased, while EGFR expression decreased following the transfection of miR-4792 mimics for 24 h. The opposite phenotype was observed for miR-4792 inhibitors (Fig. 5c). We additionally found that active pEGFR levels were highly expressed when miR-4792 expressed lowly following WM148 stimulation (Fig. 1). Furthermore, we assessed the secretion of other inflammatory factors in WM148 infected BV2 cells, and found that the levels of pro-inflammatory factors including IL-1α, IL-12, Eotaxin, GM-CSF, MCP-1/CCL2 decreased after treatment with miR-4792 mimics for 24 h (Fig. 5d). Taken together, these data highlight that in BV2 cells, miR-4792 regulates EGFR/MAPK signaling following WM148 infection and partially alleviates MAPK-mediated inflammatory responses therefore affect the production of pro-inflammatory factors following WM148 infection.
Expression of miR-4792 in patients with CM before and after treatment
We collected CSF from patients with CM before and after regular antifungal treatment. The results showed that the expression of miR-4792 in CSF significantly increased after treatment (Fig. 6a). Receiver operating characteristics (ROC) curve analysis showed the areas under curves (AUCs) of 0.75 (95% CI, 0.54–0.96) (Fig. 6b). We also found the expression of EGFR in CSF significantly decreased after treatment (Fig. 6c). Receiver operating characteristics (ROC) curve analysis showed the areas under curves (AUCs) of 0.79 (95% CI, 0.6–0.98) (Fig. 6d). These data confirmed the reciprocal relationship between miR-4792 and EGFR in vivo setting of fungal infection and showed that the CSF miR-4792 and EGFR might be served as an useful biomarker for judging the effort of the treatment of CM patients.