Long Non-coding RNA TUG1 Sponges Mir-145a-5p to Regulate Microglial Polarization After Oxygen-Glucose Deprivation

Abstract

Microglia plays a critical role in neuroinflammation after ischemic stroke by releasing diverse inflammatory cytokines. Long non-coding RNA taurine up-regulated gene 1 (lncRNA TUG1) is widely expressed in adult brain and has been reported to participate in multiple biological processes associated with nervous system diseases. However, the role of TUG1 in microglial activation remains unidentified. BV-2 microglial cells were cultured in vitro and TUG1 siRNA was used to knock down its RNA level. Microglial cells were subjected to oxygen-glucose deprivation (OGD) for 4 h following TUG1 siRNA or scramble siRNA transient transfection. After 24 h reoxygenation, TUG1 level and microglial M1/M2 phenotype, as well as releasing inflammatory cytokines and their role to viability of SH-SY5Y neuroblastoma cells were determined by quantitative real-time PCR (qRT-PCR), ELISA, immunofluorescence and western blot. In addition, miR-145a-5p, a putative microRNA to bind with TUG1 by bioinformatics analysis, was simultaneously examined, then the interaction of TUG1 with miR-145a-5p and the potential involvement of NF-κB pathway were further evaluated by RNA-RNA pull-down assay and western blot. The cellular level of TUG1 was transiently up-regulated in microglial cells 24 h after OGD treatment, with an inverse correlation to downregulated miR-145a-5p. TUG1 knockdown drove microglial M1-like to M2-like phenotypic transformation with reduced production of pro-inflammatory cytokines (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6) and incremental release of anti-inflammatory cytokine (interleukin-10, IL-10), as a result, promoted the survival of SH-SY5Y cells. Meanwhile, TUG1 knockdown prevented OGD-induced activation of NF-κB pathway as well, represented by decreased ratios of p-p65/p65 and p-IκBα/IκBα proteins. Furthermore, we found that TUG1 could physically bind to miR-145a-5p while miR-145a-5p inhibitor abolished the protective effects of TUG1 knockdown through activation of NF-κB pathway, suggesting a negative interaction between TUG1 and miR-145a-5p. Our study demonstrated that lncRNA TUG1, sponging miR-145a-5p with negative interaction, could regulate microglial polarization and production of inflammatory cytokines at a relatively early stage after OGD insult, where NF-κB pathway might be involved, possibly providing a promising therapeutic target against inflammatory injury.

Keywords: NF-κB signaling; inflammatory cytokines; microRNA-145a-5p; microglia; oxygen-glucose deprivation; phenotype; taurine up-regulated gene 1.

Figure 1

Quantification of RNAs by quantitative real-time-polymerase chain reaction (qRT-PCR), displaying transiently up-regulated taurine up-regulated gene 1 (TUG 1) concomitant with down-regulated miR-145a-5p after 4-h OGD and 24-h reoxygenation. No significant difference was observed at 12-h or 48-h reoxygenation. *P < 0.05 vs. the corresponding control. OGD, oxygen-glucose deprivation. n = 6.

Figure 2

Correlation between TUG1 and miR-145a-5p. (A) Putative binding sites revealed by bioinformatics analysis. (B) Direct binding further confirmed by biotin-labeled RNA-RNA pull-down assay. (C) Quantification of RNAs by qRT-PCR. OGD-induced up-regulation of TUG1 was sufficiently turned over by miR-145a-5p inhibitor, vice versa, OGD-induced down-regulation of miR-145a-5p was sufficiently turned over by TUG1 siRNA, suggesting a negative interaction between TUG1 and miR-145a-5p. *P < 0.05 vs. the corresponding control. NC, negative control. n = 4.

Figure 3

Microglial polarization by immunofluorescence (A) and western blot (B). TUG1 knockdown increased the number of ARG1 and CD206 positive cells (M2-like phenotype, green) as well as the protein levels, whereas decreased that of CD16 and CD68 positive cells (M1-like phenotype, red) as well as the protein levels after OGD. The effect of TUG1 knockdown on microglial polarization was reversed by miR-145a-5p inhibitor. *P < 0.05 vs. the corresponding control. 400×. Scale bar = 50 μm. n = 4.

Figure 4

Cytokine detection by ELISA and cell viability assay by CCK-8. TUG1 knockdown suppressed the production of tumor necrosis factor-α (TNF-α; A) and interleukin-6 (IL-6; B) while elevated the concentration of interleukin-10 (IL-10; C) after OGD, with enhanced SH-SY5Y cell viability after OGD (D). The effects of TUG1 knockdown on inflammatory cytokines and cell survival were abolished by miR-145a-5p inhibitor. *P < 0.05 vs. the corresponding control. n = 4.

Figure 5

NF-κB activity by western blot analysis. OGD-induced NF-κB activation was inhibited by TUG1 knockdown, represented by the decreased ratios of p-p65/p65 and p-IκBα/IκBα proteins. The effect of TUG1 knockdown was reversed by miR-145a-5p inhibitor. *P < 0.05 vs. the corresponding control. n = 4.