Knockdown of long noncoding RNA nuclear paraspeckle assembly transcript 1 could attenuate microglia activation through inhibiting autophagy

Abstract

Objective To investigate the role of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) in regulating the activation of microglias. Methods (1) Microglias BV2 were routinely cultured in vitro and NEAT1 mRNA expression was detected by real-time PCR (RT-PCR) after 0, 0.1, 0.5 and 1 μg/mL lipopolysaccharide (LPS) stimulation for 6 h; NEAT1 mRNA expression was detected by RT-PCR after 1 μg/mL LPS stimulation for 0, 6, 12 and 24 h. (2) NEAT1 siRNA (NEAT1-si) was transfected into BV2, and RT-PCR and Western blotting were employed to detect the LC3 mRNA and protein expressions. (3) The BV2 cells were divided into control group, NEAT1-si group, LPS group and NEAT1-si+LPS group; RT-PCR was used to detect the tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) mRNA expressions; morphological changes of BV2 cells were observed under inverted microscope. (4) The BV2 cells were divided into control group, negative control+Torin-1 group and NEAT1-si+Torin-1 group; Western blotting was used to detect the LC3 protein expression and LC3, TNF-α and iNOS mRNA expressions were detected by RT-PCR. Results (1) NEAT1 was significantly up-regulated in LPS-stimulated BV2 cells in dose- and time- dependent manners; significant differences were noted between each two groups (P<0.05). (2) The LC3-II mRNA expression in the NEAT1-si group was significantly decreased as compared with that in the control group (P<0.05); LC3-II/I protein ratio (0.7) in the NEAT1-si group was significantly lower than that in the control group (1.03, P<0.05). (3) As compared with those in the control group, the TNF-α and iNOS mRNA expressions in the NEAT1-si group were decreased; As compared with those in the LPS group, the TNF-α and iNOS mRNA expressions in the NEAT1-si+LPS group were significantly decreased (P<0.05). (4) LC3-II/I protein ratio in the control group, Torin-1 group and Torin-1+NEAT1-si group were 0.7, 1.14 and 0.97, respectively; LC3-II, TNF-α and iNOS mRNA expressions in the Torin-1 group were significantly higher than those in the control group (P<0.05); LC3-II, TNF-α and iNOS mRNA expressions in the NEAT1-si+Torin-1 group were significantly higher than those in the Torin-1 group (P<0.05). Conclusion Knockdown of long noncoding RNA NEAT1 could attenuate microglia activation through inhibiting autophagy, and NEAT1 maybe the key molecule for the mitigation and cure of the neuroinflammation related diseases. Key words: Nuclear paraspeckle assembly transcript 1; Autophagy; Microglia; Neuroinflammation