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Title: [miR-155-5p alleviates lipopolysaccharide-induced inflammatory damage of human SH-SY5Y neuroblastoma cells by down-regulating SOCS1].
Author: Zhou H, Zhou L, Zhang C, Zhou L, Han Y.
Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2023 Mar; 39(3):220-229. PubMed ID: 36946346.
Abstract:
Objective To explore the effects of microRNA-155-5p (miR-155-5p) on lipopolysaccharide (LPS)-induced neuroinflammatory damage of human SH-SY5Y neuroblastoma cells. Methods SH-SY5Y cells line was overexpressed miR-155-5p or transfected with negative control (miR-155-5p mimic group, mimic-NC group), down-expressed miR-155-5p or transfected with its negative control (miR-155-5p inhibitor group, inhibitor-NC group). The cells with successful transfection in the above groups were treated with LPS for 24 hours. The cells without SH-SY5Y cells transfection and those with LPS treatment were included into control group and LPS group, respectively. The activity of SH-SY5Y cells was detected by MTT assay. The apoptosis rate was detected by flow cytometry. The mRNA levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 10 (IL-10), and expression of miR-155-5p were detected by reverse transcription PCR. The levels of cleaved caspase-3 (c-caspase-3), B-cell lymphoma/leukemia-2 (Bcl2), Bcl2-associated X protein (BAX), phosphorylated nuclear factor κB p65/nuclear factor κB p65 (p-NF-κB p65/NF-κB p65) and phosphorylated p38 mitogen-activated protein kinase/p38 mitogen-activated protein kinase (p-p38 MAPK/p38 MAPK) were detected by Western blot analysis. The expression of miR-155-5p in SH-SY5Y cells was regulated by miR-155-5p mimic and miR-155-5p inhibitor. The target relationship between miR-155-5p and suppressor of cytokine signaling 1(SOCS1) was predicted by bioinformatics, which was verified by luciferase assay. SH-SY5Y cells with down-regulation of both miR-155-5p and SOCS1 were constructed (miR-155-5p inhibitor/si-SOCS1 group). The cells activity, apoptosis, mRNA expressions of inflammatory cytokines, expression of SOCS1 protein, ratios of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK were detected by the above methods. Results Compared with control group, activity of SH-SY5Y cells, expression of Bcl2 protein, level of IL-10 mRNA and expression of SOCS1 protein decreased in LPS group while apoptosis rate, expressions of c-caspase-3 and BAX proteins, and levels of TNF-α, IL-1β and IL-6 mRNA were increased, along with the increased miR-155-5p level and ratios of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK. The activity of SH-SY5Y cells, expression of Bcl2 protein, level of IL-10 mRNA and expression of SOCS1 protein increased in miR-155-5p inhibitor group, compared with LPS group, whereas decreased miR-155-5p level, apoptosis rate, expressions of c-caspase-3 and BAX proteins, levels of TNF-α, IL-1β and IL-6 mRNA, and expressions of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK proteins were observed. Compared with LPS group, activity of SH-SY5Y cells, expression of Bcl2 protein, level of IL-10 mRNA and expression of SOCS1 protein were decreased in miR-155-5p mimic group, while miR-155-5p level, apoptosis rate, expressions of c-caspase-3 and BAX proteins, levels of TNF-α, IL-1β and IL-6 mRNA, and ratios of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK proteins were increased. Targeted relationship was identified between miR-155-5p and SOCS1. Compared with miR-155-5p inhibitor group, cells activity and level of IL-10 mRNA decreased in miR-155-5p inhibitor/si-SOCS1 group, while apoptosis rate, ratios of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK, and levels of TNF-α, IL-1β and IL-6 mRNA increased. Conclusion Inhibiting miR-155-5p can alleviate neuroinflammatory damage induced by LPS, which may be related to down-regulating SOCS1 level.

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