[miR-155-5p通过下调SOCS1减轻脂多糖诱导的人SH-SY5Y神经母细胞瘤细胞的炎症损伤]。

Haiyan Zhou, Lihong Zhou, Caixia Zhang, Li Zhou, Yuhua Han
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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.</p>","PeriodicalId":23737,"journal":{"name":"Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology","volume":"39 3","pages":"220-229"},"PeriodicalIF":0.0000,"publicationDate":"2023-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"[miR-155-5p alleviates lipopolysaccharide-induced inflammatory damage of human SH-SY5Y neuroblastoma cells by down-regulating SOCS1].\",\"authors\":\"Haiyan Zhou,&nbsp;Lihong Zhou,&nbsp;Caixia Zhang,&nbsp;Li Zhou,&nbsp;Yuhua Han\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Objective To explore the effects of microRNA-155-5p (miR-155-5p) on lipopolysaccharide (LPS)-induced neuroinflammatory damage of human SH-SY5Y neuroblastoma cells. 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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. 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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. 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引用次数: 0

摘要

目的探讨microRNA-155-5p (miR-155-5p)对脂多糖(LPS)诱导的人SH-SY5Y神经母细胞瘤细胞神经炎症损伤的影响。方法SH-SY5Y细胞系过表达miR-155-5p或转染阴性对照(miR-155-5p模拟组、模拟- nc组),下调表达miR-155-5p或转染其阴性对照(miR-155-5p抑制剂组、抑制剂- nc组)。转染成功的各组细胞用LPS处理24小时。将未转染SH-SY5Y细胞的细胞和LPS处理的细胞分别分为对照组和LPS组。MTT法检测SH-SY5Y细胞活性。流式细胞术检测细胞凋亡率。采用反转录PCR检测肿瘤坏死因子α (TNF-α)、白细胞介素1β (IL-1β)、白细胞介素6 (IL-6)、白细胞介素10 (IL-10) mRNA水平及miR-155-5p表达。Western blot检测小鼠裂解型caspase-3 (c-caspase-3)、b细胞淋巴瘤/白血病-2 (Bcl2)、Bcl2相关X蛋白(BAX)、磷酸化核因子κB p65/核因子κB p65 (p-NF-κB p65/NF-κB p65)、磷酸化p38丝裂原活化蛋白激酶/p38丝裂原活化蛋白激酶(p-p38 MAPK/p38 MAPK)水平。SH-SY5Y细胞中miR-155-5p的表达受miR-155-5p mimic和miR-155-5p inhibitor的调控。通过生物信息学预测miR-155-5p与细胞因子信号传导1抑制因子(SOCS1)之间的靶标关系,并通过荧光素酶实验验证。构建miR-155-5p和SOCS1均下调的SH-SY5Y细胞(miR-155-5p inhibitor/si-SOCS1组)。采用上述方法检测各组细胞活性、凋亡、炎性因子mRNA表达、SOCS1蛋白表达、p-NF-κB p65/NF-κB p65及p-p38 MAPK/p38 MAPK比值。结果与对照组比较,LPS组SH-SY5Y细胞活性、Bcl2蛋白表达、IL-10 mRNA水平和SOCS1蛋白表达均降低,凋亡率、c-caspase-3和BAX蛋白表达、TNF-α、IL-1β和IL-6 mRNA水平升高,miR-155-5p水平升高,p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK比值升高。与LPS组相比,miR-155-5p抑制剂组SH-SY5Y细胞活性、Bcl2蛋白表达、IL-10 mRNA水平和SOCS1蛋白表达均升高,miR-155-5p水平、凋亡率、c-caspase-3和BAX蛋白表达、TNF-α、IL-1β和IL-6 mRNA水平、p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK蛋白表达均降低。与LPS组比较,miR-155-5p模拟组SH-SY5Y细胞活性、Bcl2蛋白表达、IL-10 mRNA水平和SOCS1蛋白表达降低,miR-155-5p水平、凋亡率、c-caspase-3和BAX蛋白表达、TNF-α、IL-1β和IL-6 mRNA水平以及p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK蛋白比值升高。发现miR-155-5p与SOCS1之间存在靶向关系。与miR-155-5p抑制剂组比较,miR-155-5p抑制剂/si-SOCS1组细胞活性和IL-10 mRNA水平降低,细胞凋亡率、p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK比值以及TNF-α、IL-1β和IL-6 mRNA水平升高。结论抑制miR-155-5p可减轻LPS诱导的神经炎症损伤,其机制可能与下调SOCS1水平有关。
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[miR-155-5p alleviates lipopolysaccharide-induced inflammatory damage of human SH-SY5Y neuroblastoma cells by down-regulating SOCS1].

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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