miR-136-5p effect on A20 expression in interleukin-17-stimulated astrocytes

doi:10.3969/j.issn.2095-4344.2017.16.022

Abstract

Abstract:

BACKGROUND: miRNA plays a critical regulatory role in the development and plasticity of spinal cord, and pathological changes after spinal cord injury.

OBJECTIVE: To study the effect of miR-136-5p on the A20 expression in mouse astrocytes stimulated by interleukin-17 (IL-17).

METHODS: C57BL/6 mouse astrocytes were cultured in vitro, identified by immunofluorescence staining, and then stimulated by 100 μg/L IL-17 for 0, 3, 6, 12 and 24 hours, respectively. The relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected by RT-PCR to determine the optimal stimulation time of IL-17. The mouse astrocytes were respectively stimulated by 10, 20, 50, 100 and 200 μg/L IL-7 for 6 hours, and similarly, the relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected to determine the optimal concentration of IL-17. At 6 hours after IL-17 (50 μg/L) stimulation, the mRNA expression levels of miR-136-5p and A20 in mouse astrocytes were detected by RT- PCR, and the protein expression level of A20 was detected by western blot assay. In addition, the lentiviral expression vector (miR-136-5p-inhibition) was constructed and transfected into the mouse astrocytes that were also stimulated by IL-7 to detect the expression levels of miR-136-5p, A20 mRNA and A20 protein.

RESULTS AND CONCLUSION: Compared with the blank control group, the expression level of miR-136-5p in the miR-136-5p-inhibition group was significantly decreased after 6-hour IL-17 stimulation (P < 0.05). The expression levels of A20 mRNA and protein in each group were significantly decreased after 6-hour IL-17 (50 μg/L) stimulation (P < 0.05). The expression levels of A20 mRNA and protein in the miR-136-5p-inhibition group were significantly higher than those in the blank control group (P < 0.05), while there were no significant differences in the expression level of A20 protein between blank control and negative groups (P > 0.05). To conclude, miR-136-5p makes certain effect on the expression of A20 protein in astrocytes after IL-17 stimulation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Cytokines, Viruses, Transfection, Tissue Engineering, MicroRNAs

CLC Number:

R318

Shi Xiong-zhi, Zong Shao-hui, He Ji-chen, Peng Xiao-ming, Gao Yun-bing, Deng Gui-ying. miR-136-5p effect on A20 expression in interleukin-17-stimulated astrocytes[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(16): 2587-2592.

Figures/Tables 2

2.1 小鼠星形胶质细胞的培养与鉴定提取的小鼠原代星形胶质细胞经连续传代培养后在倒置显微镜下观察，细胞形状呈不规则形，胞浆丰富，核浆较少，胞体分支较多，细胞核呈椭圆形，常在胞体一侧。用兔抗胶质纤维酸性蛋白多克隆抗体进行细胞免疫化学染色及免疫荧光染色鉴定，可见星形胶质细胞胶质纤维酸性蛋白的表达位于胞浆。根据阳性细胞率(阳性细胞数/总细胞数)×100%，统计3个视野，分别计数阳性细胞的个数和细胞总数，计算出胶质纤维酸性蛋白阳性率可达95%以上。 2.2 小鼠星形胶质细胞最佳刺激时间和浓度为了确定白细胞介素17的最佳刺激时间，选用100 μg/L的白细胞介素17分别刺激3，6，12，24 h后行RT-PCR检测白细胞介素6和肿瘤坏死因子α的mRNA表达量。结果如表1所示，白细胞介素6和肿瘤坏死因子α的mRNA丰度在3 h开始上升，6 h显著升高并到达峰值，12 h以后稍有下降，24 h仍明显高于对照组，差异有显著性意义(P < 0.05)。因此，认为6 h为小鼠星形胶质细胞最佳刺激时间。为确定白细胞介素17的最佳刺激浓度，分别选取10，20，50，100，200 μg/L的白细胞介素17刺激6 h后行RT-PCR检测白细胞介素6和肿瘤坏死因子α的mRNA表达量。结果如表2所示，在10 μg/L的质量浓度时，白细胞介素6和肿瘤坏死因子α的mRNA 丰度开始上升，剂量增加到20 μg/L时，白细胞介素6和肿瘤坏死因子α mRNA丰度显著升高，在剂量增加到200 μg/L时，mRNA的表达量依然缓慢上升。由于在50-200 μg/L之间，mRNA的表达差异无显著性意义(P > 0.05)。因此，认为50 μg/L的白细胞介素17为最佳刺激浓度。 2.3 miR-136-5p表达抑制的慢病毒表达载体的构建与测序鉴定实验构建了miR-136-5p表达抑制(LV-miR-136-5p- inhibition)的慢病毒表达载体。经测序验证，核酸序列与LV-miR-136-5p-inhibition序列完全一致。表明，LV-miR- 136-5p-inhibition慢病毒表达载体构建成功。小鼠miR-136- 5p反向互补序列：CCA TCA TCA AAA CAA ATG GAG T。"

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