Objective:

To identify the role of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) for improving the apoptosis of hippocampal neurons induced by microglia activation.

Methods:

Mouse microglial cell line (BV2 cell) was employed and treated with lipopolysaccharide. Mouse hippocampal nerve cell line (HT22 cell) was then grown in BV2 conditioned medium, and miR-146a overexpression and silencing cell lines were constructed. CCK8 and clone formation test were utilized to evaluate the proliferation ability of the transfected cells, and the level of inflammatory factors was measured by ELISA. Apoptosis was determined extensively by flow cytometry. The apoptosis-related protein and TRAF6 protein expressions were verified by Western blot. TRAF6 was identified to be the target gene of miR-146a based on double Luciferase Report. Finally, both TRAF6 and miR-146a were used to treat HT22 cells and the above indexes were detected repeatedly.

Results:

Interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 expressions in BV2 cells increased significantly. miR-146a overexpression distinctly increased the cell proliferation ability and B-cell lymphoma-2 expression ((Bcl-2, p < 0.05); meanwhile, the apoptosis rate of cells, apoptosis-related proteins (Bcl-2 associated X and cleaved caspase-3), and TRAF6 gene and protein expressions were significantly decreased (p < 0.05). However, these above results were reversed for miR-146a silence. There is a targeting relationship between miR-146a and TRAF6. Silencing TRAF6 gene can promote HT22 cells’ proliferation and inhibit apoptosis. The effect of miR-146a on HT22 cells was reversed by adding TRAF6 mimics to miR-146a overexpression cells.

Conclusion:

miR-146a can inhibit the apoptosis of hippocampal neurons caused by microglia activation via targeting TRAF6 and down-regulating its expression.

中文翻译：

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miR-146a 通过靶向 TRAF6 减轻小胶质细胞激活诱导的海马神经元凋亡。

客观的：

确定 miR-146a 和肿瘤坏死因子受体相关因子 6 (TRAF6) 在改善小胶质细胞激活诱导的海马神经元凋亡中的作用。

方法：

使用小鼠小胶质细胞系（BV2细胞）并用脂多糖处理。然后在BV2条件培养基中培养小鼠海马神经细胞系（HT22细胞），构建miR-146a过表达和沉默细胞系。采用CCK8和克隆形成试验评价转染细胞的增殖能力，ELISA检测炎症因子水平。通过流式细胞术广泛确定细胞凋亡。Western blot验证凋亡相关蛋白和TRAF6蛋白的表达。基于双荧光素酶报告，TRAF6被鉴定为miR-146a的靶基因。最后用TRAF6和miR-146a处理HT22细胞，反复检测上述指标。

结果：

BV2 细胞中白细胞介素 (IL)-1β、肿瘤坏死因子-α 和 IL-6 的表达显着增加。miR-146a 过表达显着增加细胞增殖能力和 B 细胞淋巴瘤 2 表达（（Bcl-2，p < 0.05）；同时，细胞凋亡率、凋亡相关蛋白（Bcl-2 相关 X 和裂解的 caspase -3), TRAF6 基因和蛋白表达显着降低( p < 0.05)。但上述结果因 miR-146a 沉默而逆转。miR-146a 和 TRAF6 之间存在靶向关系。沉默 TRAF6 基因可以促进 HT22 miR-146a 对 HT22 细胞的影响通过在 miR-146a 过表达细胞中添加 TRAF6 模拟物来逆转。

结论：

miR-146a可通过靶向TRAF6并下调其表达来抑制小胶质细胞激活引起的海马神经元凋亡。