BACKGROUND Vascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production. RESULTS In this study, bioinformatics analysis was used to select tumour necrosis factor α (TNFα) as a candidate inflammatory factor associated with calcification. Moreover, alizarin red staining and qRT-PCR analysis revealed that TNFα produced by BV2 cells was the key promoting factor of VSMC calcification. Interestingly, the expression of TNFα was significantly increased at the mRNA and protein levels after miR32-5p mimic treatment but significantly decreased after miR32-5p antagomir treatment. To explore the mechanism of the regulation of TNFα expression by miR32-5p, bioinformatics analysis indicated that PIKfyve was a candidate target gene of miR32-5p, and luciferase assays verified that the expression of PIKfyve was significantly repressed by miR32-5p mimics. Importantly, rescue experiments showed that the expression of TNFα in BV2 cells treated with miR32-5p antagomir and the PIKfyve inhibitor YM201636 was significantly increased. CONCLUSIONS The production of TNFα in microglia could be affected by miR32-5p targeting PIKfyve, and these results will be beneficial to reveal the mechanism of brain arterial calcification.

中文翻译：

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miR32-5p通过在微环境中上调TNFα促进血管平滑肌细胞钙化。

背景技术血管钙化通常与慢性炎症相关，并且是脑动脉僵硬的危险因素。我们以前的结果表明，miR32-5p与血管平滑肌细胞（VSMC）钙化呈正相关，但尚不清楚miR32-5p是否通过调节炎症因子的产生来促进VSMC钙化。结果在这项研究中，生物信息学分析被用来选择肿瘤坏死因子α（TNFα）作为与钙化相关的候选炎症因子。此外，茜素红染色和qRT-PCR分析表明，BV2细胞产生的TNFα是VSMC钙化的关键促进因子。有趣的是 miR32-5p模拟物处理后，TNFα的表达在mRNA和蛋白水平上显着增加，而miR32-5p antagomir处理后，TNFα的表达显着降低。为了探索miR32-5p调节TNFα表达的机制，生物信息学分析表明PIKfyve是miR32-5p的候选靶基因，萤光素酶检测证实miR32-5p模拟物显着抑制了PIKfyve的表达。重要的是，救援实验表明，用miR32-5p antagomir和PIKfyve抑制剂YM201636处理的BV2细胞中TNFα的表达显着增加。结论靶向PIKfyve的miR32-5p可能会影响小胶质细胞中TNFα的产生，这些结果将有助于揭示脑动脉钙化的机制。为了探索miR32-5p调节TNFα表达的机制，生物信息学分析表明PIKfyve是miR32-5p的候选靶基因，萤光素酶检测证实miR32-5p模拟物显着抑制了PIKfyve的表达。重要的是，救援实验表明，用miR32-5p antagomir和PIKfyve抑制剂YM201636处理的BV2细胞中TNFα的表达显着增加。结论靶向PIKfyve的miR32-5p可能会影响小胶质细胞中TNFα的产生，这些结果将有助于揭示脑动脉钙化的机制。为了探索miR32-5p调节TNFα表达的机制，生物信息学分析表明PIKfyve是miR32-5p的候选靶基因，萤光素酶检测证实miR32-5p模拟物显着抑制了PIKfyve的表达。重要的是，救援实验表明，用miR32-5p antagomir和PIKfyve抑制剂YM201636处理的BV2细胞中TNFα的表达显着增加。结论靶向PIKfyve的miR32-5p可能会影响小胶质细胞中TNFα的产生，这些结果将有助于揭示脑动脉钙化的机制。荧光素酶测定法证实miR32-5p模拟物显着抑制了PIKfyve的表达。重要的是，救援实验表明，用miR32-5p antagomir和PIKfyve抑制剂YM201636处理的BV2细胞中TNFα的表达显着增加。结论靶向PIKfyve的miR32-5p可能会影响小胶质细胞中TNFα的产生，这些结果将有助于揭示脑动脉钙化的机制。荧光素酶测定法证实miR32-5p模拟物显着抑制了PIKfyve的表达。重要的是，救援实验表明，用miR32-5p antagomir和PIKfyve抑制剂YM201636处理的BV2细胞中TNFα的表达明显增加。结论靶向PIKfyve的miR32-5p可能会影响小胶质细胞中TNFα的产生，这些结果将有助于揭示脑动脉钙化的机制。