目的 探讨METTL3调控SPRING1促进巨噬细胞脂质蓄积的作用及机制。 方法 100 ng/mL PMA诱导THP-1细胞贴壁后，50 μg/mL Ac-LDL孵育THP-1细胞。Western blot测定METTL3和SPRING1蛋白；qRT-PCR测定SPRING1mRNA水平；细胞内总胆固醇、胆固醇酯以及游离胆固醇变化用高效液相色谱法检测；SRAMP和RMBase网站分析SPRING1 mRNA上的m6A修饰位点情况；质膜红色荧光标记探针Dil-Ac-LDL观察巨噬细胞脂滴摄取情况。 结果 与对照组相比，Ac-LDL孵育后THP-1细胞METTL3和SPRING1蛋白表达上调，并且SPRING1 mRNA水平上调；过表达METTL3会使SPRING1蛋白表达上调，巨噬细胞对脂质摄取增加，细胞内Dil-Ac-LDL明显增多；反之，沉默METTL3表达，SPRING1蛋白表达下调；甲基化抑制剂环亮氨酸处理可部分抑制METTL3过表达对SPRING1表达的促进作用；生物信息学分析显示，SPRING1 mRNA存在m6A修饰位点。 结论 METTL3上调SPRING1表达，促进巨噬细胞脂质蓄积。
Abstract
Objective To investigate the effect and mechanism of METTL3 regulating SPRING1 on macrophage lipid accumulation. Methods After inducing the attachment of THP-1 cells with 100 ng/ml PMA, 50 μg/ml Ac-LDL was added to incubate THP-1 cells. The protein levels of METTL3 and SPRING1 were detected by Western blot and the mRNA levels was detected by qRT-PCR. The total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC) were detected by high performance liquid chromatography (HPLC). SRAMP and RMBase websites were used to analyze the m6A modification sites on the SPRING1 mRNA. The Oil red O staining and plasma membrane red fluorescent-labeled probe Dil-Ac-LDL were used to observe the uptake of lipid droplets in macrophages. Results Compared with the control group, the protein level of METTL3 and SPRING1 in THP-1 cells was upregulated; the SPRING1 mRNA level was also upregulated after Ac-LDL incubation. Overexpression of METTL3 upregulated the expression of SPRING1 protein, increased the uptake Dil-Ac-LDL by macrophages. While METTL3 silence obviously downregulated the expression of SPRING1 protein. Cycloleucine, as a methylation inhibitor, can partially inhibit the promotion of METTL3 overexpression on SPRING1. Bioinformatics analysis showed that there were m6A modification sites in SPRING1 mRNA. Conclusions METTL3 upregulates the expression of SPRING1 and promotes lipid accumulation in macrophages.
关键词
METTL3 /
m6A /
SPRING1 /
脂质蓄积
Key words
METTL3 /
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M6A /
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SPRING1 /
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Lipid accumulation
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基金
湖南省自然科学基金项目（2020JJ4532）；湖南省教育厅优秀青年项目（18B264）
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