Zhongguo cuzhong zazhi (Jan 2025)
甲基转移酶3对人脑血管平滑肌细胞基因表达及增殖和迁移功能的影响 Effects of Methyltransferase 3 on Gene Expression, Proliferation, and Migration Functions of Human Brain Vascular Smooth Muscle Cells
Abstract
目的 探究甲基转移酶3（methyltransferase 3，METTL3）对人脑血管平滑肌细胞（human brain vascular smooth muscle cells，HBVSMC）基因表达以及增殖和迁移功能的影响。 方法 采用小干扰RNA（small interfering RNA，shRNA）干扰技术，构建METTL3基因干扰载体并通过腺病毒感染HBVSMC，依据感染情况将其分为空白组（无病毒感染）、对照组（空载病毒感染）和干扰组（METTL3-shRNA病毒感染）。使用逆转录实时定量PCR（real-time reverse transcriptional PCR，RT-qPCR）检测各组细胞脑小血管病相关基因锌指蛋白395（zinc finger protein 395，ZNF395）、锌指蛋白548（zinc finger protein 548，ZNF548）、DIS3样外泌体3’-5’核糖核酸外切酶（DIS3-like exosome 3’-5’exonuclease，DIS3L）、白细胞介素增强子结合因子3（interleukin enhancer binding factor 3，ILF3）和G蛋白偶联受体107（G protein-coupled receptor 107，GPR107）的mRNA表达水平，通过RNA甲基化免疫共沉淀（methylated RNA immunoprecipitation，MeRIP）技术检测以上基因mRNA的N6-甲基腺苷（N6-methyladenosine，m6A）修饰水平，利用细胞计数试剂盒8（cell counting kit 8，CCK8）、Transwell小室实验检测各组HBVSMC的增殖和迁移能力。 结果 与空白组相比，对照组细胞各基因的mRNA相对表达水平未发生明显改变。与对照组相比，干扰组细胞ZNF395（P=0.02）、ZNF548（P=0.03）、DIS3L（P=0.02）和GPR107（P＜0.01）的mRNA相对表达水平，以及ZNF395（P＜0.01）和ZNF548（P＜0.01）的m6A修饰水平均降低。与对照组相比，干扰组HBVSMC的增殖和迁移能力明显增强。 结论 在HBVSMC中干扰METTL3基因表达可能通过下调靶基因ZNF395和ZNF548 mRNA的m6A修饰水平进而增强HBVSMC的增殖和迁移能力。 Abstract: Objective To investigate the effects of methyltransferase 3 (METTL3) on gene expression, proliferation, and migration functions of human brain vascular smooth muscle cells (HBVSMC). Methods The transfected HBVSMC were divided into three groups: the blank group (virus-free), the control group (empty vector), and the interference group (METTL3-shRNA) by using small interfering RNA (shRNA) interference technology and adenovirus packaging method. The mRNA expression levels of cerebral small vessel disease-related genes including zinc finger protein 395 (ZNF395), zinc finger protein 548 (ZNF548), DIS3-like exosome 3’-5’ exonuclease (DIS3L), interleukin enhancer binding factor 3 (ILF3) and G protein-coupled receptor 107 (GPR107) were detected by real-time reverse transcriptional PCR (RT-qPCR). Methylated RNA immunoprecipitation (MeRIP) was used to detect the N6-methyladenosine (m6A) modification levels of the mRNA for the genes above. The proliferation and migration abilities of HBVSMC were detected by cell counting kit 8 (CCK8) and Transwell chamber assay. Results Compared with the blank group, there was no significant change in the relative mRNA expression levels of each gene in the control group. Compared with the control group, the relative mRNA expression levels of ZNF395 (P=0.02), ZNF548 (P=0.03), DIS3L (P=0.02) and GPR107 (P＜0.01) as well as the m6A modification levels of ZNF395 (P＜0.01) and ZNF548 (P＜0.01) were decreased in the interference group. Compared with the control group, the proliferation and migration abilities of HBVSMC in the interference group were significantly enhanced. Conclusions Interfering with METTL3 expression may promote the proliferation and migration abilities of HBVSMC by down-regulating the m6A modification levels of ZNF395 and ZNF548 mRNA.
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