miR-138-5p在锰致SH-SY5Y细胞自噬中的作用
Role of miR-138-5p in autophagy in SH-SY5Y cells induced by manganese
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摘要:
目的 探讨miR-138-5p在氯化锰（MnCl2）诱导人神经母细胞瘤SH-SY5Y细胞自噬中的作用及其机制。
方法 以生理盐水处理组作为对照，0.125、0.25、0.5、1 mmol/L MnCl2染毒SH-SY5Y细胞6 h后，利用MTT法检测细胞活力；0.25和0.5 mmol/L MnCl2处理细胞6 h后，采用Western blot检测自噬相关蛋白LC3和Beclin1的表达；MnCl2染毒6 h后，使用反转录PCR和Western blot检测miR-138-5p和组蛋白脱乙酰酶SIRT1 mRNA以及蛋白表达的变化；使用miRNA模拟物转染细胞实现miR-138-5p过表达，然后0.25 mmol/L MnCl2染毒6 h，检测SIRT1 mRNA和蛋白的表达以及自噬相关蛋白LC3和Beclin1的变化。
结果 MnCl2可剂量依赖性地降低SH-SY5Y细胞活力（趋势χ2=12.42，P < 0.05）。与对照组相比，0.25、0.5 mmol/L MnCl2染毒后，自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ值和Beclin1表达水平明显增加（P < 0.05）；miR-138-5p表达下调，SIRT1 mRNA及蛋白表达上调（均P < 0.05）。miR-138-5p过表达后，相比未过表达的MnCl2染毒组，SIRT1 mRNA及蛋白表达均出现下调，自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ值及Beclin1蛋白表达也相应下调（P < 0.05）。
结论 miR-138-5p过表达可通过调节SIRT1的表达抑制锰诱导的SH-SY5Y细胞自噬。
Abstract:Objective To investigate the role and mechanism of miR-138-5p in autophagy induced by manganese chloride (MnCl2) in human neuroblastoma SH-SY5Y cells.
Methods SH-SY5Y cells were treated with 0.125, 0.25, 0.5, and 1 mmol/L MnCl2, respectively. Control cells were treated with normal saline. After 6 h of the treatment, MTT test was used to detect the cell viability of SH-SY5Y cells. After 6 h of the treatment, Western blot was used to detect the expression of autophagy related proteins LC3 and Beclin1, reverse transcription PCR (RT-PCR) and western blot were used to detect the expression of miR-138-5p and the mRNA and protein expressions of histone deacetylase SIRT1. Then the cells were transfected by miRNA mimics to induce overexpressed miR-138-5p, and treated with 0.25 mmol/L MnCl2 for 6 h to detect the expressions of SIRT1 at mRNA and protein levels and the expressions of autophagy related proteins LC3 and Beclin1.
Results MnCl2 dose-dependently suppressed the viability of SH-SY5Y cells (trend χ2=12.42, P < 0.05). The LC3-Ⅱ/LC3-Ⅰ ratio and Beclin1 expression level were significantly elevated in the 0.25 and 0.5 mmol/L MnCl2-treated cells compared with the controls (P < 0.05). MnCl2 down-regulated the expression of miR-138-5p, and up-regulated the mRNA and protein expressions of SIRT1 (P < 0.05). Furthermore, the cells with overexpressed miR-138-5p showed decreased expression of SIRT1 both at mRNA and protein levels and decreased expressions of LC3-Ⅱ/LC3-Ⅰ and Beclin1 protein (P < 0.05).
Conclusion Overexpression of miR-138-5p suppresses manganese-induced autophagy by modulating the expression of SIRT1.
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