miR-138-5p在锰致SH-SY5Y细胞自噬中的作用
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摘要
[目的]探讨miR-138-5p在氯化锰(MnCl_2)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 MnCl_2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 MnCl_2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;MnCl_2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 MnCl_2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]MnCl_2可剂量依赖性地降低SH-SY5Y细胞活力(趋势χ~2=12.42,P<0.05)。与对照组相比,0.25、0.5 mmol/L MnCl_2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MnCl_2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。
[Objective] To investigate the role and mechanism of miR-138-5 p in autophagy induced by manganese chloride(MnCl_2) in human neuroblastoma SH-SY5 Y cells. [Methods] SH-SY5 Y cells were treated with 0.125, 0.25, 0.5, and 1 mmol/L MnCl_2, 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-SY5 Y 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-138-5 p and the mRNA and protein expressions of histone deacetylase SIRT1. Then the cells were transfected by miRNA mimics to induce overexpressed miR-138-5 p, and treated with 0.25 mmol/L MnCl_2 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] MnCl_2 dose-dependently suppressed the viability of SH-SY5 Y cells(trend χ~2=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 MnCl_2-treated cells compared with the controls(P < 0.05). MnCl_2 down-regulated the expression of miR-138-5 p, and up-regulated the mRNA and protein expressions of SIRT1(P < 0.05). Furthermore, the cells with overexpressed miR-138-5 p 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-138-5 p suppresses manganese-induced autophagy by modulating the expression of SIRT1.
[Objective] To investigate the role and mechanism of miR-138-5 p in autophagy induced by manganese chloride(MnCl_2) in human neuroblastoma SH-SY5 Y cells. [Methods] SH-SY5 Y cells were treated with 0.125, 0.25, 0.5, and 1 mmol/L MnCl_2, 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-SY5 Y 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-138-5 p and the mRNA and protein expressions of histone deacetylase SIRT1. Then the cells were transfected by miRNA mimics to induce overexpressed miR-138-5 p, and treated with 0.25 mmol/L MnCl_2 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] MnCl_2 dose-dependently suppressed the viability of SH-SY5 Y cells(trend χ~2=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 MnCl_2-treated cells compared with the controls(P < 0.05). MnCl_2 down-regulated the expression of miR-138-5 p, and up-regulated the mRNA and protein expressions of SIRT1(P < 0.05). Furthermore, the cells with overexpressed miR-138-5 p 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-138-5 p suppresses manganese-induced autophagy by modulating the expression of SIRT1.
引文
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[17]ZHANG H T,MI L,WANG T,et al.PINK1/Parkin-mediated mitophagy play a protective role in manganese induced apoptosis in SH-SY5Y cells[J].Toxicol in Vitro,2016,34:212-219.
[18]CHEN P,CULBRETH M,ASCHNER M.Exposure,epidemiology,and mechanism of the environmental toxicant manganese[J].Environ Sci Pollut Res Int,2016,23(14):13802-13810.
[19]AFESEH NGWA H,KANTHASAMY A,GU Y,et al.Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells[J].Toxicol Appl Pharmacol,2011,256(3):227-240.
[20]FRANKEL L B,LUND A H.Micro RNA regulation of autophagy[J].Carcinogenesis,2012,33(11):2018-2025.
[21]WANG B,WANG D,YAN T,et al.Mi R-138-5p promotes TNF-α-induced apoptosis in human intervertebral disc degeneration by targeting SIRT1 through PTEN/PI3K/Akt signaling[J].Exp Cell Res,2016,345(2):199-205.
[22]TIAN S,GUO X,YU C,et al.mi R-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1[J].Oncotarget,2017,8(7):11071-11082.
[2]RACETTE B A,CRISWELL S R,LUNDIN J I,et al.Increased risk of parkinsonism associated with welding exposure[J].Neuro Toxicology,2012,33(5):1356-1361.
[3]LIU X H,YANG J B,LU C H,et al.Downregulation of Mfn2participates in manganese-induced neuronal apoptosis in rat striatum and PC12 cells[J].Neurochem Int,2017,108:40-51,doi:10.1016/j.neuint.2017.02.008.
[4]ALAIMO A,GOROJOD R M,MIGLIETTA E A,et al.Manganese induces mitochondrial dynamics impairment and apoptotic cell death:a study in human Gli36 cells[J].Neurosci Lett,2013,554:76-81.
[5]AVILA D S,BENEDETTO A,AU C,et al.Organotellurium and organoselenium compounds attenuate Mn-induced toxicity in Caenorhabditis elegans by preventing oxidative stress[J].Free Radic Biol Med,2012,52(9):1903-1910.
[6]BAHAR E,LEE G H,BHATTARAI K R,et al.Polyphenolic extract of Euphorbia supina attenuates manganese-induced neurotoxicity by enhancing antioxidant activity through regulation of ER stress and ER stress-mediated apoptosis[J].Int J Mol Sci,2017,18(2):300,doi:10.3390/ijms18020300.
[7]ZHANG J,CAO R,CAI T,et al.The role of autophagy dysregulation in manganese-induced dopaminergic neurodegeneration[J].Neurotox Res,2013,24(4):478-490.
[8]WEIDBERG H,SHVETS E,SHPILKA T,et al.LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis[J].EMBO J,2010,29(11):1792-802.
[9]CAO Y,KLIONSKY D J.Physiological functions of Atg6/Beclin 1:a unique autophagy-related protein[J].Cell Res,2007,17(10):839-49.
[10]QIU G,LI X,CHE X,et al.SIRT1 is a regulator of autophagy:Implications in gastric cancer progression and treatment[J].FEBS Lett,2015,589(16):2034-2042.
[11]LEE I H,CAO L,MOSTOSLAVSKY R,et al.A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy[J].Proc Natl Acad Sci USA,2008,105(9):3374-3379.
[12]隋静,张艳秋,李成云,等.微小RNA-27b对RAW264.7细胞生物学功能的作用及调控机制[J].环境与职业医学,2016,33(11):1049-1054.
[13]LAU P,BOSSERS K,JANKY R,et al.Alteration of the micro RNA network during the progression of Alzheimer’s disease[J].EMBO Mol Med,2013,5(10):1613-1634.
[14]TATRO E T,RISBROUGH V,SOONTORNNIYOMKIJ B,et al.Short-term recognition memory correlates with regional CNS expression of micro RNA-138 in mice[J].Am J Geriatr Psychiatry,2013,21(5):461-473.
[15]LIU C M,WANG R Y,SAIJILAFU,et al.Genes Dev,Micro RNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration[J].Genes Dev,2013,27(13):1473-1483.
[16]FUKUSHIMA T,TAN X,LUO Y,et al.Relationship between blood levels of heavy metals and Parkinson's disease in China[J].Neuroepidemiology,2010,34(1):18-24.
[17]ZHANG H T,MI L,WANG T,et al.PINK1/Parkin-mediated mitophagy play a protective role in manganese induced apoptosis in SH-SY5Y cells[J].Toxicol in Vitro,2016,34:212-219.
[18]CHEN P,CULBRETH M,ASCHNER M.Exposure,epidemiology,and mechanism of the environmental toxicant manganese[J].Environ Sci Pollut Res Int,2016,23(14):13802-13810.
[19]AFESEH NGWA H,KANTHASAMY A,GU Y,et al.Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells[J].Toxicol Appl Pharmacol,2011,256(3):227-240.
[20]FRANKEL L B,LUND A H.Micro RNA regulation of autophagy[J].Carcinogenesis,2012,33(11):2018-2025.
[21]WANG B,WANG D,YAN T,et al.Mi R-138-5p promotes TNF-α-induced apoptosis in human intervertebral disc degeneration by targeting SIRT1 through PTEN/PI3K/Akt signaling[J].Exp Cell Res,2016,345(2):199-205.
[22]TIAN S,GUO X,YU C,et al.mi R-138-5p suppresses autophagy in pancreatic cancer by targeting SIRT1[J].Oncotarget,2017,8(7):11071-11082.