目的探讨在炎性激活条件下的椎间盘退变(IDD)过程中miR-335-3p靶向调控趋化因子配体(CCL)5的机制及可能抑制IDD进展的方法。方法建立人髓核(NP)细胞的原代培养,肿瘤坏死因子(TNF)-α诱导NP细胞,Trizol法获取NP细胞总RNA,随后对mRNA及mi...目的探讨在炎性激活条件下的椎间盘退变(IDD)过程中miR-335-3p靶向调控趋化因子配体(CCL)5的机制及可能抑制IDD进展的方法。方法建立人髓核(NP)细胞的原代培养,肿瘤坏死因子(TNF)-α诱导NP细胞,Trizol法获取NP细胞总RNA,随后对mRNA及miRNA进行高通量测序分析。对miR-335-3p靶向结合mRNA进行预测,结合STRING富集分析miRNA靶标基因,确定TNF-α影响NP细胞的关键基因。应用The Human Protein Atlas(https://www.proteinatlas.org/)分析CCL5及其下游调控蛋白受体趋化因子受体(CCR)5基因在免疫相关细胞中的表达水平。应用Targetscan预测miR-335-3p与CCL53′UTR序列的靶向结合位点。结合promega的双荧光素酶报告系统使用turner仪器进行测量。将人NP细胞分成:Control组(NP细胞)、TNF-α组、mimic-NC组、miR-335-3p mimic组。使用Lipofectamine RNAiMAX将miR-335-3p mimic或miR-NC寡核苷酸序列转染入人NP细胞。采用实时荧光定量聚合酶链反应(qRT-PCR)方法检测miR-335-3p的表达情况。Western印迹实验检测NP细胞外基质相关蛋白的表达水平。结果与Control组比较,TNF-α组miR-335-3p基因表达水平显著下调(P<0.01),CCL5基因表达水平显著上调(P<0.01);miR-335-3p mimic组miR-335-3p表达水平较mimic-NC组显著上调(P<0.01),CCL5表达水平显著较mimic-NC组下调(P<0.01)。与Control组比较,TNF-α组生长分化因子(GDF)5和性别决定区Y框蛋白(SOX)9蛋白显著下调,而基质金属蛋白酶(MMP)2蛋白表达水平显著上调(P<0.01);与mimic-NC组比较,miR-335-3p mimic组MMP2蛋白表达水平显著下调,而GDF5和SOX9蛋白显著上调(P<0.01)。结论miR-335-3p在TNF-α诱导的炎症条件下可特异性下调CCL5,从而逆转TNF-α诱导的软骨生成因子SOX9的异常表达。展开更多
Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's dis...Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.展开更多
目的:探究miR-335-5p/ADCY3失衡对获得性再生障碍性贫血(AA)患者淋巴细胞功能的影响。方法:采集AA患者外周血标本50例,其中重型再生障碍性贫血(SAA)38例,非重型再生障碍性贫血(NSAA)12例,同时采集健康志愿者(HC)外周血标本22例。标本分...目的:探究miR-335-5p/ADCY3失衡对获得性再生障碍性贫血(AA)患者淋巴细胞功能的影响。方法:采集AA患者外周血标本50例,其中重型再生障碍性贫血(SAA)38例,非重型再生障碍性贫血(NSAA)12例,同时采集健康志愿者(HC)外周血标本22例。标本分离单个核细胞(PBMNC)后,用RT-PCR检测miR-335-5p及ADCY3 mRNA的表达量。用RNAimax转染试剂,将阴性对照miR-335-5p及miR-335-5p mimic分别转染至AA患者的PBMNC,流式细胞术检测CD4^+T和CD8^+T细胞增殖、活化及分泌细胞因子的能力。应用双荧光素酶报告基因系统验证miR-335-5p与靶基因的靶向关系。结果:同HC来源的PBMNC(HC-PBMNC)相比,SAA和NSAA来源的PBMNC中miR-335-5p的表达量均明显降低(0.08±0.01 vs 0.74±0.10,P<0.01^**;0.17±0.02 vs 0.74±0.10,P<0.01^**)。而且,SAA来源的PBMNC中miR-335-5p的表达量显著低于NSAA(P<0.01^**)。与对照组相比,体外上调miR-335-5p的表达,AA来源的PBMNC(AA-PBMNC)中,CD4+T和CD8+T细胞增殖能力均明显降低(P<0.05^*和P<0.05^*)。而且上调miR-335-5p的表达可显著抑制AA-PBMNC中CD4^+和CD8^+T细胞的活化(P<0.01^**和P<0.01^**)。此外,上调AA-PBMNC中,miR-335-5p的表达还可明显降低CD4^+TNFα^+T细胞、CD8^+IFNγ^+T细胞和CD8^+TNFα^+T细胞的比例(P<0.01、P<0.05和P<0.05)。靶基因筛查显示,ADCY3在AA-PBMNC中的表达量明显高于HC-PBMNC(1.70±0.15 vs 0.76±0.12,P<0.001)。而且,miR-335-5p可显著抑制ADCY33′UTR野生型质粒的荧光素酶活性(P<0.05)。结论:MiR-335-5p在AA-PBMNC中显著低表达,且与疾病严重程度相关。体外上调AA-PBMNC中miR-335-5p的表达量可纠正患者免疫异常状态。这些改变可能与靶基因ADCY3的抑制增强有关。展开更多
文摘目的探讨在炎性激活条件下的椎间盘退变(IDD)过程中miR-335-3p靶向调控趋化因子配体(CCL)5的机制及可能抑制IDD进展的方法。方法建立人髓核(NP)细胞的原代培养,肿瘤坏死因子(TNF)-α诱导NP细胞,Trizol法获取NP细胞总RNA,随后对mRNA及miRNA进行高通量测序分析。对miR-335-3p靶向结合mRNA进行预测,结合STRING富集分析miRNA靶标基因,确定TNF-α影响NP细胞的关键基因。应用The Human Protein Atlas(https://www.proteinatlas.org/)分析CCL5及其下游调控蛋白受体趋化因子受体(CCR)5基因在免疫相关细胞中的表达水平。应用Targetscan预测miR-335-3p与CCL53′UTR序列的靶向结合位点。结合promega的双荧光素酶报告系统使用turner仪器进行测量。将人NP细胞分成:Control组(NP细胞)、TNF-α组、mimic-NC组、miR-335-3p mimic组。使用Lipofectamine RNAiMAX将miR-335-3p mimic或miR-NC寡核苷酸序列转染入人NP细胞。采用实时荧光定量聚合酶链反应(qRT-PCR)方法检测miR-335-3p的表达情况。Western印迹实验检测NP细胞外基质相关蛋白的表达水平。结果与Control组比较,TNF-α组miR-335-3p基因表达水平显著下调(P<0.01),CCL5基因表达水平显著上调(P<0.01);miR-335-3p mimic组miR-335-3p表达水平较mimic-NC组显著上调(P<0.01),CCL5表达水平显著较mimic-NC组下调(P<0.01)。与Control组比较,TNF-α组生长分化因子(GDF)5和性别决定区Y框蛋白(SOX)9蛋白显著下调,而基质金属蛋白酶(MMP)2蛋白表达水平显著上调(P<0.01);与mimic-NC组比较,miR-335-3p mimic组MMP2蛋白表达水平显著下调,而GDF5和SOX9蛋白显著上调(P<0.01)。结论miR-335-3p在TNF-α诱导的炎症条件下可特异性下调CCL5,从而逆转TNF-α诱导的软骨生成因子SOX9的异常表达。
基金supported by the National Institute on Aging (NIA)National Institutes of Health (NIH)+3 种基金Nos.K99AG065645,R00AG065645R00AG065645-04S1 (to SK)NIH research grants,NINDS,No.R01 NS115834NINDS/NIA,No.R01 NS115834-02S1 (to LG)。
文摘Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.
文摘目的:探究miR-335-5p/ADCY3失衡对获得性再生障碍性贫血(AA)患者淋巴细胞功能的影响。方法:采集AA患者外周血标本50例,其中重型再生障碍性贫血(SAA)38例,非重型再生障碍性贫血(NSAA)12例,同时采集健康志愿者(HC)外周血标本22例。标本分离单个核细胞(PBMNC)后,用RT-PCR检测miR-335-5p及ADCY3 mRNA的表达量。用RNAimax转染试剂,将阴性对照miR-335-5p及miR-335-5p mimic分别转染至AA患者的PBMNC,流式细胞术检测CD4^+T和CD8^+T细胞增殖、活化及分泌细胞因子的能力。应用双荧光素酶报告基因系统验证miR-335-5p与靶基因的靶向关系。结果:同HC来源的PBMNC(HC-PBMNC)相比,SAA和NSAA来源的PBMNC中miR-335-5p的表达量均明显降低(0.08±0.01 vs 0.74±0.10,P<0.01^**;0.17±0.02 vs 0.74±0.10,P<0.01^**)。而且,SAA来源的PBMNC中miR-335-5p的表达量显著低于NSAA(P<0.01^**)。与对照组相比,体外上调miR-335-5p的表达,AA来源的PBMNC(AA-PBMNC)中,CD4+T和CD8+T细胞增殖能力均明显降低(P<0.05^*和P<0.05^*)。而且上调miR-335-5p的表达可显著抑制AA-PBMNC中CD4^+和CD8^+T细胞的活化(P<0.01^**和P<0.01^**)。此外,上调AA-PBMNC中,miR-335-5p的表达还可明显降低CD4^+TNFα^+T细胞、CD8^+IFNγ^+T细胞和CD8^+TNFα^+T细胞的比例(P<0.01、P<0.05和P<0.05)。靶基因筛查显示,ADCY3在AA-PBMNC中的表达量明显高于HC-PBMNC(1.70±0.15 vs 0.76±0.12,P<0.001)。而且,miR-335-5p可显著抑制ADCY33′UTR野生型质粒的荧光素酶活性(P<0.05)。结论:MiR-335-5p在AA-PBMNC中显著低表达,且与疾病严重程度相关。体外上调AA-PBMNC中miR-335-5p的表达量可纠正患者免疫异常状态。这些改变可能与靶基因ADCY3的抑制增强有关。
