Role Of MiR-19 In Aluminum-induced Neural Cell Apoptosis And Folic Acid Intervention

Aluminum(Al)is the most abundant metal existed in the biosphere,which has also been widely used in industrial applications,water treatment,food additives,pharmaceuticals and etc.Environmental exposure of humans to aluminum is ubiquitous and extensive.Aluminum is not the essential element in human body;on the contrary,it has been reported to be neurotoxic.Previous studies have postulated aluminum-induced apoptosis as the major cause of its neurotoxicity.However,the underlying molecular mechanisms by which aluminum induces apoptosis of neuronal cells remain to be elucidated.Aluminum maltolate(Al-malt)is a lipophilic Al complex with maltolate.Maltolate is a common component of human diet with high affinity for Al and the ability to facilitate the entry of Al into the brain.Therefore,Al-malt is advantageous for use in the investigation of Al neurotoxicity since it is relevant to human health.Numerous studies have demonstrated microRNAs(miRNAs)regulation as an important mechanism in gene expression.Emerging evidence has highlighted the functional importance of miRNAs in the pathogenesis of neurological diseases.Dysregulation of miR-19 alteration is involved in kinds of nervous system disease.miR-19 is a key member of the miR-17/92 cluster,which targets several critical apoptosis related genes such as PTEN and regulates cell apoptotic process.To date,however,whether miR-19 play a role in Al-induced neural cell apoptosis has not yet been investigated.Folic acid is a water-soluble vitamin,which has reported to a nutrient with neuroprotective effect.Studies have shown that folic acid supplementation could prevent neurodegenerative diseases such as Alzheimer's disease.Folic acid exhibits neuroprotective effect by preventing neural cell apoptosis.However,whether miR-19 plays a role in the protective effect of folic acid against aluminum-induced neural cell apoptosis has not yet been reported.The present study aimed to investigate the role of miR-19 in Al-malt-induced neural cell apoptosis by using in vitro SH-SY5Y cells model and in vivo SD rats model.Meanwhile,the role of miR-19 in neural protection effect of folic acid were also examined.Findings from this study could provide new insight into the molecular mechanisms of aluminum-induced neural cell apoptosis and the mechanisms by which folic acid exerts its neuroprotective function.Part ?:Modulation of miR-19 in Aluminum-induced Neural Cell ApoptosisObjectives:by using in vitro SH-SY5Y cells model and in vivo SD rats model treated with Al-malt,the present study aimed to investigate the effects of Al-malt on apoptosis of neuronal cells;observe the expression changes of miR-19 and its downstream PTEN/AKT/p53 signaling pathway with treatment of Al-malt;and explore modulation of miR-19 in neural cell apoptosis induced by Al-malt.Methods:Human SH-SY5Y cells were exposed to various concentrations of Al-malt for 72h and cell viability was examined by MTT assay;hochest 33258 staining assay and flow cytometry analysis were used to detect the neural cell apoptosis;the protein levels of caspase family,Bcl-2 family and PTEN/AKT/p53 pathway were examined by Western blotting;real-time PCR was used to determine the levels of miR-19a and miR-19b.SH-SY5Y cells were transfected with miR-19 mimics,after transfection,SH-SY5Y cells were treated with Al-malt(0.25 mM)for 72h and cell viability was measured by MTT assay;the protective effect of miR-19 in Al-malt induced apoptosis was detected by flow cytometry analysis;Western blotting was used to determine the protein levels of caspase family,Bcl-2 family and PTEN/AKT/p53 pathway.Sprague Dawley rats were exposed to Al-malt(20,100,and 200 mg/L Al)for 12 weeks;Al contents in serum and brain tissues were determined by high-resolution continuum source graphite furnace atomic absorption spectrometry;the protein levels of cleaved-caspase-9,cleaved-caspase-3 and PTEN/AKT/p53 pathway were analyzed by Western blotting;the apoptosis of rat hippocampus was determined by TUNEL assays;real-time PCR was used to determine the expression levels of miR-19a and miR-19b in rats brain;immunohistochemistry analysis illustrated the expression of PTEN in hippocampus of rats.Results1.Al-malt induces apoptosis in human SH-SY5Y cellsHuman SH-SY5Y cells were exposed to various concentrations of Al-malt for 72h.Al-malt decreased cell viability in a dose-dependent manner.Hochest 33258 staining assay revealed that compared to untreated controls,SH-SY5Y cells treated with Al-malt for 72h showed intense fluorescence in the nuclei and nuclear fragmentation,indicating the apoptotic changes.Flow cytometry analysis revealed that treatment of SH-SY5Y cells with Al-malt significantly resulted in apoptosis of the cells.Moreover,Al-malt treatment decreased the expression levels of pro-caspase-9 and pro-caspase-3,whereas the levels of cleaved-caspase-9 and cleaved-caspase-3 were significantly increased,indicating the activation of these caspases in neuron cells.Data from cell viability,hochest 33258 staining,flow cytometry and caspase activation demonstrated that Al-malt induced apoptosis in SH-SY5Y cells in a concentration-dependent manner.2.Al-malt-induced apoptosis is associated with miR-19 down-regulation and alteration of miR-19-targeted PTEN/AKT/p53 pathwayHuman SH-SY5Y cells were exposed to Al-malt(0.1,0.25 and 0.5mM)for 72h.Al-malt treatment significantly decreased the expression levels of both miR-19a and miR-19b in SH-SY5Y cells.Consistent with the reduction of miR-19,Al-malt upregulated the expression of PTEN,along with decreased level of phosphorylated(p)-AKT,increased p53 and Bax,and decreased Bcl-2.These data suggested the downregulation of miR-19 and alteration of miR-19-targeted PTEN/AKT/p53 pathway in Al-malt induced apoptosis in SH-SY5Y cells.3.miR-19 overexpression ameliorates Al-malt-induced apoptosis of SH-SY5Y cellsSH-SY5Y cells were transfected with miR-19a and miR-19b or control mimics,after transfection,SH-SY5Y cells were treated with Al-malt(0.25 mM)for 72h.Overexpression of miR-19a and miR-19b significantly improved the viability of SH-SY5Y cells treated with Al-malt.Flow cytometry analysis further revealed that compared with Al-malt treatment group,the percentages of apoptotic cells were reduced in Al-malt and transfected with miR-19 mimics groups.Western blotting also showed that the activation of caspase-9 and caspase-3 triggered by Al-malt was suppressed with miR-19a and miR-19b mimics in the cells.In addition,ectopic expression of miR-19a ameliorated the spectrum of effects associated with Al-malt treatment in the expression of PTEN,p-AKT,p53,Bax,and Bcl-2.Similar results were also observed with miR-19b mimic.Together,these data revealed the important role of miR-19 in Al-malt induced apoptosis in SH-SY5Y cells.4.Al-malt treatment increases Al contents in serum and brain tissues of ratsSprague Dawley rats were exposed to Al-malt(20,100,and 200 mg/L Al)for 12 weeks.There was no difference in terms of body weight with Al-malt treatment.Water consumption was slightly decreased in rats treated with 200 mg/L Al,and similar change was observed in food consumption.The average daily Al intakes for rats in the groups of 20,100,and 200 mg/L Al were 1.73,8.49,and 15.77 mg/kg body weight,respectively.Al-malt exposure increased Al concentrations both in serum and brain tissues in a dose-dependent manner.These data suggested the bioavailibity of Al from Al-malt complex and the entry of Al into the brain tissues.5.Al-malt induces apoptosis in brain tissue of ratsSprague Dawley rats were exposed to Al-malt for 12 weeks and the rat brain tissues were isolated and used for the analyses of Western blotting and TUNEL assays.The expression levels of cleaved-caspase-9 and cleaved-caspase-3 were up-regulated in rat brain with Al-malt treatment.Fluorometric TUNEL assay revealed the intensity of green fluorescence in rat hippocampus tissues were dose-dependently increased in Al-malt treated groups with regard to the control group,indicating Al-malt triggered apoptosis.These results demonstrated the induction of neural cell apoptosis in rats by Al-malt.6.Al-malt reduces miR-19 expression and alters PTEN/AKT/p53 pathway in rat brain tissuesSprague Dawley rats were exposed to Al-malt for 12 weeks.The expression levels of both miR-19a and miR-19b in rat brain tissues were significantly reduced in Al-malt treated groups compared with the control group.Western blotting analyses indicated that,in agreement with miR-19 down-regulation,Al-malt treatment resulted in upregulated expression of PTEN,decreased p-AKT,increased p53 and Bax,and decreased Bcl-2.Moreover,immunohistochemistry analysis illustrated that the expression of PTEN in hippocampus of rats treated with Al-malt was significantly increased when compared with the control group.These data suggested that consistent with in vitro study,Al-malt reduced miR-19 expression and altered miR-19-targeted PTEN/AKT/p53 pathway in rat brain tissues.Conclusions:In summary,the present study revealed for the first time that miR-19 modulation is critically involved in Al-induced neural cell apoptosis.Findings from this study could provide new insight into the molecular mechanisms of aluminum-induced neural cell apoptosis.Part ?:Folic Acid Protected Neural Cells against Aluminum-maltolate-induced Apoptosis by Preventing miR-19 DownregulationObjectives:by using in vitro Al-malt and/or folic acid treatment model,the present study aimed to investigate the neural protection effect of folic acid on Al-malt-induced neural cell apoptosis and the role of miR-19 on neuroprotective function of folic acid.Methods:Folic acid(1,10 and 100 ?mol/L)treated human SH-SY5Y cells for 72h and cell viability was determined by MTT assay.Treatment of SH-SY5Y cells with Al-malt in combination or not with folic acid for 72h;MTT assay was used to detect cell viability;real-time PCR was used to determine the expression levels of miR-19a and miR-19b;Western blotting examination for the protein levels of caspases family,Bcl-2 family and PTEN/AKT/p53 pathway.The SH-SY5Y cells were transfected with miR-19 inhibitors for 72h;the effect of miR-19 inhibitors on cell viability was analyzed by MTT assay;the effect of miR-19 inhibitors on the protein levels of caspases family,Bcl-2 family and PTEN/AKT/p53 pathway were examined by Western blotting.SH-SY5Y cells were treated with Al-malt and/or folic acid in the presence or absence of miR-19 inhibitors and cell viability was examined by MTT assay;the levels of caspases family,Bcl-2 family and PTEN/AKT/p53 pathway were examined by Western blotting.Results1.Folic acid preventes Al-malt-induced apoptosis of SH-SY5Y cellsSH-SY5Y cells were exposed to folic acid for 72 h.MTT assay showed that folic acid had no significant effect on cell viability of SH-SY5Y cells at concentrations up to 100 ?mol/L.Treatment of SH-SY5Y cells with Al-malt in combination with folic acid for 72h effectively prevented Al-malt-induced decrease of cell viability.Al-malt treatment resulted in decreased Bcl-2 and increased Bax,along with increased levels of cleaved-caspase-9 and cleaved-caspase-3;these alterations were reversed by the combination treatment of Al-malt and folic acid when compared with Al-malt alone treatment.These data suggested the protective effect of folic acid against Al-malt-induced apoptosis of SH-SY5Y cells.2.Folic acid attenuates Al-malt-induced downregulation of miR-19a/19b in SH-SY5Y cellsAl-malt treatment significantly decreased the expression levels of both miR-19a and miR-19b in SH-SY5Y cells,while the reduction in miR-19 expression induced by Al-malt was significantly attenuated by folic acid.Moreover,Western blotting indicated that,consistent with the reduction of miR-19,Al-malt upregulated the expression of PTEN,decreased the level of p-AKT,and increased p53 expression.Furthermore,treatment of folic acid attenuated Al-malt triggered alterations in the expression of PTEN,p-AKT and p53;and this effect was in line with folic acid ameliorated Al-malt-induced downregulation of miR-19a and miR-19b in SH-SY5Y cells.3.miR-19 inhibition induces apoptosis of SH-SY5Y cellsTransfection of miR-19 inhibitors for 72h significantly reduced the expression levels of miR-19a and miR-19b.Inhibition of miR-19a and miR-19b significantly decreased the viability of SH-SY5Y cells.Meanwhile,miR-19a inhibitor and miR-19b inhibitor resulted in increased PTEN,p53,Bax,and decreased Bcl-2,as well as increased cleaved-caspase-9 and cleaved-caspase-3 in SH-SY5Y cells.Thus,these results suggested the induction of apoptosis by miR-19 suppression in SH-SY5Y cells.4.Folic acid ameliorates Al-malt-induced apoptosis through miR-19/PTEN/AKT/p53 pathwaySH-SY5Y cells were treated with Al-malt and/or folic acid in the presence or absence of miR-19a/19b inhibitor.We again showed that Al-malt-decreased viability of SH-SY5Y cells was significantly ameliorated by folic acid;however,this protective effect of folic acid was significantly weakened by miR-19a inhibitor or miR-19b inhibitor.Moreover,the protective effects of folic acid against Al-malt-altered expression of miR-19-related apoptotic proteins,including PTEN,p-AKT,p53,caspase-9 and caspase-3,were reduced by miR-19 inhibitors.Therefore,these data suggested that folic acid suppressed Al-malt-induced apoptosis by preventing miR-19 downregulation in human SH-SY5Y cellsConclusions:In summary,the present study revealed for the first time that folic acid prevented Al-malt-induced neuronal apoptosis through modulation of miR-19/PTEN/AKT/p53 axis.Findings from this study could shed new light on the molecular mechanisms by which folic acid exerts its neuroprotective function.