The Role And Mechanism Of LncRNA NEAT1 On MPP~+-induced Autophagy Axis In SH-SY5Y Cells Targeted By MiR132-3p

Parkinson’s disease(PD)is a neurodegenerative disease characterized by bradykinesia and resting tremor.The specific pathogenesis is unclear,which may be related to multiple factors such as gene mutation,autophagy dysfunction,α-synuclein(α-syn)deposition,and mitochondrial dysfunction.Autophagy dysfunction can cause the death of dopamine neurons,which is considered to be one of the possible pathogenesis of PD.Studies have found that improving autophagy dysfunction plays an important role in the neuroprotection of PD and may have therapeutic potential for PD.Long non-codingRNAs(lncRNAs)is a transcript greater than 200 nt in eukaryotic cells,which can regulate gene expression through a variety of different mechanisms.Studies have found that lncRNAs participates in the autophagy regulatory network and mediates the regulation of autophagy-related genes.Another report shown that in the PD model constructed with SH-SY5Y cells and rats,LncRNA NEAT1 is closely related to the activation of the autophagy regulatory network.Phosphatases and tensin homologs(PTEN)are key negative regulators of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling cascade,which can regulate the PI3K/AKT pathway through dephosphorylation,which can be said to be the most important pro-survival pathway in neurons.PI3K/AKT is a classic autophagy signaling pathway.Various factors can activate PI3K to induce the production of3,4,5-triphosphatidylinositol(PIP3)and regulte AKT expression.MicroRNA132 is a type of small non-codingRNAs(miRNAs),which is rich in neural tissues,and is involved in regulating the growth of dopamine neurons,tumor formation,autophagy and apoptosis.In a study of glioma,it was found that LncRNA NEAT1 regulates the development of glioma through negative regulation of miR132.In addition,a study on Alzheimer’s disease(AD)used primary neurons and PC12 cells to prove that miR132/212 controls cell survival by directly regulating PTEN,FOXO3a and P300.These are the key components of the AKT signaling pathway.Therefore,we speculate that in MPP~+(1-methyl-4-phenylpyridinium)-induced SH-SY5Y Parkinson’s disease cell model,LncRNA NEAT1 may be involved in the regulation of cell autophagy.And this process curb nerve damage,play a neuroprotective effect.This process may be related to the activation of miR132 and PTEN/PI3K/AKT signaling pathway.In this experiment,SH-SY5Y cells treated with MPP~+were used to construct a PD cell model,and western blotting(WB)and real-time reverse transcription PCR(RT-PCR)were used to detect the expression of LncRNA NEAT1,miR132-3p,PTEN/PI3K/AKT,a-syn and autophagy-related proteins among LC3B,Beclin1 and p62.we further used lentivirus to interfere with the relative expression of LncRNA NEAT1 to investigate whether LncRNA NEAT1 regulates the autophagy process of MPP~+-induced SH-SY5Y Parkinson’s disease cell model through miR132 and PTEN/AKT/PI3K signaling pathways.Result:1.RT-PCR was used to detect the relative expression level of NEAT1 in different concentrations of MPP~+(0,0.25 m M,0.5 m M,0.75 m M,1 m M,and 1.25 m M)and different time points(0,4 h,8 h,16 h,24 h,36 h)to treat SH-SY5Y cells.Compared with the control group,the expression of LncRNA NEAT1 was significantly increased when the MPP~+concentration was 1m M,which was statistically significant(P<0.001).Compared with the control group,the expression was statistically significant at 24h(P<0.001).2.The experiment found that compared with the control group,the relative expression level of miR132-3p in MPP~+-induced SH-SY5Y Parkinson’s disease cell model decreased(P<0.001).3.The experiment found that compared with the control group,the expression levels of P-AKT and P-PI3K proteins in the MPP~+group decreased(P<0.05,P<0.001),and the expression of PTEN protein increased(P<0.01).Therefore,MPP~+treatment can down-regulate the expression of P-AKT and P-PI3K in SH-SY5Y and up-regulate the expression of PTEN.4.The experiment found that compared with the control group,the expression of a-syn and p62 protein in the MPP~+group increased(P<0.001,P<0.001),and the expression of LC3B and Beclin1 protein decreased(P<0.01,P<0.01).RT-PCR show that the relative expression of a-syn and p62 mRNA in the MPP~+group was significantly increased(P<0.001,P<0.001),while the relative expression of LC3B and Beclin1mRNA was significantly reduced(P<0.001,P<0.001).Therefore,the autophagy process is inhibited andα-syn is up-regulated in MPP~+-induced SH-SY5Y Parkinson’s disease cell model.5.RT-PCR was used to determine the virus knockout rate,and the 40MOI target virus and 40MOI blank were used for virus transfection.The virus knockdown rate was about 80%.6.The experiment found that compared with LncRNA NEAT1-NC,the relative expression of miR132-3p in the LncRNA NEAT1-NC+MPP~+group and ShRNA-LncRNA NEAT1+MPP~+group was significantly reduced(P<0.001,P<0.001),indicating that MPP~+treatment can down-regulate the expression of miR132-3p in SH-SY5Y cell.Compared with the LncRNA NEAT1-NC+MPP~+group,the relative expression of miR132-3p in the ShRNA-LncRNA NEAT1+MPP~+group increased(P<0.001).It shown that LncRNA NEAT1 can regulate the expression of miR132-3p in MPP~+-induced SH-SY5Y Parkinson’s disease cell model.7.The experiment found that compared with LncRNA NEAT1-NC,the expression of PTEN protein in the LncRNA NEAT1-NC+MPP~+and ShRNA-LncRNA NEAT1+MPP~+groups was significantly increased(P<0.001,P<0.001),while P-AKT and P-PI3K protein expression decreased(P<0.001,P<0.01;P<0.001,P<0.01).Compared with LncRNA NEAT1-NC+MPP~+group,the expression of PTEN protein in ShRNA-LncRNA NEAT1+MPP~+group was significantly reduced(P<0.001),and the expression of P-AKT and P-PI3K proteins increased(P<0.05;P<0.05).It shows that interference with the expression of LncRNA NEAT1 can regulate the expression of PTEN/AKT/PI3K in the classical pathway of autophagy.8.WB shown that compared with LncRNA NEAT1-NC,the expression of p62 andα-syn protein in the LncRNA NEAT1-NC+MPP~+group and ShRNA-LncRNA NEAT1+MPP~+group was significantly increased(P<0.001,P<0.001;P<0.001,P<0.05),the expression of Beclin1 and LC3Ⅱprotein decreased(P<0.001,P<0.001;P<0.001,P<0.05).Compared with the LncRNA NEAT1-NC+MPP~+group,the expression of p62 andα-syn protein in the ShRNA-LncRNA NEAT1+MPP~+group was significantly reduced(P<0.01,P<0.001),and the expression of Beclin1 and LC3Ⅱprotein increased(P<0.05;P<0.05).RT-PCR proved that compared with LncRNA NEAT1-NC,the expression of p62 andα-syn mRNA in the LncRNA NEAT1-NC+MPP~+group and the ShRNA-LncRNA NEAT1+MPP~+group was significantly increased(P<0.001,P<0.001;P<0.001,P<0.001),the expression of Beclin1 and LC3ⅡmRNA decreased(P<0.001,P<0.001;P<0.001,P<0.01).Compared with the LncRNA NEAT1-NC+MPP~+group,the expression of p62 andα-syn mRNA in the ShRNA-LncRNA NEAT1+MPP~+group was significantly reduced(P<0.001,P<0.001),and the expression of Beclin1 and LC3ⅡmRNA increased(P<0.001;P<0.05).The results shown that interference with the expression of LncRNA NEAT1 can regulate the autophagy process in MPP~+-induced SH-SY5Y Parkinson’s disease cell model and play a vital role in resisting nerve damage.9.The experiment found that compared with NC,miR132-3p down-regulate the expression of h-NEAT1-3UTR-WT.In summary,the above results show that LncRNA NEAT1 can reduce nerve cell damage by inducing the autophagy process of SH-SY5Y Parkinson’s disease cell model.miR132-3p participates in the neuroprotective effect of LncRNA NEAT1.This process may be related to the activation of the PTEN/AKT/PI3K signaling pathway.