| Background Alzheimer’s disease(AD)is a common central nervous system disease,which characterized by a nervous disorder,progressive debilitating memory impairment,behavioral damage and cognitive dysfunction.Course of AD is chronic,with the extension of course,patient condition aggravating gradually,until loss of independence.Its main pathological features include senile plague,neuronal fibrous tangles(NFT)and massive neuronal cells death with uncontrolled microglial cell activatives.Even though the exact etiologies of AD remain unknown,convincing evidence shows neuroinflammation and neuronal apoptosis are causatively implicated in the pathogenesis and development of AD,all of which related to Aβ peptides.micro RNA(mi RNA)are a class of endogenous and evolutionarily non-coding RNA,20-24 nucleotides in length which cause m RNA degradation or translation inhibition by binding to the 3’-untranslated region(UTR).mi R-137 located on human chromosome 1p22,and has been shown to be enriched in the central nervous system such as hippocampal dentate gyrus and molecular layer,which associated with the plasticity of brain and the generation of new neurons.mi RNA play an important role in many diseases,especially significant in cancer and diseases of the nervous system.It can regulate mature neurons induced the occurrence of schizophrenia,and have been observed to participate in the onset of AD.TNFAIP1 was originally identified gene whose expression can be induced by TNF-α and interleukin-6(IL-6)in umbilical vein endothelial cells.As a member of PDIP1 family,it contains BTB/POZ and PCNA structural domain.TNFAIP1 protein can be distributed in the cytoplasm or nuclei,and the level of expression exist differences in different organizations,which is necessary to maintain normal cell growth.TNFAIP1 protein structure is highly conservative,and mainly participated in DNA synthesis,DNA repair,or cell apoptosis.It plays an important role in the development of AD,while we haven’t know more about the relationship between mi R-137 and TNFAIP1 with the development of AD.Objective We examined expression of mi R-137 and TNFAIP1 in Cortical neurons and Neur2a(N2a),and forecasted target genes of mi R-137,meanwhile we analyzed the effects of mi R-137 overexpression in neurotoxicity which induced by Aβ,and the mechanism of mi R-137 targeted combination TNFAIP1 inhibited neurotoxicity induced by Aβ.Aimed at understanding the mechanisms of mi R-137 in neurotoxicity induced by Aβ,while providing a reliable basis for clinical diagnostics and targeted therapy of AD.Method(1)We designed and synthesized primers of mi R-137,TNFAIP1,U6 and GAPDH,cortical neurons and N2 a cells were treated with indicated concentration of Aβ25-35(0,5,10 and 20μM).We verified the expression of mi R-137 and TNFAIP1 m RNA after culture for 24 h by fluorescence quantitative PCR method.Then using the westernblot assayed the expression of TNFAIP1 protein.(2)We use bioinformatics software screening target genes of mi R-137’s,and constructed pmir GLO reporter vector with wide type and mutate TNFAIP1 3’UTR,the two were transfected into 239 TN cells with mi R-137,and luciferase reporter system were used for detecting the expression of luciferase cells.We used the q RT-PCR and westernblot to detect the expression of TNFAIP1 m RNA and protein after transfected with mi R-137,anti-mi R-137,mi R controls,anti-mi R controls.(3)N2a cells were treated with indicated concentration of Aβ25-35(0,5,10 and 20μM),and then mi R-control and mi R-137 were introduced into N2 a cells,in order to bulid mi R-137 overexpression cells lines.MTT assays were performed to analyze the cell viability,FCM and Caspase-3 activity detection was used to assess cell apoptosis,ELISA and westernblot assays were performed to analyze NF-kB viability and expression of protein.(4)Si-control and si-TNFAIP1 were introduced into N2 a cells,westernblot assays were performed to analyze expression of TNFAIP1 protein.Then N2 a cells were treated with 20μM PBS and Aβ25-35 respectively,followed which si-control,si-TNFAIP1,mi R-control,mi R-137,mi R-137+pc DNA3.1 and mi R-137+pc DNA-TNFAIP1 were transfected into N2 a cells treated with Aβ25-35.MTT assays were performed to analyze the cell viability,FCM and Caspase-3 activity detection was used to assess cell apoptosis,ELISA were performed to analyzeNF-kB viability.Results(1)Fluorescence quantitative PCR results showed that mi R-137 expression was significantly decreased by Aβ25-35 in a dose-dependent manner in mouse cortical neurons and N2 a cells(P<0.05).Additionally,Aβ25-35 treatment markedly increased TNFAIP1 expressions at both m RNA and protein levels in a dose-dependent manner in mouse cortical neurons and N2 a cells(P<0.05),as demonstrated by q RT-PCR and western blot.(2)Bioinformatics prediction results show that TNFAIP1 is a target gene of mi R-137..Luciferase reporter assay demonstrated that introduction of mi R-137 dramatically suppressed the luciferase activity of the luciferase reporter plasmids containing the wild-type 3’UTR of TNFAIP1 as compared with mi Rcontrol group(P<0.05),but not that of the luciferase reporter plasmids containing the mutated fragment in the 3’UTR of TNFAIP1.Fluorescence quantitative PCR and westernblot results showed that mi R-137 significantly reduced the expression levels of TNFAIP1 m RNA and protein in cortical neurons and N2 a cells,while anti-mi R-137 had the opposite effect(P<0.05).(3)MTT results exhibited that restoration of mi R-137 expression in N2 a cells significantly reduced Aβ25-35 induced cell viability in a dose-dependent manner relative to mi R-control-treated cells(P<0.05).Flow cytometry analysis and Caspase-3 activity were conducted to determine apoptosis,the research shows that the apoptotic rates and Caspase-3 activity of the two groups were markedly increased after treatment with different doses of Aβ25-35,meanwhile the apoptotic rates and Caspase-3 activity of mi R-137-treated cells is lower than control group(P<0.05).ELISA and westernblot assay demonstrated NF-kB activity of the two groups were markedly increased after treatment with different doses of Aβ25-35,while NF-kB activity of mi R-137-treated cells is lower than control group(P<0.05).(4)Western blot verifying the transfection efficiency showed that si-TNFAIP1 led to a significant decrease of TNFAIP1 level while pc DNA-TNFAIP1 resulted in a marked increase of TNFAIP1 level.MTT assay demonstrated that Aβ25-35 treatment remarkably suppressed cell viability in N2 a cells,TNFAIP1 knockdown or exogenous expression of mi R-137 significantly relieved Aβ25-35-induced cell viability suppression(P<0.05),while the cell viability was partially reversed by TNFAIP1 overexpression.Flow cytometry analysis and Caspase-3 activity were conducted to assay apoptosis,and the result shows Aβ25-35 treatment remarkably increased apoptotic rates and Caspase-3 activity in N2 a cells,TNFAIP1 knockdown or exogenous expression of mi R-137 significantly relieved Aβ25-35-induced cell apoptotic rates and Caspase-3 activity improvment(P<0.05),while forced expression of TNFAIP1 markedly abolished these effects(P<0.05).ELISA assay revealed that 20 μM Aβ25-35 treatment activated NF-k B in N2 a cells,whereas TNFAIP1 knockdown or mi R-137 overexpression blocked Aβ25-35-induced activation of NF-k B(P<0.05),which was partially recuperated due to TNFAIP1 overexpression(P<0.05).Conclusion Aβ25-35 treatment led to an obvious decrease of mi R-137 expression and a marked increase of TNFAIP1 expression.TNFAIP1 was a direct target of mi R-137 in N2 a cells.Mi R-137 overexpression contributed to Aβ25-35-induced cell toxicity and apoptosis through activation of NF-k B pathway by targeting TNFAIP1. |
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