| Control of seizures is always a key link in the treatment of epilepsy,but neuronal damage after seizures,glial cell proliferation,mossy fiber germination(MFS),and other aggravated abnormal network formation cannot be ignored.Neuronal injury is the result of seizures,the cause of epilepsy recurrence,and refractory epilepsy formation.Although scholars all over the world have finish much work on the aspect of epilepsy frequent seizures and epilepsy after the neuronal damage,they have certain understanding on the damage mechanism but still no specific mechanism nor the method on how to deal with it.Therefore,how to reduce neuronal damage after seizures,prevention and treatment of epilepsy,and control of epilepsy are equally important.On the premise of investigating the relationship between miR-182,miR-124 and APLN,this study is to explore the mechanism of APLN in epilepsy and neuronal injury after epileptic seizure,so as to lay the theoretical foundation for alleviating neuronal injury after epilepsy attack and preventing epilepsy Lay.Part one: Mi R-182 and miR-124 target gene prediction and preliminary identification Objectives:1.The target genes of miR-124 and miR-182 were predicted according to the analysis of miRNAs target gene bioinformatics database.2.To study the differential expression of miRNA-182/124 in the blood of patients with epilepsy and normal control,and to analyze the correlation between miR-182/124 and APLN in patients with epilepsy.Methods:1.The target gene of miRNAs could be predicted by bioinformatics software,and the results were analyzed by Target,NCBI,miRbase,micrriorna.org and other bioinformatics software.2.Patients with epilepsy and healthy control group of 30 cases were randmisly selected,5ml peripheral blood was collected for patients or healthy control.Fluorescence quantitative PCR detection of miRNA-182/124,APLN,in patients with epilepsy and healthy control in the peripheral blood of the expression changes.Results:1.According to the analysis of miRNAs target gene bioinformatics database,miR-124 and miR-182 were selected to predict the target gene of APLN.2.With the methods of real-time fluorescence quantitative detection,we found that the expression of miR-124 in epileptic patients was significantly lower than that of normal group(P < 0.05);the expression trend also miR-182 in the epilepsy group(P < 0.05),the amount is lower than the control group expression in epileptic patients;however,the APLN gene in epileptic patients was significantly higher than that in normal group,and the difference was extremely significant(P < 0.01).The general trend was that the expression levels of miR-124 and miR-182 in the epilepsy group and the normal group were all negatively correlated with the expression of APLN gene.Conclusions:1.According to the analysis of miRNAs target gene biological information database,it is predicted that APLN may be the target gene of miR-124 and miR-182.2.The application of q PCR technology,according to the control group in the blood of healthy people and to detect the expression results of miR-182,miR-124 and APLN in patients with epilepsy,showed that the high expression of miR-124 in blood and miR-182 in patients with epilepsy may have inhibitory effect on the expression of APLN.Part two: the verification of miR-182 and miR-124 with the target genes of APLN Objectives:To construct miR-128/124 expression vector and double luciferase reporter gene vector,and to verify the relationship between miR182/124,APLN,and the effect of miR182/124 on apoptosis of neurons.Methods:1.The expression vector and interference vector of miR-124-3p and miR-182-5p were designed by miRBase database.UCSC database and NCBI database were used to design of double luciferase reporter gene vector2.The luciferase activity of miR182/124-and target gene APLN were detected by double luciferase reporter gene.3.QPCR and Western blot technology were used to verify the relationship between miR182/124 and APLN protein expression.4.The effect of miR182/124 on the apoptosis of neurons was detected by flow cytometry.The expression of Bax,Casepase-3 and Bcl-2 were detected by Western blot Results:1.The expression of PBI-CMV3-APLN overexpression vector,no-load,miR-182 mimics and APLN sh RNA vectors were transfected into rat hippocampal neuron epilepsy cells respectively.The vectors could be efficiently expressed in rat neurons,Proof of success.After transfection of for 48 h,the apoptosis of hippocampal neurons was detected by flow cytometry.The results showed that the transfection of PBI-CMV3-APLN expression vector,miR-182 mimics and APLN sh RNA,no-load(silencing APLN carrier)apoptosis levels were 4%,15.55%,17.90%,18.89%,the results show that APLN could significantly.inhibit the epileptic cell apoptosis.2.The expression of APLN gene in hippocampus was detected by q-PCR,and the expression of APLN overexpression group was detected by q-PCR.The expression of APLN gene in hippocampus was detected by q-PCR.The APLN expression in the APLN silence group and the miR-182 overexpression group was lower than that in the untreated epilepsy rats,but the APLN silencing group and the miR-182 group were significantly higher than those in the untreated group There was no significant difference in the expression group.3.The epilepsy of hippocampal neuron cells were transfected with PBI-CMV3-APLN expression vector,miR-182,mimics and APLN sh RNA load(APLN silencing)carrier,each carrier can be efficiently expressed in neuronal cells in rats.Western blot was carried out at 48 h.The results showed that: m Glu R1 protein overexpression group-PBI-CMV3-APLN < sh NC < miR-182 < mimics APLN sh RNA(p<0.05)(10-A,10-B);p-AKT-PBI-CMV3-APLN overexpression group > sh NC > mimics > miR-182 APLN sh RNA(p<0.05)(10-A,10-C);Bax-PBI-CMV3-APLN overexpression group miR-182 < sh NC < mimics APLN sh RNA(p<0.05)(10-A,10-D);Caspase3-PBI-CMV3-APLN overexpression group miR-182 < sh NC < mimics < APLN sh RNA(p<0.05)(10-A,10-E);Bcl-2-PBI-CMV3-APLN overexpression group > sh NC > mimics > miR-182 APLN sh RNA(p<0.05)(10-A,10-F).4.Western blotting tissue in hippocampus of epileptic rats to detect cell survival promoting factor Bcl-2 protein overexpression of APLN genome,epileptic rats control over expression of miR-182 in mimics group,epileptic rats showed reduced expression of APLN gene silence overall trend group,and epileptic rats miR-182 mimics compared with group and epileptic rats to silence APLN genome,the expression of Bcl-2 protein had no obvious difference.However,the promotion of apoptosis gene Bax,apoptosis factor Caspase3 and metabotropic glutamate receptor 1(m Glu R1)in the above vector sequence showed a general upward trend.Conclusions:1.Double luciferase reporter gene test results showed that miR-182 mimics could be combined with the APLN gene,indicating that APLN and miR-182 mimics target relationship existed.Mi R-124 was also able to bind to the wild-type target gene Apelin,but may not be binding.2.Mi R-182 could be used to regulate the expression of APLN gene at m RNA level and protein level by real-time fluorescent quantitative detection and Western blot analysis,but miR-124 can not directly down regulate the expression level of APLN gene.3,Mi R-182 can promote the apoptosis of neurons,and can lead to the down-regulation of Bcl2 expression of Bax and Caspase-3 expression;the same miR-124 can promote cell apoptosis,but may be regulated by other media factors.Part three: The modulatory mechanisms of APLN on neuronal protection after epilepsy attack in vivo and in vitro.Objectives:1.To establish neuronal cell model and rat model of epilepsy.2.To investigate the effect of APLN on epileptic disease.3.To investigate the regulatory mechanism of miR-182-APLN after epilepsy attack.Methods:1.Magnesium free cells of epilepsy hippocampal neuron cells model was further microscopy,cells cultured;cells to epileptic rat hippocampal neurons were transfected with PBI-CMV3-APLN expression vector,miR-182,mimics and APLN sh RNA load.The apoptosis rate of hippocampal neurons in each vector was detected by flow cytometry.2.To establish a rat model of epilepsy induced by PTZ,The APLN overexpression plasmid,APLN silencing plasmid,miR-182 mimetic plasmid and control plasmid were injected into the rat mode.Nine rats in each group,each group injected corresponding plasmid.The m RNA expression of hippocampus was detected by q-PCR..3.The expression level of m RNA in hippocampus of epileptic rats in each group was detected by real-time fluorescence PCR.The effects of APLN on glutamate receptor 1(m Glu R1)and the regulation of apoptosis-related factors Bax,Caspase-3,Bcl-2 and p-Akt were detected by western blot in the nerve cell model and rat model of epilepsy.Result:1.The expression of PBI-CMV3-APLN overexpression vector,no-load,miR-182 mimics and APLN sh RNA vectors were transfected into rat hippocampal neuron epilepsy cells respectively.The vectors could be efficiently expressed in rat neurons,Proof of success.After transfection of for 48 h,the apoptosis of hippocampal neurons was detected by flow cytometry.The results show that the transfection of PBI-CMV3-APLN expression vector,miR-182 mimics and APLN sh RNA,no-load(silencing APLN carrier)apoptosis levels were 4%,15.55%,17.90%,18.89%,the results show that APLN can significantly.inhibit the epileptic cell apoptosis.2、The expression of APLN gene in hippocampus was detected by q-PCR,and the expression of APLN overexpression group was detected by q-PCR.The expression of APLN gene in hippocampus was detected by q-PCR.The expression of APLN gene in hippocampus was detected by q-PCR.The APLN expression in the APLN silence group and the miR-182 overexpression group was lower than that in the untreated epilepsy rats,but the APLN silencing group and the miR-182 group were significantly higher than those in the untreated group There was no significant difference in the expression group.3.The epilepsy of hippocampal neuron cells were transfected with PBI-CMV3-APLN expression vector,miR-182,mimics and APLN sh RNA load(APLN silencing)carrier,each carrier can be efficiently expressed in neuronal cells in rats.48 h,Western blot.The results showed that: m Glu R1 protein overexpression group-PBI-CMV3-APLN < sh NC < miR-182 < mimics APLN sh RNA(p<0.05)(10-A,10-B);p-AKT-PBI-CMV3-APLN overexpression group > sh NC > mimics > miR-182 APLN sh RNA(p<0.05)(10-A,10-C);Bax-PBI-CMV3-APLN overexpression group miR-182 < sh NC < mimics APLN sh RNA(p<0.05)(10-A,10-D);Caspase3-PBI-CMV3-APLN overexpression group miR-182 < sh NC < mimics < APLN sh RNA(p<0.05)(10-A,10-E);Bcl-2-PBI-CMV3-APLN overexpression group > sh NC > mimics > miR-182 APLN sh RNA(p<0.05)(10-A,10-F).4.Western blotting tissue in hippocampus of epileptic rats to detect cell survival promoting factor Bcl-2 protein overexpression of APLN genome,epileptic rats control over expression of miR-182 in mimics group,epileptic rats showed reduced expression of APLN gene silence overall trend group,and epileptic rats miR-182 mimics compared with group and epileptic rats to silence APLN genome,the expression of Bcl-2 protein had no obvious difference.However,the promotion of apoptosis gene Bax,apoptosis factor Caspase3 and metabotropic glutamate receptor 1(m Glu R1)in the above vector sequence showed a general upward trend.Conclusion:1.APLN inhibition of m Glu R1 can reduce the nerve cell damage caused by nerve cell toxicity,but also reduce the nerve excitability to relieve seizures through observing from our epilepsy model experiments both in vitro and in vivo.APLN may play an anti-apoptotic neuroprotective role through PI3K/p-AKT pathway.APLN can also down-regulates the expression of Bax and Caspase-3,up-regulates the expression of Bcl-2 which inhibits the neuronal damage caused by apoptosis and protects the nerve injury after epileptic seizures.2.It is suggested that miR-182 could deteriorate neuron injury after epilepsy through down-regulating APLN expression;whereas inhibition of miR-182 could up-regulate APLN expression so as to protect neuron damage after epilepsy attack.Accordingly,miR-182/APLN may be a promising target for epilepsy treatment. |
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