microRNA-204调控惊厥性脑损伤的机制研究

发布时间：2018-04-28 02:45

本文选题：miRNA + miR-204　；参考：《中南大学》2014年博士论文

【摘要】：惊厥是婴幼儿期最常见的急危重症,惊厥持续发作可产生严重神经系统后遗症,影响患儿智力发育和身心健康,甚至危及患儿生命。小儿惊厥的发病率很高,约5-6%的小儿曾有过一次或多次惊厥发作史,早产儿甚至高达22.7%～25%。若惊厥发作频繁或持续时间较长,约有15%～30%患儿将最终转变为癫痫。发育期惊厥能够抑制大脑的生长发育,改变神经环路,提高神经元兴奋性,加重大脑损伤,导致视觉能、空间能、学习记忆和情感障碍,若不及时干预,将严重危害到患儿的生命及远期生活质量,给家庭与社会带来沉重负担。就我国人口众多这一国情来说,所面临床的困难较其他国家更为严峻。因此,探索发育期惊厥的发生发展机制及开发新抗惊厥药物成了我们目前迫切需要解决的医学难题。本研究以发育期反复惊厥大鼠为模型,开展惊厥发病机制的相关研究。三氟乙醚吸入致惊是经典的发育期惊厥模型。在先前的实验中,本研究应用TaqMan MicroRNA Arrays对发育期反复惊厥后大鼠海马内niRNAs表达谱进行检测,在766种已知miRNA中筛选出惊厥后大鼠海马内具有表达差异的miRNA,结果发现,与正常对照组比较,发育期反复惊厥后大鼠海马内有20个已知miRNAs表达下调0.5倍以上,9个niRNAs表达明显上调2倍以上。其中miR-204在海马表达丰富,在反复惊厥后24h表达显著上调至正常组的7倍左右。因此,本实验将进一步观察miR-34b-5p、-204、-582-3p、-672在大鼠海马及大脑皮质间的差异性表达,同时选取miR-204做为研究对象,更深入探索其在惊厥性脑损伤中的作用及相关机制。第一部分反复惊厥性脑损伤后脑组织内特异性miRNAs表达的改变实验目的：探索发育期反复惊厥后大鼠海马及大脑皮质内特异性miRNAs的表达变化及可能机制。实验方法：1)21日龄SD大鼠70只,将实验仔鼠随机分为2大组,即对照组及惊厥组,每组又随机分为5个时间点,每时间点各6只大鼠。其中惊厥组通过三氟乙醚反复吸入(每天1次,连续6天)制作发育期反复惊厥动物模型,而对照组则经历相同步骤,但不吸入三氟乙醚,最终入组大鼠60只。各组大鼠分别于末次惊厥后2h、6h、24h、72h及7d断头取脑,分离海马及大脑皮质组织。2)分离组织后,提取各时间点海马及大脑皮质组织总RNA,随后进行miR-34b-5p、 miR-204、miR-582-3p、miR-672的逆转录,实时定量PCR检测两组织中四种miRNAs的表达。3)体外应用海人酸(100μmol/l)干预体外培养的PC12神经元样细胞,实时定量PCR检测干预后Oh及24h时细胞内miR-204的表达。4)应用公共靶基因预测软件starBase (http://starbase.sysu. edu.cn/starbasevl/clipSeq Intersection.php)对在脑组织中具有丰富表达的miR-204靶基因进行预测,进一步筛选出参与惊厥性脑损伤过程的靶基因。实验结果：海马及大脑皮质内miR-34b-5p及miR-204在末次惊厥后2h均开始表达上调(p0.05),6h时海马及皮质内miR-34b-5p的表达较2h时下调(p0.05),但仍明显高于正常对照组(p0.05),而miR-204仅在海马内表达较2h时下降,6h后皮质内的miR-204表达较2h升高。海马及大脑皮质内miR-34b-5p及miR-204的表达均在末次惊厥后24h均达顶峰,随后逐渐下降,72h恢复正常,7d时海马内miR-204的表达,大脑皮质内miR-34b-5p及miR-204的表达均较对照组显著下调(p0.05),而海马内的miR-34b-5p的表达则呈上调趋势。不同的是,末次惊厥后不同时间点miR-582-3p及miR-672在海马内的表达均呈下降趋势,而在大脑皮质内的表达均上调,在24h时达到高峰(p0.05),随后逐渐下降,至7d时达正常水平,与对照组相比无明显差异(p0.05)。不仅如此,我们还发现,反复惊厥后海马及大脑皮质内miR-34b-5p及miR-204的表达在不同时间点具有高度相关性,而miR-582-3p及miR-672在不同部位的相关性并不明显。因此可以推测,miRNAs在惊厥性脑损伤中的表达具有组织差异性。为了进一步探索特异性miRNA调控发育期惊厥性脑损伤的具体机制,本实验选取了在大鼠海马内具有丰富表达的miR-204作为研究目标。结果发现,miR-204在海人酸干预后的PC12神经元样细胞的表达较对照组显著上调(p0.05)。进而我们选用starBase对三种靶基因预测软件(Targetscan、PITA和PicTar)结果进行综合分析。为了降低假阳性率,选择同时出现在三种预测软件中的靶基因,并进一步筛选出参与了发育期惊厥性脑损伤过程的靶mRNAs。结果发现,在信息沉默因子1(SIRT1)的3'-UTR可能存在与miR-204的结合位点,而前者是一种NAD依赖性的去乙酰化酶,广泛参与了脑损伤后神经细胞凋亡及突触可塑性的调控过程。因此,我们推测SIRT1可能是miR-204调控发育期惊厥性脑损伤的靶位点之一。结论：1)大鼠海马及大脑皮质内niR-34b-5p、miR-204、miR-582-3p及miR-672的表达在发育期惊厥后均发生了改变,且具有组织相关性,从体外水平验证miRNAs参与了惊厥性脑损伤过程。2)SIRT1可能是miR-204调控惊厥性脑损伤过程的靶基因之一。第二部分惊厥性脑损伤后大鼠神经元凋亡及突触可塑性的变化实验目的：从体内及体外水平探索惊厥后神经元凋亡及神经元突触可塑性的改变,以及SIRT1参与惊厥后性脑损伤的调控机制。实验步骤：1)如前述,制作发育期反复惊厥大鼠模型,在末次惊厥后2h、6h、24h、72h及7d断头取脑,分离海马组织；2)提取各时间点对照组及惊厥组的总蛋白及RNA,应用Western blot检测各实验组大鼠海马GAP-43及p53的蛋白表达,qRT-PCR及Western blot检测各实验组大鼠SIRT1mRNA及蛋白含量；3)应用TUNEL法检测惊厥后2h、6h、24h、72h及7d大鼠海马神经元的凋亡；4)体外培养PC12神经元样细胞,应用海人酸(100μmo1/l)干预模拟体外惊厥模型,24小时后收集细胞,应用Western blot分别检测干预后Oh及24h细胞内GAP-43、SIRT1及p53的蛋白含量；5)应用TUNEL法检测不同浓度(0、50、100、250μmol/l)海人酸干预后PC12神经元样细胞的凋亡。实验结果：1)与对照组相比,海马内SIRT1的蛋白水平在惊厥后2h明显下降(p0.05),至6h时进一步下调(p0.05),72h时达最低值(p0.05),7d时逐渐恢复正常(p0.05)；与之相反,惊厥后2h海马内p53的表达较对照组明显上调(p0.05),至6h时进一步上升(p0.05),24h达顶峰(p0.05),72h时维持高水平表达,与24h比较并无统计学意义(p0.05),至7d时逐渐恢复正常,但仍显著高于正常组(p0.05)；而GAP-43在末次惊厥后6h表达开始下调(p0.05),至72h时达最低值(p0.05),7d时逐渐恢复正常,而在惊厥后2h与对照组相比并无显著差异(p0.05)。2)惊厥后2h,海马内神经元凋亡较对照组显著增多,6h进一步增加,至72h时达顶峰,7d时逐渐下降,各实验组与对照组比较差异均具有统计学意义(p0.05)。3)海人酸干预24h后PC12神经元样细胞内SIRT1、GAP-43的蛋白水平均较Oh时显著下调,而p53的蛋白水平则显著升高(p0.05)。4)不同浓度海人酸干预后PC12神经元样细胞均发生了凋亡,且呈剂量依赖性,在250μmol/l时凋亡最显著,各剂量组之间差异均具有统计学意义(p0.05)。结论：本实验从体内及体外实验证实,惊厥性脑损伤在诱发神经元凋亡的同时,还抑制了神经元突触的生长与延伸。不仅如此,惊厥性脑损伤还显著抑制了SIRT1的表达,其机制可能与其下游因子p53发生去乙酰化有关,通过上调凋亡相关因子p53的表达,加速细胞的凋亡。第三部分miR-204通过SIRT1调控发育期惊厥性脑损伤的机制实验目的：从生物学水平验证SIRT1是miR-204参与惊厥性脑损伤调控的靶点以及其相关机制。实验步骤：1)体外化学合成miR-204mimics、inhibitors及相应的阴性对照；2)如前所述,原代培养PC12神经元样细胞,将细胞随机分为5组：即正常对照组,miR-204mimics干预组、miR-204mimics阴性对照组、miR-204inhibitors干预组及miR-204inhibitors阴性对照组；3)应用瞬时转染体外制作miR-204过表达及低表达模型,实时定量PCR检测转染成功率；4)分别应用Western blot及实时定量PCR技术检测各实验组内SIRT1蛋白及mRNA表达的变化；5)制作miR-204过表达及低表达模型,应用海人酸干预各组PC12神经元样细胞,随后TUNEL法检测各实验组细胞的凋亡率,Western blot检测各组细胞GAP-43的蛋白表达；6)应用Western blot检测5组细胞内p53的蛋白表达；7)体外瞬时转染SIRT1siRNA,制作PC12神经元样细胞的SIRT1沉默模型,应用、Western blot检测转染成功率；8)应用Western blot检测SIRT1抑制后]miR-204inhibitors对PC12神经元样细胞内p53蛋白表达的变化。实验结果：1)转染了mimics后的PC12神经元样细胞内miR-204的表达较空白对照组显著上升,而转染了inhibitors后的PC12神经元细胞内miR-204的表达明显低于空白对照组,差异具有统计学意义(p0.05),提示转染成功。2)miR-204过表达组细胞内SIRT1蛋白表达水平较对照组明显下调,而在低表达组内显著上(p0.05),但在mRNA水平变化并不显著,与对照组相比无统计学差异(p0.05)；3)miR-204低表达组细胞内p53表达水平较对照组明显下调,差异具有统计学意义(p0.05)；4)miR-204过表达组细胞在海人酸诱导惊厥后凋亡较正常组显著上调,而在低表达组细胞凋亡明显减少,差异具有统计学意义(p0.05)；5)miR-204达组细胞在海人酸诱导惊厥后神经元GAP-43的水平显著下调,而过表在低表达组则GAP-43的表达较对照组明显上调,差异具有统计学意义(p0.05)；6)转染了SIRTl siRNA后的PC12神经元样细胞内SIRT1的表达较正常对照组及阳性对照组均显著下调,差异具有统计学意义(p0.05)；7)在同时转染了miR-204inhibitors+SIRT1siRNA的PC12神经元样细胞内我们发现,p53蛋白表达水平与正常对照及相应的阳性对照组比较显著下调(p0.05)。结论：miR-204通过负调控SIRT1的蛋白表达实现对惊厥性脑损伤的调控。SIRT1介导miR-204调控惊厥性脑损伤过程可能与影响其下游因子p53的活化有关。综上所述,可以初步得出结论：miR-34b-5p、-204、-582-3p、-672均参与了发育期反复惊厥性脑损伤的调控,miR-204在反复惊厥后的海马及大脑皮质中表达均较对照组显著上调,通过负调控SIRT1的表达调控惊厥后神经元的凋亡有突触可塑性的改变,其机制可能与负性调控凋亡相关因子p53的表达有关。因此,下调miR-204的表达能在转录后水平减弱了对SIRT1的抑制,使其表达显著增高,抑制惊厥性脑损伤诱发的神经元凋亡,促进轴突的生长与延伸,进而延缓脑损伤进程,为今后治疗惊厥性脑损伤提供了新的靶点。
[Abstract]:Convulsion is the most common severe severe disease in infantile period. The persistent seizures can produce severe neurological sequelae, affecting the mental development and physical and mental health of the children, even endangering the life of children. The incidence of convulsion in children is very high, about 5-6% of children have once or many times of convulsion history, preterm infants even up to 22.7% to 25%. if convulsions hair With frequent or longer duration, about 15% to 30% children will eventually change into epilepsy. Developmental seizures can inhibit the growth and development of the brain, change the nerve loop, improve the neuronal excitability, aggravate the brain damage, and lead to visual, spatial, learning, memory and emotional obstacles. If not intervened in time, it will seriously jeopardize the life of the child and The long-term quality of life has brought a heavy burden to the family and the society. As far as our country's population is concerned, the difficulty of the bed is more severe than that of other countries. Therefore, exploring the mechanism of development and the development of new anticonvulsant drugs have become the medical problems that we are urgently needed to solve.
The present study conducted a related study of the mechanism of convulsion in rats with recurrent seizures. Three fluoro ethyl ether inhalation induced convulsion was a classic model of developmental seizures. In previous experiments, this study used TaqMan MicroRNA Arrays to detect the niRNAs expression profiles in the hippocampus of rats after recurrent seizures and in the 766 known miRNA. The expression of miRNA in hippocampus of rats after convulsion was screened. It was found that, compared with the normal control group, 20 known miRNAs expressions were down regulated by 0.5 times in hippocampus after recurrent seizures, and the expression of 9 niRNAs was up to above 2 times. The expression of miR-204 was abundant in the hippocampus, and the expression of 24h was up to a significant increase after repeated convulsion. 7 times of the normal group. Therefore, this experiment will further observe the differential expression of miR-34b-5p, -204, -582-3p, -672 in the hippocampus and cerebral cortex of rats, and select miR-204 as the research object, and explore its role and mechanism in the brain damage of convulsion.
Part one changes in the expression of specific miRNAs in brain tissue after recurrent convulsive brain injury
Objective: To explore the expression and possible mechanism of specific miRNAs in hippocampus and cerebral cortex of rats after recurrent seizures.
Methods: 1) 70 rats of 21 day old SD rats were randomly divided into 2 groups, namely the control group and the convulsive group. Each group was randomly divided into 5 time points and 6 rats at each time point. The convulsions group was repeatedly inhaled by three fluorine ether (1 times a day for 6 days), and the control group underwent phase synchronization. Suddenly, three fluorine ether was not inhaled, and 60 rats were finally enrolled in the group. After the last convulsion, the rats were divided into 2h, 6h, 24h, 72h and 7d head to take the brain, and the hippocampus and cerebral cortex.2 were separated. The total RNA of hippocampus and cerebral cortex was extracted at each time point. Then miR-34b-5p, miR-204, miR-582-3p, miR-672 were reverse transcriptase, real-time quantitative PCR was used to detect the expression of four kinds of miRNAs in two tissues.) in vitro application of sea human acid (100 mu mol/l) to PC12 neuron like cells cultured in vitro, and real-time quantitative PCR detection of miR-204 expression in Oh and 24h when Oh and 24h were detected by PCR. We predicted the target genes with abundant expression in the brain tissue, and further screened out the target genes involved in the process of convulsive brain injury. MiR-204
The results showed that the expression of miR-34b-5p and miR-204 in the hippocampus and cerebral cortex began to increase (P0.05) after the last convulsion, and the expression of miR-34b-5p in hippocampus and cortex was lower than that of 2H (P0.05) at 6h, but it was still significantly higher than that in the normal control group (P0.05), but the expression of miR-204 in the hippocampus was lower than that in the normal control group (P0.05). The expression of miR-204 in the hippocampus was lower than that of 2h in the hippocampus, and the expression in the cortex after 6h was higher than that in the 6h. The expression of miR-34b-5p and miR-204 in hippocampus and cerebral cortex all reached the peak of 24h after the last convulsion, and then gradually decreased and 72h returned to normal. The expression of miR-204 in the hippocampus and the expression of miR-34b-5p and miR-204 in the cerebral cortex were significantly lower than those of the control group (P0.05), while the expression of miR-34b-5p in the hippocampus was up trend. The expression of miR-582-3p and miR-672 in the hippocampus decreased in different time points after the last convulsion, while the expression in the cerebral cortex was up up, reached the peak at 24h (P0.05), and then gradually decreased to 7d to the normal level, no significant difference compared with the control group (P0.05). Not only that, but also, we found repeated convulsions in Houhai. The expression of miR-34b-5p and miR-204 in the horse and cerebral cortex is highly correlated at different time points, while the correlation of miR-582-3p and miR-672 in different parts is not obvious. Therefore, it is possible to speculate that the expression of miRNAs in the brain damage of convulsive brain is different. In order to further explore the convulsive brain damage of the specific miRNA in the regulation of the developmental period of the brain The specific mechanism of injury was selected in the experiment of miR-204 with rich expression in the hippocampus of rats. The results showed that the expression of PC12 neuron like cells in the miR-204 after the sea human acid drying was significantly higher than that of the control group (P0.05). Then we selected the results of starBase for three target gene prediction software (Targetscan, PITA and PicTar). In order to reduce the false positive rate, select the target genes that appear in the three prediction software and further screen out the target mRNAs. that participates in the process of brain damage in the development period. It is found that in the 3'-UTR of information silencing factor 1 (SIRT1), there may be a binding site with miR-204, while the former is a NAD dependence. Acetyltransferase is widely involved in the regulation of neuronal apoptosis and synaptic plasticity after brain injury. Therefore, we speculate that SIRT1 may be one of the target sites for miR-204 to regulate the development of convulsive brain damage.
Conclusion: 1) the expression of niR-34b-5p, miR-204, miR-582-3p and miR-672 in the hippocampus and cerebral cortex of rats changes after the development of convulsion, and has tissue correlation. It is demonstrated that miRNAs participates in.2 in the process of convulsive brain injury in vitro. SIRT1 may be one of the target genes in the process of regulating convulsive brain damage in miR-204.
The second part is the change of neuronal apoptosis and synaptic plasticity after convulsive brain injury in rats.
Objective: To explore the changes in neuronal apoptosis and neuronal synaptic plasticity after convulsion in vivo and in vitro, as well as the regulatory mechanism of SIRT1 involved in sexual brain injury after convulsion.
The experimental steps: 1) as mentioned above, the rat model of recurrent seizures was made in the development period. After the last convulsion, 2h, 6h, 24h, 72h and 7d broke the head, separated the hippocampus; 2) the total protein and RNA of the control group and the convulsive group were extracted, and the protein expression of the GAP-43 and p53 in the hippocampus of the rats in the experimental groups was detected by Western blot, qRT-PCR and Western detection. SIRT1mRNA and protein content of rats in the experimental group were measured; 3) the apoptosis of hippocampal neurons in 2H, 6h, 24h, 72h and 7d rats after convulsion was detected by TUNEL; 4) cultured PC12 neuron like cells in vitro, and used sea human acid (100 micron) to simulate the model of convulsion in vitro, collect the cells after 24 hours, and detect the Oh and finely after the intervention respectively with Western blot. Intracellular GAP-43, SIRT1 and p53 protein content; 5) TUNEL method was used to detect the apoptosis of PC12 neuron like cells after different concentrations (0,50100250 mol/l mol/l) of kainic acid.
The results were as follows: 1) compared with the control group, the protein level of SIRT1 in the hippocampus decreased significantly (P0.05) after convulsion (P0.05), further down to 6h (P0.05), 72h at the lowest value (P0.05) and 7d when 7d gradually returned to normal (P0.05). On the contrary, the p53 expression in the hippocampus was up significantly up after convulsion (P0.05), up to 24. H reached the peak (P0.05) and 72h at a high level. There was no statistical significance compared with 24h (P0.05), and gradually recovered to normal at 7d, but it was still significantly higher than that in normal group (P0.05), and GAP-43 began to decrease (P0.05) after the last convulsion (P0.05), to 72h (P0.05), and gradually returned to normal at the time of 72h, and compared with the control group after convulsion. No significant difference (P0.05).2) after convulsion, 2h, neuronal apoptosis in the hippocampus increased significantly compared with the control group, 6h increased further, reached the peak at 72h, and decreased gradually when 7d, and the differences in the experimental group and the control group were statistically significant (P0.05).3). Sea human acid was involved in SIRT1 in PC12 neuron like cells after 24h, and the protein level of GAP-43 was significantly higher than that of the control group. Down regulation, while the protein level of p53 increased significantly (P0.05).4) the apoptosis of PC12 neuron like cells after different concentrations of sea human acid was in a dose-dependent manner. The apoptosis was most significant at 250 u mol/l, and the difference between each dose group was statistically significant (P0.05).
Conclusion: in this experiment, the experiment in vivo and in vitro proved that the seizure brain injury also inhibited the growth and extension of neuron synapse at the same time, and inhibited the growth and extension of neuron synapse. Not only so, the seizure brain injury also significantly inhibited the expression of SIRT1, and its mechanism may be related to the deacetylation of the downstream factor p53, by up regulation of apoptosis related factors. The expression of subp53 accelerates the apoptosis of cells.
The third part is the mechanism of miR-204 regulating the development of convulsive brain injury through SIRT1.
Objective: to verify that SIRT1 is the target and mechanism of miR-204 in the regulation of convulsive brain injury from biological level.
Experimental steps: 1) in vitro chemical synthesis of miR-204mimics, inhibitors and corresponding negative control; 2) as mentioned earlier, the primary cultured PC12 neuron like cells were randomly divided into 5 groups: the normal control group, the miR-204mimics intervention group, the miR-204mimics negative control group, the miR-204inhibitors intervention group and the miR-204inhibitors negative control group. 3) using transient transfection in vitro to make miR-204 overexpression and low expression model, real-time quantitative PCR detection of transfection success rate; 4) use Western blot and real-time quantitative PCR technique to detect the changes of SIRT1 protein and mRNA expression in each experiment group; 5) make miR-204 overexpression and low expression model, use sea human acid to intervene PC12 neuron sample in each group Cell apoptosis rate was detected by TUNEL method, Western blot was used to detect the protein expression of GAP-43 in each group; 6) Western blot was used to detect the protein expression of p53 in the 5 groups; 7) transient transfection of SIRT1siRNA in vitro, SIRT1 silent model of PC12 neuron like cells, application, Western blot test success rate of transfection; 8) Western blot was used to detect the changes of p53 protein expression in PC12 neuron like cells after inhibition of SIRT1 by SIRT1.
The results were as follows: 1) the expression of miR-204 in the PC12 neuron like cells after mimics transfection was significantly higher than that in the blank control group, and the expression of miR-204 in PC12 neurons transfected after inhibitors was significantly lower than that in the blank control group. The difference was statistically significant (P0.05), and the expression of SIRT1 protein in the miR-204 overexpression group was shown in the transfected.2). The expression level in the low expression group was significantly lower than that in the control group (P0.05), but there was no significant difference between the mRNA level and the control group (P0.05); 3) the expression level of p53 in the miR-204 low expression group was significantly lower than that in the control group (P0.05), and 4) the miR-204 overexpressed group cells were in the sea people. After acid induced convulsion, apoptosis was significantly higher than that in the normal group, but the apoptosis decreased significantly in the low expression group, and the difference was statistically significant (P0.05); 5) the level of GAP-43 in the neurons of the miR-204 group was significantly down down after the sea human acid induced convulsion, while the expression of GAP-43 in the low expression group was up significantly higher than that in the control group. The difference was statistically significant. Learning significance (P0.05); 6) the expression of SIRT1 in PC12 neuron like cells after transfected with SIRTl siRNA was significantly lower than that in the normal control group and the positive control group. The difference was statistically significant (P0.05); 7) we found that the expression level of p53 protein and the normal pair in the PC12 neuron like cells transfected to miR-204inhibitors+SIRT1siRNA at the same time. The corresponding positive control group was significantly lower than that of the control group (P0.05).
Conclusion: miR-204 can regulate the expression of SIRT1 protein by regulating the expression of SIRT1 in rats.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R720.597

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