The Potential Roles For MicroRNAs And Tumor Necrosis Factor Alpha In Epileptogensis

Part one:Expressions of Tumor Necrosis Factor Alpha and MicroRNA-155in Immature Rat Model of Status Epilepticus and Children with Mesial Temporal Lobe Epilepsy ObjectiveRecently, the role of inflammation has attracted great attentions in the pathogenesis of mesial temporal lobe epilepsy (MTLE). MicroRNAs (miRs), an important participant in the pathogenesis of MTLE, have been recognized gradually. In this study, we investigated the dynamic expression patterns of tumor necrosis factor alpha (TNF-a) and microRNA-155(miR-155) in the hippocampi of immature rat models and children with MTLE. Furthermoe, the direct correlation between them was confirmed in cultured astrocytes in vitro.MethodsMTLE models were induced by lithium-pilocarpine by immature rats. Western blot and real-time quantitative PCR (qPCR) methods were used to detect the expression levels of TNF-a and miR-155. Expression was monitored in the acute, latent, and chronic stages of disease (2h and3and8weeks after induction of lithium-pilocarpine status epilepticus, respectively), and in control hippocampal tissues corresponding to the same timeframes. Similar expression methods were applied to hippocampi obtained from children with MTLE and normal controls. In vitro cultured astrocytes level, qPCR was applied to detect the changes in expression of TNF-a and miR-155after stimulation (MRP8, LPS) and inhibition (Lenalidomide) of TNF-a.ResultsThe expressions of TNF-a and miR-155were significantly upregulated in the seizure related acute and chronic stages of MTLE in the immature rat model and also in children with MTLE. Modulation of TNF-a expression, either by stimulation using myeloid related protein (MRP8) or lipopolysaccharide (LPS), or inhibition using lenalidomide on astrocytes, leads to the same dynamic changes in miR-155expression. ConclusionThis study is the first time to focus on the dynamic expression pattern of miR-155in the immature rat model of lithium pilocarpine and children with MTLE, and to confirm their direct relationship at the astrocyte level. TNF-a and miR-155, having similar expression patterns in the three stages of MTLE development, and the direct relationship at the astrocyte level is highly suggestive of direct interactive relationship during MTLE development. Therefore, modulation of the TNF-a-miR-155axis may be a novel therapeutic target for treatment of MTLE. Part two:Effects of MRP8, LPS and Lenalidomide on the Expressions of TNF-a, Brain-enriched and Inflammation-related microRNAs in the Primary Astrocyte CultureObjectiveAstrocytes are specialized glial cells that outnumber neurons by over five fold, are recognized as a heterogeneous class of cells with many important and diverse functions in healthy and diseased central nervous system (CNS). MicroRNAs (miRNSs) are small, noncoding RNAs that regulate the posttranscriptional expression of protein-coding mRNAs, which may have key roles in astrocytes activation in response various stimuli.MethodsQuantitative real time PCR (qPCR) was used to detect changes in the expressions of brain-enriched miRNAs (124,134,9,132, and138), inflammation-related miRNAs (146a,21,181a,221, and222) and tumor necrosis factor alpha (TNF-a) in the astrocytes after stimulation with myeloid related protein8(MRP8) and lipopolysaccharides (LPS). Further, the same brain-enriched and inflammation-related miRNAs in the obove were tested for the first time after inhibiting the expression of TNF-a in the astrocytes by using TNF-a inhibitor, lenalidomide.ResultsThe expression levels were up-regulated significantly after stimulation of the astrocytes with MRP8or LPS, reversely, the expression of iRNA-138down-regulated. TNF-a inhibition with lenalidomide leads to the opposite expressions of the tested miRNAs.ConclusionThe results showed that expressions of brain-enriched and inflammation-related miRNAs in the astrocytes are significantly changed in response to activation with different pro-inflammtory stimuli and also with inhibition of TNF-a. These miRNAs may play a role in activation of the astrocytes and may be a novel target for cell-specific therapeutic interventions in multiple CNS diseases. Part Three:Dynamic Expression Patterns of Inflammation-related miR-181a, miR-221, and miR-222in an Immature Rat Model and Children with Mesial Temporal Lobe EpilepsyObjectiveAccumulating clinical and experimental evidences strongly supports the relevance of inflammation in the pathophysiology of human epilepsy. Recently, microRNAs (miRs) start to be a novel player in the mesial temporal lobe epilepsy (MTLE) pathogenesis in mature and developing brains. This study aimed to investigate the dynamic expression patterns of inflammation-related miR-181a, miR-221, and miR-222in the hippocampi of an immature rat model in the three stages of MTLE development and in children with MTLE.MethodsqPCR was performed to detect the changes of miR-181a、 miR-221and miR-222expression. Expressions were monitored in the three stages of MTLE development (2h,3and8weeks after induction of lithium-pilocarpine status epilepticus, respectively), and in control hippocampal tissues corresponding to the same timeframes. Similar expression method was applied to hippocampi obtained from children with MTLE and normal controls.ResultsmiR-181a was downregulated in the acute stage, then nearly equal to control in latent stage and finally upregulated in chronic stage. miR-221and miR-222were downregulated in the three stages. In children miR-181a was upregulated and miR-221, miR-222were downregulated.ConclusionThis is the first study to examine the dynamic expression patterns of inflammation-related miR-181a, miR-221and miR-222in the immature rat model and children with MTLE. Modulation of these inflammation-related miRs expressions may be new targets for antiepileptic and anticonvulsant therapies in the developing brains.