The Role And Mechanism Of Feedback Loop Of MiR-133b And ERK In Methamphetamine-Induced Neurotoxicity

Methamphetamine (METH) is a kind of psychomotor stimulants with neurotoxic properties, which has neurotoxic effect on animal and human in different brain regions. Recent studies have shown that a lot of neurotoxic mechanisms involved in METH-induced damage. For example, damage of excitability toxicity, oxidative stress-mediated injury, mitochondrial dysfunction, and neuronal cell apoptosis and so on. Nevertheless, neurotoxic mechanism induced by METH, especially the related molecular mechanisms is not clear, and still needs further researches and studies.A latest study has reported that METH could induce small RNA (microRNA, miRNA) expression, which regulated the function of target genes at the post-transcriptional level and activated related intracellular signaling pathways leading to toxic damage. MicroRNAs are a class of ～22 nucleotide (nt) non-coding small RNAs which can bind to 3’untranslated regions (UTRs) of their mRNA targets and cause mRNA degeneration or gene silencing. MiRNA regulate gene activity through post-transcriptional control to adjust to a number of biological processes. For instance, cell differentiation, growth, apoptosis and tumor occurrence and metastasis and the like. MiR-133b is a newly discovered miRNA, which was detected on chromosomes 1. A current report demonstrated that miR-133b is normally enriched in the midbrain and regulates differentiation and maturation of dopaminergic neuron by a negative feedback loop mechanism. It is generally known that METH acts on dopaminergic neurons and results in biological effects. More work need to be done to proof that whether there are any relations in miR-133b and neurotoxic effects of METH.Extracellular signal regulated kinase 1 and 2 (ERK1/2) are two of the mitogen-activated protein kinases, which are key proteins that transmit extracellular signals into the cell nucleus by combining with the surface receptor. ERK 1/2 are considered as an integration point of multiple cellular processes such as differentiation and proliferation of cell, maintenance of cell shape, construction of cytoskeletal proteins, apoptosis of neuron, malignant transformation of cell, and so on. Moreover, a wealth of studies have found that central stimulants taken part in the regulation of spatial learning and memory and cognitive function by activating the ERK pathway. MiR-133b increased ERK phosphorylation and participated in occurence and metastasis of tumor, suggesting that there is a regulatory relationship between miR-133b and ERK. But the role and regulatory relation of miR-133b and ERK in METH-induced toxic injuries are uncertain.To investigate the role of miR-133b and ERK and their regulation mechanism in METH-induced neurotoxicity, we have designed the following experiments:(1) PC12 cells were treated with METH of different concentrations (100μM,200 μM,400 μM, 800 μM, and 1600 μM), and the morphological changes were observed under inverted microscope. MTT and LDH assays were performed to determine the cell viability. (2): Intracellular reactive oxygen species (ROS) and the changes of mitochondrial membrane potential (MMP) were detected to analyse cellular oxidative damage. (3): Neuron apoptosis were examined by acridine orange staining method and Hoechst 33342/PI double staining. (4):To further investigate the molecular mechanism of neurotoxicity induced by METH. The expressions of miR-133b and ERK were tested by qRT-PCR and Western blot technology. (5):MiR-133b mimic or miR-133b inhibitor was transfected into PC 12 cells to observe the damage of PC 12 cells and the expression change of ERK. (6):PC 12 cells were pretreated with U0126 which was used to inhibit the ERK phosphorylation before exposed to METH. Then we observed the injury of PCI2 cells and the expression change of miR-133b. Finally, we discussed the function and regulation relation of miR-133b and ERK in METH-caused neurotoxicity.Result showed that:(1) PC 12 cells were treated with METH at five doses (100 μM,200μM,400 μM,800μM, and 1600μM). METH treatments could damage PC 12 cells in a dose-dependent manner. When PC 12 cells were exposed to METH, the cells changed to round with areas of lucency and the neurites slowly disappeared until the network was collapsed with the increasing concentration. MTT and LDH displayed that the cell viability was reduced and LDH enzyme release was increased with METH concentration enrichment (P< 0.01). From the results above,800 μM of METH was chose as the optimal METH damage concentration. When PC 12 cells were exposed to METH, the ROS level was increased, the MMP was decreased and the neurons apoptotic rate was higher. The results of qRT-PCR have proved that the expression level of miR-133b came down in injury group. T-ERK expression was stable, but the expression of p-ERK was significantly increased (P< 0.05). (3) When the expression of miR-133b was intervened by miR-133b mimic or miR-133b inhibitor before treating with METH. In the mimic group, apoptotic rate and phosphorylated ERK expression were downgraded, and MMP was apparent recovery (P< 0.01), suggesting that overexpression of miR-133b down-regulated the expression of phosphorylated ERK and weakened the toxic effects of METH. On the contrary, green/red fluorescence ratio, apoptotic cells and phosphorylated ERK were increased in miR-133b inhibitor group (P< 0.01), accounting that suppression miR-133b expression could activate ERK phosphorylation and aggravate the neurotoxicity of METH. Applying U0126 before inducing cell damage, the recovery of MMP was obvious and the apoptotic rate of PC 12 cells was decreased. At the same time, the result of qRT-PCR showed that miR-133b expression was elevated. All this indicated that activated ERK could significantly reduce miR-133b expression and lead to neural injunes.This study indicates that METH induced neurotoxic effects through down-regulating of miR-133b expression and up-regulating of ERK phosphorylation. About regulation relationship of miR-133b and P-ERK, we found that miR-133b inhibited ERK phosphorylation. Meanwhile, phosphorylated ERK could down-regulated the expression level of miR-133b. In brief, the miR-133b/ERK signaling pathways play an important role in METH induced toxic damage.