The Role Of MicroRNA Expression Profiling Changes In Lead-induced Neurotoxicity

【Background】Lead is one of the most common environmental heavy metal toxins harmful to humanhealth. The main target organ of lead is the central nervous system. Lead can seriouslyaffect children’s growth and intellectual development. The development of lead poisoningis very occult, which mostly dues to long-term and low-dose chronic exposure. However,there is no particular effective treatment yet. The metabolism of lead, which has a longhalf-life, is very slow in human body and the chronic cumulative toxicity effect of leadcontinues from childhood to adulthood and even old age and causes neurodegenerativediseases eventually. Therefore, it’s an urgent task to uncover the mechanism of leadneurotoxicity especially the molecular biology mechanism of the negative impact of leadon learning and memory, propose effective measures for the diagnosis, treatment,prevention and control of lead poisoning.microRNA negatively regulate the translation of their target mRNAs throughcomplementary paring in the post-transcriptional level. They are highly expressed in thebrain and play important roles in neural development, synapse plasticity, as well aslearning and memory. The transient nature of miRNA, their localized expression indendrites, their capacity to respond in an activity-dependent manner, and the fact that asingle miRNA can simultaneously regulate many genes and a single gene can be simultaneously regulated by many miRNA make brain-specific miRNA ideal candidatesfor the fine-tuning of gene expression associated with neural plasticity and memoryformation.Therefore, we assume that miRNA may play important roles in lead-inducedneurotoxicity and damage to learning and memory.【Objective】With the establishment of a model of lead-induced injuryies in learning and memory,and hippocampal neurons, to study the change of miRNA expression profiling; verifywhether miRNA are involved in lead-induced neurotoxicity effects; reveal the role and themechanisms of miRNA in lead-induced neurotoxicity; provide experimental basis for theprevention and treatment of lead poisoning.【Methods】1. To establish lead-exposed animal models through water drinking and blood leadmonitoring; establish lead-exposed cell model through in vitro cultured PC12cells.2. To evaluate rats’ spatial learning and memory ability through monitoring hiddenplatform latency and target quadrant residence time in the water maze test; use TUNEL,Western blot, and immunohistochemical staining methods to detect hippocampal tissueinjury following lead exposure.3. Use microarray analysis to detect the changes of miRNA expression profiles inlead-exposed hippocampus and verify the changes by qRT-PCR technology.4. Use bioinformatics methods to predict miRNA target genes and verify thepredicted results by Western blot.5. Use miRNA inhibitors to establish "Loss of function" models and explore themechanism of miRNA in lead-induced neurotoxicity. 【Results】1. Lead exposure could cause impaired spatial learning and memory ability of SDrats.Rats in tested groups (including100ppm,200ppm,300ppm groups) exposed to leadexposure through eight weeks of drinking water showed no statistical difference in waterintake and body weight changes compared to control rats. However, the blood lead levelsincreased significantly in rats exposed to lead and the spatial learning and memory abilitywas significantly impaired as shown by Morris water maze. Western blot results showedthe decreased expression of glutamate receptors and NMDA receptors related to learningand memory in hippocampus.2. Lead exposure could induce neural damage in hippocampus.TUNEL assay in the300ppm drinking water lead exposed8w rat hippocampus and10μmol/L lead exposed48h PC12cell model were all positive, which showed that leadcould induce increased apoptosis in hippocampal neurons and PC12cells, suggesting thatlead-induced neuronal damage may be the cause of changes in learning and memoryabilities.3. Lead exposure induced changes of miRNA expression profling in hippocampus.In miRNA microarray results, lead exposed rats’ hippocampus showed sevenmiRNA’s expression level significantly changed compared to that of the control group. Ofwhich, the expression of miR-204, miR-211, miR-448, miR-449a, miR-34c, and miR-34bwere increased more than1.5times, and the expression level of miR-494was reduced toone sixth of that of the control. qRT-PCR technology validated the results. Then weselected three miRNA, miR-204, miR-211, and miR-448, with whose expression changesgreater than other miRNA, and use qRT-PCR technology to validate their expression inhippocampus of2w,4w,6w, and8w respectively and the results showed that the threemiRNA’s expression were greatly increased.4. Bioinformatics technical analysis showed the target genes of significantly differentially expressed miRNA were related to apoptosis, LTP/LTD and calcium signalingpathways.We used bioinformatics technical analysis, including DIANA, TargetScan, andmiRNA three web sites to predict target genes of miRNAs. The results showed that thetarget genes were related to apoptosis, axon guidence, calcium signaling pathway,LTP/LTD, MAPK signaling pathway, Notch signaling pathway, and neurodegenerativediseases. Interestingly, miR-204，miR-211and miR-448had two common target genes,apoptosis-related Bcl-2and calcium channel related Itpr1. Western blot analysis andqRT-PCR verified the results (animal and PC12cells), which was asistent with thepredicted results.5. The inhibited expression of miR-204, miR-211, and miR-448could protect cellsfrom the effects of lead exposure to some extent.We used miRNA inhibitor to establish "Loss of function" model, and validated thefunction of miRNA on lead-induced PC12cells viability and apoptosis changes by MTT,TUNEL methods, and found that interference with miRNA expression of PC12cells canprotect the cells from lead damage to some extent. Of which, interference the expressionof miR-448could increase the cell viability by20%.【Conclusions】1. Lead exposure may lead to impaired learning and memory function by causingapoptosis to hippocampal neurons.2. Lead exposure can cause changes of miRNA expression profiling in hippocampus.miRNA may play important roles in lead-induced neurotoxic effects.3. Significantly differentially expressed miR-204, miR-211, and miR-448cannegatively regulate their common target genes, apoptosis related Bcl-2and Ca2+channelrelated Itpr1, in lead-induced cytotoxic effects.4. Interference with the expression of miR-204, miR-211, and miR-448can protectPC12cells from the damage of lead at some extent.