The Effect And Its Mechanism Of MiR-210on The Damage Effect Of Pc12Cells Exposed To Hypoxia Or Cyanide

BackgroudHypoxic enviorment would influence the normal physiological processes, decrease themental and physical operational capacity, even induce acute high altitude cerebral edema,lung edema, cardiovascular and cerebrovascular ischemic injury and some otherhypoxia-related diseases. Due to the highest oxygen consumption, the central nerve systemis very sensitive to hypoxia. The energy-consuming nervous system is more dependent onmitochondrion function, so the mitochondrial disorders is a key factor of nervous systemdysfucntions. Hypoxia could influence the expresion of various miRNAs, among which theover expression of miR-210is stable and significant. ISCU1/2and COX10, which areclosely associated with mitochondrion function, are the targets of miR-210. The currentresearch used rat pheochromocytoma cell line PC12, human bronchial epithelial cell line16HBE and human umbilical vein endothelial cell HUVEC as hypoxia-exposure cellmodels, and the different effects of hypoxia on different cells were studied. The influenceand the potential mechanisms of hypoxia or cyanide on nervous cell survival, the exprssionof miR-210, its regulatory mechanism to cell exposed to extracellular orintracellular hypoxia as well as ite influence on mitochondial functions were Investigated.Research contents1. PC12cell,16HBE cell and HUVEC cell were exposed to1%oxygen. Theexperimental group were divided into12h hypoxia group,24h hypoxia group and48hhypoxia group. The morphological alteration of the cells exposed to hypoxia was observed.CCK-8was used to detect the survival rates of cells exposed to hypoxia. We used qRT-PCRto detect the dynamic expression of miR-210. In addition, PC12cells were transfected withmiR-210mimics or inhibitor to study the protective effect of miR-210on neural cells.2. Flow Cytometry was used to measure the changes of mitochondrial membranepotential in PC12cells. High Performance Liquid Chromatography was used to detect the contents of ATP, ADP and AMP in PC12cells exposed to hypoxia. These two parameterscould reflect the changes of mitochondral function. Western Blot was used to measure theexpression of ISCU1/2and COX10, which would show the influence of hypoxia onmitochondrial respiratory chain.3. We observed the morphological changes of sodium cyanide treated PC12cells andused MTT to evaluate the cytotoxicity. qRT-PCR wasused to detect the dynamic expressionof miR-210in PC12cells exposed to sodium cyanide. Western Blot was used to measurethe expression of ISCU1/2and COX10, and the differences between extracellular orintracellular hypoxia were studied.Research results1. Hypoxia exposure decreased the survival rates of PC12,16HBE and HUVEC cells,among which the24h hypoxia group and48h hypoxia group showed lower survival ratecompared with the control group. After12h hypoxia, PC12cell swelled. After24h hypoxia,the density and the refractivity of cells were decreased, the amount and the length of cellprotrusions also decreased. The predonimant cell shapes changed from polygonal to bipolar.16HBE cells shrank and the density of cells decreased after hypoxia for24h. HUVECshowed lowered density, fuzzy boundaries and decreased refractivity. All of these threekinds of cells exhibited large amount of cell loss after hypoxia for48h.2. Rhodamine123staining and High Performance Liquid Chromatography analysisshowed a decreased mitochondrial membrane potential and ATP contents in PC12cells,while the ADP and AMP contents were obviously elevated, which meant that themitochondrial respiratory function and oxidative phosphorylation were disturbed byhypoxia, and the activity of ATP synthase complex in mitochondral inner membrance wasinhibited, which resulted in the decreased synthesis of ATP, and aggravated the decrease ofcell ATP content.3. The qRT-PCR results showed an over expression of miR-210in thehypoxia-exposure cells, among which16HBE cell responded early and strong (the highestexpression was found at12h hypoxia), PC12cell response was gradually strengthened (thehighest expression was found in48h group), and HUVEC cell showed a moderatelyelevated expression. Western Blot results showed the expression of ISCU and COX10, asthe target genes of miR-210, decreased dramatically and the expression of both proteins showed a time-effect relationship.4. After the expression of miR-210was elevated or inhibited in PC12cell by transienttransfection of nuleotide oligos, the miR-210mimic transfected cells showed an enhancedsurvival rates after exposed to hypoxia for24h or48h, while the miR-210inhibitor-treatedcells showed the opposite results. So miR-210could protect cell when they were exposed tohypoxia.5. Sodium cyanide (1mM) treated PC12cells displayed the highest induction ofmiR-210expression. The expression of ISCU1/2and COX10decreased accordingly, whichconfirmed the consistency of extracellular and intracellular hypoxia.Research conclusionsBased on the above results, we drew the conclusion as follows:1. Our research for the first time comparatively investigated the miR-210expressionpattern on different cells exposed to hypoxia. The bronchial epithelial cells responded earlyand strong (the highest expression was found at12h hypoxia), PC12cell response wasgradually strengthened (the highest expression was found in48h group), and HUVEC cellshowed a moderately elevated expression (12h-24h).2. Hypoxia environment decreased the survival rates of PC12,16HBE and HUVECcells, and hypoxia-induced miR-210would protect neural cells exposed to hypoxia.3. PC12cells exposed to hypoxia showed mitochondrial function disorders, decreasedmembrane potential, and declined ATP synthesis.4. Hypoxia-induced miR-210participated in cell responses to hypoxia by regulatingthe expression of its target genes, ISCU1/2and COX10.5. It was showed that cyanide treatment-induced neual cell intracellular hypoxiainhibited the proliferation of the cells, simulated the expression of miR-210and supressedthe expressions of ISCU1/2and COX10.