The Regulation Mechanism Of TFPI-2 In Chemical Hypoxia Of SH-SY5Y Cells

Objectives: Hypoxia damage takes part in a variety of brain diseases,such as cerebral infarction,hematencephalon,brain tumor,high altitude cerebral edema and so on.It is reported that hypoxia can cause the expression change of a series of function protein and gene.Of these,hypoxia-inducible factor is regarded as the most important nuclear transcription factors to date,which can regulate the expression of various genes.Hypoxia signaling pathways regulated by HIF participate in the pathological process of energy metabolism,cell cycle regulation,angiogenesis apoptosis and tumor invasion.So far,brain hypoxic injury mechanisms and regulatory pathways have not been completed yet.New biomarkers or regulatory factors can provide us novel targets for neuroprotection,prevention and treatment of diseases,which would be of great significance.To explore new biomarkers related to hypoxia,our research team used protein microarray,gene microarray and miRNA microarray to detect difference factors in cortex of hypoxic rats.We found that two factors,TFPI-2 and miR-92a-2,had targeted relationship: TFPI-2 is positively correlated with hypoxia,while miR-92a-2 was negative correlation with hypoxic.Tissue Factor Pathway Inhibitor-2,belonging to the family of serine protease inhibition,is a native anticoagulant and inhibitor of endogenous Tissue Factor.TFPI-2 is a broad spectrum serine protease inhibitor,which can inhibit the activity of variety of protease,such as plasmin,matrix metalloproteinases,etc,and maintain the integrity of the extracellular matrix.It is reported that TFPI-2 participated in the regulation of angiogenesis and apoptosis in invasion and metastasis of many kind of tumors.However,the change of TFPI-2 and related regulation mechanism during the process of hypoxia and the relationship between TFPI-2 and HIF pathway is still unclear.Cobalt chloride is a common hypoxic revulsant in cytochemistry,which can activate HIF-induced hypoxic pathway and simulate cell hypoxic environment.In this study,we aim to establish cell hypoxic damge model of SH-SY5 Y neuroblastoma cells with cobalt chloride.Furthermore,we would explore the change of TFPI-2 and related regulation mechanism in hypxia process.Methods: 1 The influence on cell activity and cell morphology of SH-SY5 Y cells induced by CoCl₂ 1.1 The change of cell shape was observed with HE staining by inverted microscope at 6 hours and 24 hours after hypoxia induced by CoCl₂?0,100,200,400?mol/L?.1.2 SH-SY5 Y cells was treated with different concentrations of CoCl₂?0,50,100,150,200,250,300,350,400,450,500?mol/L?.Cell viability was detected by MTT asssy.1.3 SH-SY5 Y cells was treated with 100?mol/L CoCl₂.Then cell viability was detected at different time?0h,6h,12 h,24h,48 h and 72h?by MTT asssy.2 Assess the expression level of TFPI-2 after Co Cl₂-induced hypoxia 2.1 Western blot was used to detect the expression of HIF-1?,TFPI-2 and VEGF in SH-SY5 Y cells at different time?1,2,4,6 and 8h?after hypoxia.Besides,the variation trend of gene expression was analyzed after hypoxia.2.2 Real-time PCR was used to detect the mRNA expression level change of HIF-1?,TFPI-2 and VEGF after hypoxia with 100?mol/L CoCl₂ for 8 hours.2.3 The expression of miR-92 a was detected by Real-time PCR after 8 hours induced by 100?mol/L CoCl₂.3 Influence of YC-1 pretreatment to TFPI-2 expression after hypoxia in SH-SY5 Y cells 3.1 SH-SY5 Y cells were pretreated with 10?mol/L or 100?mol/L YC-1 for 2 hours.Then the cells were co-treated with 100?mol/L CoCl₂ for 8 hours.EDU was used to detect the proliferative activity of each group.3.2 The protein expression of TFPI-2,HIF-1? and VEGF in different groups was detected by Western blot.Results:1 Hypoxia induce changed the morphology of cells and decrease cell survival rate.The cell morphology changed after hypoxia,with neurites thinner and shorter,nucleolus swelling.With the increase of hypoxia time and CoCl₂ concentration,cells crimped and apoptosis was observed.As MTT assay showed,Cell viability decreased with the increase of hypoxia time and CoCl₂ concentration.2 CoCl₂-induced hypoxia can decrease mi R-92 a level in SH-SY5 Y cells,and increase mRNA and protein expression level of TFPI-2.As Western blot showed,the protein expression of HIF-1? and VEGF began to increase at 2 hours after Co Cl₂ treatment,and maintained stable at 6-8 hours.CoCl₂ treatment could effective active HIF-induced hypoxia pathway,establishing hypoxia cell model.The protein expression of TFPI-2 increased at 4hours after CoCl₂ treatment?P<0.05?.HIF-1? and TFPI-2 expression gradually rised with the hypoxic time prolonged.Their variation tendency was similar.Real-time PCR showed that the mRNA expression of TFPI-2 and VEGF was amplified after hypoxia,whereas the level of miR-92 a was declined?P< 0.05?.3 YC-1 pretreated could attenuate the inhibition of CoCl₂ on cell proliferation,and reduced expression level of TFPI-2 in SH-SY5 Y cells after hypoxia.Cell proliferation was decreased after hypoxia,and YC-1 pretreated increased cell proliferation compared with the hypoxia group detected by EdU assay?P< 0.05?.As Western blot showed,compared with the hypoxia group,the increase expression of HIF-1? was suppressed in YC-1 pretreatment group.Compared with the hypoxia group,the protein expression of TFPI-2 and VEGF decreased in a concentration-dependent manner?P< 0.05?.Conclusion:In this study,SH-SY5 Y cells hypxia model induced by cobalt chloride was successfully established.We confirmed that hypoxia can increase he mRNA and protein expression of TFPI-2,and that maybe regulated through the HIF-1—VEGF pathway.