The Study On The Mechanism That HDAC Regulates MKK7 Expression And Glioma's Invasion

Objective:1.To explore whether MKK7 or MKK4 regulates the activity of JNK-c-Jun pathway;2.To explore whether or not that HDACs regulate the expression of MKK7 or MKK4;3.To explore which member or members of HDACs regulate the expression of MKK7 or MKK4;4.To explore the mechanism that HDACs regulate MKK7 or MKK4;5.To explore whether the intervention of HDACS or the activity of JNK-c-Jun can influence the migration and invasion of glioma;6.To explore whether HDACs` expression is related to the expression of MKK7 or MKK4.Method:1.Use small interfering RNA to knock down MKK7 or MKK4,Western Blot detect the expression of p-JNK/JNK and p-c-Jun/c-Jun.2.Use HDAC inhibitors,TSA,SAHA,M344,VPA to treat different glioma cells,detect the expression of p-JNK/JNK and p-c-Jun/c-Jun.RT-PCR detect the RNA expression of MKK7.3.Use small interfering RNA of HDACs,specific HDAC inhibitors and overexpression of HDACs to treat glioma cells,Western blot detect the expression of MKK7,p-JNK/JNK,p-c-Jun/c-Jun.RT-PCR detect the RNA expression of MKK7.Use the technology of reporter gene to detect the fluorescent value of MKK7.4.Use cell immunochemistry,the separationof cytoplasma and nuclear to detect the distribution of HDACs;.ChIP Assay to detect whether HDACs locate in the promoter of MKK7;Truncation or mutantion of repoter gene to detect the change of MKK7 fluorescein and transcription factor that regulate the expression of MKK7.;small interfering RNA and overexpressing the transcription factor of MKK7,Western blot detect the expression of MKK7 in order to verify it.;CHX?MG132 was used to detect it's effect on MKK7.5.Use Small interfering RNA,overexpression of MKK7 or HDACs,and specific HDAC inhibitors to treat glioma cells,then use Transwell Test to detect the impact of these treating metheds on the migration and invasion of glioma.6.Use inmmunohistochemical staining to detect the expression and the correlation of HDACs and MKK7 in human`s glioma tissue samples.Result:1.It is MKK7 instead of MKK4 that regulates the activity of JNK-c-Jun in glioma.Only after knock down MKK7 can suppress the activity of JNK-c-Jun2.HDACs regulate the activity of JNK-c-Jun by regulating the expression of MKK7 In glioma.After HDAC inhibitors,TSA,SAHA,M344,VPA treated,the expression of p-JNK,and p-c-Jun,MKK7 RNA were suppressed.while there was no change in the expression of JNK and c-Jun.3.HDAC4 regulates MKK7 by transcriptionUse small interfering RNA of HDAC4,specific HDAC4 inhibitors and overexpression of HDAC4 to treat glioma cells,the expression of MKK7,p-JNK p-c-Jun and mRNA of MKK7 were suppressed.As well as the change of MKK7 fluorescein4.The transcription factor SP1 mediates negative regulation of MKK7 in the case that HDAC4 is deactivated.When truncated repoter gene of MKK7 promoter to-149 bp,the expression ofmkk7fluorescein was dubbled as well as SP1 site was mutated before it.Knock down SP1,the expression of MKK7 increased,while,over expressed sp1,the expression of MKK7 was suppressed.Knock down SP1 in the case that HDAC4 is deactivated,,the expression of MKK7 still increased.5.HDAC6 mediates the stability of MKK7.When treated with CHX,MKK7 degraded quickly,enven to be half after two hours.While,MG132 can prevent it.6.Knock down MKK7 suppressed the migration and invasion of glioma cells;suppress the activity of HDAC4/HDAC6 suppress the migration and invasion of glioma cells.Knock down MKK7 the number of cell that passed the cabinet was decreased.(n=3,P<0.05),After suppressed the activity of HDAC4/HDAC6 orJNK-c-Jun,the number of cell that passed the cabinet was decreased(n=3,P<0.05).7.In human`s high-grade glioma samples,the expression of HDAC4/6 is positively-corelated with the expression of MKK7.inmmunohistochemical staining display that he expression of HDAC4/6 is positively-corelated with the expression of MKK7 In human`s high-grade glioma samplesConclution:1.It is MKK7 instead of MKK4 that regulates7.s the activity of JNK-c-Jun in glioma.2.HDAC4 regulates the expression of MKK7 by transcription in glioma cells.3.HDAC6 mediates the stability of MKK7 in glioma cells.4.Transcription factor SP1 mediates the MKK7 negative regulation in the case that HDAC4 is deactivated.5.suppressed the activity of HDAC4/HDAC6 or JNK-c-Jun,can the ability of migration and invasion of glioma cells be reduced.6.The expression of HDAC4/6 is positively-corelated with the expression of MKK7 in human`s high-grade glioma samples.