Studies On Effects Of HDACs And WT1Gene On Apoptosis And Development-Related Gene Expression In Swine Testis Cells And Porcine Kidney Cells

Hsitone acetylation was firstly discovered as an epigenetic modification, whichhas a close relationship with the level of acetylation and the gene expression. Theresearch on the histone acetylase (HATs) and histone deacetylase (HDACs) make it abreakthrough in understanding the role of the histone acetylation. The acetylation isrelated to the activation of gene transcription while the deacetylation has connectionwith the inhabiting gene transcription. The histone deacetylase in mammals can bedivided into three categories according to homology yeast. TypeIandIIare quitesensitive to the inhibitors trichostatin A (TSA). TypeIII requests the NAD+as acofactor. Studies have showed that the inhibitors trichostatin A (TSA), the SAHAand the valproate perform as the histone acetylation inhibitors all can increase thelevel of histone acetylation. The scriptaid is a novel histone deacetylase inhibitor,which can induce the hyper histone acetylation. More importantly, the scriptaid isless poisonous to cells compared with the TSA. Hence it is widely used in theresearch related to the cell functions.Currently, the research on the histone deacetylase mainly focuses on theapplication in the cancer. The study of the histone deacetylase applied in the urinary,reproductive system is quite little;especially the study on influence of the HDACs onthe mammalian testis growth has not been reported. Some research showed that theinhibiting of TypeIHDACcan lead to the kidney’s developmental block and cellapoptosis in mice. Pigs in anatomy, physiology and metabolism is very close tothose in human, and drug metabolism, organ size and function are also similarbetween pigs and human, which are used to carry on the research animal xenograft transplantation test. This study was designed to test the expression level of HDAC1gene and HDAC2gene by using scriptaid with different concentrations to treat swinetestis cells and porcine kidney cells, investigate the influence on the urogenital cellproliferation and apoptosis, research the impact of HDAC1and HDAC2geneexpression on the gene expression in kidney and reproductive system.Wilms’s tumor gene1mainly expressed in human embryonic urogenital systemwas found when perform the molecular biology research on the hereditary renaltumors.TheWT1gene expresses lower in mature kidney cells. In earlier study, downregulating WT1gene can inhibit both urogenital cell growth and apoptosis gene. Thisstudy found that histone deacetylase inhibitors scriptaid also can inhibit the renal andtesticular apoptosis and growth gene. This studyproposed hypothesis that the histonedeacetylase inhibitors andWT1gene have a synergistic effect on testicular cells andcarry out the experience to verify the hypothesis. The mainresults of this experimentare as follows:1. After treating the swine testicular cells (ST) by scriptaid, the expression ofHDAC1gene and HDAC2gene in TypeIHDAC showed a downward trend with thescriptaid concentration increased. The increase the concentration of scriptaid alteredthe ST cell shape. It is proved that the scriptaid can inhibit the cell proliferation bythe MMT method. The cell apoptosis was tested by the flow cytometry, showing thatST cell would apoptosis with the scriptaid concentration increasing. The fluorogenicquantitative PCR tested the change of the gene expression related to ST cellapoptosis, showing that scriptiad can lead to the anti-apoptotic Bcl-2genedown-regulated, while the expression of pro-apoptotic Bax gene up-regulated,indicating the histone deacetylase maintain the balance of apoptotic gene expression,keeping the ST cells activity.2. In porcine kidney cells (PK), the cells were treated with differentconcentrations of scriptaid, showing the down regulating ofHDAC1and HDAC2gene expression. In BrdU cell immunofluorescence staining, the cells proliferation inthe treated group were significantly inhibited, the expressions of related development gene SF1, GNDF, SRY are also inhibited, the apoptosis-related genes are alsoaffected. Scriptaid can lead to the anti-apoptotic Bcl-2gene down-regulated, whilethe expression of pro-apoptotic Bax gene up-regulated.3. The experiment in this paper showed that he expression level of WT1genedecreased while the expression level of Histone H4increased by treating the ST cellswith different concentrations of scriptaid, which was tested by fluorogenicquantitative PCR.The histone deacetylase inhibitors can increase histone acetylationof ST cells and inhibit the expression of WT1gene. Down-regulated of WT1geneexpression can cause urinary and reproductive system development related geneexpression was significantly down regulated, that histone deacetylase inhibitors mayinhibit the expression of WT1gene and expression of growth related genesdown-regulated in ST cells.