Cloning Of Danio Rerio Hdac8 Gene And Its Roles In Zebrafish ZF4 Cells

The results of pre-laboratory studies showed that the expression of zebrafish epithelial enzymes changed to different degrees under low-temperature stress conditions,and the expression of genes such as hdac8 and kdam4A was reduced in ZF4 cells of zebrafish after low-temperature treatment compared to the control group.This may imply that the regulation of epigenetic mechanisms plays a crucial role in the adaptation of fish to stressful environments such as low temperatures.Histone deacetylation enzymes?HDACs?,as an important member of the epigenetic class of enzymes,can play an important role in cellular transcriptional regulation by deacetylating endonuclear histones,HDAC8 is a Zn²⁺-dependent class I HDAC enzyme with a size of 42 k Da and consists of 377 amino acids^[1],which can be easily identified from other HDAC enzymes because of its unique structure and functional specificity^[2].Recent studies on HDAC8 have mostly focused on its role as a therapeutic target in pathophysiology,and it has been reported that RNA interference?RNAi?-mediated down-regulation of HDAC8 helps to inhibit the proliferation of human cervical,colon and lung cancer cell lines,while up-regulation of HDAC8 promotes the proliferation of hepatocellular carcinoma cells and inhibits apoptosis^[3].It has been found that HDAC8 enzyme activity also has an important effect on wild-type and mutant p53 expression^[4],and it has also been found that decreased or inhibited HDAC8 expression can affect the proliferation of cells with p53 mutations,a phenomenon that clearly points to the effect of HDAC8inhibitors on p53 mutation carrier tumors^[1].However,little research has been done on the role of HDAC8 in fish growth development or stress tolerance.This experiment is expected to observe what changes occur in ZF4 cells under normal growth conditions by knocking down the hdac8 gene in Zebrafish ZF4 cells,and by analyzing these changes to find the function of the hdac8 gene in fish adapted to low temperature and other stressful environments,thus laying the foundation for the study of the epigenetic mechanism of fish adapted to low temperature and other stressful environments.We designed and synthesized sh RNA for zebrafish HDAC8,constructed two recombinant plasmids,pLKO.1-hdac8-sh RNA and pLKO.1-negative control?nc?,using EcoRI and AgeI,and co-transfected them into HEK-293T cells with lentiviral packaging plasmids p CMV-d R8.91 and p CMV-VSVG,respectively.We collected HEK-293T cells packed with virus and infected them with zebrafish ZF4 cells,and after 24 h of incubation at 28°C in an incubator containing 5%CO₂,cellular RNA was extracted and RT-q PCR assay showed successful knockdown of hdac8 in ZF4 cells.Subsequently,we examined ZF4 cell viability using Taipan Blue staining and further examined the expression of apoptosis-related genes bax,bcl2,and caspase-3.bax and caspase-3proteins have a positive effect on apoptosis and their elevated expression increases the level of apoptosis in cells.In contrast,elevated Bcl2 protein expression inhibits apoptosis.Meanwhile,we cloned the full-length cDNA of zebrafish hdac8 and constructed the overexpression plasmid of zebrafish hdac8 using the pLVX-IRES-NEO plasmid with EcoRI and Xba I digestion and transfected it into HEK-293T cells to detect its expression.By Western Blotting experiments we found that the overexpressing plasmid of hdac8 was efficiently expressed in HKE-293T cells,and the plasmid we constructed can be used for subsequent experiments.It was found that normal expression of hdac8 plays an important role in the growth of ZF4 cells and the survival of cells knocking down hdac8 was significantly reduced compared to control?nc?cells?p<0.05?.At the same time,we detected that the expression of both the intracellular apoptosis-related genes bax and caspase-3 were significantly up-regulated in knockdown hdac8 compared to the control group,suggesting that the increase in cell death in knockdown hdac8 may be caused by up-regulating the level of apoptosis.In conclusion,normal expression of hdac8 is essential for maintaining intracellular homeostasis,and its regulatory effect on the level of apoptosis may play an important role in adaptation under various stresses such as low temperature.The finding that hdac8has a regulatory effect on the level of apoptosis in zebrafish provides a basis for later studies related to apoptosis in zebrafish and provides new ideas for the study of the mechanism of fish adaptation under stressful environments.