Regulation Of SZNF Expression And Genes Regulated By SZNF Related To Cyadox

Cyadox, as the new variety of quinoxaline, has the advantages of low toxic and side-effect, high security, fast absorption eliminated quickly and residue-free, while especially shows the excellent effects in antibacterial activity and growth promotion, our lab have finished various experiments on cyadox, but its pharmacological mechanism still remains unclear. In2008, our lab obtained8genes related to cyadox pharmacological action from swine liver by using DDRT-PCR, including a transcription regulation factor-zinc finger(SZNF). SZNF, as the member of CCHC type zinc finger family, can regulate gene expression on the post-transcriptional level through combination to mRNA of target genes. We speculate that cyadox could probably promote growth and display antibacterial activity mediated by SZNF through regulating gene expression on the post-transcriptional level. Thus, research on SZNF is significantly important to show the regulation network and mechanism of cyadox. The preliminary research on SZNF has finished, but how cyadox regulates the expression of SZNF and the physiological functions of SZNF, as well as the tissue distribution, are under clarified. This research designs as follows:.1. The related signalling pathways of SZNF expression induced by cyadox in PK-15cellsFirstly, the signaling pathways related to SZNF expression induced by cyadox in PK-15cells have been studied in order to understand the mechanism of SZNF expression.The time-effect and dose-effect relationship of SZNF expression induced by cyadox in PK-15cells were detected by real time quantitative PCR. The result indicated that the up-regulation of SZNF expression induced by cyadox showed low-dose with the long-time effect and high-dose with the short-time effect, and SZNF mRNA arrived the peak level when2μM cyadox had incubated PK-15cells for4hours. After PK-15cells incubated by cyadox and the specific inhibitors of single pathways, the JNK, NF-κB and JAK2/STAT5B single pathways were demonstrated as the primary regulation pathways of SZNF expression induced by cyadox through RT-qPCR and Western blot technology, while the PI3K/Akt and P38single pathways were the secondary ones. When exploring the relationship between time and space of these single pathways, the TAKI/NF-κB and JAK2/STAT5B single pathways had been activated when PK-15cells were incubated by cyadox for1hour,2hours later, myD88/TAK1&ASK1/JNK&NF-κB and JAK2/PI3K/Akt/NF-κB single pathways joined in to transfer the cell singles into nucleus and up-regulated the SZNF expression by cooperating with the previous ones.2. The related signalling pathways of SZNF expression induced by cyadox in swine liver cellsIn order to understand the mechanism of SZNF expression in swine liver cells, the signaling pathways related to SZNF expression induced by cyadox have been studied. When detecting the time-effect and dose-effect relationship of SZNF expression induced by cyadox in swine liver cells, the result demonstrated that the trend of SZNF expression were still consistent. Through RT-qPCR and Western blot technology, JAK2/STAT1, PI3K/Akt, TGF-β/Smad3and P38single pathways had been found to play a primary roles in the regulation of SZNF expression induced by cyadox in swine liver cells, while the JNK and NF-κB single pathways played an additional role. In addition, the JAK2/STAT1, JAK2/PI3K/Akt and PI3K/Akt signal pathways activated when swine liver cells had been incubated by cyadox for0.5h, and the TGF-β/Smad3, TGF-β/PI3K/Akt and myD88/TRAF2/TAK1&ASK1/P38signal pathways joined in at1h later. These signal pathways cooperated with each other to transfer the regulation signals with the help of the TGF-(3/JNK1/2signal pathway, and finally raised the expression of SZNF.3. The mRNA tissue expression profile of SZNF in swine in vivo The SZNF mRNA expression level of different swine tissues had been detected by RT-qPCR technology in order to obtain the tissue expression profile of SZNF. The result showed that SZNF in central nervous system were the highest, such as hypothalamus, pituitary, bone marrow and so on; and SZNF were much higher in the tissues which have strong secretory function, such as adrenal, than in the liver, duodenum and kidney, etc.4. The research on the nucleotide sequence binding with SZNF by RIP-Seq technologyIn order to obtain the nucleotide sequence binding with SZNF and its biological function, we had finished the high-throughput sequence detection on the nucleic acid binding with SZNF by RIP-Seq technology. The results demonstrated that118differential genes had been detected in blank PK-15cells, while87differential genes from the PK-15cells which had been incubated by cyadox. In addition, there were45differential genes which combined with SZNF increasingly induced by cyadox versus blank sample, while93differential genes which combined with SZNF decreasingly. SZNF was found to be more easily combined with nucleic acid full of G or C/T through the SZNF binding motif detect.12differential genes from RIP-Seq results were verified by RT-qPCR technology, and found the RIP experiment was repeatable and the data from sequencing were reliable. After that, through go ontology analysis of differential genes from RIP-Seq, SZNF were demonstrated to participate in several physiological processes, such as cell proliferation, differentiation, inflammatory reaction, the cell cycle and so on. Meanwhile cyadox could regulate some genes to express mediated by SZNF in the post-transcriptional level, which were related to growth, MLXIP, CKS2for example, and some ones related to inflammatory reaction, LGALS3for example, finally showed its antibacterial activity and growth promotion function.In conclusion, we have originally detected the molecular mechanism of SZNF expression and its biological function in PK-15cells and swine liver cells, also the first time for the SZNF tissue expression profile. The results showed that the expression of SZNF were regulated by JAK/STAT signaling pathway in both types cells; and SZNF was highly expressed in central nervous system and the tissues full of secretion function. In addition, SZNF could participate in some of physical process, including proliferation, differentiation and inflammatory reaction by binding with mRNA of target genes. Further more, this research originally studied the molecular mechanism of drug action of cyadox in PK-15cells and swine liver cells. We have demonstrated that cyadox could up-regulate the expressions of MLXIP, CKS2, etc, and down-regulate the expressions of Bax, etc in the post-transcriptional level by up-regulating the SZNF level, and finally showed its pharmacological effects.