Ctenopharyngodon Idella P53 Mediates Between NF-?B And PKR At The Transcriptional Level

As an important tumor suppressor,p53 is also an important transcriptional regulator that can exert its different physiological functions,including apoptosis,cell cycle regulation and anti-virus,etc.,by regulating a series of different target genes.In addition,NF-?B is an important transcription factor that can regulate a series of gene expression involved in cell proliferation,differentiation,inflammation,innate immunity and apoptosis and other physiological processes.PKR is an important protein in the innate immune antiviral signaling pathway,and the mechanism is thoroughly understood.Furthermore,the role of p53 in innate immunity has also been gradually reported in fish.However,there are few reports on the correlation between p53,NF-?B and PKR.Therefore,this dissertation mainly aims to study the transcriptional regulation of p53 mediates between NF-?B and PKR,which further demonstrated the importance of p53 in innate immune response in fish.Here,we cloned and characterized the full length of cDNA sequence of grass carp(Ctenopharyngodon idella)p53(Cip53)and its promoter sequence.The full length of Cip53 cDNA is 1879 bp along with 146 bp of 5'UTR,617 bp of 3'UTR and the largest open reading frame(1116 bp)encoding a poly-peptide of 317 amino acids with a well conserved DNA binding domain(DBD)and a oligomerization domain(OD)in C-terminal.Then,we cloned the 991 bp sequence located at the upstream region of transcriptional start site as the promoter sequence,which contains one putative Rel-A,IRF3,XBP-1 and p53 binding sites.In addition,we analyzed the expression of Cip53 in different tissues(brain,eyes,skin,gill,spleen,kidney)and CIK cells under poly I:C stimulation by qRT-PCR.The results showed that Cip53 was ubiquitously expressed in all tested grass carp tissues and was up-regulated in 6h,24 h and 48 h.And the expression of Cip53 in CIK cells was significantly up-regulated under stimulation with poly I:C after 6h.To further understand the roles of fish p53 in the transcriptional control of NF-?B and PKR,gel mobility shift assays were employed to analyze the interaction of Cip65 protein with the promoter of grass carp Cip53 and Cip53 protein with Ci PKR promoter.The results demonstrated the high affinity interaction between Cip65 and Cip53 promoter.Similarly,Cip53 b ound to Ci PKR promoter with high affinity.In conclusion,these results indicate d that Cip65 can regulate the transcription of Cip53,and Cip53 is the potentia l regulator of CiPKR.Furthermore,we constructed the luciferase reporter gen e recombinant plasmid of pGL-Cip53 with pcDNA3.1-Cip65 and pGL-Ci PKR with pcDNA3.1-Cip53.The dual-luciferase reporter assays showed that Cip65 a ctivates Cip53 promoter and Cip53 activates Ci PKR promoter,respectively.To further investigate the role of p53 in p65-p53-PKR transcription pathway,siRNA was designd to decrease mRNA level of p53.When Cip53 was knockdown in CIK cells,the mRNA levels of Cip65 and CiPKR were decreased.It was also shown that p53 is involved in the antiviral transduction pathway mediated by NF-?B and PKR.Taken together,by analysing the transcriptional mechanism of Cip53,we consider that p53 plays pivotal roles in the immune response.