The Regulation And Mechanism Analysis Of Transcription Factor KLF4 On The Porcine Epidemic Diarrhea Virus Resistance

Porcine epidemic diarrhea virus(PEDV)is the main pathogen for viral diarrhea in pigs.It is the premise and urgent demands to identify functionally resistant genes and regulatory elements and elucidate the underlying mechanisms for establishing novel strategies from the perspective of host for preventing PEDV infection.In the early stage,our team had conducted transcriptome sequencing on mRNA level of the jejunum tissues of PEDV-infected piglets and normal piglets and obtained multiple differentially expressed genes(DEGs).Based on biological functions and preliminary verification,we found that transcription factor KLF4 plays a role in the process of PEDV infecting porcine small intestinal epithelial cells(IPEC-J2).However,the specific mechanism is remained unclearly.In the study,we focused on porcine intestinal epithelial cell line(IPEC-J2).Hence,the mRNA level of KLF4 in IPEC-J2 infected with PEDV at different time was measured.The cell line of KLF4 knockout was built and was used to investigate the effect of KLF4 on IPEC-J2 basic functionality and the replication of PEDV.The SNPs in the KLF4 promoter were identified and analyzed to reveal the molecular mechanism of KLF4 gene expression.The RNA-seq was conducted to analyzed gene expression alterations in KLF4 gene knockout,wild type cells and infected with PEDV,and screened genes targeted by KLF4.Finally,the key genes selected was verified,the KLF4 gene regulatory network was constructed.The results of this study were shown as follows:1?The mRNA expression level of KLF4 gene in IPEC-J2 cells infected with PEDV at different time(12,24,48 and 72 h)were analyzed using qRT-PCR.The result showed that the KLF4 gene expression was significantly up-regulated in IPEC-J2 after infected with PEDV,and show a rise trend.Interestingly,the protein level of KLF4 was similar to its gene expression by western blot.KLF4 gene knockout IPEC-J2 cell was used to investigate the effect on the cell proliferation and the alteration of cell cycle protein.Our results indicated that the cell viability of KLF4 knockout cell was significantly decreased(P<0.01)after incubated 12,24 and 48 hours than that of wildtype.And the population of IPEC-J2 cells in G1 phase was significantly increased(P<0.01),and that of in S phase was dramatically decreased(P<0.01).The protein expression of CDK4 and PCNA was down-regulated,and the protein expression of P21 was up-regulated significantly.We also detected the PEDV copies of KLF4 gene knockout cells and wildtype infected with PEDV at different times by qRT-PCR.Our found that the PEDV copies have no difference at the 2,4,6 hours infected with PEDV.Interestingly,our results also showed that KLF4 gene knockout induced a significant increase in PEDV copies of IPEC-J2 cells infected with PEDV at 12 hours(P<0.01)and 24 hours(P<0.05).Moreover,the core promoter region of KLF4 gene in pigs was identified,and PEDV infection significantly increased the KLF4 gene promoter activity(P<0.01),C-base insertion mutations at positions-195 and-217 in the promoter region of KLF4 gene in Large White pigs were found,base insertion resulted in a significant up-regulation of promoter activity(P<0.01),and after PEDV infection,the mutant promoter activity was still significantly higher than that of wild type(P<0.01).2?Transcriptome sequencing was performed on PEDV-infected KLF4 knockout cells,wild-type cells,and virus-uninfected knockout and wild-type cells for gene differential expression analysis between experimental groups(KLF4 knockout cells vs wild-type cells;PEDV-infected KLF4 knockout cells vs PEDV-infected wild-type cells).A total of 4246 genes were identified that were differentially expressed between the KLF4 knockout and wildtype groups(P<0.05),among which the expression of 1619 genes was up-regulated and that of 2627 genes was down-regulated;A total of 2295 genes were identified that were differentially expressed between the KLF4 knockout and wildtype cells infected with PEDV(P<0.05),among which the expression of 1372 genes was up-regulated and that of 923 genes was down-regulate.The differentially expressed genes selected from virus-uninfected KLF4 knockout and wild-type IPEC-J2 cells were mainly enriched in GOterms such as positively regulated lipid localization,regulated of lipid transport,organic hydroxy compound transport,extracellular space and gated channel activity,and were mainly involved in signaling pathways such as hematopoietic cell lineage and steroid biosynthesis.The differentially expressed genes selected from PEDV-infected KLF4 knockout and wild-type IPEC-J2 cells were mainly enriched in GOterms such as protein binding,response to cytokine,immune system process and apical part of cell,and were mainly involved in signaling pathways such as cytokine-cytokine receptor interaction and intestinal immune network for IgA production.Furthermore,important candidate genes including,ACOD1 C3?CD59?IL-19?IL1A?MX1?OAS1.RIPK3 and TLR7 were screened based on gene biological function.The RT-qPCR was conducted to verify the transcriptomic analysis results.We confirmed that these genes expression trends were consistent with transcriptome sequencing results.3,We detected the expression differences of TLR3,TLR7,TLR8 and TLR9 genes before and after PEDV infection of IPEC-J2 cells,the expression levels of the above genes were significantly up-regulated after PEDV infection(P<0.01),while in PEDV-infected KLF4 knockout IPEC-J2 cells,the expression levels of the above genes were significantly lower than those in PEDV-infected wild-type IPEC-J2 cells(P<0.01).The interrelationship between KLF4 and TLRs was predicted by String software,and the results showed that KLF4 could act directly on TLR9 and generate a regulatory network acting on other TLRs through TLR9.The expression differences of MyD88,MAP3K7 and TAB2 genes were verified for the differentially expressed genes selected by genes involved in the regulatory network combined with transcriptome sequencing,and they were found to be consistent with the expression trend of TLR9 and other genes.It was speculated that KLF4 gene may affect the secretion of cytokines and ultimately affect cell resistance by affecting the signal transduction of TLRs and reducing the expression of MyD88 and other genes.