Effects Of MiR-204 On CPB2 Toxin-induced Apoptosis And Inflammatory Response In IPEC-J2 Cells By Targeting BCL2L2

Piglet diarrhea is a common intestinal infectious disease in pig production,which seriously affects the healthy development of the pig industry.Clostridium perfringens type C(C.perfringens type C)is one of the common bacterial pathogens that cause diarrhea in young piglets.It mainly produces α and β toxins that damage the intestinal health of piglets and cause diarrhea in piglets.In recent years,although commercial vaccines and antibiotics can prevent and treat piglet diarrhea to a certain extent,with the appeal of green non-antibiotic breeding,new challenges have been raised to the prevention and treatment of piglet diarrhea.Therefore,from the perspective of genetics,carrying out research on disease resistance breeding has become an important means to prevent and treat piglet diarrhea.Micro RNAs(miRNAs)are a class of non-coding small RNA molecules with the length about 22 nt that can regulate gene expression at the transcription or post-transcriptional level and participate in the regulation of various biological processes.In our early study,we established 7-day-old landrace × large white crossbred piglets infected with C.perfringens type C resistant and susceptible individuals.Then we used high-throughput sequencing technology to perform small RNA sequencing on piglet ileum tissues and the results revealed that ssc-miR-204 was significantly differentially expressed between the resistant group and the susceptible group,and was highly expressed in the susceptible group.It was hypothesized that miR-204 might play an important regulatory role in C.perfringens type C infection in piglets.Therefore,this study took miR-204 as the research object,the intestinal porcine epithelial cells(IPEC-J2)were treated with Clostridium perfringens beta2(CPB2)toxin.A various of experimental techniques such as qRT-PCR,Western Blot,CCK-8,Ed U staining,flow cytometry,lactate dehydrogenase(LDH)activity detection,double luciferase reporter gene assay,overexpression and RNA interference etc.were used to preliminary exploration the regulation role of miR-204/BCL2L2 regulatory axis in piglet infection with C.perfringens type C.The main results are as follows:1.miR-204 expression patterns and functional study: miR-204 was significantly up-regulated expression in the susceptible group ileum tissues of piglets infected with C.perfringens type C(P<0.01);the tissue expression profiles showed that miR-204 was significantly highly expressed in the kidney,liver,thymus,lymph and ileum of piglets.miR-204 was also significantly up-regulated expression after CPB2 toxin treatment in IPEC-J2 cells(P<0.01).Compared with the negative control group,miR-204 mimic inhibited the proliferation,promoted apoptosis,increased LDH activity,increased the expression of pro-inflammatory factors TNF-α,IL-6,IL-8 and IL-1β,and also down-regulated Bcl2 protein expression while up-regulating Bax protein expression of IPEC-J2 cells.2.miR-204 bioinformatics analysis and targeting BCL2L2 gene relationship validation:miR-204 target genes prediction were performed using Target Scan,miRDB and Pic Tar softwares,and 114 common target genes were obtained.miR-204 target genes could be significantly enriched in 15 GO functions and 13 KEGG signaling pathways.Bioinformatics prediction revealed binding sites between the miR-204 mature sequences in seed region and the 3’UTR region of BCL2L2 gene.Double luciferase reporter gene assay confirmed that miR-204 may directly target with BCL2L2 gene.Further detection by qRT-PCR and Western Blot revealed that miR-204 could negatively regulate the expression of BCL2L2 gene.3.miR-204 participates in CPB2 toxin-induced IPEC-J2 cell apoptosis and inflammatory response by targeting BCL2L2 gene: Firstly,the effects of BCL2L2 gene knockdown and overexpression on CPB2 toxin-induced apoptosis and inflammatory response in IPEC-J2 cells were analyzed.The results showed that knockdown of BCL2L2 promoted apoptosis,increased LDH activity and inflammatory factors expression in IPEC-J2 cells,while overexpression of BCL2L2 showed the opposite expression trends.In addition,co-transfection of pcDNA3.1 and pcDNA-BCL2L2 with miR-204 mimic revealed that overexpression of BCL2L2 attenuated the apoptotic and inflammatory responses caused by miR-204 mimic.In summary,after CPB2 toxin treatment of IPEC-J2 cells,up-regulated expression of miR-204 promoted apoptosis and inflammatory response of CPB2 toxin induced IPEC-J2 cells by directly targeting BCL2L2.miR-204 and its target genes may play an important role in the process of C.perfringens type C infection leading to diarrhea in piglets.The results of this study may provide a basis for the action mechanism of miRNAs in the regulation of C.perfringens type C piglet diarrhea.