| In modern large-scale pig production,oxidative stress is an important factor affecting the healthy growth of weaned piglets.As an important organ of oxidative stress,the structure and function of the intestine have an important impact on the growth performance of piglets.Oxidative stress can affect the expression and regulation of related circRNA,miRNA and genes in the piglet's intestine,causing cell oxidative damage,affecting DNA synthesis,and reducing the growth performance of piglets.As a new type of non-coding RNA,circRNA is stable in cells and is not easily degraded.It can also bind miRNA through competition to regulate the expression of its target genes.The role of circRNA in the mechanism of intestinal oxidative stress in weaned piglets is still unclear.Therefore,in this study,Landrace piglets were used as the research object.The oxidative stress model of piglets was established by intraperitoneal injection of Diquat(DQ,Diquat),and a large number of circRNA,miRNA and mRNA related to oxidative stress were discovered through transcriptome sequencing.Subsequently,we used DQ to induce small intestinal epithelial cells(IPEC-J2)to construct an oxidative stress cell model,and used the cell model to test the ce RNA regulatory relationship of circGLI3-miR-339-5p-VEGFA.The specific results are as follows:(1)The oxidative stress response induced by DQ destroyed the intestinal structure of weaned piglets.The height of small intestinal villi of weaned piglets in the oxidative stress group was significantly reduced,and the depth of crypts was significantly increased;the superoxide dismutase(superoxide dismutase,in serum of piglets in the oxidative stress group)SOD),glutathione peroxidase(GSH-PX)and total antioxidant capacity(T-AOC)were significantly lower than the control group,diamine oxidase(DAO)The content of malondialdehyde(MDA)was significantly higher than that of the control group(P<0.01).(2)Transcriptomics analysis of piglet jejunum mucosa.Circular RNA sequencing of 6samples were completed.After sequencing quality control,a total of 133.70 Gb Clean Data was obtained.The Clean Reads of each sample was sequenced with the designated reference genome.The comparison efficiency ranged from 99.98% to 99.99%.A total of 2559 circular RNAs were detected.Using |log2(Fold Change)| ?2.0 and FDR <0.05 as the criteria for screening differential circRNAs,751 differentially expressed circular RNAs were screened,of which 432 were up-regulated and 319 were down-regulated;Through the GO enrichment analysis of genes derived from differential circular RNAs,the GO enrichment analysis of genes derived from differentially expressed circRNAs mostly focuses on the cellular processes,metabolic processes,biological regulation and stimulus responses of biological processes,and the related molecular functions are mainly Catalytic activity and protein binding;KEGG enrichment analysis shows that the enriched signal pathways are RNA degradation,protein synthesis and oxidative phosphorylation in the endoplasmic reticulum,etc.;the small RNA sequencing of 6 samples was completed,and a total of 201.39 M Clean Reads were obtained.2708 miRNAs were detected.Using |log2(Fold Change)| ?1 and FDR<0.01 as the screening criteria,43 differentially expressed miRNAs were screened,of which 19 were up-regulated miRNAs and 24 were down-regulated miRNAs.GO analysis of the target genes of differentially expressed miRNAs indicated that most differentially expressed genes are enriched in cellular processes,metabolic processes,stimulus responses,immune system processes,catalytic activity,and protein binding;the results of KEGG annotations for differentially expressed miRNAs target genes show that,the main enrichment signal pathways for target genes are peroxisomes,endocytosis,protein synthesis in the endoplasmic reticulum,spliceosomes,ubiquitin-mediated proteolysis and RNA transport.In addition,the mRNA sequencing of 6 samples was completed,and a total of 133.69 Gb Clean Reads were obtained.The Clean Reads of each sample reached 16.02 Gb,and the Q30 base percentage of each sample was not less than 93.24%.The comparison efficiency of Reads of each sample with the reference genome was from 94.31% to 95.65%.A total of 164 expressed mRNAs were screened,including 60 up-regulated genes and 104 down-regulated genes.GO enrichment analysis of differential genes revealed that the differential genes were mainly annotated to cell processes,nitrogen compound metabolism processes,biological regulation,catalytic activity,and cells Internal protein synthesis.Further KEGG enrichment analysis was performed on the pathways where the differential genes are located.The pathways with a higher percentage of enrichment are glutathione metabolism,pentose and glucuronate conversion,amino acid biosynthesis,carbon metabolism and DNA copy.In addition,in the expression levels of 11 circRNAs,13 micro RNAs and 10 mRNAs among the identified differential genes were verified by real-time fluorescent quantitative PCR(qRT-PCR),and the results were basically consistent with the trend of high-throughput sequencing results.After the treatment of DQ,miR-339-5p was expressed in the jejunum,ileum,spleen and liver tissues of Landrace piglets and Dapulian piglets,and there were significant differences between the control group and the treatment group.Moreover,the expression levels in the jejunum,ileum and spleen tissues of Landrace and Dapulian pigs were significantly lower than those of the control group(P<0.01);VEGFA was expressed in the jejunum,ileum,and ileum of Landrace piglets and Pulian piglets.It was expressed in both spleen and liver tissues;moreover,the expression in the ileum and jejunum of Landrace piglets was significantly up-regulated,and it was significantly up-regulated in the ileum and spleen tissues of Dapulian piglets(P<0.05).(3)The IPEC-J2 cell oxidative stress model was successfully constructed.After treated with IPEC-J2 cells,it was determined that the concentration of 50% survival rate of IPEC-J2 cells was 250.7 ?mol/L DQ.Standards for oxidative stress.At the same time,it was observed through an optical microscope that the DQ treatment destroyed the integrity and tight connectivity of IPEC-J2 cells,which caused the original diamond-shaped cells to change to a round shape,and they floated off the wall.(4)Overexpression of circGLI3 can promote the proliferation of IPEC-J2 cells,increase the proportion of S-phase cells(P<0.01),and reduce the generation of ROS after IPEC-J2 is subjected to oxidative stress;at the same time,circGLI3 can promote the increase of GSH-PX activity and increase T-AOC level in IPEC-J2 cells reduces the MDA content in IPEC-J2 cells,and reduces the levels of intracellular inflammatory factors IL-2,TNF-? and DAO,thereby reducing oxidative damage.Overexpression of miR-339-5p can inhibit the proliferation of IPEC-J2,cause G0/G1 phase arrest of IPEC-J2 cells,and reduce the proportion of cells in S phase;in addition,miR-339-5p can cause SOD and GSH-PX activity significantly decrease,reduce the total antioxidant capacity of IPEC-J2 cells,promote a significant increase in MDA content,and increase the levels of intracellular inflammatory factors IL-2,IL-6,IL-1?,TNF-?and DAO,thereby exacerbating oxidative damage.(5)The target genes of circGLI3 are predicted by RNAhybrid and Target Scan to be miR-339-5p,and the target genes of miR-339-5p was VEGFA;the construction of a dual luciferase reporter gene vector successfully verified that circGLI3 can bind miR-339-5p,Mi R-339-5p targets the 3'UTR region of VEGFA.Overexpression of circGLI3 can down-regulate the expression of miR-339-5p(P<0.01),and significantly up-regulate the expression of VEGFA gene and protein;inhibiting the expression of miR-339-5p can also significantly up-regulate the expression of VEGFA;co-transfection of circGLI3 Overexpression vector and miR-339-5p mimics can reduce the inhibitory effect of miR-339-5p mimics on the proliferation of IPEC-J2 cells;co-transfection of circGLI3 overexpression vector and miR-339-5p mimics can reverse miR-339-5p mimics To reduce the proportion of S phase cells of IPEC-J2,while reducing the proportion of cells in G0/G1 phase;co-transfection of circGLI3 overexpression vector and miR-339-5p mimics can also reverse the effect of miR-339-5p mimics on IPEC-J2 cells The increase of ROS content.Subcellular co-localization of circGLI3 and miR-339-5p,miR-339-5 is located in the nucleus and cytoplasm,and the area where circGLI3 functions is mainly in the cytoplasm.In summary,we have constructed an individual oxidative stress model for Landrace piglets and a cellular oxidative stress model for IPEC-J2,and found that oxidative stress significantly disrupted the piglet's intestinal structure and the redox balance in the body,leading to abnormal expression of circRNA,miRNA and mRNA in jejunal mucosal tissue.According to the results of high-throughput sequencing and the prediction analysis of RNAhybrid and Target Scan,we constructed a ce RNA regulatory network composed of circGLI3,miR-339-5p and VEGFA,and verified that circGLI3 regulates regulation by directly binding miR-339-5p.The expression of target gene VEGFA in turn affects IPEC-J2 cell proliferation,cell cycle,ROS content,and changes in the levels of antioxidant enzymes and inflammatory factors related to oxidative stress.This study reveals the ceRNA molecular mechanism of oxidative stress in the intestinal tract of piglets,and provides a theoretical basis for further research on the effect of oxidative stress on intestinal function. |
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