| Piglet diarrhea is a common intestinal disease in intensive swine production,which seriously affects the healthy growth and economic efficiency of swine industry.As a common pathogen,C.perfringens type C could settle and multiply quickly in the intestines of piglets,release a variety of toxins and damage the intestinal mucosal epithelium and endothelial cells,resulting in piglet diarrhea.CPB2 is one of the key pathogenic toxin produced by C.perfringens type C,which promotes diarrhea and speeds up systemic infections through blood circulation.Currently,it is limited that only from the perspective of vaccines and antibiotics to prevent and treat the piglet diarrhea,so that improving the resistance of piglets to bacteria and toxins on a genetic basis is an effective way to solve piglet diarrhea.LncRNA and miRNA have been considered to play an important role in the regulation of bacterial infection.However,the underlying regulatory function and molecular mechanism in piglet diarrhea infected by C.perfringens type C are still unclear.In this study,a recombinant CPB2 protein was firstly generated by Escherichia coli expression system,and then a damage model of CPB2 treated IPEC-J2 was established.Combined with RNA-seq and q RT-PCR analysis,some differentially expressed lnc RNA were screened,and the regulatory function of lnc RNA ALDB-898(ALDBSCG0000000898)in the process of CPB2-induced IPEC-J2 damage was evaluated by CCK8,Ed U,LDH,ROS,flow cytometry,TUNEL,WB,ELISA and FITC-Dextran 4 assays;The subcellular location of ALDB-898 was detected by the separation of cytoplasmic and nuclear and RNA-FISH assays;The ce RNA network of lnc RNA/miRNA/m RNA was constructed through the analysis of RNA-seq and prediction of target gene,the target relationship of ssc-miR-122-5p(ssc-micro RNA-122-5p)and ALDB-898 or OCLN(occludin)was verified by dual luciferase reporter assay;q RT-PCR and WB were performed to detect the expression of ALDB-898,ssc-miR-122-5p and OCLN in tissues and cells and their expression correlation;Moreover,the regulatory function of ssc-miR-122-5p in the process of CPB2-induced IPEC-J2 damage were evaluated;Finally,a rescue experiment was used to verify the functional mechanism of ALDB-898/ssc-miR-122-5p/OCLN to regulate the IPEC-J2 damage caused by CPB2.The main results were as follows:1.The pET-28a-CPB2 recombinant plasmid was successfully constructed,and the CPB2 recombinant protein was produced and purified by Escherichia coli expression system.SDS-PAGE and WB assays proved that the CPB2 recombinant protein has high purity,good integrity and specificity.IPEC-J2 was treated with different concentrations of CPB2 for different times,cell apoptosis and damage with different degrees was obviously observed,which was dose-and time-dependently increased by treatment with CPB2,indicating that CPB2 recombinant protein obtained in this study has good biological activity.The cell viability of IPEC-J2 was decreased to about 50% at a dose of 20 ?g/m L for 24 h treatment.An injury model of CPB2-treated IPEC-J2 was successfully constructed under this condition.2.According to bioinformatics analysis,6 lnc RNAs connected with infectious diarrhea of C.perfringens type C were screened,and 6 lnc RNAs were detected in the ileum of diarrhea piglets(resistance(IR)and susceptibility(IS))and IPEC-J2 treated with CPB2 by using q RT-PCR.The results showed that the expression of ALDB-898 was significantly reduced in the tissues and cells.Tissue expression profile results showed that ALDB-898 was expressed with high abundance in jejunum and ileum,followed by lymph,kidney and lung,while the expression level was lower in liver and stomach,and there was almost no detection in the thymus,heart,duodenum and spleen,indicating that ALDB-898 is a tissue-specific lnc RNA.Moreover,we found that the expression of ALDB-898 in IPEC-J2 was significantly decreased with the increase of CPB2 concentration and induced-time.In addition,overexpression of ALDB-898 significantly reduced the IPEC-J2 damage induced by CPB2 through promoting cell proliferation,inhibiting cell apoptosis,toxicity and inflammation,and reducing the permeability of cell monolayers,while interference of ALDB-898 aggravated the toxic effects of CPB2 to IPEC-J2.3.Separation of cytoplasmic and nuclear and RNA-FISH results declared that ALDB-898 was mainly located in the cytoplasm.Combined RNA-seq,q RT-PCR and software predictions,a ce RNA network with ALDB-898 as the regulatory center was constructed,and a ce RNA regulatory relationship pairs(ALDB-898/ssc-miR-122-5p/OCLN)were obtained.The dual luciferase reporter assay further proved that ssc-miR-122-5p has a targeting relationship with ALDB-898 and OCLN,respectively.Pearson correlation analysis indicated that ALDB-898 expression was negatively correlated with ssc-miR-122-5p,ssc-miR-122-5p was negatively correlated with OCLN,and ALDB-898 was positively correlated with OCLN.Overexpression of ALDB-898 reduced the expression of ssc-miR-122-5p and increased the OCLN expression level in IPEC-J2 treated with CPB2,while overexpression of ssc-miR-122-5p reversed the positive regulation of OCLN by ALDB-898.The expression of ssc-miR-122-5p was significantly up-regulated in ileum of diarrhea piglets(IR and IS)and in IPEC-J2 treated with CPB2.Moreover,the level of ssc-miR-122-5p in IPEC-J2 was dose-and time-dependently increased by CPB2 stimulation.In addition,overexpression of ssc-miR-122-5p markedly intensified cell damage of IPEC-J2 induced by CPB2 through accelerating cell apoptosis,promoting LDH activity and the production of pro-inflammatory factors,and increasing cell permeability.Furthermore,the expression of OCLN at m RNA and protein levels were significantly down-regulated in ileum of diarrhea piglets(IR and IS)and in IPEC-J2 treated with CPB2 and were remarkably decreased with increase of CPB2 dose and treatment time in IPEC-J2.The rescue experiments proved that overexpression of ssc-miR-122-5p partially reversed the protective effect of ALDB-898 on IPEC-J2 injury induced by CPB2,while the overexpression of OCLN significantly inhibited the reversed effect of ssc-miR-122-5p on ALDB-898,so as to restore the protective effect of ALDB-898 and reduce the damage of CPB2 on IPEC-J2.In conclusion,CPB2-induced IPEC-J2 injury model was successfully constructed,and then we discovered that the toxic action of CPB2-treated IPEC-J2 was inhibited and promoted respectively by ALDB-898 and ssc-miR-122-5p,and further revealed that ALDB-898 has the ability to capture ssc-miR-122-5p to enhance OCLN expression and promote intestinal epithelial barrier function,and reduce cell apoptosis and inflammation,ultimately inhibit excessive damage of CPB2 to IPEC-J2.LncRNA ALDB-898/ssc-miR-122-5p/OCLN plays an inhibitory effect on the piglet diarrhea caused by C.perfringens type C infection,and could be used as a molecular target for the prevention and control of diarrhea in piglets.Overall,this study would provide a vital reference for the molecular disease-resistant breeding of bacterial diarrhea in piglets. |
PDF Full Text Request