Inhibition of ssc-microRNA-140-5p ameliorates the Clostridium perfringens beta2 toxin-induced inflammatory response in IPEC-J2 cells via the ERK1/2 and JNK pathways by targeting VEGFA.

Piglet diarrhea and even death due to Clostridium perfringens (C. perfringens) type C infection have led to huge economic losses in the pig industry worldwide. C. perfringens beta2 (CPB2) toxin is the main virulence factor for this pathogen. MiR-140-5p can exacerbate toxin-induced toxicity of toxin to cells by promoting oxidative stress. However, the role of pig miR-140-5p (ssc-miR-140-5p) in piglet diarrhea caused by C. perfringens type C has not been studied. Here, we study investigated the function of ssc-miR-140-5p by generating an in vitro CPB2-induced injury model in intestinal porcine epithelial (IPEC-J2) cells. Our results revealed that transfection with an ssc-miR-140-5p inhibitor significantly increased the viability of CPB2-induced IPEC-J2 cells, decrease the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS), and inhibit inflammatory responses and apoptosis. In addition, vascular endothelial growth factor A (VEGFA) was identified as a direct target of ssc-miR-140-5p by luciferase reporter assay. Western blot analysis showed that inhibition of ssc-miR-140-5p could activate the ERK1/2 signaling pathway and inhibit the JNK signaling pathway. In summary, we showed that down-regulation of ssc-miR-140-5p ameliorated CPB2-induced inflammatory responses in IPEC-J2 cells via the ERK1/2 and JNK signaling pathways by targeting VEGFA.

Luo R ，Yan Z ，Yang Q ，Huang X ，Gao X ，Wang P ，Wang W ，Xie K ，Gun S ... -  《-》

MicroRNA-21-5p targets PDCD4 to modulate apoptosis and inflammatory response to Clostridium perfringens beta2 toxin infection in IPEC-J2 cells.

Clostridium perfringens (C. perfringens), a toxin-producing enteric pathogen, causes a variety of intestinal infections in humans and animals. C. perfringens beta2 (CPB2) toxin has been considered to be a strong virulence factor for C. perfringens infectious enteric diseases (CPED). Altered levels and functions of microRNA-21-5p (miR-21-5p) have been associated with apoptosis and inflammation response in pathological processes. However, little is known about its functional mechanism in CPED. Here, we found that miR-21-5p expressed in multiple tissues of pig, had a highest level in jejunum, and significantly upregulated in intestinal porcine epithelial cells (IPEC-J2) exposed to CPB2 toxin. Noteworthily, transfection of CPB2-treated IPEC-J2 cells with miR-21-5p mimic increased cell viability and Bcl2 expression, as well as reduced cytotoxicity, apoptosis rates and Bax level. Moreover, overexpression of miR-21-5p significantly suppressed the levels of interleukin (IL)-6, IL-8, TNF-α, IL-1β and nuclear factor-kappa B (NF-κB p65) activity induced by CPB2 toxin, whereas that of the IL-10 was increased in IPEC-J2 cells. On the contrary, transfection of miR-21-5p inhibitor promoted CPB2-induced cell apoptosis and inflammation. Furthermore, we validated that programmed cell death 4 (PDCD4) was strikingly downregulated in CPB2-treated IPEC-J2 cells. PDCD4 exhibited opposing effects to those of miR-21-5p mimic on IPEC-J2 cells, and restoration of PDCD4 expression counteracted the suppressive effect of miR-21-5p on CPB2-induced apoptosis and inflammatory response. Collectively, our findings demonstrated that miR-21-5p was involved in regulating the immune response triggered by CPB2 toxin and contributed to protective effects in CPB2-induced CPED cell model by targeting PDCD4.

Gao X ，Huang X ，Yang Q ，Zhang S ，Yan Z ，Luo R ，Wang P ，Wang W ，Xie K ，Gun S ... -  《-》

LncRNA ALDB-898 modulates intestinal epithelial cell damage caused by Clostridium perfringens type C in piglet by regulating ssc-miR-122-5p/OCLN signaling.

Diarrhea of piglets caused by Clostridium perfringens type C (C. perfringens type C) infection is a global problem afflicting piglet production. Long noncoding RNA (LncRNA) and microRNA (miRNA) have emerged as critical regulators of this pathological process, but the underlying molecular mechanisms remain unclear. In this study, we first observed the expression changes of ALDBSSCG0000000898 (ALDB-898) and ssc-miR-122-5p in infected ileum tissue of piglets with C. perfringens type C, and then used C. perfringens beta2 toxin (CPB2) to induce intestinal porcine epithelial cells (IPEC-J2) to construct an injury model. Cytometry kit 8 (CCK-8), lactate dehydrogenase (LDH), real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, flow cytometry and fluorescein isothiocyanate-dextran 4 (FITC-Dextran 4) flux assays were performed to study the effect of ALDB-898 and ssc-miR-122-5p in apoptosis, inflammation and intestinal barrier damage and inflammatory in IPEC-J2 cells induced by CPB2. In addition, dual-luciferase reporter gene analysis was performed to confirm the relationship between ssc-miR-122-5p and ALDB-898 or ssc-miR-122-5p and occludin (OCLN), respectively. There were lower expression levels of ALDB-898 and OCLN and higher expression levels of ssc-miR-122-5p in diarrhea piglets caused by Clostridium perfringens type C. ALDB-898 and OCLN were significantly decreased and ssc-miR-122-5p was increased in IPEC-J2 after exposure to the CPB2 in a dose- and time-dependent manner. ALDB-898 overexpression mitigated CPB2-induced cell injury by promoting viability, restraining apoptosis, cytotoxicity, and inflammatory response, as well as weakening the destruction of the intestinal barrier. Further mechanisms disclosed that ALDB-898 functioned as a competing endogenous RNA (ceRNA) via binding to ssc-miR-122-5p, and OCLN was a target of ssc-miR-122-5p. Importantly, the ssc-miR-122-5p mimic led to abolishing the protective function of ALDB-898 on CPB2-induced IPEC-J2 cell damage, and the addition of OCLN reversed the negative impact of ssc-miR-122-5p, thereby restoring the protection of ALDB-898. Our data showed that ALDB-898 could enhance the expression of OCLN through competitive binding ssc-miR-122-5p to suppress CPB2-induced damage. The ALDB-898/ssc-miR-122-5p/OCLN signaling may be a candidate therapeutic pathway for diarrhea of piglets.

Gao X ，Yang Q ，Zhang S ，Huang X ，Yan Z ，Wang P ，Gun S ... -  《-》

lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis.

Xie K ，Yan Z ，Yang Q ，Huang X ，Wang P ，Gao X ，Li J ，Gun S ... -  《Cells》

Epigenetic upregulation of ssc-miR-124a following treatment with Clostridium perfringens beta2-toxin attenuates both apoptosis and inflammation in intestinal porcine epithelial cells.

Clostridium perfringens (C. perfringens) is a globally recognized zoonotic pathogen. It has been reported that the beta2-toxin produced by C. perfringens can cause a variety of gastrointestinal diseases and even systemic inflammation. MicroRNA-124a (miR-124a) has been reported to play important roles in the host response to pathogenic infection. Although C. perfringens beta2-toxin induced injury in intestinal porcine epithelial (IPEC-J2) cells has been established, the underlying molecular mechanism is not completely unraveled. Here we show that a significant upregulation of ssc-miR-124a in IPEC-J2 cells after beta2-toxin stimulation was associated with the MiR-124A-1 and MiR-124A-2 gene promoter demethylation status. Importantly, overexpression of ssc-miR-124a significantly increased cell proliferation and decreased apoptosis and cytotoxicity in beta2-toxin treated IPEC-J2 cells. Transfection of IPEC-J2 cells with ssc-miR-124a mimic suppressed beta2-toxin induced inflammation. On the contrary, ssc-miR-124a inhibitor promoted aggravation of cell apoptosis and excessive damage. Furthermore, rho-associated coiled-coil-containing protein kinase 1 (ROCK1) was identified as the direct target gene of ssc-miR-124a in IPEC-J2 cells and its siRNA transfection reversed the promotion of apoptosis and aggravation of cellular damage induced by ssc-miR-124a inhibitor. Overall, we speculated that the miR-124A-1/2 gene was epigenetically regulated in IPEC-J2 cells after beta2-toxin treatment. Upregulation of ssc-miR-124a may restrain ROCK1, and attenuate apoptosis and inflammation induced by beta2-toxin that prevent IPEC-J2 cells from severe damages. We discover a new molecular mechanism by which IPEC-J2 cells counteract beta2-toxin-induced damage through the ssc-miR-124a/ROCK1 axis partially.

Gao X ，Yang Q ，Zhang S ，Huang X ，Yan Z ，Wang P ，Luo R ，Wang W ，Xie K ，Gun S ... -  《-》