The CPB2 toxin was extracted and purified according to Luo’s method (Luo et al., 2020). Briefly, a recombinant plasmid containing the CPB2 gene, pET-28a-CPB2, was constructed transformed into BL21 E. coli cells for expression. The expressed CPB2 protein was purified using High Affinity Ni-Charged Resin FF (GenScript, Nanjing, China) and concentrated using PEG6000. Finally, a ToxOut™ Rapid Endotoxin Removal Kit (AmyJet Scientific, Wuhan, China) was used to remove the endotoxin.

Intestinal porcine epithelial cell line-J2 cells were provided by the BeNa Culture Collection (Beijing, China). Cells were cultured using Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% double antibodies (penicillin and streptomycin) at 37°C in an atmosphere of 5% CO₂. The CPB2 group of cells was treated with the CPB2 toxin (20 μg/ml). Three replicates were set up for each group.

Total RNA was extracted from the control and CPB2-treated cells using RNAiso™ Reagent (Invitrogen, Carlsbad, CA, USA). The total RNA quantity and purity were assessed using a Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, Santa Clara, CA, USA), respectively. The RNA samples were of high quality with RNA integrity numbers > 7. Ribosomal RNA was removed using a Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, CA, USA) and then reverse transcribed into cDNA. The cDNA libraries were constructed and sequenced on an Illumina NovaSeq™ 6000 platform (Illumina, San Diego, CA, USA) using 2 × 150 bp paired-end sequencing. The raw data obtained from IPEC-J2 samples were stored in the NCBI SRA database (accession number: PRJNA749943).

The raw data were processed using FastQC software.¹ This step produced clean data by removing the reads with shifted connectors and low quality sequences (quality score < Q30). The reads were mapped against the pig genome assembly (Sscrofa 11.1) and the alignment results were assessed using HISAT2² (Kim et al., 2015). StringTie software³ was used to estimate gene expression levels according to fragments per kilobase of transcript per million mapped reads (FPKM) (Trapnell et al., 2010). After all transcripts were obtained, transcripts smaller than 200 bp and known mRNAs were removed and lncRNA prediction was performed on the remaining transcripts using CPC, Pfam-sca, and CNCI (Kong et al., 2007; Sun et al., 2013). LncRNAs with fold change (FC) ≥ 2 and p < 0.05 were regarded as significantly differentially expressed.

Long non-coding RNA target genes were predicted using both cis and trans methods. LncRNA cis-regulatory target genes were mainly predicted based on positional relationships. The mRNAs that were differentially expressed within 100 kb upstream and downstream of the lncRNA were considered to be lncRNA cis-regulatory target genes. The trans-regulated target genes of lncRNAs were analyzed using RIsearch (Wenzel et al., 2012) software. Screening conditions included the formation of secondary structure free energy between the lncRNA and mRNA sequences (energy < −11) and the Pearson correlation coefficient (r > 0.95 or r < −0.95). Subsequently, GOSeq Release 2.12 and KO-BAS (V2.0) (Kanehisa et al., 2007; Young et al., 2010) were used to perform GO and KEGG pathway enrichment analyses of the target genes of the lncRNAs, respectively.

Total RNA was extracted from IPEC-J2 cells (1 × 10⁷) according to the method described in section “2.3 RNA-sequencing (RNA-seq) and data analysis.” Cytoplasmic and nuclear RNAs of IPEC-J2 cells were isolated using a PARIS™ Kit reagent kit (Ambion, Austin, TX, USA) according to the instructions provided by the supplier. Total RNA was reverse transcribed into cDNA using Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 (Accurate Biotechnology, Hunan, China). The qRT-PCR assay was performed according to the instructions of SYBR Green assay (Accurate Biotechnology, Hunan, China) and performed on a Roche LightCycler 480 (Roche Applied Science, Mannheim, Germany). Gene expression levels were normalized to that encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to determine the relative expression using the 2^–ΔΔCt method (Ljvak, 2001). The PCR primers for transcripts and genes are listed in Table 1.

The green fluorescent FAM-labeled probe for lncRNA EN-9075 (lncRNA-FISH probe mix) was designed by GenePharma (Shanghai, China) and detected using a fluorescent in situ hybridization kit (GenePharma) according to the manufacturer’s instructions. In short, IPEC-J2 cells were inoculated overnight in 48-well plates at a density of 1 × 10⁴ cells/well and then treated with CPB2 toxin for 24 h. IPEC-J2 cells were then fixed with 4% (v/v) paraformaldehyde for 15 min at room temperature. The IPEC-J2 cells permeabilized in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 for 15 min at room temperature and then blocked with pre-hybridization buffer for 30 min at 37°C. The IPEC-J2 cells were treated with the FAM-labeled probe in hybridization buffer (10 μL, 10 μM probe, and 90 μL hybridization buffer) the dark at 37°C for 14 h. The cells were then washed three times with wash buffer (4 × SSC/2 × SSC) in the dark and stained with 4′, 6-diamidino-2-phenylindole for 15 min. The IPEC-J2 cells were washed three times with PBS and then observed under a fluorescent microscope at 200× magnification (Olympus IX71, Tokyo, Japan).

The small interfering RNA targeting lncRNA EN-90756 (si EN-90756) and the negative control siRNA (si NC) were obtained from GenePharma. IPEC-J2 cells (1 × 10⁵ cells/ml) were grown overnight in six-well plates until they were 70% confluent for transfection. Lipofectamine 2000 (Invitrogen) was mixed with Opti-MEM medium (Invitrogen, CA, USA) and left for 5 min. Then, the solution was added to si EN-90756, si NC (1:1 ratio) diluted with Opti-MEM medium, mixed and left for 20 min. The prepared mixtures were added to IPEC-J2 cells separately and transfection was completed after 24 h.

Intestinal porcine epithelial cell line-J2 (density of 5 × 10³/well) were seeded in 96-well plates, with three replicates for each group. After treatment with CPB2 toxin, cell viability was determined according to the instructions of Cell Counting Kit-8 (Beyotime, Shanghai, China). IPEC-J2 cells (1 × 10⁵ cells/ml) were grown in six-well plates and treated with CPB2 toxin or PBS. Cell proliferation was assayed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 [5-ethynyl-2′-deoxyuridine (EdU), Beyotime]. Cell proliferation was observed under a fluorescence microscope at 200× magnification (Olympus IX71).

Intestinal porcine epithelial cell line-J2 cells (1 × 10⁵ cells/ml) were grown in six-well plates, transfected with si NC or si EN-90756 for 24 h and then treated with CPB2 toxin for 24 h. Cells were collected and washed twice with PBS. Then, 1 ml of pre-cooled 70% ethanol was added to the cells, mixed with gentle shaking, and fixed at 4°C for 12 h. The ethanol was discarded, the cells were washed using PBS and collected by centrifugation. Then, the cells were incubated with Annexin V-fluorescein isothiocyanate and propidium iodide for 20 min at 25°C in the dark. Apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Intestinal porcine epithelial cell line-J2 cells were transfected with si NC or si EN-90756 and treated with CPB2 toxin or PBS and then assayed for mitochondrial membrane potential (Δψm) according to the instructions of the mitochondrial membrane potential assay kit with JC-1 (Beyotime). Briefly, cells were incubated with JC-1 staining solution at 37°C for 20 min, washed two times with JC-1 staining buffer (1×) and then observed under a fluorescent microscope (Olympus IX71).

Total IPEC-J2 cell proteins were extracted using Radioimmunoprecipitation assay (Beyotime). The extracted proteins were quantified using a bicinchoninic acid protein analysis kit (Beyotime). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) was used to separate equal amounts of proteins, which were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4°C.

The primary antibodies comprised those recognizing: MX dynamin like GTPase 1 (MX1) (bsm-51528m, Bioss, Woburn, MA, USA; 1:1,000); Janus kinase 1 (JAK1) (bs-1439R, Bioss; 1:1,000); phosphorylated (p-JAK1) (bs-3238R, Bioss; 1:1,000); signal transducer and activator of transcription 3 (STAT3) (GB11176, Servicebio, Wuhan, China; 1:1,000); p-STAT3 (bs-1658R, Bioss; 1:1,000); and GAPDH (GB15002, Servicebio; 1:2,000). After incubation with horseradish peroxidase-labeled secondary antibodies (GB23303, Servicebio; 1:5,000) or (GB25301, Servicebio; 1:5,000), the immunoreactive protein bands were observed using the enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).

The results were statistically analyzed using SPSS v.21 software (IBM Corp., Armonk, NY, USA). All experiments had three replicates, and the experimental data are expressed as the mean ± standard deviation (SD). Differences were determined using Student’s t-test (two-tailed test). P-values < 0.05 were considered statistically significant.

Six cDNA libraries of IPEC-J2 cells from the control and CPB2 groups were sequenced and Pearson’s correlation coefficient between the two groups ranged from 0.98 to 0.99, indicating a high degree of similarity in expression patterns between the samples from the same group (Figure 1A). After quality control of the raw data, we obtained approximately 8.36 Gb of high quality data and the average GC content of the six libraries was approximately 53%; and the Q30 (sequencing error rate less than 0.001) per sample ranged from 95.34 to 96.14% (Supplementary Table 1). Over 70% of the clean reads were mapped to the porcine reference genome, containing approximately 60% of the unique mappings (Supplementary Table 2).

A total of 6,558 lncRNAs were identified in IPEC-J2 cells in the control and CPB2 groups, among which 39 were significantly up-regulated and 10 were significantly down-regulated lncRNAs between the two groups of IPEC-J2 cells [p-value < 0.05 and | (log2 (FC)| ≥ 1)] (Figure 1B and Supplementary Table 3).

Ten significantly differentially expressed lncRNAs were randomly selected for qRT-PCR validation of the accuracy of the sequencing results. The expression levels of lncRNAs LOC106507243, LOC106508476, and EN-41166 were down-regulated after CPB2 treatment, while lncRNAs EN-47080, EN-41190, EN-47438, EN-46898, EN-45823, LOC102162336, and EN-48701 were up-regulated after CPB2 treatment (Figure 1C). The results of qRT-PCR were consistent with the RNA-seq results, demonstrating that the sequencing results were reliable and reproducible.

To explore their functions, the 49 differentially expressed lncRNAs were subjected to target gene prediction. Consequently, genes associated with the immune response and viral defense (e.g., CXCL2, MX1, OASL) were identified among the target genes (Figure 2A) of the significantly differentially expressed lncRNA EN-90756. Further enrichment analysis revealed that significantly enriched GO terms were defense response to virus, negative regulation of viral genome replication, negative regulation of apoptotic process, and regulation of cell cycle (Figure 2B). The KEGG enrichment analysis identified Legionellosis, influenza A, Salmonella infection, and cellular senescence signaling pathways were significantly enriched (Figure 2C). Interestingly, the MX1 gene was enriched in the defense response to virus, cellular response to type I interferon, and hepatitis C, influenza A, and measles signaling pathways. These results suggest that lncRNA EN_90756 might be involved in the invasion of IPEC-J2 cells by CPB2 toxin through the regulation of target genes.

The expression pattern of lncRNA EN-90756 in CPB2-treated cells was determined at different time points (0, 12, 24, and 36 h) of CPB2 toxin treatment. The results showed that lncRNA EN-90756 expression was significantly up-regulated during CPB2 toxin treatment and reached its highest level at 24 h (Figure 3A). The nuclear/cytosol separation assay showed that lncRNA EN-90756 was mainly located in the cytoplasm, with a small amount in the nucleus (Figure 3B). RNA-FISH results also confirmed that the green fluorescent signal of lncRNA EN-90756 was distributed in both the nucleus and the cytoplasm, but was mainly located in the cytoplasm (Figure 3C). These results suggest that lncRNA EN-90756 might be an important regulator in the regulation of C. perfringens type C infection.

Long non-coding RNA EN-90756 was knocked down to investigate its role in CPB2-treated IPEC-J2 cells. The expression level of lncRNA EN-90756 was significantly reduced after transfection with si EN-90756 (Figure 4A). Cell viability assays showed that knockdown of lncRNA EN-90756 in CPB2-treated IPEC-J2 cells significantly reduced cell viability (to 78.75%) (Figure 4B). EdU analysis revealed that more EdU-positive cells were observed in the CPB2 and si NC + CPB2 groups, while fewer EdU-positive cells were observed in the si EN_90756 + CPB2 group (Figures 4C, D). These results indicated that down-regulation of lncRNA EN-90756 inhibited the proliferation of IPEC-J2 cells.

JC-1 staining showed that after knocking down lncRNA EN-90756, the fluorescence turned almost entirely green in CPB2-treated IPEC-J2 cells, reflecting the collapse of the Δψm (Figure 5A). This suggested that knockdown of lncRNA EN-90756 exacerbated CPB2-induced loss of Δψm in IPEC-J2 cells. Caspase 3 and Caspase 8, are key proteins in apoptosis, and qRT-PCR revealed that the mRNA expression levels of Caspase 3 and Caspase 8 were significantly elevated in CPB2-induced IPEC-J2 cells after knockdown of lncRNA EN-90756 (Figures 5B, C). Furthermore, the results of flow cytometry showed that the apoptosis rate was 27.35% in the CPB2 group and 37.74% in the si EN_90756 + CPB2 group (Figure 5D). These results suggested that down-regulation of lncRNA EN-90756 promoted apoptosis in IPEC-J2 cells.

Long non-coding RNA EN-90756 knockdown resulted in significant up-regulation of the expression of target gene MX1 (Figure 6A) and its protein level (Figures 6B, C) in CPB2-induced IPEC-J2 cells. The JAK-STAT signaling pathway is involved in a variety of important biological processes, including cell proliferation and apoptosis. To explore the effect of lncRNA EN-90756 on the JAK-STAT pathway, we examined the levels of JAK1 and STAT3 proteins and their phosphorylation. The results showed that JAK1 and p-JAK1 protein levels were significantly down-regulated in CPB2-induced IPEC-J2 cells after knockdown of lncRNA EN-90756. STAT3 and p-STAT3 proteins were barely detected in CPB2-induced IPEC-J2 cells after knockdown of lncRNA EN-90756 (Figures 6D, E). The results suggested that lncRNA EN-90756 regulates MX1 expression and affects JAK-STAT pathway activation in CPB2-induced IPEC-J2 cells.