The sick FMDs were looked after by us all day long. We take blood as soon as they got worse. When the sick FMDs died, an autopsy was performed by veterinarian to determine the PF. The 17 FMD venous blood samples were taken at the Chongqing Institute of Medicinal Plant Cultivation in Chongqing, China (Table 1; Supplementary Figure S1). Fresh blood samples were promptly kept in RNAlater (Ambion Inc., Austin, TX, USA). The PF and healthy blood groups of FMD were taken between January 2021 and December 2022 in Chongqing, China. All animal experiments were approved by Chongqing Three Gorges University and Chongqing Institute of Medicinal Plant Cultivation. The collected blood samples are packaged, sealed, labeled, and immediately refrigerated at 4°C before being sent to the laboratory. For long-term storage, the blood should be rapidly frozen at −40°C immediately after collection and then stored at −80°C for no more than three months.

The total RNA was extracted using TRIzol reagent and then processed with RNase-free DNase I. A whole RNA pool was collected the venous blood of 17 FMD individuals to create an mRNA library or a small RNA library by using an Illumina HiSeq2500 platform (24, 25). Principal components analysis (PCA) of mRNAs and miRNA expression were performed in the nine samples and eight samples, respectively. The known miRNAs were identified by aligning them against the miRBase (26), and the novel miRNAs were predicted by using miranda (27). Expression levels of genes and miRNAs estimated as FPKM (fragments per kilobase million) and TPM (transcripts per million) indices, respectively, which were used to determine DEGs and DEmiRNAs between PF and healthy blood groups. The unigenes with P_adj < 0.05 and |log₂(fold change) | >1.0 were taken as the threshold for DEGs. DEmiRNAs were identified with the thresholds of p < 0.05 and | log₂ (fold change) | >2.0.

The target genes of DEmiRNAs in the two groups were predicted by using RNA hybridization (28), MiRanda (29), and PITA (Probability of Interaction by Target Accessibility, 30), and the results from the three algorithms were intersected. The potential target genes predicted by the above two softwares were combined, and the intersecting components were included as a set of candidate target genes. DEGs and DEmiRNA target mRNAs were further used to fulfil the GO and KEGG enrichment analyses. The p < 0.05 was designated as the threshold for significance.

A set of 1,811 immune-related genes was obtained from ImmPort. Additionally, 306 apoptosis-related genes were recognized from the molecular signature database (MSigDB), which were intersected with the DEGs of the PF and healthy blood groups to regard as immune DEG set and apoptotic DEG set. In the typical immune pathway of this study, these immune DEGs were uploaded to STRING and obtain their interaction information (31, 32). The key potential regulatory networks of immune-related DEGs (or apoptosis-related DEGs) in the immune pathway (or apoptotic pathway) were pictured using the cytoscape software. It has been reported that DEGs and DEmiRNAs have potential negative regulatory relationships. On the basis of this idea, we researched the expression correlation of immune DEGs (or apoptotic DEGs) and DEmiRNAs by using the PCC (Pearson correlation coefficient). The negatively coexpressed DEmiRNA–DEG pairs with PCC < −0.7 and p < 0.05 were screened to construct miRNA-gene networks.

Six immune hub DEGs (TLR2, TLR4, CXCL13, IL18, AKT1, and ITGB2) and six immune hub DEmiRNAs (let-7f-5p, let-7d, miR-30b-3p, miR-25-5p, miR-149-5p, and miR-760) were chosen for differential expression analysis. The primers for six immune-related DEGs and six DEmiRNAs were provided in Supplementary Table S1. Each sample’s mRNA and miRNA was reverse transcribed using PrimeScript™ RT reagent Kit (TaKaRa) and a miScript II RT Kit (Qiagen, Hilden, Germany). Quantitative PCR of the immune DEGs and immune DEmiRNAs were performed by using TB GreenR Premix Ex TaqTM II (Takara) and miScript SYBR Green PCR Kit (Qiagen), respectively. GAPDH and U6 snRNA were used as reference gene and miRNA, respectively. The relative expression of six DEGs and DEmiRNAs was computed using the 2^−ΔΔCt method. The mean ± SE of three tests were presented in the data. Statistical significance was assessed by using t-tests as follows: *p ≤ 0.05 and **p ≤ 0.01.

To investigate the changes of gene expression profiles in the PF pathological changes of FMD body and to compare their differences with healthy blood groups, nine cDNA libraries were built from the two blood groups of PF and healthy FMD. The unprocessed data were stored in the NCBI Sequence Read Archive (SRA) with the accession number PRJNA916839. After quality filtering, a total of 400,157,758 and 423,446,384 clean reads, representing a total of 62.12 and 80.09 Gb nucleotides, were generated for the PF and healthy blood groups, respectively (Supplementary Table S2). Approximately 86.33% of the clean reads was mapped to the FMD reference genome (33), with a match ratio ranging from 78.40 to 90.83% (Supplementary Table S3). After eliminating the low level of genes and transcripts, the reads were organized into 19,445 known genes and 4,907 annotated transcripts. A PCA was conducted, revealing that the samples from the PF and healthy blood groups formed two separate clusters based on the first principal component (PC1), which accounted for 66% of the variance (Supplementary Figure S2). This indicated that the sequencing data was suitable for further analysis. A mean of 11,884 (70.92%) and 12,229 (74.62%) unigenes with FPKM values greater than 0.5 were acquired from the PF and healthy blood group of FMD, respectively (Supplementary Figure S3). A detailed overview was described in Table 2.

A total of 2,744 DEGs (1,657 upregulated and 1,087 downregulated genes) were identified in the PF blood groups compared to the healthy blood groups. In GO enrichment analysis, the DEGs in the PF blood group were found to be associated with various immune functions, including immune receptor activity (GO:0140375), adaptive immune response (GO:0002250), innate immune response (GO:0045087), regulation of immune response (GO:0050776), immune effector process (GO:0002252), immune system development (GO:0002520), regulation of immune system process (GO:0002682), activation of immune response (GO:0002253), cell activation involved in immune response (GO:0002263), immune response-regulating signaling pathway (GO:0002764), and myeloid cell activation involved in immune response (GO:0002275) (Supplementary Table S4). The 2744 DEGs were related to 116 KEGG pathways (Supplementary Table S5), in which the most common immune pathways were CD molecules, immune disease, the immune system, cytokine receptors, T-cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine–cytokine receptor interaction, the intestinal immune network for IgA production, and NOD-like receptor signaling pathway.

Among 2744 DEGs, 240 immune DEGs were identified and verified in the PF blood groups compared to the healthy blood groups, including 186 upregulated and 54 downregulated genes (Supplementary Table S6). The significant expression patterns of immune-related DEGs between PF and healthy blood groups are shown in Supplementary Table S6. In addition, we conducted a hierarchical cluster analysis of top 60 immune DEGs across the nine samples, which indicated that immune-related DEG expression levels in the PF groups could be robustly separated from those in the healthy groups (Figure 1A). The upregulated immune DEGs gathered into one group, and the downregulated immune DEGs gathered into another group. Similarly, PF and healthy blood groups were gathered separately (Figure 1A), thus highlighting gene expression differences between PF and healthy blood groups. The GO analysis revealed that the 240 immune DEGs were significantly enriched in BP including regulation of immune response, adaptive immune response, cell activation involved in immune response, leukocyte activation involved in immune response, cytokine production involved in immune response, immune system process, regulation of immune effector process, innate immune response, immune effector process; CC including receptor complex, extracellular region, and plasma membrane signaling receptor complex, and MF including immune receptor activity, cytokine receptor activity, signaling receptor binding, and molecular transducer activity (Supplementary Table S7). Collectively, these terms were all strongly linked to immunological function. Furthermore, the analysis of KEGG showed that these 240 immune DEGs were significantly enriched in various pathways related to the immune function. These pathways included the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, cytokine–cytokine receptor interaction, Th17 cell differentiation, Th1 and Th2 cell differentiation, CD molecules, immune system and immune disease (Supplementary Table S8).

In the PF blood group, 56 apoptosis-related DEGs (42 upregulated and 14 downregulated genes) were discovered. The significant expression patterns and distribution of the apoptotic DEGs between PF and healthy blood groups are shown in Supplementary Table S9. Furthermore, the analysis of GO revealed that these 56 DEGs related to apoptosis were significantly enriched in BP including regulation of mitochondrial membrane permeability involved in apoptotic process, positive regulation of apoptotic process, negative regulation of extrinsic apoptotic signaling pathway, apoptotic signaling pathway, apoptotic mitochondrial changes, leukocyte apoptotic process, glial cell apoptotic process, and execution phase of apoptosis; CC including proteasome accessory complex, organelle lumen, and membrane-enclosed lumen; and MF including cysteine-type endopeptidase activity involved in apoptotic signaling pathway, cysteine-type endopeptidase activity involved in the execution phase of apoptosis, cysteine-type endopeptidase inhibitor activity involved in apoptotic process, and peptidase activator activity involved in apoptotic process (Supplementary Table S10). An interesting point was that these terms were also related to the process of apoptosis. Furthermore, the KEGG analysis demonstrated that these 56 apoptosis-related DEGs were obviously enriched in apoptosis, cell growth, and death, B-cell receptor signaling network, NOD-like receptor signaling pathway, and NF-kappa B-signaling pathway (as shown in Supplementary Table S11).

In our analysis, we projected a total of 7,658 mRNA-mRNA pairs in the BIPF and healthy blood groups. The apoptosis-related DEGs in the apoptosis, Toll-like receptor signaling pathway, MAPK signaling pathway, T-cell receptor signaling pathway, cell growth and death, and PI3K-Akt signaling pathway were piped to STRING to create PPI networks (Figures 1B, C); the hub genes identified in one of the MCODE models in the PPI network including TLR4, IKBKB, AKT1, MYD88, TP53, JUN, IRF3, IRF7, MAPK1, NGF, and IGF1R major belonged to apoptosis, Toll-like receptor signaling pathway, and MAPK signaling pathway (Figure 1B); the other one MCODE model including TP53, AKT1, IKBKB, PIK3CB, MAPK1, PIK3CG, IGF1R, TLR4, NGF, and CASP7, which were mainly involved in T-cell receptor signaling pathway, cell growth and death, and PI3K-Akt signaling pathway (Figure 1C).

Among the KEGG pathways of immune-related DEGs in the PF and healthy blood groups, we observed that the majority of interaction networks were associated with CD molecules, cytokine–cytokine receptor interaction, NOD-like receptor signaling pathway, TNF signaling pathway, NF-kappa B-signaling pathway, immune system, immune disease, Th17-cell differentiation, Th1 and Th2 cell differentiation (Figures 1D, E; Supplementary Figures S4, S5). The immune-related DEGs in the NF-kappa B-signaling pathway, CD molecules, NOD-like receptor signaling pathway, cytokine–cytokine receptor interaction, Th1 and Th2 cell differentiation, and TNF signaling pathway were inputted into the STRING database to create PPI networks (Figure 1D); the hub genes identified in one of the MCODE models in the PPI network including TLR4, TYK2, CD40, LCK, ZAP70, CD86, TLR2, CCR7, TLR3, IRF3, IRF7 major belonged to CD molecules, NF-kappa B-signaling pathway, and NOD-like receptor signaling pathway (Figure 1B); the other one MCODE model including TYK2, IL6R, CCR7, CSF3R, CXCL13, LCK, IL4R, PIK3CB, SOCS3, IL2RA, LTA, CD40, TNFSF11, and IL27RA were mainly in cytokine–cytokine receptor interaction, Th1 and Th2 cell differentiation, and TNF signaling pathway (Figure 1E). The most prevalent immune pathways associated with DEGs were involved in immune disease and immune system, in which interaction network of the related DEGs were built in Supplementary Figure S4; The hub genes, including TLR4, LCK, ZAP70, CD86, ITGB2, ICOS, CD28, CD3E, CD40, TLR2 major belonged to the immune disease, functioned as core nodes in the regulatory network (Supplementary Figure S4); The hub genes, containning PIK3CB, HCK, VAV1, ITK, TYK2, CCR7, FGR, IKBKB, IRF7, IRF3, PIK3CG, AKT1, and TLR3 were mainly in the regulatory network of immune system pathway (Supplementary Figure S4).

Eight short RNA libraries were created using blood samples from two groups: PF and healthy individuals of FMD. The counts of raw reads and clean reads of high-throughput sRNA were listed in Supplementary Table S12. Out of all the clean reads, about 99.08% were successfully aligned to the FMD reference genome. The unique match ratio ranged from 72.45% to 77.89% (Supplementary Table S13). Initially, the sRNA data were adjusted to mitigate the impact of technical noise. A PCA was conducted, revealing that the samples from the PF and healthy blood groups formed two independent clusters. This distinct cluster was mainly based on the PC1, which accounted for 47% of the total variance (Supplementary Figure S6). The unprocessed data have been stored in the NCBI SRA with the accession number PRJNA667837. The clean reads were annotated using the FMD reference genome and categorized into miRNAs, tRNAs, rRNAs, and other categories by performing a blast analysis using Rfam and miRBase. The unannotated RNAs were used to recognize novel miRNAs. The majority of miRNAs had lengths ranging from 21 to 23 nt, and the majority of the sequencing reads had a length of 22 nucleotides in the eight miRNA libraries. The research discovered a total of 3,206 miRNAs, out of which 1,407 miRNAs (700 known and 707 novel) were shown to be co-expressed in both PF and healthy blood groups. An average of 707 (69.77%) and 546 (64.12%) miRNAs with TPM > 0.5 were obtained in the PF and healthy blood group of FMD, respectively (Supplementary Figure S7). A heatmap of DEmiRNAs in the eight samples was plotted, which showed an obvious separation of DEmiRNAs and group clusters (Figure 2A).

Three hundred fifty-six DEmiRNAs (332 upregulated and 24 downregulated miRNAs) were identified from a comparison of the PF and healthy blood sRNA libraries in the M. berezovskii. In the DEmiRNAs, 123 known and 233 novel mature miRNAs were identified. Among 356 DEmiRNAs, 53 immune-associated known DEmiRNAs and 55 immune-associated novel DEmiRNAs were identified and verified (Supplementary Table S14). The GO analysis of the DEmiRNA target immune genes were significantly enriched in the immune term including adaptive immune response, negative/positive regulation of immune response, activation of the immune response, regulation of immune system process, cytokine receptor activity, ligand-activated transcription factor activity, G protein–coupled peptide receptor activity, and activation of the immune effector process (Supplementary Table S15). KEGG analysis of these DEmiRNA-target immune genes that they were involved in the Toll-like receptor signaling pathway, antigen processing and presentation, RIG-I-like receptor signaling pathway, HIF-1 signaling pathway, Th1 and Th2 cell differentiation, TGF-beta signaling pathway, immune system, immune disease, and NF-kappa B-signaling pathway (Supplementary Table S16).

The interaction networks of immune-related DEGs (or apoptosis-related DEGs) and DEmiRNAs, which regulated the FMD immune and apoptosis response, were predicted by the STRING and pictured by cytoscape software. The interaction network of DEmiRNAs with their target immune-related DEGs (or apoptosis-related DEGs) has also been constructed and demonstrated in the critical signal pathway (Figure 2A). In the immune disease, the network contained 97 molecules and 173 interactions, including 88 DEmiRNAs and nine immune DEGs (Figure 2A), in which the known DEmiRNAs, such as miR-149-5p, miR-185, miR-204-3p, miR-324-3p, miR-671-5p, and miR-760, related to immune disease, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included TG, C3, NOD2, TLR4, TNFSF11, IL18, and TNFSF13B as the core node genes. In the immune system, the network contained 132 molecules and 320 interactions, including 104 DEmiRNAs and 28 immune DEGs (Figure 2B), in which the known DEmiRNAs, such as let-7d-5p, miR-324-3p, miR-185, miR-204-3p, miR-193a-5p, miR-671-5p, miR-760, and miR-1842-5p related to immune system, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included CXCR2, IL1R2, FGR, IL1RAP, IL15RA, C3, NOD1, NOD2, TLR2, TLR4, CCL3, IL18, and TNFSF13B as the core node genes. In the Toll-like receptor signaling pathway, the network contained 31 molecules and 34 interactions, including 27 DEmiRNAs and four immune DEGs (Supplementary Figure S8), in which the known DEmiRNAs, such as miR-23b-5p, miR-491, miR-491-5p, and miR-760 related to Toll-like receptor signaling pathway, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included TLR2, TLR4, CD14, and CCL3 as the core node genes.

In the NF-kappa B-signaling pathway, the network contained 42 molecules and 79 interactions, including 36 DEmiRNAs and 6 immune DEGs (Figure 2B), in which the known DEmiRNAs, such as miR-138a, miR-193a-5p, miR-204-3p, miR-23b-5p, miR-491, miR-491-5p, miR-331-3p, and miR-760 related to NF-kappa B-signaling pathway, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included LTA, TLR4, CD14, TNFSF11, TNFSF13B, and LTBR, as the core node genes. In the NOD-like receptor signaling pathway, the network contained 59 molecules and 72 interactions, including 54 DEmiRNAs and five immune DEGs (Supplementary Figure S9), in which the known DEmiRNAs, such as let-7d-5p, miR-149-5p, miR-185, miR-193a-5p, miR-204-3p, miR-23b-5p, miR-185, miR-210, miR-491, miR-491-5p, miR-671-5p, miR-877-5p, miR-760, and miR-1842-5p related to NOD-like receptor signaling pathway, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included IL18, NOD1, NOD2, TLR4, and NLRX1, as the core node genes. In the apoptosis signaling pathway, the network contained 136 molecules and 214 interactions, including 118 DEmiRNAs and 18 apoptosis-related DEGs (Figure 2B), in which the known DEmiRNAs, such as miR-138a, miR-877-5p, miR-149, miR-760, miR-138, miR-149-5p, miR-877, miR-671-5p,miR-339-5p, miR-326, miR-324-3p, miR-296-3p, miR-34a-5p, and miR-23b-5p, related to apoptosis signaling pathway, functioned as hub miRNAs in the regulatory network, and the target immune DEGs of these miRNAs included MCL1, MAPK8, MAPK1, FOS, IL3RA, TP53, PIK3R3, PIK3CB, CASP7, CSF2RB, NTRK1, IKBKB, and JUN, as the core node genes.

In the study, six immune-related hub DEGs and six immune-related hub DEmiRNAs were chosen for qRT-PCR verification. The results showed that the FPKM of the six immune-related DEGs and the TPM of six DEmiRNAs from RNA-seq (p < 0.05; Figures 3A, 4A) were consistent with those from the RT-qPCR data (Figures 3B, 4B) based on the expression trends between the PF and healthy blood groups, which shown the high reliability of the RNA-seq results in the study.

Innate immunity is the first line of defense, which can mount resistance to reinfection. In the study, a differential expression analysis was conducted by utilizing mRNA-seq. 240 immune-related DEGs (54 downregulated and 186 upregulated) and 56 apoptosis-related DEGs (14 downregulated and 42 upregulated) were identified in the PF and healthy blood group of FMD. Genes rarely function alone, which form regulatory networks with other molecules to perform biological functions. At this point, immune-related DEGs and apoptosis-related DEGs were built to elucidate the regulatory relationships of DEGs. In the study, some immune-related DEGs (CXCL13, CCL3, ISG20, and IL1R2) were important to the innate immune response, which were upregulated in the PF blood groups. The interaction between CXCL13 and its receptor (CXCR5) has been implicated in the pathogenesis of numerous diseases and the immune responses of healthy organisms (34, 35). The downregulation of CXCL13 may recruit immune cells to the site of infection (34) and eliminate the proinflammatory response (36). CLL3 gene highly expressed in several autoimmune diseases (37–39), which attracted a variety of leukocytes in vitro (40, 41) and thus played protective roles in all kinds of infectious diseases (42–45). Furthermore, it has been documented that CCL3 can serve as a protective barrier against Chlamydia infection; conversely, CCL3 deficiency would render an individual more vulnerable to contracting infectious diseases (38, 39). ISG20, a protein induced by interferon, participated in the innate immune response and has been found to play a role in inflammatory responses and viral infections (46). In addition, the upregulation of ISG20, an exonuclease gene, could inhibit the DNA and RNA virus replication (47). NFKBIE, ISG20, and IL1R2 were crucial in regulating inflammation and host defense (47, 48). CCL23, IRF1, and IRF7 were identified for immune-related DEGs, which had high expression level. The chemokine CCL23 was secreted by different immune cells, which bound its receptor CCR1 and involved in immune response (49, 50). IRF7 could involve in regulation of type I interferon to against pathogens infections and the innate immune response (51). In our study, the upregulation of ISG20, CCL23, IRF7 suggested that the strong innate immune response in the FMD had been activated by the PF.

To further clarify the function of immune DEGs and apoptotic DEGs, KEGG pathway enrichment analysis was performed. In our study, the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine–cytokine receptor interactions, CD molecules, immune disease, and immune system were significantly immune-related pathways, which was conducted to elucidate further the function of immune-related DEGs. An examination of the topology of interaction networks involving immune-related DEGs (apoptosis-related DEGs) revealed that the majority of key DEGs were members of the chemokine, interleukin, and TLR families. In this study, five TLR genes (TLR1, TLR2, TLR3, TLR4, and TLR7) were identified for immune DEGs, which participated in pathogens reorganization. In TLRs, TLR1, TLR2, TLR4, and TLR7 were significantly upregulated in the PF, whereas TLR3 was significantly downregulated in the PF. While TLR5, TLR6 had no differences in the PF and healthy blood comparisons. TLRs, which stand for pattern recognition receptors, were identified as their role in recognizing pathogens and activating immune responses (52, 53). TLR4, TLR5, TLR6, TLR7, and TLR8 can identify viral proteins and extracellular bacterial and fungal cell wall components (54), which were vital to the immune responses of the host against a variety of invading pathogens (53). TLR2 and TLR3 were able to induce the immune response by recognizing glycoproteins from various viruses (55, 56). TLR4 participated the innate immunity of rodents against the respiratory syncytial virus (57). TLR7 and TLR8 have the capability to identify single-stranded RNA of viruses that are abundant in guanosine and uridine (58). The upregulation of TLR2 and TLR4, which function as recognition receptors for the immune disease, played a crucial role in the innate immune system of FMD. Interleukin-18 (IL-18) was a highly potent pro-inflammatory cytokine that regulated innate and acquired immune responses and involved in the host’s defense against infections (59). In our study, TLR2, TLR4, and IL18 were identified as hub gene, TLR2 and TLR4 were significantly upregulated, which promoted IL18 production. This research was consistent with previous reports (60). Chemokines were very important for the role of the innate immune system and control the migration and positioning of immune cells, which were a defense against infection and inflammation. CXCL13 and CX3CR1 are both members of the chemokine family. CXCL8/IL-8 was Proven to directly bind to Mycobacterium tuberculosis and potentially enhance the host resistance to infection (61). IL-1R2 was an endogenous inhibitor that prevented the transduction of the IL-1 signal (62). Prior research has demonstrated that IL1R2 exhibited efficacy as an in vivo anti-inflammation and can cure various diseases (63, 64). It was the assertion that IL1R2 secretion cannot eradicate the acute inflammatory response and restrict the inflammatory reaction to alleviate disease (65). Consequently, IL1R2 upregulation may play a significant role in maintaining the physiological equilibrium of FMD inflammatory.

The regulatory mechanisms of the adaptive immune system was regulated by T cells, B cells, and their antigen-specific receptors (TCR and BCR) (66). Innate immune response can resist the pathogen, while adaptive immune response can finally clear the infection and achieve long-lasting and highly specific protection and sustained by memory T cells (67, 68). CD3E, CD3G, NFKBIE, TRBV4-1, TRBV12-3, TRAV26-1, IL4R, and IL2RA were found to be enriched in the Th1 and Th2 cell division pathways, respectively, in the study. TRAV14DV4, TRAV38-2DV8, and TRDC were also significantly downregulated in the PF blood groups in the study. The TCR family, comprising TRBV4-1, TRBV12-3, TRAV26-1, TRAV14DV4, TRAV38-2DV8, and TRDC, was essential for adaptive immune initiation and facilitated the recognition of an array of antigens (69). CD3E and CD3G were present on the surface of T lymphocytes, which performed important functions in the adaptive immune response (70). IL4R was expressed on eosinophils, macrophages, lung fibroblasts, T and B lymphocytes, which was over-expressed and played significant role in the immune system (71–74). IL2RA is mainly expressed on mature T cells and some other activated hematopoietic cell, which played essential roles in immune regulation of multiple diseases (75).

A variety of interleukins (IL2, IL4, IL5, IL6, IL7, IL10, IL13, IL14, and IL21) were found to participate in the proliferation and differentiation of B cells (76–80). In the study, only IL18, IL27, and IL36a had significantly different expression and were significantly upregulated in the PF blood groups. IL18 regulated both Th 1 and Th2 responses, which acted synergistically with IL12 in the Th1 paradigm, whereas IL18 with IL2 and without IL12 it can induce Th2 cytokine production from CD4⁺ T cells, natural killer (NK) cells, NKT cells, as well as from Th1 cells (81). IL-27, a member of the cytokine superfamily IL-6/IL-12, significantly regulated immune response (82). IL-27 was discovered to play a broad anti-inflammatory function in infectious and chronic immune-mediated diseases (83–85). IL36 is implicated in both the development and advancement of inflammatory and fibrotic disorders (1–7). Indeed, emerging evidence indicated that IL-36 could mediate the relationship between inflammation and fibrosis (86–88). IL-36a was a members of the IL36 cytokine family (89, 90). In addition to stimulating humoral immunity, IL27 could suppressed T-cell responses in autoimmune conditions and thus induced inflammation and cell death in certain situations (91). IL-18 is a crucial factor in facilitating the synthesis of specific cytokines (92, 93). The observation that the expression of IL18 and the receptor of IL18 (IL18RAP) was significantly increased in the PF blood group compared to the healthy blood group and the expressions of the two genes were negative. More research is required to determine whether the down expression of IL18 affected the effectiveness of the immune responses.

MiRNAs regulated many cellular processes and signaling pathways, including embryogenesis, cell proliferation and differentiation, apoptosis, and disease onset. In doing so, miRNAs functioned as potent inhibitors of protein translation via the degradation of mRNAs (94, 95). Nevertheless, the hub miRNAs accountable for the aberrant regulation of immune response and apoptosis in the PF blood group of FMD still need to be further study. In our research, total 282 DEmiRNAs were targeted by the 217 immune DEGs with immunity, and 39 apoptotic DEGs were identified as miRNAs associated with both immunity and apoptosis. A total of 25 genes shared by immune-related DEGs and apoptosis-related DEGs that were identified in the PF and healthy blood groups. DEmiRNA–DEGs interaction networks were constructed based on the principle that miRNAs restrained protein translation by binding to the 3′UTR of target mRNAs and degrading them. In the study, the immune DEGs-DEmiRNA (and apoptotic DEGs-DEmiRNA) interaction network was first constructed in the PF blood group of FMD. In the apoptosis-related DEG–DEmiRNA network of apoptosis pathway, elevated expression of 42 apoptosis-related DEmiRNAs could induce the down expression of MAPK1, MAPK8, TP53, PIK3R3, and NTRK1, while the above 42 apoptosis-related DEmiRNAs could induce the upregulation of IL3RA, CAPN1, CASP7, LMNB1, FOS, NGF, AKT1, JUN, PIK3CB, MCL1, CAPN2, CSF2RB, and IKBKB (Figure 2E). In the immune-related DEG–DEmiRNA network of Toll-like receptor signaling pathway, elevated expression of miR-760, miR-23b-5p, miR-491, and miR-491-5p could induce the down expression of CCL3; while the above 4 apoptosis-related DEmiRNA could induce the upregulation of CD14, TLR2, and IL18 (Figure 2D). In the immune-related DEG–DEmiRNA network of immune disease pathway, elevated expression of 24 immune-related DEmiRNAs could induce the down expression of CCL3 and TNFSF11, while the above 24 immune-related DEmiRNAs could induce the upregulation of TLR2, NOD2, TLR4, C3, TG, IL18, TNFSF13B, and TG (Figure 2B). In the immune-related DEG–DEmiRNA network of immune disease pathway, elevated expression of miR-331-3p, miR-491, miR-491-5p, miR-760, miR-23b-5p, miR-1386, miR-204-3p, miR-193a-5p, and miR-138a could induce the down expression of TNFSF11 and LTA; while the above nine immune-related DEmiRNAs could induce the upregulation of CD14, TLR4, TNFSF13B, and LTBR (Figure 2B). In the immune-related DEG–DEmiRNA network of immune system pathway, downregulated expression of let-7d-5p could induce the upregulated expression of TNFSF11 and LTA, while the above nine immune-related DEmiRNAs could induce the upregulation of CD14, TLR4, TNFSF13B, and LTBR (Figure 2C). The integrated analysis of DEmiRNA–DEG interaction networks results indicated that most hub mRNAs are members of the chemokine, interleukin, and TLR families. It was confirmed that upregulation of hub mRNAs, including CCL4, CXCL10, TLR2, TLR4, and TLR7, occurred in the PF blood groups.

Investigations into the role of miRNAs in the pathogenesis of PF are still rarely. The differential expression of let-7f-5p between the PF and healthy blood groups was identified in the study, suggesting that let-7f-5p may serve as a biomarker for the investigation of the PF mechanism. This result was accordance with previous report (96). It has been reported that the let-7 family miRNAs were demonstrated to participate in the regulation of PF (97). These studies further support the hypothesis that PI3K and the let-7 family may have significant functions in the PF. It has been reported that Let-7f-5p prevents the PF by modulating cellular reactive oxygen species, mitochondrial DNA damage, and cell apoptosis (98). A prior investigation has validated the notion that the target genes of let-7f could enhance the transcriptional program of PF in a model of lung fibrosis induced by bleomycin (99). Notably, the target gene PIK3CA of let-7f-5p was an essential element of the PI3K/Akt pathway, which was instrumental in the pathogenesis of PF (14). It has been demonstrated that vascular endothelial growth factor, reactive oxygen species, and COX2 are involved in the PF in the downstream of the PI3K/AKT signaling pathway (100, 101). It has been believed that COX-2 was shown to regulate the expression of Fas receptor in pulmonary fibroblasts (102). Notably, we found that the downregulated let-7f-5p might induce the upregulation of SLC6A4, POR, and ATP13A3. The results of the integrated analysis of DEmiRNA–DEGs indicated that 29 miRNAs (18 upregulated and 11 downregulated) and 267 genes) formed miRNA-target gene pairs (Figure 2), these complex network of which potentially regulate neuronal cell proliferation, immune cell death, epilepsy, neurodevelopmental disorders, and Wnt/β-catenin and PTEN signaling.