Fig 2: miR-33-5p negatively regulates JNK1 in DF-1 cells. (A) The KEGG enrichment result of target genes of miR-33-5p; (B) Alignment of JNK1 3'UTR in different species; (C) The secondary structure of the RNA duplex of miR-33-5p and the JNK1 3’UTR target site; (D) The JNK1 expression was detected by qPCR and Western blot in MG-infected DF-1 cells and CP-II cells, GAPDH was used as the internal control; (E) The relative expression level of JNK1 in chicken embryo lungs from chicken embryo lungs on days 6-9 post-infection (equivalent to days 15-18 of eggs hatching) using qPCR, GAPDH was used as the internal control; (F) psiCHECK-2 dual-luciferase reporter vector containing the 3'UTR (wild-type or mutant) of JNK1; (G) DF-1 cells were co-transfected with Luc-JNK1 3'UTR (wild-type or mutant) and miR-33-5p mimics. At 24h post-transfection, the cells were assayed for both firefly and renilla activity through a dual-luciferase glow assay; (H) The mRNA and protein expression of JNK1 after treatment with miR-33-5p mimic/miR-33-5p inhibitor, GAPDH was used as the internal control. (*p < 0.05, **p < 0.01).

Fig 3: miR-33-5p inhibits MG infection-induced inflammation and apoptosis and reduces pMGA1.2 expression through JNK1. DF-1 cells were transfected with the indicated RNA oligonucleotides and infected with MG. (A) The expression levels of pro-inflammatory cytokines (TNF-α and IL-1β) after treatment with co-transfection of overexpression JNK1 vector and miR-33-5p mimics were detected by qPCR and Elisa; (B) The expression levels of pro-inflammatory cytokines (TNF-α and IL-1β) after treatment with co-transformation of SP600125 and miR-33-5p inhibitor were detected by qPCR and Elisa; (C) CCK-8 kit was used to detect cell proliferation at 12, 24, 48h post-infection; (D) The cells were stained with Annexin V- PI and analyzed by flow cytometer after 24h post-infection; (E) The relative mRNA expression level of apoptosis marker genes after treatment with co-transfection of overexpression JNK1 vector and miR-33-5p mimics or co-transformation of SP600125 and miR-33-5p inhibitor was detected by qPCR; (F) The protein expression of apoptosis marker genes treatment with co-transfection of overexpression JNK1 vector and miR-33-5p mimics or co-transformation of SP600125 and miR-33-5p inhibitor was detected by Western blot; (G) Western blot was used to detect the adhesion protein pMGA1.2 of MG treatment with co-transfection of overexpression JNK1 vector and miR-33-5p mimics or co-transformation of SP600125 and miR-33-5p inhibitor. GAPDH was used as the internal control. Data was expressed as the mean ± SD of three independent experiments, each with three replicates. (*p < 0.05, **p < 0.01).

Fig 4: Lnc90386 regulates MG infection through miR-33-5p/JNK1. DF-1 cells were transfected with oligonucleotides and infected with MG. (A)The expression levels of pro-inflammatory cytokines (TNF-a and IL-1ß) after treatment with co-transfection of overexpression Lnc90386 vector and miR-33-5p mimics were detected by qPCR and Elisa; (B) The expression levels of pro-inflammatory cytokines (TNF-a and IL-1ß) after treatment with co-transformation of Si-Lnc90386 and miR-33-5p inhibitor were detected by qPCR and Elisa; (C) CCK-8 kit was used to detect cell proliferation at 12, 24, 48h post-infection; (D) The cells were stained with Annexin V- PI and analyzed by flow cytometer after 24h post-infection; (E) The relative mRNA expression level of apoptosis marker genes after treatment with co-transfection of overexpression Lnc90386 vector and miR-33-5p mimics or co-transformation of Si-Lnc90386 and miR-33-5p inhibitor was detected by qPCR; (F) The protein expression of apoptosis marker genes treatment with co-transfection of overexpression Lnc90386 vector and miR-33-5p mimics or co-transformation of Si-Lnc90386 and miR-33-5p inhibitor was detected by Western blot and qPCR; (G) Western blot was used to detect the adhesion protein pMGA1.2 of MG treatment with co-transfection of overexpression Lnc90386 vector and miR-33-5p mimics or co-transformation of Si-Lnc90386 and miR-33-5p inhibitor. GAPDH was used as the internal control. Data was expressed as the mean ± SD of three independent experiments, each with three replicates. (*p < 0.05, **p < 0.01).

Fig 5: Changes of placental ryanodine receptor/C-Jun N-terminal kinase/Y-box protein 1 (RYR/JNK/YB-1) pathway and histone acetylation of ATP-binding cassette, sub-family B, member 1a/b (abcb1a/b) promoter region of prenatal caffeine exposure-induced intrauterine growth retardation rat model. A Relative mRNA expression levels of RYRs, n=10–12; B Relative protein expression of placental JNK, p-JNK, and E1A binding protein P300 (P300) was standardized by GAPDH, and relative protein expression of nucleus YB-1 was standardized by H3, n=3; C, D Histone 3 Lysine 9 (H3K9) and histone 3 Lysine 14 (H3K14) acetylation levels in the promoter region of abcb1a and abcb1b, n=3. Mean ± S.E.M. *P<0.05, **P<0.01 vs. control