Fig 2: Map3k9 is the target gene of miR-20a to suppress Treg differentiation in EAE. A The expression of Map3k9 was upregulated in CD4+ T cells with miR-20a knockdown. B Sequences of WT and mutant 3’ UTR of Map3k9 and miR-20a mimics used in the luciferase assays. C Relative luciferase activity of the reporter carrying the WT or mutant Map3k9 3’ UTR or NC 3’ UTR co-transfected with miR-20a mimics or NC mimics into HEK-293 cells. The activity of Renilla luciferase was used to eliminate variances between groups. D, E Flow cytometric analysis of the CD4+ T cells transfected with NC or miR-20a antagomirs, or miR-20a antagomirs + Map3k9 siRNA after stimulated with MOG35-55 peptide in Treg-polarizing condition for 5 days. The percentages of CD25+Foxp3+ cells are shown in the CD4+ gate. The flow cytometry plots shown are representative of 4 independent experiments. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 using unpaired (A, C) and paired (E) Student’s t-test

Fig 3: miR-144-3p protects GCs against cisplatin-induced apoptosis by targeting MAP3K9 via the p38 MAPK pathway. A Western blotting to detect the protein levels of cleaved caspase-3 and p38 MAPK pathway-related molecules in cisplatin injured COV434 cells. B Western blotting to determine the level of MAP3K9 in COV434 cells transfected with MAP3K9 siRNA and scrambled control oligonucleotide. C Cleaved caspase-3 and p38 MAPK pathway-related proteins levels were analyzed by western blotting in miR-144-3p antagomir and MAP3K9 siRNA co-treated COV434 cells. GCs, granulosa cells. *P < 0.05; **P < 0.01, two-tailed Student’s t test

Fig 4: Map3k9 is upregulated in GCs of POF mice. A Bioinformatics prediction combined with mRNA microarray analysis were performed to identify the potential target genes of miR-144-3p. B Differentially expressed gene pathway enrichment analysis in the POF group and the control group. C Real-time PCR was used to evaluate the relative expression of target genes of miR-144-3p in the POF group and the control group. D Protein levels of MAP3K9 in both groups were detected using western blotting. E Immunohistochemistry analysis of the protein expression of MAP3K9 in both groups. Control group, n = 10; POF group, n = 10. GCs: granulosa cells; POF: premature ovarian failure. *P < 0.05; **P < 0.01, two-tailed Student’s t test. Scale bar, 100 μm

Fig 5: MAP3K9 is a target of miR-144-3p. A Real-time PCR showing that miR-144-3p expression was significantly decreased in cisplatin injured COV434 cells. B MAP3K9 mRNA and protein levels increased significantly, as analyzed using real-time PCR and western blotting, respectively. C Real-time PCR and western blotting were performed to detect the mRNA and protein levels of MAP3K9 in the injured COV434 cells transfected with miR-144-3p agomir and antagomir. D Predicted target sites and relevant mutation sequences are shown. Luciferase reporter assay results demonstrating that overexpression of miR-144-3p could reduce the site-specific luciferase activity of the MAP3K9 gene with wild type 3′ untranslated region (UTR). *P < 0.05; **P < 0.01, two-tailed Student’s t test