Fig 2: YY1 is essential for Xist expression and functions as an upstream regulator for Xist-miR-23b-3p/miR-29a-3p-Stx17 axis during PF formation.a Schematic representation for YY1 binding motif within the proximal promoter region of Xist, with respective primer sets to amplify YY1 binding region. The upstream control region is also represented (top). Bottom: ChIP analysis of YY1 binding in newborn mouse ovaries at 0.5 dpp, and 4.5 dpp. The dashed line represents the basal IgG binding. b, c Relative Yy1 mRNA (b), and YY1 protein (c) expression in perinatal ovaries at indicted time. d, e Relative Yy1 mRNA (top), and YY1 protein (bottom) expression in cultured newborn ovaries transfected with siYy1 or control (d), pcDNA3.1-Yy1, and NC control (e). f Relative Xist mRNA expression in cultured newborn ovaries transfected with siYy1 or siRNA control (top), pcDNA3.1-Yy1 or NC control (bottom). g, h Representative DDX4 immunofluorescence images (g) and quantification of follicles (h) in newborn ovaries transfected under indicated condition. Scale bars: 50 µm. i WB analysis of STX17 expression in newborn ovaries treated under indicated condition. j Proposed model for Xist-miR-23b-3p/miR-29a-3p-STX17 axis in regulating oocyte loss in the perinatal mouse ovaries. Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: STX17 is involved in the mechanism of miR-34a-mediated autophagy activity. (A) Positions of the putative miR-34a binding sites in 3'UTR of SNAP29, RAB7L1, STX17 and EPG5. (B and C) RT-qPCR assay revealed SNAP29, RAB7L1, STX17 and EPG5 mRNA expression after miR-34a mimic treatment. (D and E) The STX17 protein expression was detected using a western blot assay. (F) Transfection of bladder cancer cells with STX17-3'-UTR plasmid (1 µg/µl) and miR-34a mimic (100 nM) for 24 h, then relative luciferase/Renilla activities were measured. (G-I) The basal levels of STX17 protein and mRNA expression in control stable and miR-34a stable cells were detected using western blotting and RT-qPCR assay, respectively. All data are expressed as the mean ± SD in triplicate samples. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 relative to the control group. STX17, syntaxin 17; miR, microRNA; UTR, untranslated region; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; Con, control; ns, not significant.

Fig 4: STX17 overexpression rescues miR-34a-inhibited autophagy activity. (A) BC cells were transfected with vector or STX17 plasmid (1 µg/µl) for 24 h. The overexpressing efficiency of STX17 was verified using western blot assays in 5637 and T24 cells. (B and C) Co-transfection of BC cells with miR-34a mimic and STX17 plasmid for 24 h, the LC3-I/II and p62 protein expression were then analyzed through western blotting. All data are expressed as the mean ± SD in triplicate samples. *P<0.05, **P<0.01 and ****P<0.0001 relative to the control group. STX17, syntaxin 17; miR, microRNA; BC, bladder cancer.

Fig 5: MiR-34a confers chemosensitivity in BC cells. (A-D) The miR-34a stable cells were transfected with or without STX17 plasmid (1 µg/µl) for 24 h followed by exposing to various concentrations of chemotherapeutic drugs, including cisplatin (0-50 µM), doxorubicin (0-200 µg/ml), epirubicin (0-200 µM) and mitomycin C (0-200 nM). After 24 h of treatment, resazurin-based cell viability was measured. (E-H) The miR-34a stable cells were treated with chemotherapeutic drugs, including cisplatin (1.5 µM), doxorubicin (0.1 µg/ml), epirubicin (0.1 µM) and mitomycin C (12.5 nM) for 7 days. Cell survival was detected by colony formation assay. All data are expressed as the mean ± SDs in triplicate samples. *P<0.05, **P<0.01, and ****P<0.0001 relative to the control group. miR, microRNA; BC, bladder cancer; STX17, syntaxin 17.