CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

Abstract

Although HCO(3)(-) is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO(3)(-) acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO(3)(-) on preimplantation embryo development can be suppressed by interfering the function of a HCO(3)(-)-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO(3)(-) or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO(3)(-) removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO(3)(-) to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

Figure 1

Involvement of CFTR in HCO₃⁻-dependent preimplantation embryo development. (A) Effects of HCO₃⁻ on preimplantation embryo development. Embryos were cultured in different concentrations of HCO₃⁻. Transition from two-cell to four-cell embryos were significantly inhibited by removing (0 mM) or reducing extracellular (12.5 mM) HCO₃⁻ (0 mM group: 6/108 embryos; 12.5 mM group: 46/106 embryos; 25 mM group: 95/110 embryos; 50 mM group: 83/105 embryos). (B) CFTRinh172 (inh172) significantly reduced four-cell (n = 4, 12/85 embryos) and blastocyst formation (0/85 embryos) in embryo culture containing 25 mM HCO₃⁻ compared to the DMSO-treated vehicle control (four-cell: 57/88 embryos; blastocyst: 30/88). (C) Embryos obtained from Cftr^+/− mice (H × H) on 3.5 dpc have reduced percentage of expanded blastocysts (31/68 embryos) as compared to those obtained from Cftr^+/+ mice (W × W) (60/79 embryos). (D) Cftr^−/− embryos, as indicated by lacking CFTR immunoreactivity (arrow), exhibit a remarkable decrease in blastocyst formation rate (5/13 embryos) as compared to CFTR-positive embryos (38/48 embryos). (E) Embryos were categorized into two types according to the developmental stages and ZO-1 fluorescence: type A blastocysts showed continuous and well-organized ZO-1 expression only at the cell junction, while type B blastocyst showed disrupted expression of ZO-1 at the cell junction (arrow head), and diffused localization in cytoplasm. (F) Embryos from Cftr^+/+ and Cftr^+/− mice were classified by ZO-1 expression patterns (green) in blastocyst stages. Nuclei were counterstained by DAPI. Embryos obtained from Cftr^+/− mice have a significantly reduced percentage of type A blastocyst. (G) Embryos obtained from Cftr^+/− mice (H × H) on 1.5 dpc have reduced expanded blastocyst formation (60/122 embryos) after in vitro culture for 72 h as compared to those obtained form Cftr^+/+ mice (W × W) (87/126 embryos). Data are presented as mean ± SEM; one-way ANOVA (A); t-test (B-E); ^*P < 0.05 and ^***P < 0.001. Scale bar, 50 μm.

Figure 2

Involvement of miR-125b in CFTR-regulated embryo development. (A) Real-time PCR results showing high miR-125b expression in two-cell and four-cell embryos, but significantly reduced in morula and blastocyst stages (n = 4, 100 embryos/group). (B) MiR-125b expression in two-cell embryo was inhibited by CFTRinh172 treatment (compared to DMSO-treated vehicle control) and in HCO₃⁻-free condition (n = 4, 100 embryos/group). (C) Knockdown of miR-125b inhibited four-cell embryo formation (n = 4, 80 embryos/group). (D) Impaired four-cell and blastocyst formation by removal of HCO₃⁻ and CFTR inhibition was rescued by injection of miR-125b precursor (pre-miR-125b) (n = 3); HCO₃⁻ free + pre-miR-nc group: four-cell (15/62 embryos), blastocyst (0/62 embryos); HCO₃⁻ free + pre-miR-125b group: four-cell (36/61 embryos), blastocyst (21/60 embryos); Vehicle + pre-miR-nc group: four-cell (46/60 embryos), blastocyst (25/60 embryos); Vehicle + pre-miR-125b group: four-cell (55/64 embryos), blastocyst (38/64 embryos); CFTRinh172 + pre-miR-nc group: four-cell (10/60 embryos), blastocyst (0/60 embryos); CFTRinh172 + pre-miR-125b group: four-cell (33/63 embryos), blastocyst (19/63 embryos). Data are presented as mean ± SEM; One way ANOVA (A, B); t-test (C, D); ^**P < 0.01, ^***P < 0.001, ns – not significant. Scale bar, 50 μm.

Figure 3

p53 and p21 are the downstream targets of CFTR-HCO₃⁻-miR-125b pathway. (A) Immunofluorescent results showing increased expression of p53 (green) and p21 (red) in HCO₃⁻-free condition or after CFTRinh172 treatment (5-10 embryos/group). (B) Western blot results showing increased expression of p53 and p21 in two-cell embryos from Cftr^+/− mice (H × H) compared to those from Cftr^+/+ (W × W) mice. (C) Western blot results showing upregulation of p53 and p21 by CFTRinh172 or HCO₃⁻-free condition in two-cell embryos. (D) Western blot results showing that transfection of anti-miR-125b oligo leads to increased expression of p53 and p21, while transfection of miR-125b precursor leads to decreased expression of p53 and p21 in two-cell embryos. Scale bar, 50 μm.

Figure 4

Involvement of CFTR and HCO₃⁻ in human embryo development and regulation of miR-125b-p53 cascade. (A) Decreased four-cell and blastocyst formation in HCO₃⁻-free condition or after CFTRinh172 (inh172) treatment (n = 3; HCO₃⁻-free group: four-cell (9/60 embryos), blasocyst (0/60 embryos); Vehicle group: four-cell (30/63 embryos), blastocyst (27/63 embryos); CFTRinh172 group: four-cell (6/62 embryos), blastocyst (0/62) ). (B, C) Real-time PCR showed expression of miR-125b, p53 and p21 under HCO₃⁻-free condition (B) or after CFTRinh172 treatment (C) (n = 4 100 embryos/group). Inhibition of CFTR or cultured in HCO₃⁻-free condition reduced the expression of miR-125b and increased the expression of p53 and p21. Data are represented as mean ± SEM; One way ANOVA (A); t- test (B, C); ^**P < 0.01, ^***P < 0.001.

Figure 5

Involvement of sAC/PKA/NF-κB cascade in HCO₃⁻-dependent miR-125b activation. (A) Involvement of sAC in the HCO₃⁻-dependent embryo development. HCO₃⁻-dependent four-cell formation was significantly inhibited by addition of sAC inhibitor KH7 (10/86 embryos) and PKA inhibitor H89 (14/80 embryos) compared to DMSO-treated vehicle control (60/77 embryos (KH7); 56/75 embryos (H89)). (B) miR-125b expression in two-cell embryo was inhibited by both KH7 and H89 treatments compared to DMSO-treated vehicle control. (C, D) Immunofluorescent staining showing the localization of NF-κB p65 subunit in both nucleus and cytoplasm in 25 mM HCO₃⁻ condition (23/30 embryos). HCO₃⁻-free (3/20 embryos), 10 μM CFTRinh172 (4/28 embryos), 10 μM KH7 (7/30 embryos), 20 μM PKA inhibitor H89 (3/27 embryos) (C) and 10 μM NF-κB inhibitor AKβBA (4/24 embryos) (D) treatments triggered the extrusion of NF-κB p65 from nucleus to the cytoplasm compared to DMSO-treated vehicle control group (20/26 embryos (KH7); 26/30 embryos (AKβBA)); statistics showing HCO₃⁻-free condition, CFTRinh172, H89 and AKβBA significantly reduced the percentage of p65 nuclear positive embryos. (E) Real-time PCR results showing that AKβBA inhibited miR-125b expression in two-cell embryos (n = 4 100 embryos/group). Data are represented as mean ± SEM. One way ANOVA (C); t-test (A, B, D); ^**P < 0.01, ^***P < 0.001. Scale bar, 50 μm.

Figure 6

Working model for the regulation of early embryo development by CFTR/HCO₃⁻-dependent activation of miR-125b. CFTR mediates the influx of HCO₃⁻ ion directly and/or indirectly by cooperating with an anion exchanger. The influx of HCO₃⁻ activates sAC, an enzyme that converts ATP to cAMP, which in turn activates PKA, triggering the nuclear shuttle of NF-κB a transcription factor known to regulate the expression of miR-125b. Induction of miR-125b expression by CFTR-mediated HCO₃⁻ influx maintains the dormancy of p53, which is required for early embryo development.