Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy

Abstract

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

Figure 1. Delivery of miRNAs into damaged GCs via exosomes.

(a,b) qRT-PCR analysis of expression levels of miR-146a and miR-10a in AFSCs and NIH-3T3 (a) and their corresponding exosomes (b). Error bars represent s.e.m. n = 3. *P < 0.05; unpaired t test. (c) A representative micrograph shows that PKH26 labeled-AFSC-derived exosomes (30 μg ml⁻¹ of exosomes proteins) (red) were incorporated into the cytoplasm of damaged GCs. Nucleus was stained by DAPI (blue). Scale bar, 40 μm. (d) The levels of miR-146a and miR-10a in damaged GCs cultured with AFSC-derived exosomes at different time points compared to 0 h (Ctrl). Error bars represent s.e.m. n = 3. *P < 0.05; unpaired t test. (e) The sequence alignment of miR-146a and its predicted target sites of the mouse Irak1 and Traf6 mRNA 3′-untranslated region (3′-UTR), and miR-10a and its putative target sites of the mouse Bim mRNA 3′-UTR. (f) The expression levels of Irak1, Traf6, Bim and Casp9 in damaged GCs cultured with AFSC-derived exosomes at different time points compared to 0 h (Ctrl). Error bars represent s.e.m. n = 3. *P < 0.05; unpaired t test.

Figure 2. Down-regulation of miR-146a and/or miR-10a impaired the effects of AFSC-derived exosomes on damaged GCs in vitro.

(a,b) qRT-PCR analysis showed that the expression of miR-146a and miR-10a was knocked down in AFSCs (a) and AFSC-derived exosomes (b) when AFSCs were transfected with miR-146a or/and miR-10a inhibitors. Error bars represent s.e.m. n = 3. Different characters (a,b) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (c) The fold change of relative cell number of damaged GCs cultured with AFSC-derived exosomes with various transfections at different time points. Error bars represent s.e.m. n = 6. Different characters (a–c) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (d) The percentage of apoptotic cells of damaged GCs cultured with AFSC-derived exosomes with various transfections at different time points. Error bars represent s.e.m. n = 3. Different characters (a–c) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test.

Figure 3. MiR-10a recapitulates the effects of AFSC-derived exosomes on damaged GCs in vitro.

(a–d) The expression levels of Irak1 (a), Traf6 (b), Bim (c) and Casp9 (d) of damaged GCs cultured with liposomes with various cargos at different time points. Error bars represent s.e.m. n = 3. Different characters (a–c) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (e) The relative cell number (fold change) of damaged GCs cultured with liposomes with various cargos at different time points. Error bars represent s.e.m. n = 6. Different characters (a–c) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (f) The percentage of apoptotic cells of damaged GCs cultured with liposomes with various cargos at different time points. Error bars represent s.e.m. n = 3. Different characters (a–c) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test.

Figure 4. MiR-10a exerts beneficial effects on ovaries of CTx-mice.

(a) The percentage of apoptotic cells in the ovaries of each group at different time points. Error bars represent s.e.m. n = 3. Different characters (a,b) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (b) Atretic follicles were counted in the ovarian sections of each group at different time points. Error bars represent s.e.m. n = 3. Different characters (a,b) represent significant differences (P < 0.05) among each group; Tukey’s multiple comparisons test. (c) A schematic diagram of the restorative mechanism of AFSCs on damaged GCs. AFSCs can restore the fertility and prevent POF in CTx treated mice possibly by delivering miR-146a, miR-10a and other potential miRNAs via exosomes to damaged GCs, and in turn down-regulating the pro-apoptotic genes. The dotted line represents the putative interactions between Irak1/Traf6 and the apoptotic pathway and that the potential miRNAs contribute to anti-apoptosis.