1. Identification of PMSCs and its Exos
After osteogenic differentiation of PMSCs was induced, calcium nodules were formed via Alizarin Red staining. After adipogenic differentiation was induced, Oil Red O staining showed obvious lipid droplets in the PMSCs (Fig. 1a). The results of flow cytometry demonstrated that the PMSCs were positive for MSC markers, such as CD90, CD73, and CD105, and negative for hematopoietic markers, such as CD34 (Fig. 1b). The TEM results showed that the Exos were in the shape of round or oval membrane vesicles, and their peak particle size was about 117 nm according to NTA (Fig. 1c). The Exos markers CD9 and HSP70 were well expressed according to WB (Fig. 1d). Among the biomolecules carried by MSC-Exos, miRNAs received the most attention due to their important role in regulating gene expression23. Therefore, RNA-seq analysis of PMSC-Exos was conducted, and miR-21-5p was found to have the highest content of 16.395% (Fig. 1e). Therefore, miR-21-5p was chosen as the focus of the subsequent mechanism research.
2. Therapeutic effect of PMSC-Exos on POI, in which miR-21-5p plays an important role
The miR-21-5p inhibitor of PMSCs was transfected to knock down miR-21-5p to explore its role in treating PMSC-Exos (Fig. 2a). Then, in-Exos were obtained. A POI model was constructed with cisplatin-induced OGCs, and Exos and in-Exos were cocultured with OGCs separately to explore the therapeutic effect of PMSC-Exos. First, the expression difference of apoptosis-related proteins was detected by WB. The results showed that compared with the cisplatin group, the cisplatin + Exos group had downregulated Bax expression and upregulated Bcl2 expression. When the miR-21-5p in Exos was knocked down, the expression levels of Bax and Bcl2 in the cisplatin + in-Exo group was upregulated and downregulated, respectively, compared with those in the cisplatin + Exos group (Fig. 2b,c). Flow cytometry analysis showed that compared with the proportion of apoptotic cells in the control group, that in the cisplatin group significantly increased. Moreover, the proportion of apoptotic cells in the cisplatin + Exos group significantly decreased compared with that in the cisplatin group. However, compared with the cisplatin + Exos group, the cisplatin + in-Exos group demonstrated an increase in apoptosis rate (Fig. 2d,e). This finding suggested that PMSC-Exos inhibited cisplatin-induced apoptosis of OGCs, in which miR-21-5p played an important role. The effects of Exos on the proliferation and migration of OGCs were examined by CCK-8 and wound-healing assay, respectively. The results showed that Exos could promote the proliferation and migration of granulosa cells. When miR-21-5p was knocked down, this promoting effect weakened (Figs. 2f-h). These results indicated that PMSC-Exos can inhibit apoptosis of granulosa cells and promote their proliferation and migration, in which miR-21-5p plays a strong protective role.
3. MiR-21-5p regulates apoptosis of OGCs by targeting PTEN
MiRNAs control the expression of post-transcriptional target genes, mainly by inhibiting translation or promoting mRNA degradation in the cytoplasm24. Identifying a gene that miR-21-5p targets is necessary to further explore its regulation on the downstream mechanism. Therefore, the target gene PTEN of miR-21-5p was identified by referring to literature21,25 and the database22.
Dual luciferase reporter gene assays were performed to demonstrate that miR-21-5p could indeed target PTEN. The results showed that miR-21-5p mimics significantly inhibited the luciferase activity of the PTEN wild-type 3ʹ UTR reporter gene, whereas vectors containing PTEN mutated 3ʹ UTR significantly eliminated this inhibition (Fig. 3a,b). After miR-21-5p mimic and inhibitor were transfected, the PTEN RNA and protein expression levels were detected by qRT-PCR and WB, respectively. The results showed that miR-21-5p mimic could up-regulate the RNA and protein levels of PTEN, whereas miR-21-5p inhibitor could down-regulate them (Figs. 3c-e). The changes in miR-21-5p and PTEN after Exo/in-Exo treatment were also examined. The qRT-PCR results showed that the expression of miR-21-5p in the Exos group significantly increased, whereas the RNA level of PTEN decreased. Compared with the Exos group, the in-Exos group showed a significant reduction in the content of miR-21-5p, whereas the RNA level of PTEN increased (Figs. 3f,g). This finding indicated that Exos can deliver the most abundant miR-21-5p into OGCs, and the miR-21-5p entering cells could target PTEN and inhibit its expression. In addition, the WB results showed that cisplatin induction increased the PTEN protein levels. Compared with the cisplatin group, the cisplatin + Exos group had significantly decreased PTEN expression. However, the PTEN in the cisplatin + in-Exos group was higher than that in the cisplatin + Exos group (Fig. 3h,i). Next, miR-21-5p was transfected into cisplatin-induced OGCs, and the results found that miR-21-5p reversed the cisplatin-induced up-regulation of PTEN (Fig. 3j,k). All in all, the results fully confirmed that the miR-21-5p delivered by PMSC-Exos can bind to the 3ʹ UTR region of PTEN to inhibit its expression.
4. MiR-21-5p-PTEN axis regulates the AKT/mTOR signaling pathway
PTEN can inhibit the activation of the PI3K/AKT/mTOR pathway, thereby inhibiting cell growth and proliferation26. Therefore, in the present study, miR-21-5p was hypothesized to regulate the AKT/mTOR pathway by targeting PTEN. First, WB detected the phosphorylation changes in the AKT/mTOR pathway genes after Exos/in-Exos treatment. The results showed that cisplatin significantly inhibited the activation of the AKT/mTOR pathway. Compared with the cisplatin group, the cisplatin + Exos group showed a significantly increase in the phosphorylation level of AKT/mTOR pathway proteins, and when miR-21-5p was inhibited, the therapeutic effect of Exos weakened (Fig. 4a,b). Then, WB was used to detect the phosphorylation changes in the pathway after transfection with miR-21-5p mimic/inhibitor, the results showed that the phosphorylation levels of the AKT/mTOR pathway genes in the miR-21-5p mimic group increased compared with those in the NC mimic group. However, the phosphorylation levels of the AKT/mTOR pathway genes in the miR-21-5p inhibitor group decreased compared with those in the NC inhibitor group (Fig. 4c,d). The transfection of miR-21-5p into cisplatin-induced OGCs alone reversed the cisplatin inhibition of the AKT/mTOR pathway (Fig. 4e,f).
Next, two siRNAs of PTEN were synthesized to reduce the expression of PTEN. The si-PTEN-1 and si-PTEN-2 detected by qRT-PCR and WB could significantly reduce the expression of PTEN (Figs. 5a-c). Then, the miR-21-5p inhibitor was co-transfected with si-RNAs, and the miR-21-5p mimic was co-transfected with AKT pathway inhibitor (LY294002)27. The protein expression levels of PTEN and the AKT/mTOR pathway were detected by WB. The results showed that simultaneous knockdown of PTEN could reverse the PTEN increase and AKT/mTOR phosphorylation decrease caused by miR-21-5p inhibitor. However, LY294002 could reverse the PTEN decrease and AKT/mTOR phosphorylation increase induced by miR-21-5p mimic to a certain extent (Fig. 5d-f). The above results fully proved that miR-21-5p promotes the AKT/mTOR pathway by inhibiting the expression of PTEN.
5. MiR-21-5p delivered by PMSC-Exos inhibits apoptosis of OGCs and promote proliferation and migration via the PTEN/AKT/mTOR axis
The results demonstrated that miR-21-5p can regulate the PTEN/AKT/mTOR axis, and the next step is to verify the functional regulation of this signaling axis on OGCs. After transfecting miR-21-5p mimic/inhibitor separately, the cell apoptosis, proliferation and migration were detected by WB, CCK8 and Wound-Healing Assay, respectively. The results showed that miR-21-5p mimic could promote the expression of Bcl-2 and inhibit the expression of Bax. On the contrary, miR-21-5p inhibitor could inhibit the expression of Bcl-2 and promote the expression of Bax (Fig. 6a,b). Moreover, miR-21-5p mimic could promote the proliferation and migration of granulocyte cells, while miR-21-5p inhibitor played the opposite role (Figs. 6c-e). Then, to more fully confirm that miR-21-5p could inhibit the apoptosis of OGCs in POI, miR-21-5p mimic was transfected under cisplatin induction. As shown in Fig. 6f,g, the results of flow cytometry showed that miR-21-5p could effectively inhibit cisplatin-induced apoptosis. Similar results were found in WB. Compared with the cisplatin group, the cisplatin + miR-21-5p group demonstrated an increase in Bcl-2 expression and a decrease in Bax expression (Fig. 6h,i). Finally, WB was used to detect the changes in apoptosis-related protein expression levels among different groups. The results showed that the Bcl-2 decrease and Bax increase caused by miR-21-5p inhibitor were rescued by siRNA-PTEN. LY294002 also alleviated the increase in Bcl-2 and the decrease in Bax caused by miR-21-5p mimic (Fig. 6j,k).
In summary, a large amount of miR-21-5p carried by PMSC-Exos can inhibit apoptosis of OGCs and promote proliferation and migration by regulating the PTEN/AKT/mTOR axis (Fig. 7).