Stem cell exosomes with miR-644-5p may reduce ovarian cell death by controlling p53

The isolation of BMSCs was based on methods established in previous studies [15]. BMSCs were resuspended in DMEM medium containing 10% FBS, and the cells were evenly distributed in the culture dish by shaking gently, and then statically cultured at 37 °C, 5% CO₂.

To observe the effect of miR-664-5p carried by BMSC-derived exosomes on granulosa cells, we transfected shRNA-miR-664-5p into BMSCs and isolated the exosomes from BMSCs for the subsequent experiments.

BMSCs were identified by cell differentiation potential and surface markers. The specific method was to add 10% FBS, 5 μg/mL insulin, 0.1 μM dexamethasone, 0.2 mM vitamin C, and 10 mM β-glycerophosphate to IMDM to obtain an osteogenic induction medium. During the process of osteogenic differentiation, calcium ions could precipitate and form calcium nodules, and the potential of osteogenic differentiation of cells could be discriminated by the color reaction of alizarin red with calcium and the production of deep red-colored compounds. The specific method was to add 10% FBS, 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM IBMX, and 0.1 mM indomethacin to IMDM to obtain an adipogenic induction medium. The solution was changed once every 3 days and differentiated for 12 days. During the process of adipogenic differentiation, oil droplets appeared in the cytoplasm of cells. The oil droplets were subjected to target staining by oil red O staining to discriminate the potential of adipogenic differentiation of cells. In addition, flow cytometry was used to detect the expression of BMSC surface markers CD34 and CD90.

The above BMSCs isolated were cultured in DMEM supplemented with 10% FBS and expanded to the third passage to extract exosomes. Specifically, the supernatant was collected and centrifuged at 3000g for 15 min, and the supernatant was mixed with the ExoQuick exosome precipitation solution. After centrifugation of the ExoQuick mixture at 1500g for 30 min, the supernatant was gently aspirated. Exosomes were isolated from the remaining ExoQuick solution by centrifugation at 1500g for 5 min. Finally, the exosomes were resuspended in PBS. The isolated BMSC-derived exosomes were identified based on the morphological characteristics observed by TEM and the detection of exosomes surface markers by Western blot.

Fifteen C57BL/6 mice were from the Experimental Animal Center of Henan Province and divided into three groups after 6–7 weeks of feeding. The control group (n = 5) was intraperitoneally injected with normal saline. POF group (module group, n = 5) was intraperitoneally injected with 5 mg/kg cisplatin, and 100 μL PBS was injected into the tail vein on the 1st, 5th, and 10th day after modeling. The POF + BMSC exosome group (treatment group, n = 5) was intraperitoneally injected with 5 mg/kg cisplatin, and then exosomes (125 μg dissolved in 100 μL PBS) were injected into the tail vein on the 1st, 5th, and 10th day after modeling. Fifteen days later, the mice were sacrificed and the blood from the eyeball and ovarian tissues were taken for the subsequent experiments. Our research was approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

The collected ovarian tissue was fixed overnight in a Bouin solution (containing 5% acetic acid, 9% formaldehyde, and 0.9% picric acid), embedded in paraffin and serially sectioned ( 4 μM). After HE staining, the morphological structure of the ovary was observed under an optical microscope.

Mouse ovarian tissue was fixed, embedded in paraffin, and cut into 4 μm thick. After deparaffinization and rehydration, the sections were taken, then incubated with the antibody, and then immunohistochemically tested according to the kit supplier’s instructions.

The mice were euthanized to collect the ovarian tissue, and a 30-gauge needle was used to pierce the follicles and granulosa cells were obtained under a stereoscopic microscope. Two microliters of MEM-α medium (containing 3.7 mg/mL NaHCO₃, 10% (v/v) FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin) was added to granulosa cells and placed them in an 37 °C incubator with 5% CO₂. Two microliters of non-adherent cells were aspirated every 3 days and then 2 mL of fresh medium was added to continue the culture.

In order to clarify the function of exosomes to protect granulosa cells from cisplatin injury, 10 μg/mL cisplatin, granulosa cells, and 10 μg/mL exosomes were co-cultured for 48 h to detect granulosa cell viability or cell apoptosis, respectively.

Cisplatin-induced granulosa cell apoptosis was determined by flow cytometry using a FITC Annexin V/PI Apoptosis Detection Kit according to the manufacturer’s protocol. Briefly, cells were seeded in 6-well plates at a density of 4 × 10⁵. After 48 h, the cells were washed two to three times with cold PBS and stained with FITC Annexin V and propidium iodide (PI) for 15 min at room temperature using the Annexin V-FITC Apoptosis Detection Kit. The stained cells were analyzed using flow cytometry.

After the cells were seeded in 96-well plate at a density of 4 × 10⁴ for 48 h, 5 mg/mL of MTT was added to the mouse ovarian granulosa cells and incubated at 37 °C for 4 h. Subsequently, the medium was discarded, and then incubation was continued for 6 min at room temperature after adding DMSO. Absorbance was measured at 570 nm with a μQuant microplate reader (BioTek Instruments, Winooski, VT).

After exosomes were treated with 100 μg/mL RNase A for 1 h at 37 °C, 400 U/mL RNase inhibitor was added to terminate the reaction. Finally, the exosomes were washed by centrifugation.

Ovarian granulosa cells were lysed by placing them in a whole lysis buffer containing a protease inhibitor cocktail (Sigma). Protein concentration was measured using the Pierce BCA Protein Assay (Thermo). SDS-PAGE gel electrophoresis was used to separate proteins and transfer to PVDF membranes at 4 °C. The membrane was then blocked with 5% skim milk for 1 h and then the membrane was incubated with primary antibody overnight at 4 °C. Subsequently, the membrane was further incubated with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature, and finally, the band intensity was quantified using Image Lab software (Bio-Rad Laboratories).

The concentration of E2 in the mouse eye serum samples taken above was determined according to the ELISA kit supplier’s instructions (R&D Quantikine, R&D Systems Inc., Minneapolis, MN, USA).

Twenty-one overlapping miRNAs were screened by Target scan and microT-CDS, and miRNAs with the most significant differences in expression between BMSCs and 3 T3 exosomes were identified by qRT-PCR. Total RNA from exosomes was extracted using Trizol reagent. Total RNA was reverse transcribed into cDNA using the PrimeScriptTM RT kit. Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan) and Applied Biosystems 7500 Fast Real Time PCR System. The 2^-ΔΔCT formula was performed to calculate the relative expression levels.

Luciferase reporter plasmid containing the trp53 3′UTR was constructed and the trp53 3′UTR containing the mmu-miR-664-5p binding site mutation was cloned into the same reporter plasmid. The miR-664-5p mimic (miR-664-5p overexpression sequence) was subjected to lipofection with Lipofectamine 2000 (Invitrogen) and the above recombinant vectors were transfected into mouse ovarian granulosa cells. Subsequently, luciferase activity was measured using a Dual-Glo Luciferase kit (Promega) 48 h after transfection.

SPSS 13.0 was used for statistical analysis, and the data were presented as mean ± standard deviation (SD). When two groups were compared, the Student’s t test was used to analyze the differences between the groups. When multiple groups were compared, the differences among the groups were assessed by using one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference. All experiments were performed three times.

We identified the isolated BMSCs by detecting the pluripotent differentiation potential and surface markers of the cells. Microscopic observation showed that BMSCs formed calcium nodules after osteogenic differentiation induction, and the radioactive center was orange-red after staining with alizarin red. After BMSCs were induced to differentiate into adipogenesis, fine lipid droplets appeared in the cells (Fig. 1a). These data indicated that BMSC had both the ability of osteogenic and adipogenic differentiation. Flow cytometry results showed that BMSCs were immunopositive for markers of mesenchymal stromal stem cells namely CD90 and immunonegative for hematopoietic markers namely CD34 (Fig. 1b). BMSC-derived exosomes were identified by morphological observation and marker protein detection. By transmission electron microscopy, membranous vesicles of uniform size, round or oval shape, with clear margins and double lipid membranes surrounding them can be seen. Western blot results showed that the expression of exosome surface marker protein CD63 was significantly higher than BMSC lysate (Fig. 1c).

The therapeutic effect of BMSC-derived exosomes on POF was evaluated by injecting BMSC-derived exosomes into a POF mouse model (protocols were shown in Fig. 2a). HE staining ovarian tissue experiment showed that the mice had large and abundant follicles, abundant follicular fluid, and multiple corpus luteum in the control group. The follicles in the POF group were few and mostly primitive or initial follicles, and the atresia follicles formed by granule cell damage increased, and the interstitial increased. Compared with the POF group, the atresia follicles in the POF + exosome group decreased, while the corpus luteum increased (Fig. 2b). Immunohistochemistry detection of cleaved caspase 3 was performed on the ovarian tissues. Compared with the control group, the expression of c-caspase3 was significantly up-regulated in the POF group, while this up-regulation of c-caspase3 was inhibited after exosome injection (Fig. 2c). Western blot was performed to detect the expression level of P53 in the ovarian tissue. Results showed that the expression of P53 in the POF group was increased compared with the control group, while the expression of P53 was dramatically lower in the POF + exosome group than that in the POF group (Fig. 2d). The concentration of E2 in the serum was analyzed by ELISA. It was found that the concentration of E2 in the POF group decreased compared with the control group, and the concentration of E2 in the POF + exosome group was increased relative to the POF group (Fig. 2e). From the above experimental results, it could be seen that BMSC-derived exosomes could improve POF.

Flow cytometry analysis showed that compared with the control group, the proportion of early apoptotic cells was increased in the cisplatin group, while this increase was reversed by BMSC-derived exosomes. This indicated that BMSC-derived exosomes inhibited the damage of granulose cells to cisplatin. When BMSC-derived exosomes were treated with RNase, the proportion of early apoptotic cells was increased compared with cisplatin + BMSC exosome group, indicating that the RNA in BMSC-derived exosomes inhibited granulose cells damage to cisplatin (Fig. 3a). The same therapeutic result was reached in the cell viability assay (Fig. 3b). Western blot was performed to detect the expression difference of apoptosis-related proteins. Results showed that compared with the cisplatin group, the expression of P53 protein and c-caspase3 protein was down-regulated and the expression of Bcl2 was up-regulated in the cisplatin + BMSC exosome group. When BMSC-derived exosomes were treated with RNase, the expression of P53 protein and c-caspase3 protein were up-regulated and the expression of Bcl2 was down-regulated compared to cisplatin + BMSC exosome group (Fig. 3c). These results consistently showed that the RNA in BMSC-derived exosomes had a strong protective effect on cisplatin-induced ovarian granulosa cell damage.

Two databases, Target scan and microT-CDS, were used to search for the miRNA intersection predicted to target p53. A total of 21 miRNA intersections were found. We chose the top 10 miRNA from total miRNAs to verify their expression level by using qRT-PCR. The results indicated that miR-664-5p was highly expressed in BMSC-derived exosomes (Fig. 4a). Therefore, miR-664-5p was selected as the follow-up study object. From the results of dual-luciferase reporter assay, it was found that miR-664-5p bound to the 3′UTR of p53, which resulted in decreased relative luciferase activity in response to miR-664-5p mimic (Fig. 4b). This experimental result confirmed that miR-664-5p targeted p53 of cells. When miR-664-5p was overexpressed in mouse ovarian granulosa cells, cisplatin damage to granulosa cells was inhibited, and the expression of p53 protein was down-regulated (Fig. 4c). Briefly, we found that miR-664-5p targeted p53 of cells and inhibited granulosa cells damage to cisplatin.

To further validate the protective role of miR-664-5p carried by exosome on granulosa cells, we isolated exosomes from BMSCs after miR-664-5p knockout and observed its effect on granulosa cell apoptosis. Results showed that the proportion of early apoptotic granulosa cells induced by cisplatin was increased, which reversed the protective effect of BMSC-derived exosomes on cells (Fig. 5a). Western blot was used to detect the expression of p53 and Bcl2 in granulosa cells. Knockout of miR-664-5p in BMSC reversed the inhibition of p53 and the promotion of Bcl2 expression in granulosa cells by BMSC-derived exosomes (Fig. 5b). The above experiments confirmed that miR-664-5p in BMSC-derived exosomes had a protective effect on granulosa cells.

Not applicable.