Exosomal miR-27 negatively regulates ROS production and promotes granulosa cells apoptosis by targeting SPRY2 in OHSS

Abstract

Ovarian hyperstimulation syndrome (OHSS) is one of the most dangerous iatrogenic complications in controlled ovarian hyperstimulation (COH). The exact molecular mechanism that induces OHSS remains unclear. In recent years, accumulating evidence found that exosomal miRNAs participate in many diseases of reproductive system. However, the specific role of miRNAs, particularly the follicular fluid-derived exosomal miRNAs in OHSS remains controversial. To identify differentially expressed follicular fluid exosomal miRNAs from OHSS and non-OHSS patients, the analysis based on miRNA-sequence was conducted. The levels of 291 miRNAs were significantly differed in exosomes from OHSS patients compared with normal control, and exosomal miR-27 was one of the most significantly down-regulated miRNAs in the OHSS group. By using MiR-27 mimic, we found it could increase ROS stress and apoptosis by down-regulating the expression of p-ERK/Nrf2 pathway by negatively regulating SPRY2. These data demonstrate that exosomal miRNAs are differentially expressed in follicular fluid between patients with and without OHSS, and follicular fluid exosomal miR-27 may involve in the pathological process of OHSS development.

Keywords: OHSS; exosome; follicular fluid; miRNAs.

FIGURE 1

Characterization of exosomes isolated from the human follicular fluid. A, Follicular fluid‐derived exosomes were examined by negative staining on transmission electron microscope. (scale bar, 200 nm). B, Nanoparticle tracking analysis profile of exosomes indicated the distribution of diameter size. The red line and black line represent OHSS group and normal control group. (C,D) Western blots of positive exosomal markers of exosomes (Alix, Tsg101, and CD 9) and two negative marker of exosomes (Calnexin and GM130). ExoA:OHSS group; ExoB: normal control group. GCs: human granulosa cells

FIGURE 2

Normal control group exosomes inhibits cell viability and promotes apoptosis in KGN cells. (A,B) Edu staining analysis of KGN cells. KGN cells were transfected with OHSS group and normal control group exosomes for 24, 48 and 72 hours. (scale bar, 50 μm). (C,D) KGN cells were transfected with exosomes for 48 h, and apoptotic cells were measured by Caspase3/7 activity kit. Relative caspase3/7 activity was calculated by average positive cell ratio (positive cells / total cells) and standardized the data with PBS group as the internal control. The results were shown as means ± SD (n = 3). (scale bar, 50 μm). (E,F) Western blot analysis of cleaved caspase 3 protein level(normalized to TUBULIN) after transfection with two groups exosomes for 48 h. The results were shown as means ± SD of three independent experiments. *<0.05.

FIGURE 3

The expression of miRNA‐27 in two groups of exosomes. A, Heat map of differentially expressed exosomal miRNAs from OHSS and normal control group. B, Venn diagrams of differentially expressed miRNAs. C, The Volcano map showing the distribution of differential miRNAs between the OHSS group and normal control group according to their P values and fold‐changes. Candidates with P <0.05 and |log 2(fold‐change)| ≥1 are considered differential. Red point indicated the 78 up‐regulated miRNSs, blue point indicated the 213 down‐regulated miRNAs in OHSS group. Black point represented the miRNAs that were no difference between OHSS and normal control samples, green point represented miR‐27. D, Exosomal miR‐27‐3p expression in OHSS and normal control groups of exosomes. (1.18 ± 0.24 vs 21.06 ± 2.60) (Mean ± SD, **<0.01,t‐test, n = 3). E, Relative expression of miR‐27 in exo‐free follicular fluid and exosomes. (0.11 ± 0.04 vs 1.007 ± 0.03) (Measn ± SD, **<0.01, t‐test, n = 3)

FIGURE 4

Exosomal miRNA‐27‐3p could target SPRY2. A, Venn figure of the number of predicted target genes of miR‐27‐3p from miRNA target prediction software (TargetScan, miRDB, miRWalk and miRTarBase). Red dotted box represented the common predicted targets. B, The four predicted targets were verified by human granulosa cells mRNA in two groups. (Mean ± SD, *<0.01,t‐test, n = 3). C, D, Targeting of SPRY2 by miR‐27‐3p was confirmed by a dual‐luciferase reporter assay. Luciferase activity in each group of 293T cells. Compared with the miR‐27‐3p‐NC + SPRY2‐3’UTR, miR‐27‐3p‐NC + SPRY2‐3’UTR Mut and miR‐27‐3p mimics + SPRY2‐3’UTR Mut groups. Mean ± SD, Student's t‐test. * <0.05, n = 3

FIGURE 5

SPRY2 interacts with EGFR in granulosa cells. A, The mRNA expression of SPRY2 and EGFR in two groups of granulosa cells. SPRY2:1.01 ± 0.05 vs 7.4 ± 0.80; EGFR:0.99 ± 0.03 vs 1.02 ± 0.05. (normal control vs OHSS, *<0.05, ** <0.01, n = 3). B, Western blot of SPRY2 and EGFR expression in two groups of granulosa cells. SPRY2:1.01 ± 0.04 vs 3.35 ± 0.13; EGFR:0.99 ± 0.03 vs 1.46 ± 0.14. (normal control vs OHSS * <0.05, ** <0.01, n = 3). C, The distribution of SPRY2 and EGFR in granulosa cells was showed by immunofluorescence. (scale bar, 20 μm). D, Immunocoprecipitation showed that there was interaction between SPRY2 and EGFR. (E,F) The mRNA and protein expression of SPRY2 in KGN cells after transfected with si‐SPRY. ** <0.01, n = 3. (G,H) The protein expression of SPRY2, EGFR, ERK, p‐ERK in the KGN cells after the expression of SPRY2 was knock down. * <0.05, n = 3

FIGURE 6

Exosomal miR‐27‐3p inhibited expression SPRY2 in KGN cells. A, The expression level of MiR‐27 in granulosa cells was increased by adding different concentrations of exosomes. * <0.05, ** <0.01, n = 3. B, Effect of normal control group exosome, miR‐27 mimic, miR‐27 inhibitor and miR‐27 NC on KGN cell miR‐27 expression. * <0.05, ** <0.01, n = 3. (C, D) The protein expression of SPRY2, EGFR, ERK and p‐ERK in KGN cells after transfection with normal control group exosome, miR‐27 mimic, miR‐27 inhibitor. * <0.05

FIGURE 7

miR‐27‐3p regulates KGN cells Nrf2 expression and promotes KGN cells apoptosis. (A,B) Protein levels of ERK, p‐ERK, Nrf2, Keap1 and cleaved caspase 3 in KGN cells after miR‐27 mimic or inhibitor transfection for 48 h. (C,D) Effect of miR‐27 mimic and mimic‐27 inhibitor on ROS release in KGN cells. Fluorescence intensity of H2DCFDA (green colour) was determined by Image J software. Each value in the graph represents the relative units(fluorescence/cell numbers). (mean ± SD, ** < 0.01, n = 3). (scale bar, 50 μm). (E,F) Tunel staining and number of Tunel‐positive cell in each group. *<0.05, ** < 0.01, n = 3. (scale bar, 50 μm)