miR-21 and Pellino-1 Expression Profiling in Autoimmune Premature Ovarian Insufficiency

Abstract

Background: Premature ovarian insufficiency (POI) represents the hypergonadotropic hypoestrogenic symptoms that result in the loss of ovarian follicles. 5-30% POI cases are suggested to be involved in autoimmune etiology. MicroRNA-21 (miR-21) plays a vital role in ovarian folliculogenesis via regulating and interacting with multiple target genes. Here, we conduct the target prediction of miR-21, identify the expression and correlation of miR-21 and its putative target Pellino-1 (Peli1), and confirm their relationship with clinical characteristics in autoimmune POI.

Methods: Bioinformatic analysis was conducted to screen the miR-21 putative target gene. Autoimmune POI mouse models were established by ZP3 immunization. Serum miR-21, Peli1 mRNA of peripheral blood mononuclear cells (PBMCs) and regulatory T cells (Tregs), general status, spleen Tregs ratio, inflammatory factors, ovarian endocrine function, and ovarian structure were evaluated. For autoimmune POI patients, serum miR-21, PBMCs Peli1 mRNA levels, general data, immune parameters, hormone levels, and ultrasound examinations were obtained. The correlations of miR-21 with Peli1 and clinical characteristics in patients were analyzed.

Results: Peli1 was selected based on four microRNA prediction databases and literature retrieval. In mouse models, serum miR-21 level, PBMCs and Tregs Peli1 mRNA, and spleen Tregs ratio were 0.61 ± 0.09, 0.12 ± 0.12, 0.27±0.23 and 4.82 ± 0.58, respectively, lower than those in the control group. In patients, miR-21 level (0.60 ± 0.14) and Peli1 mRNA (0.30 ± 0.14) were lower than those in the control group (1.01 ± 0.07 and 1.63 ± 0.54); miR-21 was positively related with Peli1, AMH, E₂, the size of the uterus, and ovarian volume and negatively related with FSH, LH, and the number of positive immune parameters (AOAb, EMAb, ACL, ANA, ds-DNA, ACA, IgG, IgA, IgM, IgE, C3, and C4).

Conclusions: Low expressions of miR-21 and Peli1 were detected in autoimmune POI mice and patients. Positive correlation between miR-21 and Peli1 was observed in autoimmune POI patients, suggesting that miR-21 and Peli1 might be associated with the pathogenesis of autoimmune POI.

Figure 1

Ovary function and structure of mice. (a) The levels of E₂ and AMH decreased while the level of FSH significantly increased in the autoimmune POI group. (b) Ovarian structure was observed under light microscopy (×40) after embedding and staining. Follicles of every stage were legible in the control group. And the amounts of follicles decreased in autoimmune POI mice. (c) Follicle counts were observed under light microscopy. The amounts of follicles of every stage obviously are lower in the autoimmune POI group.

Figure 2

Immunologic function and Peli1 expression in autoimmune POI mouse models. (a) The levels of inflammatory factors were tested using the ovary homogenate by the ELISA assay. In the autoimmune POI group, the expression of IFN-γ increased while IL-10 decreased. (b) Flow cytometry analysis was performed for Treg cell sorting. The ratio of Treg cells in the autoimmune POI group was apparently lower. (c–e) qRT-PCR was performed to detect the relative expressions of Peli1 mRNA and miR-21. Peli1 mRNA quantitation in both PBMCs (c) and spleen Treg cells (d) and serum miR-21 (e) were significantly lower in the autoimmune POI group.

Figure 3

miR-21 target genes in four databases.

Figure 4

Ovary structure and immune parameters of patients. (a) Antral follicles are observed in the ovary of a normal-cycling woman while no follicle images are seen in an autoimmune POI patient under ultrasonography. (b) Antibodies and immunity factors were considered immune parameters. Two or more positive immune parameters were found in 85.5% of autoimmune POI patients. (c) Ovarian volume was measured with ultrasound examination and decreased in the autoimmune POI group. (d) Peli1 mRNA was measured by qRT-PCR in PBMCs isolated from the peripheral blood of each participant of both groups. The relative expression of Peli1 mRNA in autoimmune POI patients is dramatically lower than normal-cycling women.