Downregulation of MicroRNA-222 Reduces Insulin Resistance in Rats with PCOS by Inhibiting Activation of the MAPK/ERK Pathway via Pten.

Polycystic ovary syndrome (PCOS), characterized by the dysfunction of endocrine metabolism, is a common disease among women. Insulin (INS) resistance (IR) is considered as an obstruction to effective PCOS treatment. Here, we aimed to explore the mechanism by which microRNA-222 (miR-222) affects IR in PCOS via Pten. Quantitative reverse transcription-polymerase chain reaction and western blot assays indicated that miR-222 expression was higher in the peripheral blood of PCOS patients with IR than in PCOS patients without IR, while Pten expression was lower. Further mechanistic analysis identified Pten as a target gene of miR-222. Moreover, PCOS rat models were established through the administration of dehydroepiandrosterone and were subsequently treated with miR-222 agomir, miR-222 antagomir, or Pten overexpression plasmid. The inhibition of miR-222 improved ovarian morphology, enhanced the production of serum sex hormones (follicle-stimulating hormone [FSH], luteotropic hormone [LH], estradiol 2 [E2], prolactin [PRL], and testosterone [T]), increased the levels of glucose metabolism indicators (homeostasis model of assessment for IR [HOMA-IR], blood glucose [BG]120min, and INS120min), and reduced the production of progesterone in the PCOS rats. Notably, miR-222 downregulation resulted in the inactivation of the mitogen-activated protein kinase (MAPK)/ERK pathway by upregulating Pten. Collectively, miR-222 inhibition might reduce IR in PCOS by inactivating the MAPK/ERK pathway and elevating Pten expression, which indicates miR-222 as a promising target for PCOS treatment.

Introduction

Polycystic ovary syndrome (PCOS) is regarded as the most prevalent endocrine metabolic disorder, affecting 6%–9% of reproductive-age women, according to the National Institutes of Health criteria. In PCOS, ovaries contain multiple small cysts and are characterized by increased deposition of small follicles of 4–7 mm in diameter and hypertrophied theca internal layers. Nevertheless, PCOS tends to influence many organ systems and leads to different health complications, such as hirsutism, acne, menstrual dysfunction, and damage profiles for cardio-metabolic risk factors. Intriguingly, approximately 60%–70% of PCOS patients exhibit insulin (INS) resistance (IR), which can be attributed to body mass, race, or age. In animal reproduction and some fields of human reproductive medicine, INS and INS-mediated uptake of glucose are crucial to ovarian cells. The correlation of IR with PCOS is yet to be comprehensively understood, as there is no deficiency in traditional INS pathways, including INS binding and INS receptor expression. Particularly, women with PCOS exhibit a significantly higher risk for IR, dyslipidemia, impaired glucose tolerance, and type 2 diabetes mellitus. Thus, these findings described above imply the presence of an underlying correlation between IR and PCOS, which requires further elucidation.

Notably, microRNAs (miRNAs) are a large group of small noncoding RNAs that regulate the expression of their numerous target genes, typically at the posttranscriptional level, thereby playing a significant role in physiological and pathological processes. miRNAs suppress protein expression through imperfect base pairing with the 3′ untranslated region (3′ UTR) of the target mRNA in mammalian cells, which results in the reduction of translation. Depending on the biological functions of their target genes, miRNAs may have a tumor-promoting or tumor-suppressing effect on cancer cells. miR-221 and miR-222 are overexpressed in radioresistant tumor cell lines. Pten (phosphatase and tensing homolog) is a tumor suppressor that is altered in human cancers and may regulate cell growth and apoptosis. Moreover, Pten is also involved in the modulation of the division and proliferation of granulosa cells in human ovaries. Intriguingly, Pten has been identified as a direct target of miR-222 by bioinformatics analysis. Hence, despite IR being a recognized feature of PCOS, the underlying molecular mechanisms that reduce cellular INS sensitivity remain unclear. In the present study, we selected 168 patients with PCOS, among which 76 patients showed normal ovulation. Additionally, we further verified our results in vivo by inducing a rat model of PCOS to examine the role of miR-222 in the IR of PCOS.

Results

miR-222 Was Highly Expressed in Peripheral Blood of PCOS Patients with IR, while Pten Showed Reciprocal Trends

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine miR-222 and Pten expression in the peripheral blood of PCOS patients in the IR and non-IR groups. Compared with the control group, the PCOS patients in the IR and non-IR groups exhibited increased miR-222 expression and decreased Pten mRNA expression (Figure 1A); the IR group showed higher miR-222 expression and lower Pten mRNA expression than that in the non-IR group. Based on the correlation analysis, miR-222 expression was negatively correlated with the mRNA expression of Pten in the IR group (Figure 1B).

Figure 1

The Expression of miR-222 and Pten in the Peripheral Blood of PCOS Patients with IR

(A) miR-222 expression and Pten mRNA expression in peripheral blood of PCOS patients in the IR and non-IR groups detected by qRT-PCR. (B) The correlation of miR-222 expression and Pten mRNA expression in PCOS patients with IR evaluated by Pearson’s coefficient. In (A), data comparison was conducted by one-way ANOVA. ∗p < 0.05, versus the control group; #p < 0.05, versus the non-IR group. miR-222, microRNA-222; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; IR, insulin resistance; PCOS, polycystic ovary syndrome.

miR-222-Targeted Pten

Based on previous studies, there are miR-222-binding sites in PCSK9, MIA3, ABCG2, PUMA, DKK2, and Pten. To further evaluate it in the present study, miR-222 mimic or negative control (NC) mimic was transfected into 293A cells. qRT-PCR results showed that, compared with that in the NC mimic group, the expression of PCSK9, MIA3, ABCG2, PUMA, DKK2, and Pten was decreased in the miR-222 mimic group and that among these, the difference in expression of Pten was the most significant (Figure 2A). Pten was identified as the target gene of miR-222 following retrieval of the TargetScan database (www.targetscan.org) (Figure 2B). The results of the dual-luciferase reporter assay showed that the co-transfection of the miR-222 mimic and Pten-WT resulted in a decrease in luciferase activity compared to that with the remaining treatments (p < 0.05; Figure 2C), indicating that Pten was a direct target gene of miR-222.

Figure 2

The Targeting Relationship between Pten and miR-222

(A) mRNA expression of PCSK9, MIA3, ABCG2, PUMA, DKK2, and Pten after miR-222 mimic transfection determined by qRT-PCR. (B) miR-222-specific binding sites on Pten predicted by TargetScan. (C) Targeting the relationship between Pten and miR-222 confirmed by dual-luciferase reporter assay. ∗p < 0.05, versus the NC mimic group. miR-222, microRNA-222; NC, negative control; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Inhibition of miR-222 Attenuated the Morphological Changes in the Ovaries of PCOS Rats

Thereafter, morphological changes in the ovaries of PCOS rats were observed. As shown in Figure 3, numerous follicles and corpus luteum cysts were observed at each growth stage in the ovaries of the normal group; the granulosa cells had an orderly arrangement, with intact morphological integrity. However, the number of granulosa cells was lower in the experimental model group; the number of granulosa cell layers was decreased, and the follicles were cystic dilated. The changes in the NC and miR-222 agomir + Pten groups were not significant compared with those in the model group. The miR-222 agomir and miR-222 agomir + empty vector groups presented with increased numbers of cystic follicles and decreased numbers of follicles and corpus luteum cysts compared with the model group, while the normal and miR-222 antagomir groups exhibited the opposite trend by showing an increased number of granulosa cell layers. Taken together, these findings suggest that the inhibition of miR-222 expression can attenuate the morphological changes in the ovaries of PCOS rats.

Figure 3

Morphology of Ovarian Tissues in Rats of Each Group Observed by H&E Staining

Arrows indicate cystic follicles (200×).

Inhibition of miR-222 Decreased Serum Sex Hormone Production and Glucose Metabolism in PCOS Rats

The potential influence of miR-222 on serum sex hormone production (Table 1) and glucose metabolism (Table 2) was investigated in PCOS rats. After treatment, the expression of the homeostasis model of assessment for IR (HOMA-IR) in the normal group was lower than 2.69, indicating non-IR in the normal group. Compared with the normal group, the model, NC, and miR-222 agomir groups showed increased expression of follicle-stimulating hormone (FSH), luteotropic hormone (LH), estradiol 2 (E2), prolactin (PRL), testosterone (T), HOMA-IR, blood glucose (BG)120min, INS120min, gonadotropin-releasing hormone (GnRH), INS autoantibody (IAA), islet cell antibody (ICA), and glutamic decarboxylase antibody (GAD) (p < 0.05). The miR-222 agomir group showed an increasing trend, whereas the miR-222 antagomir group showed a decreasing trend compared with the model group (p < 0.05). Moreover, compared with the normal group, progesterone (P) expression was decreased in the model, NC, miR-222 agomir, and miR-222 agomir + Pten groups. On the other hand, the miR-222 antagomir group did not exhibit any significant difference (p > 0.05). However, compared with the model group, P expression was lower in the miR-222 agomir and miR-222 agomir + empty vector groups and higher in the miR-222 antagomir group (p < 0.05). These results collectively demonstrated that the miR-222 antagomir can improve serum sex hormone production and glucose metabolism in PCOS rats.

Table 1

Inhibition of miR-222 Improves the Production of Serum Sex Hormones of the PCOS Rats

Group	FSH	LH	E2	P	PRL	T	HOMA-IR	GnRH
	(mIU/mL)	(mIU/mL)	(ng/L)	(ng/mL)	(μg/L)	(μg/L)
Normal	2.04 ± 0.67a	1.50 ± 0.31a	94.01 ± 21.78a	34.71 ± 6.82a	13.56 ± 3.51a	0.13 ± 0.03a	2.13 ± 0.11a	11.51 ± 1.12a
Model	2.79 ± 0.71b	2.24 ± 0.62b	129.76 ± 23.62b	17.21 ± 1.96b	23.11 ± 2.75b	0.29 ± 0.03b	3.79 ± 0.42b	21.12 ± 2.05b
NC	2.82 ± 0.77b	2.21 ± 0.71b	127.13 ± 19.73b	18.34 ± 1.93b	22.94 ± 2.79b	0.31 ± 0.04b	3.71 ± 0.43b	22.91 ± 2.07b
miR-222 agomir	3.65 ± 0.72c	2.95 ± 0.97c	159.76 ± 23.62c	12.11 ± 1.02c	28.11 ± 2.75c	0.39 ± 0.07c	4.54 ± 0.79c	29.99 ± 2.73c
miR-222 antagomir	2.07 ± 0.66a	1.51 ± 0.68a	102.41 ± 16.23a	32.63 ± 3.21a	14.57 ± 1.97a	0.17 ± 0.07a	2.06 ± 0.22a	12.57 ± 1.09a
miR-222 agomir + empty vector	3.67 ± 0.78c	2.99 ± 0.99c	162.45 ± 24.03c	12.05 ± 1.07c	28.11 ± 2.75c	0.41 ± 0.08c	4.53 ± 0.78c	28.11 ± 2.75c
miR-222 agomir + Pten overexpression	2.85 ± 0.78b	2.23 ± 0.67b	132.87 ± 21.57b	19.07 ± 1.87b	21.07 ± 2.64b	0.32 ± 0.06b	3.74 ± 0.41b	21.78 ± 2.01b

PCOS, polycystic ovary syndrome; FSH, follicle-stimulating hormone; LH, luteotropic hormone; E2, estradiol 2; P, progesterone; PRL, prolactin; T, testosterone; HOMA-IR, homeostasis model assessment for insulin resistance; GnRH, gonadotropin-releasing hormone; NC, negative control.

compared with the Model group P < 0.05.

compared with the Normal group P < 0.05.

compared with the NC group P < 0.05.

Table 2

Inhibition of miR-222 Improves Glucose Metabolism of the PCOS Rats

Group	IAA (μg/mL)	ICA (μg/mL)	GAD (g/L)	BG120min (mmol/L)	INS120min (mU/L)
Normal	0.18 ± 0.08a	0.12 ± 0.06a	0.17 ± 0.08a	5.08 ± 0.47a	11.51 ± 1.12a
Model	1.34 ± 0.12b	1.25 ± 0.09b	1.31 ± 0.11b	6.12 ± 0.51b	21.12 ± 2.05b
NC	1.31 ± 0.11b	1.24 ± 0.08b	1.33 ± 0.12b	6.15 ± 0.52b	22.91 ± 2.07b
miR-222 agomir	2.47 ± 0.18c	2.81 ± 0.21c	3.71 ± 0.26c	6.79 ± 0.56c	29.99 ± 2.73c
miR-222 antagomir	0.16 ± 0.06a	0.11 ± 0.06a	0.18 ± 0.09a	5.11 ± 0.49a	12.57 ± 1.09a
miR-222 agomir + empty vector	2.45 ± 0.16c	2.84 ± 0.22c	3.74 ± 0.27c	6.81 ± 0.58c	28.11 ± 2.75c
miR-222 agomir + Pten	1.36 ± 0.13a	1.27 ± 0.07a	1.35 ± 0.14a	6.18 ± 0.47a	21.98 ± 2.01a

IAA, insulin autoantibody; ICA, islet cell antibody; GAD, glutamic decarboxylase antibody; BG, blood glucose; INS, insulin; NC, negative control.

compared with the Model group P < 0.05.

compared with the Normal group P < 0.05.

compared with the NC group P < 0.05.

Inhibition of miR-222 Inactivated the Mitogen-Activated Protein Kinase (MAPK)/ERK Pathway by Upregulating Pten in PCOS Rats

Furthermore, qRT-PCR and western blot assays were performed to explore the effects of miR-222 on the MAPK/ERK pathway. According to our results in Figure 4A, compared with the normal group, miR-222 expression in the model, NC, miR-222 agomir, and miR-222 agomir + empty vector groups was significantly increased (p < 0.05), whereas no significant difference was observed in the miR-222 antagomir group (p > 0.05). Compared with the model group, miR-222 expression was further increased in the miR-222 agomir and miR-222 agomir + Pten groups, but it was decreased in the miR-222 antagomir group (all ps < 0.05). Compared with the normal group, the mRNA expression of Pten was significantly decreased in the model, NC, miR-222 agomir, miR-222 agomir + empty vector, and miR-222 agomir + Pten groups (all ps < 0.05), whereas no significant change was observed in the miR-222 antagomir group (p > 0.05). Compared with the model group, the mRNA expression of Pten in the miR-222 agomir group was further decreased, while the mRNA expression of Pten was increased in the miR-222 antagomir group (p < 0.05; Figure 4B). The results of the western blot assay indicated that the changes in Pten protein expression in these groups were consistent with the changes in Pten mRNA expression in these groups (Figures 4C and 4D). There was no significant difference in the protein expression of Erk1/2 among all groups (p > 0.05). However, the expression of phosphorylated Erk1/2 was further increased in the model, NC, miR-222 agomir, and miR-222 agomir + empty vector groups compared with that in the normal group (p < 0.05), whereas no significant change was observed in the miR-222 antagomir group (p > 0.05). Compared with the model group, the level of phosphorylated Erk1/2 was significantly increased in the miR-222 agomir group, but it was decreased in the miR-222 antagomir group (all ps < 0.05; Figures 4C and 4D). Hence, these aforementioned findings suggested that the inhibition of miR-222 could downregulate the expression of MAPK/ERK pathway-related genes by increasing the expression of Pten.

Figure 4

The Expression of Pten and the MAPK/ERK Pathway in Ovarian Tissues of Rats after Transfection

(A) miR-222 expression in the ovarian tissues of rats measured by qRT-PCR. (B) Pten mRNA expression in the ovarian tissues of rats evaluated by qRT-PCR. (C) The protein bands of Pten and Erk1/2 and phosphorylated Erk1/2 in the ovarian tissues of rats assessed by western blot assay. (D) Grayscale value statistics of bands in (C). Data comparison was conducted by one-way ANOVA. ∗p < 0.05, versus the normal group; #p < 0.05, versus the model group. miR-222, microRNA-222; NC, negative control; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Discussion

PCOS is one of the most prevalent metabolic and reproductive disorders and is characterized by hyperandrogenism and IR. In the present study, we established a rat PCOS model to determine the mechanism by which miR-222 influenced IR in PCOS by regulating Pten.

Our present study found that PCOS patients in the IR and non-IR groups exhibited increased miR-222 expression and reduced Pten mRNA expression but that the IR group showed higher miR-222 expression and lower Pten mRNA expression than the non-IR group, indicating that miR-222 expression was negatively linked to Pten mRNA expression in the IR group. A previous study identified Pten as a direct target gene of miR-222, whereas it has also been identified in many other types of human cancers. Furthermore, Pten is known as a key factor in the regulation of a wide range of physiologically relevant activities and cellular mechanisms, such as metabolism and mammalian chromosome stability.

Notably, the involvement of miRNAs in the development of various cancers has been described in cumulative studies; for instance, miR-222 has been observed in colorectal, lung, breast, and papillary thyroid cancers and in lymphoma, hepatocellular carcinoma, and glioblastoma. A previously reported study indicated that Pten can negatively regulate IR in peripheral tissues, such as skeletal muscle and adipose tissues, but its downregulation resulted in improved INS sensitivity, which was consistent with one of our findings that the IR group exhibited higher miR-222 expression and lower Pten mRNA expression than the non-IR group.

Interestingly, we also demonstrated that the inhibition of miR-222 decreased serum sex hormone production and glucose metabolism in PCOS rats, since the miR-222 antagomir led to reduced production of FSH, LH, E2, PRL, T, HOMA-IR, BG120min, INS120min, GnRH, IAA, ICA, and GAD but elevated production of P. Moreover, miR-222 has been identified as a regulator of estrogen receptor α (ERα) expression in estrogen-induced IR. The role of miR-222 in the reduction of hyperglycemia in a mouse model of INS-deficient diabetes via pancreatic β-cell proliferation has also been indicated in a previous study.

Furthermore, qRT-PCR and western blot assays were used to investigate the effects of miR-222 on the MAPK/ERK pathway. Notably, our results demonstrated that inhibition of miR-222 led to inactivation of the MAPK/ERK pathway by increasing the expression of Pten. Consistently, previous studies have reported that basal phosphorylation of ERK is significantly higher in isolated PCOS muscle than in controls. Moreover, abnormally high ERK expression has also been reported in the theca cells of PCOS patients. Thus, these findings collectively support our results, indicating that the miR-222 inhibitor group exhibits a decreased number of cystic follicles and an increased number of follicles and corpus luteum cysts, whereas the number of granulosa cell layers was increased. Additionally, the miR-222 inhibitor group showed decreased miR-222, Erk1/2, and phosphorylated Erk1/2 levels, whereas Pten expression was increased. Consistently, Nelson-Degrave et al. demonstrated that the complete absence of INS was maintained in theca cells, while ERK1/2 phosphorylation was decreased in PCOS theca cells compared with normal theca cells.

In summary, the present findings demonstrated that miR-222 inhibition could potentially attenuate IR in PCOS by upregulating Pten expression and inactivating the MAPK/ERK pathway, implying that miR-222 could be a potential therapeutic target for combating IR in PCOS patients. However, there are several limitations to this study. First, further studies are required to determine whether there is a more specific role of miRNA in this setting and to understand the specific mechanisms. Moreover, in addition to IR, there are other factors in the cases we collected that could not be properly evaluated in our study. Therefore, future research needs to be conducted with more appropriate cases to study the role of IR more precisely.

Materials and Methods

Study Subjects

The total number of enrolled female patients was 168, and all of them were diagnosed with PCOS by gynecological examination and transvaginal-pulsed color Doppler ultrasound from The First Clinical Medical College of China Three Gorges University from January 2012 to March 2015. These patients included 96 IR patients aged 18 to 46 years (mean age: 31.5 ± 8.6 years) and 72 non-IR patients aged 18 to 49 years (mean age: 31.5 ± 9.2 years). None of the patients described earlier was administered hormone drugs before diagnosis. The diagnostic criteria for PCOS met at least two of the following conditions: (1) ovarian dysfunction, such as amenorrhea (absence of menstrual periods over the past 3 cycles or more than 6 months) and oligomenorrhea (menstrual cycle >35 days and anovulation over 3 months every year); (2) clinical manifestations of hyperandrogenemia and signs of excess androgen, such as increased testosterone and dehydroepiandrosterone (DHEA) sulfate (DS) concentrations; coarse hair appearing in the upper lip, jaw, abdominal midline, and other areas; alopecia; and acne; and (3) polycystic ovaries, with 12 or more follicles in one or both ovaries with a 2- to 9-mm diameter and/or ovarian volume > 10 mL. However, other etiologies, such as congenital adrenal hyperplasia, hyperprolactinemia, and premature ovarian failure, were excluded. According to the international criteria, the resistance index of the HOMA-IR was calculated as ([fasting glucose (mmol/L)] × [fasting INS (μU/mL)])/22.5; an index value ≥2.69 was considered to indicate IR. None of the aforementioned patients had a family history of the disease, i.e., fallopian tube dysfunction or male infertility. Patients with genital malformation and ovarian dysgenesis were also excluded. Seventy-six patients (mean age: 35.6 ± 8.6 years) who underwent surgery for fallopian tube obstruction were selected as controls. Peripheral venous blood was collected from all subjects for subsequent experiments. The characteristics of the subjects are depicted in Table 3. The present study was authorized by the ethics committee of The First Clinical Medical College of China Three Gorges University, and all of the study subjects voluntarily joined the present study and signed informed consent before the study.

Table 3

The Characteristics of the Subjects

Variable	PCOS		Control (n = 76)
	IR (n = 96)	Non-IR (n = 72)
Age (years)	31.5 ± 8.6a	31.5 ± 9.2b	35.6 ± 8.6
BMI (kg/m2)	26.7 ± 1.9a,c	22.8 ± 2.0	21.4 ± 1.8
Waist-hip ratio (WHR)	0.91 ± 0.13a,c	0.86 ± 0.12	0.85 ± 0.12
Systolic pressure (mmHg)	120.1 ± 15.3a,c	109 ± 10.2	104 ± 11.7
Diastolic pressure (mmHg)	76 ± 8.2a	74 ± 5.8b	71.1 ± 4.5
Fasting blood glucose (mmol/L)	5.41 ± 0.73a,c	4.45 ± 0.56	4.27 ± 0.48
Fasting insulin (mU/L)	22.75 ± 4.90a,c	11.49 ± 2.15b	9.13 ± 1.38
HOMA-IR	5.37 ± 0.82a,c	2.23 ± 0.25b	1.73 ± 0.32
High-density lipoprotein (mmol/L)	1.29 ± 0.18a	1.34 ± 0.31	1.42 ± 0.24
Low-density lipoprotein (mmol/L)	2.10 ± 0.57a	2.34 ± 0.51	2.49 ± 0.38
Triglyceride (mmol/L)	2.14 ± 0.31a,c	1.18 ± 0.22	0.89 ± 0.14
Free testosterone	4.1 ± 1.2a,c	2.6 ± 0.7b	1.3 ± 0.2
Total testosterone (ng/mL)	0.97 ± 0.13a,c	0.72 ± 0.08b	0.51 ± 0.09
Ovarian volume (cm3)	11.32 ± 1.64	10.76 ± 0.87	6.76 ± 0.52

PCOS, polycystic ovary syndrome; IR, insulin resistance; HOMA-IR, homeostasis model assessment for insulin resistance.

p < 0.05, the IR group versus the control group

p < 0.05, the non-IR group versus the control group

p < 0.05, the IR group versus the non-IR group.

Cell Culture

The 293A cells (National Infrastructure of Cell Line Resource, Peking Union Medical College) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 100 μg/mL penicillin-streptomycin and 10% fetal bovine serum (FBS), followed by incubation in a humid environment containing 5% CO2 at 37°C. The cells were seeded onto a 24-well plate 24 h before transfection in DMEM containing 10% FBS.

Experimental Animals and Grouping

In total, 120 Sprague-Dawley (SD) female rats aged 21 days (purchased from the Shanghai Experimental Animal Center) were included as PCOS models in the present study, and an additional 20 rats were selected as normal controls (normal group). All rats were housed in an experimental environment for 1 week of adaptive feeding. The rats in the PCOS model underwent hypodermic injection of DHEA (6 mg/100 g, dissolved in 0.2 mL soybean oil) for 21 consecutive days, while rats in the normal group underwent hypodermic injection of 0.2 mL soybean oil. Vaginal smears were observed for 4 consecutive days beginning on the 16th day, and the continuous appearance of keratinocytes was considered successful model establishment. The 120 successfully modeled rats were randomly divided into 6 groups (n = 20 for each group) as follows: model group (normal saline via tail vein injection), miR-222 antagomir group (tail vein injections of miR-222 antagomir, 2.5 mg/kg/day), miR-222 agomir group (tail vein injections of miR-222 agomir, 2.5 mg/kg/day), NC group (tail vein injections of NC plasmid, 2.5 mg/kg/day), miR-222 agomir + empty vector group (tail vein injections of miR-222 agomir and pcDNA3 plasmid), and miR-222 agomir + Pten group (tail vein injections of miR-222 agomir and pcDNA3-Pten plasmid). The normal group was injected with normal saline. Thereafter, the rats were administered for 28 consecutive days. The miR-222 antagomir, miR NC, and miR-222 agomir rats were purchased from Shanghai GeneBio (Shanghai, China) (Table 4).

Table 4

The Oligo Sequences for Transfection

Group	Sequences
miR-222 antagomir	5′-AGCUACAUCUGGCUACUGGGU-3′
miR NC	5′-AAAAGAGACCGGUUCACUGUGA-3′
miR-222 agomir	5′-AGCUACAUCUGGCUACUGGGU-3′
	5′-CCAGUAGCCAGAUGUAGCUUU-3′

miR-222, microRNA-222.

Detection of Serum Sex Hormones and Glucose Metabolism-Related Indicators

Ten rats were randomly selected from each group and anesthetized after the last injection. Thereafter, 5 mL blood was obtained via the inferior vena cava, and chemiluminescence detection (Bayer, Leverkusen, Germany) was conducted to assess the levels of serum LH, FSH, E2, P, PRL, T, and GnRH.

Glucose tolerance testing was performed in 10 rats randomly selected from each group. The rats underwent fasting for 12 h after the last injection. After anesthetization with 3% pentobarbital sodium, 1 mL orbital venous blood was extracted. Then, 120 min after intraperitoneal injection of 50% hypertonic glucose solution, 1 mL orbital venous blood was extracted again. By using an automated biochemical analyzer (Hitachi, Tokyo, Japan) and a chemiluminescence detection system (Bayer, Leverkusen, Germany), the levels of BG, INS, IAA, ICA, and GAD were measured after 20 min of injections under fasting conditions, and the resistance index of HOMA-IR was calculated as (fasting INS × fasting glucose)/22.5.

Hematoxylin and Eosin (H&E) Staining

After index detection, 6 rats in each group were euthanized, and then their ovaries were excised. The ovaries were fixed in 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), and soaked in xylene to clear the tissues. After being washed with distilled water to absorb xylene, the tissues were dewaxed with melted paraffin in an incubator and transferred into a mold containing melted paraffin. After the liquid paraffin was cooled to form blocks, the paraffin tissues were fixed on a specimen table, sliced, and dewaxed with ethanol to obtain tissue sections. The obtained sections were stained in H&E solution for 5 min and stained in 0.5% eosin for 2 min, dehydrated with alcohol, cleared with xylene, mounted with neutral balsam, and finally observed under a microscope (Olympus, Tokyo, Japan).

qRT-PCR

The rats in each group were euthanized by cervical dislocation. Briefly, 7 rats in each group were selected, and their ovaries were excised and stored in liquid nitrogen. Total RNA was isolated from peripheral blood and ovaries of rats using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, reversely transcribed into cDNA with the Primer Script RT Reagent Kit (TaKaRa Biotechnology, Dalian, China), and stored at −20°C for subsequent experiments. qPCR was conducted using the ABI 17500 Sequence Detection System (Life Technologies, Grand Island, NY, USA), U6 was used as an internal reference for miR-222, and GAPDH was used as the reference for Pten. The qRT-PCR conditions included one cycle of predenaturation at 95°C for 5 min and 40 cycles of denaturation at 90°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 40 s. The following primers for qRT-PCR were used: miR-222: 5′-ATCCAGTGCGTGTCGTG-3′ (forward) and 5′-TGCTAGCTACATCTGGCT-3′ (reverse); U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse); Pten: 5′-GGGACGAACTGGTGTAATGA-3′ (forward) and 5′-CGCCTCTGACTGGGAATAG-3′ (reverse); GAPDH: 5′-AGCCACATCGCTCAGACAC-3′ (forward) and 5′-CTCGCTCCTGGAAGATGGT-3′ (reverse). Moreover, all the primers used in this study were purchased from Sangon Biotech (Shanghai, China). Finally, each sample was measured in triplicate. Relative expression levels were calculated using the 2−ΔΔCt method.

Western Blot Assay

After index detection, rats were euthanized by cervical dislocation, and 7 rats from each group were selected for ovarian tissue collection. Total protein was extracted from the tissues with the addition of cell lysis buffer (KeyGen Biotech, Nanjing, China). The protein concentration was estimated with the Bradford method (KeyGen Biotech, Nanjing, China). An aliquot of 50 μg total protein per sample was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA) and blocked with 5% skim milk powder at 37°C for 1 h. The membrane was then incubated overnight at 4°C with rabbit anti-human Pten (ab31392, 1:1,000) Erk1/2 (ab17942, 1:1,000), phosphorylated Erk1/2 (ab201015, 1:400), and GAPDH polyclonal antibodies (ab37168, 1:1,000). All of the aforementioned antibodies were purchased from Abcam (Cambridge, UK). After three washes with PBS with Tween 20 (PBST), each wash lasting for 5 min, the membrane was incubated with secondary horseradish-peroxidase-labeled goat anti-rabbit immunoglobulin G (1:4,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody at room temperature for 1 h. The chemiluminescence reagent was added dropwise onto the membrane, and further incubation was carried out for 3 to 5 min followed by drying and development under dark conditions. The relative protein expression levels are indicated as the ratio of the grayscale value of the target band to that of the internal reference GAPDH band. Each experiment was performed in triplicate with the average obtained.

Luciferase Reporter Gene Assay

Based on the TargetScan database, target genes of miR-222 were obtained, while Pten was verified as the direct target gene of miR-222. qRT-PCR was performed to detect the 6 target genes reported in previous studies, and Pten, the target gene showing the most significant differential expression, was selected as the direct target gene of miR-222. The binding sites of miR-222 to Pten were predicted using the prediction of bioinformatics software based on the rules and principles of the interaction between the confirmed miRNA, the target gene sequence, and the online target gene prediction software miRGen v.3.0. When the 293A cells reached approximately 70% confluence, the full-length sequence of Pten in the 3′ UTR was subjected to clonal amplification, and the PCR products were cloned into the pMIR-REPORT Luciferase vector (Promega, Beijing, China) (pMIR/Pten-WT vector). The target genes and the binding site of miR-222 were predicted based on bioinformatics for site-directed mutagenesis with the construction of the pMIR/Pten-mut vector. The pRL-TK vectors (TaKaRa Biotechnology, Dalian, China) expressing Renilla luciferase were used as an internal reference. PMIR/Pten-WT and pMIR/Pten-mut were co-transfected with the miR-222 mimic and NC sequences, respectively (Table 5), into 293A cells for luciferase activity detection according to the manufacturer’s instructions (Promega, Beijing, China).

Table 5

The Gene Sequences of the Recombinant Plasmids

Recombinant Plasmid	Target Gene Sequences
miR-222 mimic	5′-AGCUACAUCUGGCUACUGGGU-3′ (sense)
	5′-CCAGUAGCCAGAUGUAGCUUU-3′ (antisense)

miR-222, microRNA-222.

Statistical Analysis

All data were processed by SPSS v.21.0 statistical software (IBM, Armonk, NY, USA). Measurement data were displayed as the means ± standard deviation, in which the differences between the two groups were analyzed with the use of the t test and differences among multiple groups were analyzed using a one-way analysis of variance (ANOVA). Correlation analysis was performed with Pearson’s coefficient. The level of significant difference was set as p <0.05.

Author Contributions

H.Y., X.-J.L., Y.H., Y.-H.L., C.-H.L., and Q.W. designed the study. H.Y., X.-J.L., and Y.H. collated the data, carried out data analyses, and produced the initial draft of the manuscript. H.Y. and C.-H.L. conducted the experiments. Y.-H.L., C.-H.L., and Q.W. contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

Conflicts of Interest

The authors declare no competing interests.