Application of coreceptor coactivator 4 as a target in screening and preparing medicine for treating ovulation disorder of polycystic ovary syndrome

Abstract

The application provides application of a nuclear receptor coactivator 4 as a target point in screening of a medicine for treating ovulation disorder of polycystic ovary syndrome. The application also provides application of a reagent for detecting the nuclear receptor coactivator 4 in preparation of a kit for diagnosing ovulation disorder of polycystic ovary syndrome. The application also provides use of the nuclear receptor coactivator 4 in preparation of a marker for diagnosing ovulation disorder of polycystic ovary syndrome. The application also provides application of the nuclear receptor coactivator 4 as a diagnostic marker in preparation of a kit for diagnosing ovulation disorder of polycystic ovary syndrome. The application also provides an interfering RNA, and a nucleotide sequence of the interfering RNA is 5'-GACCUUAUUUAUCAGCUUATT-3'. The application finds that NCOA4 can improve inhibition of ERK1 / 2 phosphorylation and reduction of C / EBPbeta expression caused by androgen.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a nuclear receptor coactivator 4. Specifically, it relates to the application of nuclear receptor coactivator 4 as a target in the screening and preparation of drugs for treating ovulation disorders in polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women and the most common reproductive endocrine disorder in women of reproductive age, affecting over 50 million people in my country. PCOS is characterized by infrequent or absent ovulation, clinically or biochemically hyperandrogenemia, and polycystic ovarian morphology, leading to female infertility. The pathogenesis of ovulation disorders in PCOS is currently unclear, and the treatment of infertility caused by PCOS remains a challenge in clinical practice.

[0003] PCOS exhibits extremely high heterogeneity. Current treatments for PCOS patients desiring fertility primarily involve lifestyle modifications, but the effectiveness remains poor, with many patients still unable to ovulate spontaneously after treatment. Consequently, these PCOS patients with ovulation disorders require ovulation induction and assisted reproductive technologies, but conventional ovulation induction treatments are still not entirely effective for PCOS patients. Currently, low oocyte maturity, low fertilization rates, high miscarriage rates, and a high incidence of complications such as ovarian hyperstimulation syndrome place a significant psychological and socioeconomic burden on patients. Therefore, there is an urgent need to explore therapeutic targets that address the mechanisms underlying ovulation disorders in PCOS.

[0004] The gene ID of nuclear receptor coactivator 4 (NCOA4) is 8031 ​​(https: / / www.ncbi.nlm.nih.gov / gene / ?term=8031). Also known as androgen receptor coregulatory factor 70, it enhances the transcriptional activity of the androgen receptor (AR). Previous studies have found its involvement in the development and progression of prostate cancer. Hyperandrogenism is a core cause of PCOS, and elevated local androgen concentrations in the ovaries of PCOS patients are a major cause of ovulation disorders. Studies have shown that elevated local androgen concentrations in the ovaries of PCOS patients lead to increased ovarian granulosa cell death, resulting in impaired follicle development and thus polycystic ovarian changes in PCOS patients. Ferrophagy, a newly discovered type of programmed cell death, was found to be elevated in the ovaries of PCOS patients, consistent with previous research findings. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides the application of nuclear receptor coactivator 4 as a target in the screening and preparation of drugs for treating ovulation disorders in polycystic ovary syndrome (PCOS). The application of nuclear receptor coactivator 4 as a target in the screening and preparation of drugs for treating ovulation disorders in PCOS can solve the technical problem of poor efficacy of existing drugs in treating ovulation disorders in PCOS.

[0006] This invention provides the application of nuclear receptor coactivator 4 as a target in the screening and preparation of drugs for treating ovulation disorders in polycystic ovary syndrome, wherein the gene ID of nuclear receptor coactivator 4 is 8031.

[0007] This invention also provides the application of reagents for detecting nuclear receptor coactivator 4 in the preparation of kits for diagnosing ovulation disorders in polycystic ovary syndrome.

[0008] This invention also provides the use of nuclear receptor coactivator 4 in the preparation of markers for diagnosing ovulation disorders in polycystic ovary syndrome.

[0009] This invention also provides the application of nuclear receptor coactivator 4 as a diagnostic marker in the preparation of a kit for diagnosing ovulation disorders in polycystic ovary syndrome.

[0010] The present invention also provides an interfering RNA with the nucleotide sequence 5'-GACCUUAUUUAUCAGCUUATT-3' (SEQ ID NO.1).

[0011] The present invention also provides the application of the above-mentioned interfering RNA in the preparation of a drug for treating ovulation disorders in polycystic ovary syndrome.

[0012] This invention reveals that androgen-induced elevation of NCOA4 expression in ovarian granulosa cells leads to decreased expression of ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4), a major inhibitor of ferroptosis. Furthermore, this study found that NCOA4 can alleviate androgen-induced inhibition of ERK1 / 2 phosphorylation and decreased C / EBPβ expression. Animal experiments further validated that blocking NCOA4 can improve the reduction in the number of corpora lutea, the number of ovulations, and the quality of oocytes in PCOS rats. This study identifies an important signaling pathway for the pathogenesis of ovulatory disorders in PCOS and identifies NCOA4, a key molecular target in the pathogenesis of PCOS, which could serve as a potential therapeutic target for future treatment of ovulatory disorders in PCOS.

[0013] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention provides a more precise target NCOA4 in the pathogenesis of ovulation disorders in polycystic ovary syndrome (PCOS). By targeting NCOA4-mediated hyperandrogenism and the NCOA4-regulated ferroautophagy signaling pathway, it is possible to reverse the pathogenesis of ovulation disorders in PCOS, providing a new perspective for future research on the molecular mechanisms of PCOS pathogenesis and treatment. Attached Figure Description

[0014] Figure 1 The expression levels of NCOA4 protein and FTH1 and GPX4 protein are increased and decreased in ovarian granulosa cells of PCOS patients. (A) Representative graph of NCOA4, FTH1 and GPX4 protein expression in ovarian granulosa cells of non-PCOS patients and PCOS patients. (B) NCOA4 statistical graph. (C) FTH1 statistical graph. (D) GPX4 statistical graph. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

[0015] Figure 2 In Yuan dynasty ovarian granulosa cells, the effect of inhibiting NCOA4 on the expression of androgens-induced iron autophagy-related proteins was investigated. (A) The expression levels of NCOA4, FTH1, and GPX4 proteins were measured after adding different concentrations of the androgen DHT. (B) The expression levels of FTH1 and GPX4 proteins were measured after knocking down NCOA4 and adding the androgen DHT.

[0016] Figure 3 In Yuan dynasty granulosa cells, inhibition of NCOA4 improved the effect of androgens on ovulation-related genes. (A) Expression levels of p-ERK / ERK protein were detected after NCOA4 knockdown and the addition of androgens (DHT). (B) Protein expression levels of C / EBPβ were detected after NCOA4 knockdown and the addition of androgens (DHT).

[0017] Figure 4 Local injection of knockdown lentivirus into the ovaries of PCOS rats improved ovulation dysfunction. (A) Ovarian morphology (B) Number of corpus luteum (C) Number of ovulations (D) Oocyte quality. Detailed Implementation

[0018] Example 1

[0019] In one embodiment of the present invention, the reagent for inhibiting or downregulating the expression level of NCOA4 is NCOA4 interfering RNA (siRNA), and the sequence of the interfering RNA is as follows:

[0020] 5'-GACCUUAUUUAUCAGCUUA-3'.

[0021] The above sequence was obtained using the following method:

[0022] 1. The nucleotide sequence of the NCOA4 gene (gene ID: 8031) was scanned to ensure that its 3' end was dTdT; the first AA sequence was searched starting 75 bp downstream of the promoter, and the 19 bases following this sequence were used as the target sequence.

[0023] 1. Analyze the obtained sequence to ensure that its GC content is approximately 50%.

[0024] 2. Compare the obtained sequences with the NCBI gene database using BLAST.

[0025] 3. TT was added to the 3' end to increase the stability of the siRNA, resulting in the following sequence of the interfering RNA:

[0026] 4.5'-gaccuuauuuaucagcuuatt-3' (SEQ ID NO.1). (The first to eighth "t"s in the appended sequence listing represent uracil in the RNA sequence, and the ninth and tenth "t"s represent thymine in the DNA sequence).

[0027] Example 2

[0028] This study included 12 PCOS patients (n=12) and 12 non-PCOS patients aged 20-35 years who underwent IVF-ET treatment at a reproductive medicine center. All participants underwent ovulation induction using a flexible antagonist protocol, and oocytes were retrieved 36 hours after hCG triggering. PCOS patients were included according to the Rotterdam criteria revised in 2004 (meeting at least two of the three criteria).

[0029] Example 3

[0030] Western blotting was used to detect protein expression: Tissue was lysed on ice using protein lysis buffer (RIPA to extract total cellular protein). Protein concentration was determined using a BCA protein quantification kit. 30 μg of sample was separated by SDS-PAGE electrophoresis. Proteins were then transferred to a PVDF membrane, blocked with 5% skim milk-TBST on a shaker at room temperature for 2 hours, incubated with primary antibody on a shaker at 4°C overnight, and then incubated with secondary antibody on room temperature for 1 hour. Fluorescence was excited using ECL luminescent agent. Chemiluminescence was then performed in a G-BOX. The following antibodies were used: anti-p-ERK (CST), total-ERK (CST), anti-C / EBPβ (Santa Cruz), anti-GAPDH (Proteintech), FTH1 (Abcam), GPX4 (Abcam), and GAPDH (Proteintech).

[0031] Example 4

[0032] After extracting human progeny granulosa cells, the expression of ferrophagy-related proteins NCOA4, FTH1, and GPX4 in progeny granulosa cells from both groups of patients was detected by Western blotting.

[0033] The results showed that the iron autophagy carrier protein NCOA4 was elevated in ovarian granulosa cells of PCOS patients compared to the control group, while the iron storage protein FTH1 and the iron autophagy inhibitor protein GPX4 were decreased compared to the control group. Figure 1 As shown, the level of ferrophagy in ovarian granulosa cells is significantly higher in PCOS patients than in the control group.

[0034] Example 5

[0035] After extracting progenitor ovarian granulosa cells and seeding them, different concentrations of androgens were added and cultured for 24 hours. The protein expression of ferrophagy-related proteins NCOA4, FTH1, and GPX4 in the progenitor granulosa cells was then detected. It was found that the ferrophagy carrier protein NCOA4 was increased in ovarian granulosa cells after androgen addition compared to the control group, while the iron storage protein FTH1 and the ferrophagy inhibitor protein GPX4 were decreased, suggesting that androgens can induce ferrophagy in progenitor ovarian granulosa cells. After knocking down NCOA4 by liposome transfection with si-NCOA4, and then simultaneously adding the androgen DHT, the expression of NCOA4, FTH1, and GPX4 was again detected. It was found that the ferrophagy carrier protein NCOA4 was decreased compared to the androgen-only group, while the iron storage protein FTH1 and the ferrophagy inhibitor protein GPX4 were increased, suggesting that knocking down NCOA4 can block androgen-induced ferrophagy in progenitor ovarian granulosa cells. Figure 2 As shown.

[0036] Example 6

[0037] Liposome transfection of si-NCOA4 to knock down NCOA4, followed by the addition of androgens (DHT), revealed that the expression of ERK signaling pathways and C / EBPβ in ovulation-related pathways was inhibited by androgen addition, and C / EBPβ expression was decreased compared to the control group. Knocking down NCOA4 could improve these conditions. Figure 3 As shown.

[0038] Example 7

[0039] Three-week-old female SD rats were divided into three groups: a control group, a PCOS model group, and a PCOS+siNOCA4 model group. The PCOS model group received subcutaneous injections of DHEA (6 mg / 100 g body weight) for 21 days. In the PCOS+siNOCA4 model group, after rats were approved for implantation, a 1 cm incision was made at the dorsal projection of the ovary. Small interfering virus (containing 5'-GACCUUAUUUAUCAGCUUATT-3' (SEQ ID NO.1) small interfering RNA) was injected into the ovarian capsule using a microsyringe, and simultaneously, DHEA (6 mg / 100 g body weight) was administered subcutaneously.

[0040] Eight days before the end of the modeling process, vaginal smears were taken daily to observe the estrous cycle to determine the success of the modeling before the rats were sacrificed. After sacrifice, the ovarian morphology of each group of rats was observed. It was found that compared with the control group, the number of corpora lutea in the PCOS group was significantly reduced, but the number of corpora lutea increased significantly after treatment with siNOCA4. Simultaneously, ovulation induction experiments were performed on the rats. After successful modeling, rats were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) 300 IU / kg body weight. Forty-eight hours after PMSG injection, rats were intraperitoneally injected with hCG 300 IU / kg body weight. Sixteen hours after hCG injection, oocytes were collected from the fallopian tubes.

[0041] Compared with the control group, the number of ovulations in the PCOS group was significantly reduced, and the oocyte quality was significantly decreased. The number of ovulations and the oocyte quality in the PCOS+siNOCA4 model group were significantly increased compared with the PCOS group. Figure 4 As shown.

[0042] Statistical methods used in the above embodiments: One-way ANOVA was used to test whether the differences in the means among the groups were statistically significant; the significance level was p<0.05. SPSS 19.0 statistical software was used for data statistics and analysis.

Claims

1. Application of nuclear receptor coactivator 4 as a target in screening drugs for the treatment of ovulation disorders in polycystic ovary syndrome.

2. The use of an interfering RNA in the preparation of a drug for treating ovulation disorders in polycystic ovary syndrome, wherein the nucleotide sequence of the interfering RNA is 5'-GACCUUAUUUAUCAGCUUATT-3'.

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