Application of reagent for promoting or up-regulating expression quantity of FOXO4 and related medicine

Abstract

The invention relates to application of a reagent for promoting or up-regulating the expression quantity of FOXO4 and a related medicine. The invention relates to the field of reproductive medicine, and provides application of a reagent for promoting or up-regulating the expression quantity of FOXO4 in preparation of a medicine for treating ovulation disorder of polycystic ovarian syndrome and related medicines. Aiming at an important signal channel of PCOS ovulation disorder attack, an important molecular target FOXO4 in the PCOS pathogenesis is found, it is found that feedback regulation loop imbalance between FOXO4 and AR possibly plays an important role in occurrence and development of PCOS ovulation disorder, and the FOXO4-AR can be used as a treatment target of PCOS ovulation disorder in the future, and has a good application prospect in treatment of PCOS ovulation disorder. And a new thought is provided for research and treatment methods of PCOS pathogenesis molecular mechanisms.

Description

Technical Field

[0001] The present invention relates to the field of reproductive medicine, and particularly to the application of a reagent for promoting or up-regulating the expression level of forkhead box transcription factor FOXO4 (forkhead box O 4) in a therapeutic drug for polycystic ovary syndrome, and a drug containing the above reagent. Background Art

[0002] Polycystic Ovary Syndrome (PCOS) is the most common endocrine and metabolic disease in women of reproductive age, with an incidence rate of about 5%-20%. It is an endocrine disorder syndrome characterized by oligomenorrhea or anovulation, hyperandrogenism, insulin resistance, and polycystic ovarian changes. For women of reproductive age with PCOS, ovulation disorder is the main cause of infertility, and PCOS accounts for about 75% of infertility related to ovulation disorders, which troubles many women of reproductive age. The pathogenesis of PCOS ovulation disorder has not been clearly defined yet, and the treatment of infertility caused by PCOS is still a difficult problem clinically.

[0003] Clinically, the treatment of PCOS mainly focuses on a certain clinical symptom, and individualized symptomatic treatment measures are taken. For PCOS patients with fertility requirements, the treatment methods mainly include lifestyle adjustment, menstrual cycle regulation, relief of hyperandrogenism symptoms, insulin sensitizers, ovulation induction treatment, surgical treatment, and assisted reproductive technology, etc. However, because the etiology of PCOS has not been clarified, each treatment method has certain limitations.

[0004] PCOS is an extremely complex endocrine and metabolic disease. Currently, the clinical treatment of PCOS mainly focuses on a certain clinical symptom. For PCOS patients with fertility requirements, a series of treatments such as lifestyle and menstrual cycle adjustment are carried out first, but many patients still cannot ovulate spontaneously, and then ovulation induction and assisted reproductive technology are used. For infertility, although ovulation induction and assisted reproductive technology can solve the ovulation disorders of most PCOS patients, conventional ovulation induction treatment is prone to ovarian hyperstimulation syndrome, and the egg quality, fertilization rate, implantation rate, and abortion rate are all affected to a certain extent. In view of the limitations of various treatment methods, the need to explore the molecular mechanism of PCOS pathogenesis to find more critical treatment targets is still urgent. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to regulate the expression level of FOXO4, a relatively precise target in the pathogenesis of PCOS ovulation disorder, and reverse the pathogenic characteristics of the imbalance of the feedback regulation loop between FOXO4 and AR, thereby reversing the pathogenesis of PCOS ovulation disorder.

[0006] The present application provides the use of a reagent for promoting or upregulating the expression level of FOXO4 in the preparation of a drug for treating anovulation in polycystic ovary syndrome.

[0007] Furthermore, the above use is to take FOXO4 as an intervention target for treating anovulation in polycystic ovary syndrome.

[0008] Furthermore, the reagent for promoting or upregulating the expression level of FOXO4 includes but is not limited to:

[0009] A recombinant vector;

[0010] A protein that specifically binds to FOXO4;

[0011] A FOXO4 activator, upregulator or protein degradation pathway blocker.

[0012] Furthermore, the recombinant vector includes one or more combinations of a FOXO4 overexpression plasmid vector and a FOXO4 overexpression viral vector.

[0013] Furthermore, the drug uses the reagent for promoting or upregulating the expression level of FOXO4 or the recombinant vector as an active ingredient.

[0014] A drug for treating anovulation in polycystic ovary syndrome uses the reagent for promoting or upregulating the expression level of FOXO4 or the recombinant vector as an active ingredient.

[0015] The improvement of the present application brings the following advantages: The present invention targets an important signaling pathway for the onset of PCOS anovulation, finds an important molecular target FOXO4 in the pathogenesis of PCOS, and discovers that the imbalance of the feedback regulation loop between FOXO4-AR may play an important role in the occurrence and development of PCOS anovulation. This invention can be used as a future treatment target for PCOS anovulation, providing new ideas for the research on the molecular mechanism of PCOS pathogenesis and treatment methods. Description of the Drawings

[0016] Figure 1 (A-I) show the expression levels of FOXO1, FOXO3, FOXO4, AR, CCND1, CDKN1A, C / EBPβ, HAS2, and CYP11A1 in granulosa cells of PCOS and non-PCOS patients (n = 30:30).

[0017] Figure 2 (A-D) show the effects of knocking down FOXO4 on cell proliferation and cell cycle in KGN cells; (E-H) show the effects of overexpressing FOXO4 on cell proliferation and cell cycle in KGN cells.

[0018] Figure 3(A-D) Effects of adding FSK on cell proliferation and cell cycle in KGN cells; (E-F) Detection of the expression of FOXO4, CCND1, and CDKN1A after knocking down FOXO4 and / or adding FSK.

[0019] Figure 4 (A-B) Detection of the expression of FOXO4, C / EBPβ, HAS2, and CYP11A1 after knocking down FOXO4 in KGN cells; (C-D) Detection of the expression of FOXO4, C / EBPβ, HAS2, and CYP11A1 after overexpressing FOXO4 in KGN cells; (E-F) Detection of the expression of FOXO4, C / EBPβ, HAS2, and CYP11A1 after knocking down FOXO4 and / or adding FSK.

[0020] Figure 5 (A-D) Effects of knocking down AR on cell proliferation and cell cycle in KGN cells; (E) Detection of the binding of AR to the promoter region of CCND1 by ChIP assay.

[0021] Figure 6 (A) Spearman analysis of the correlation between the expression abundance of FOXO4 and the total testosterone level in follicular fluid; (B-C) Detection of the expression of AR and FOXO4 after adding DHT to KGN cells; (D) Detection of the expression of AR and FOXO4 after adding Flutamide (AR antagonist) and DHT simultaneously; (E-F) Detection of the expression of AR and FOXO4 after knocking down AR in KGN cells; (G) Detection of the binding of AR to the promoter region of FOXO4 by ChIP assay.

[0022] Figure 7 (A-B) Detection of the expression of AR and FOXO4 after adding DHT to KGN cells; (C) Detection of the expression of FOXO4 and AR after knocking down FOXO4 and / or adding MG132; (D-E) Detection of the binding between FOXO4 and AR proteins by Co-IP after knocking down and overexpressing FOXO4.

[0023] Figure 8 Expression patterns of FOXO4, AR, CDKN1A, and CCND1 in ovarian granulosa cells of the rat superovulation model.

[0024] Figure 9Construction of FOXO4 knockdown model by local injection of lentivirus-mediated FOXO4 shRNA into rat ovaries. (A) Estrous cycles of two groups of rats; (B) Body weights, ovarian weights and ovarian pictures of two groups of rats; (C) Immunohistochemistry showing the distribution of FOXO4 in rat ovaries; (D) Serum progesterone levels of two groups of rats; (E) HE staining of ovarian sections of two groups of rats; (F) Expressions of FOXO4, AR, CCND1 and CDKN1A in ovaries of two groups of rats.

[0025] Figure 10 DHEA-induced PCOS-like rat model combined with superovulation model. (A) Schematic diagram of constructing rat model; (B) Estrous cycles of two groups of rats; (C) Body weights and ovarian weights of two groups of rats; (D) Serum testosterone and progesterone levels of two groups of rats; (E) HE staining of ovarian sections of two groups of rats; (F-G) Expressions of AR, FOXO4, CCND1 and CDKN1A in ovarian granulosa cells of two groups of rats; (H-J) Number of retrieved eggs and MII oocyte rate of two groups of rats during superovulation. Specific implementation manners

[0026] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] Since the etiology of polycystic ovary syndrome has not been clarified, it is still difficult to cure. Currently, for the related infertility of PCOS, although ovulation induction and assisted reproductive technologies can solve the ovulation disorders of most PCOS patients, the risk of ovarian hyperstimulation syndrome increases, and the egg quality, fertilization rate, implantation rate and abortion rate are all affected to a certain extent. Currently, the target research on the pathogenesis of PCOS has received extensive attention. The present invention aims at the molecular mechanism of PCOS ovulation disorder, combines the important signal pathways involved in the regulation during ovulation, finds the key molecule FOXO4 in the pathogenesis of PCOS ovulation disorder, and in the future, molecular targeted inhibitors against FOXO4 can be developed for the treatment of PCOS ovulation disorder.

[0028] Based on a large number of differentially expressed genes in the published sequencing results of PCOS granulosa cells, it is predicted by bioinformatics analysis that upstream regulatory factors such as AR and FOXO family may jointly participate in the regulation of differentially expressed genes in PCOS. The present invention finds that FOXO4 is lowly expressed in granulosa cells of PCOS patients, and the expressions of FOXO1 and FOXO3 have no obvious differences. Therefore, it is speculated that FOXO4 may participate in the pathogenesis of PCOS ovulation disorder by regulating the functions of granulosa cells.

[0029] Forkhead box O (FOXO) transcription factors are a subgroup of forkhead box transcription factors and key regulatory molecules in many signaling pathways in the body. They are involved in regulating many pathological and physiological processes such as cell proliferation, cycle, differentiation, metabolism, apoptosis, and inflammatory responses. A study (DOI: 10.1093 / humrep / dex320) showed that the expression of FOXO4 was upregulated in granulosa cells of patients with clinical pregnancy, while the expressions of FOXO1 and FOXO3 were not different. This study proposed that the expression level of FOXO4 in granulosa cells could be used as one of the marker molecules for evaluating oocyte quality and pregnancy outcome.

[0030] The present invention's research found that the expression of cell cycle-related molecule CCND1 was upregulated and the expression of CDKN1A was downregulated in PCOS granulosa cells; the expressions of ovulation-related molecules C / EBPβ, HAS2, and CYP11A1 were downregulated. Further findings showed that FOXO4 affected granulosa cell proliferation and cycle function by regulating the expressions of cell cycle-related molecules CCND1 and CDKN1A; moreover, FOXO4 affected the ovulation process by regulating the expressions of ovulation-related molecules C / EBPβ, HAS2, and CYP11A1; suggesting that FOXO4 may be involved in the occurrence of PCOS ovulation disorder by regulating granulosa cell proliferation function and ovulation gene expression.

[0031] Hyperandrogenemia is the core of PCOS pathogenesis. The increased local androgen level in the ovary is the main cause of polycystic ovarian changes. Androgens mainly exert their effects through the androgen receptor (AR), and the androgen action mediated by AR is an important link in the pathogenesis of PCOS. The present invention found that the expression of AR is increased in granulosa cells of PCOS patients, which is consistent with previous research results. It was also found that AR affects granulosa cell proliferation and cell cycle function by regulating the expression of CCND1 and CDKN1A; previous studies found that AR inhibits the ovulation process by inhibiting the expression of ovulation-related molecules such as C / EBPβ; suggesting that AR is also involved in the occurrence of ovulation disorders in PCOS by regulating granulosa cell proliferation function and ovulation gene expression. The present invention found that the abundance of FOXO4 expression in PCOS granulosa cells is negatively correlated with the total testosterone level in follicular fluid, and the androgen action mediated by AR is related to the downregulation of FOXO4. The present invention further found that AR regulates its transcription and expression by binding to the FOXO4 promoter; FOXO4 regulates its ubiquitin-proteasome pathway by binding to AR protein, thereby affecting its protein level expression; suggesting that there is a mutual regulatory balance between AR and FOXO4, and the hyperandrogenic state disrupts the balance of the loop regulation between the two. Through animal models, the molecular mechanism by which FOXO4 downregulation participates in the occurrence of ovulation disorders in PCOS by affecting granulosa cell function, as well as the molecular mechanism by which hyperandrogen-related FOXO4 downregulation and AR upregulation participate in the occurrence of ovulation disorders in PCOS, were verified.

[0032] Example 1

[0033] PCOS patients (n = 30) and non-PCOS patients (n = 30) who were scheduled to undergo IVF-ET in the Reproductive Medicine Center of Guangdong Women and Children's Hospital were recruited. After 34 - 36 h after hCG triggering, follicular fluid was obtained by puncturing the ovary under the guidance of transvaginal ultrasound. Sample criteria: aged 20 - 39 years, using the antagonist protocol for ovulation induction, and the diagnosis of PCOS conforms to the Rotterdam diagnostic criteria.

[0034] Granulosa cells were extracted from follicular fluid. By extracting RNA and reverse transcribing RNA into cDNA, real-time PCR was performed to detect the expression of target genes at the mRNA level; the results showed that compared with non-PCOS patients, the mRNA expression levels of FOXO4, CDKN1A, C / EBPβ, HAS2, and CYP11A1 in granulosa cells of follicular fluid from PCOS patients decreased, while the expression of AR and CCND1 increased, as Figure 1 shown.

[0035] Example 2

[0036] Using the human ovarian granulosa cell carcinoma cell line (KGN cell line), FOXO4 was knocked down by liposome transfection of FOXO4 siRNA, and FOXO4 was overexpressed by plasmid transfection. The results suggested that FOXO4 inhibited cell proliferation and cell cycle progression by inhibiting CCND1 and promoting CDKN1A expression ( Figure 2 ). Adenylate cyclase activator (forskolin, FSK) that mimics the LH peak effect was added, and the results showed that FSK inhibited cell proliferation, the proportion of cells in the G0-G1 phase of the cell cycle increased, and the proportion of cells in the S phase decreased ( Figure 3 A-B); FSK inhibited the expression of AR and CCND1 and promoted the expression of FOXO4 and CDKN1A ( Figure 3 C-D); moreover, after knocking down FOXO4 and simultaneously giving FSK to mimic the LH peak, it was found that knocking down FOXO4 could inhibit the induction of FSK on the expression of CCND1 and CDKN1A ( Figure 3 E-F).

[0037] FOXO4 siRNA target sequence 5'-CGUCCACGAAGCAGUUCAAAUTT-3'

[0038] Furthermore, by knocking down and overexpressing FOXO4, the results suggested that FOXO4 had a positive regulatory effect on the ovulation-related genes C / EBPβ, HAS2, and CYP11A1; after knocking down FOXO4 and simultaneously giving FSK to mimic the LH peak, it was found that knocking down FOXO4 could inhibit the induction of FSK on the expression of C / EBPβ, HAS2, and CYP11A1, as Figure 4 shown.

[0039] By transfecting AR siRNA to knock down AR, the results suggested that AR promoted cell proliferation and cell cycle progression by promoting CCND1 and inhibiting CDKN1A expression. Through ChIP experiments, it was proved that AR could bind to the promoter region of CCND1, as Figure 5 shown.

[0040] AR siRNA target sequence 5'-GAAAUGAUUGCACUAUUGAUU-3'

[0041] Example 3

[0042] Through spearman correlation analysis, it was found that the abundance of FOXO4 expression in granulosa cells of PCOS patients was negatively correlated with the total testosterone level in follicular fluid ( Figure 6 A). DHT intervention was added to KGN cells, and it was found that DHT inhibited FOXO4 and promoted the expression of AR. Pretreatment with the AR inhibitor (flutamide) for 24 hours could inhibit the induction of DHT on the expression of FOXO4 and AR ( Figure 6B-D). The results showed that the down-regulation of FOXO4 expression in granulosa cells of PCOS patients was related to hyperandrogenism, and DHT inhibited the expression of FOXO4 through the action mediated by AR.

[0043] Furthermore, in the KGN cell line, AR was knocked down by transfection with AR siRNA. The results showed that AR inhibited the expression of FOXO4 mRNA and protein levels. The ChIP experiment confirmed that AR could bind to the promoter region of FOXO4 ( Figure 6 E-G).

[0044] Furthermore, it was found that after knocking down FOXO4, the AR mRNA level remained unchanged, while the AR protein level increased significantly. Moreover, adding MG132 (a proteasome inhibitor) while knocking down FOXO4 could inhibit the regulatory effect of FOXO4 on the AR protein level ( Figure 7 A-C); in addition, the Co-IP experiment showed that there was a binding relationship between FOXO4 and AR proteins ( Figure 7 D-E); the results suggested that FOXO4 might regulate the expression of AR protein level by affecting its proteasome pathway through binding to AR.

[0045] In summary, there is a feedback regulatory loop between FOXO4 and AR, and the hyperandrogenic state in PCOS may disrupt the balance of the loop regulation between the two.

[0046] Example 4

[0047] Construct a rat superovulation model: Superovulate SD female rats, and detect the expression of FOXO4, AR, CCND1, and CDKN1A in ovarian granulosa cells at different time points after hCG. The results showed that the LH surge could inhibit the expression of AR and CCND1, and promote the expression of FOXO4 and CDKN1A, verifying the induction effect of FSK simulating the LH surge on the expression of related molecules in the cell model, as Figure 8 shown.

[0048] Example 5

[0049] Construct a rat ovarian local FOXO4 knockdown model: Randomly divide clean-grade SD rats (6 weeks old) into 2 groups, with 5 rats in each group. After anesthesia, make an incision on the back, isolate the ovaries, and locally inject lentivirus-mediated FOXO4 shRNA (10 ul, 1×10 9 TU / ml) and control empty lentivirus to construct a FOXO4 knockdown model, and then suture the wound. Start vaginal smear on the 14th day to observe the estrous cycle of rats. After 3 weeks, sacrifice the 2 groups of rats. Statistically analyze the body weight and ovarian weight of rats, perform HE staining on ovarian sections, and collect rat serum. The results showed that the estrous cycle of the FOXO4 knockdown group was disordered ( Figure 9A), The progesterone level decreased in the FOXO4 knockdown group ( Figure 9 D), HE staining showed that the number of ovarian cystic follicles increased and the number of corpora lutea decreased in the FOXO4 knockdown group, which was similar to the ovarian cystic changes in the PCOS-like rat model ( Figure 9 E). In addition, the expressions of FOXO4 and CDKN1A decreased, while the expressions of AR and CCND1 increased in the ovaries of the FOXO4 knockdown group ( Figure 9 F), verifying the regulatory relationship between FOXO4 and the expressions of AR, CCND1, and CDKN1A in cell experiments.

[0050] Example 6

[0051] PCOS-like rat model combined with superovulation model: A PCOS-like rat model was constructed using DHEA. By detecting the estrous cycle, serum testosterone, and progesterone levels of rats, and HE staining of ovarian sections showed polycystic-like changes in the ovaries, etc., to screen and construct a successful PCOS-like rat model ( Figure 10 B, D, E). The results showed that the expression of AR increased, the expression of FOXO4 decreased, and the expression of CCND1 increased in the ovarian granulosa cells of the DHEA group ( Figure 10 F - G), verifying that the expression of related molecules in the PCOS-like rats modeled by DHEA was basically consistent with the results of clinical samples, and the down-regulation of FOXO4 expression in PCOS granulosa cells was related to hyperandrogenism. Further, superovulation was performed on the rats in the DHEA group and the control group, and it was found that the number of retrieved eggs and the MII oocyte rate in the DHEA group were significantly decreased ( Figure 10 H - J), verifying that the abnormal ovulation process induced by the LH surge in a hyperandrogenic environment was related to the pathogenesis of PCOS ovulation disorder.

[0052] The present invention targets the important signaling pathway for the pathogenesis of PCOS ovulation disorder, finds the important molecular target FOXO4 in the pathogenesis of PCOS, and discovers that the imbalance of the feedback regulation loop between FOXO4 and AR may play an important role in the occurrence and development of PCOS ovulation disorder. This invention can be used as a future treatment target for PCOS ovulation disorder, providing new ideas for the research on the molecular mechanism of PCOS and treatment methods.

[0053] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. The use of reagents that promote or upregulate the expression of FOXO4 in the preparation of drugs for the treatment of ovulatory disorders caused by polycystic ovary syndrome.

2. The use according to claim 1, characterized in that: The application is to use FOXO4 as an intervention target for treating ovulatory disorders in polycystic ovary syndrome.

3. The use according to claim 1, characterized in that: The reagents for promoting or upregulating the expression of FOXO4 include but are not limited to: A recombinant vector; Proteins that specifically bind to FOXO4; FOXO4 activators, upregulators, or protein degradation pathway blockers.

4. The use according to claim 3, characterized in that: The recombinant vector includes one or more combinations of a FOXO4 overexpression plasmid vector and a FOXO4 overexpression viral vector.

5. The use according to any one of claims 1 to 4, characterized in that: The drug uses an agent or a recombinant vector that promotes or increases the expression of FOXO4 as an active ingredient.

6. A drug for treating ovulation disorder in polycystic ovary syndrome, characterized in that: The active ingredient is an agent or a recombinant vector that promotes or upregulates the expression of FOXO4.

Images