Application of ENC1 gene in diagnosis of ovarian function decline

Abstract

The invention provides application of an ENC1 gene in diagnosis of ovarian function decline, and belongs to the technical field of biomedicine. According to the invention, ovarian tissue single cells of mice of different ages are used for sequencing, screening and verification show that an Enc1 expression level is negatively related to the age in the ovarian of the mice, and human ovarian transcriptome sequencing public data also shows that the ENC1 gene is obviously differentially expressed in an old group and a young group; the ENC1 transcription and protein expression level can be used for early warning and diagnosing the state of female ovarian function decline. The invention further proves the correlation between ENC1 and ovarian senescence through experiments, and explores related mechanisms. On one hand, a new theoretical perspective is provided for researching the molecular mechanism of ovarian aging, a scientific basis is provided for early recognition of ovarian aging and injury, and a theoretical basis is laid for research on development of drugs for protecting ovarian functions.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of ENC1 gene in diagnosing ovarian dysfunction. Background Art

[0002] The ovary is an important reproductive endocrine organ in women. Compared with other organs, the aging process of the ovary is significantly advanced, which is mainly manifested by a decrease in ovarian sex hormone levels and a decrease in ovarian reserve function. This process not only causes a series of clinical symptoms such as infertility and menopausal syndrome, but is also closely related to the occurrence and development of a variety of chronic aging-related diseases, such as cardiovascular disease, osteoporosis, endocrine disorders, etc., which has a profound impact on women's physical and mental health. Despite the serious consequences of ovarian aging, the current understanding of the molecular mechanisms that regulate ovarian aging is still limited, which directly leads to the lack of effective biomarkers and molecular targets for early warning, diagnosis and intervention of ovarian aging.

[0003] In current clinical practice, the evaluation of ovarian function mainly focuses on indicators that reflect ovarian endocrine function, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), inhibin B, etc.; in addition, ultrasound monitoring of the number of follicles and ovarian blood flow, CT, magnetic resonance imaging and other imaging methods are also often used for auxiliary diagnosis.

[0004] The process of ovarian aging is affected by a combination of factors, including genetic background, environmental factors, medical intervention, immune status, personal lifestyle, and social and psychological factors. The interweaving of these factors makes the accurate evaluation of ovarian function and the early diagnosis of ovarian aging face major challenges. With the rapid development of molecular biology technology, the focus of research has gradually shifted to the molecular level, dedicated to the discovery and identification of new biomarker molecules. The goal of these studies is to reveal the molecular mechanism of ovarian aging in order to discover more potential biomarkers and thus improve the ability to evaluate and predict ovarian aging.

[0005] Ectodermal neural cortex 1 (ENC1) is a kelch-related gene that has been shown to play a key role in regulating multiple biological processes such as cell proliferation, differentiation, and apoptosis. Studies have shown that ENC1 is overexpressed in various types of cancer, including breast cancer, lung cancer, pancreatic cancer, and colorectal cancer, and its upregulation is associated with poor prognosis. However, there is currently no research on the role of the ENC1 gene in aging and ovarian aging. Summary of the invention

[0006] The purpose of the present invention is to provide an application of ENC1 gene in diagnosing ovarian dysfunction, so as to fill the gap in the prior art in the association between ENC1 gene expression and ovarian dysfunction.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a use of an ENC1 gene as a biomarker in the preparation of a product for diagnosing ovarian dysfunction.

[0009] In a second aspect, the present invention provides a use of a reagent for detecting the expression level of the ENC1 gene in the preparation of a product for diagnosing ovarian dysfunction.

[0010] In the present invention, single-cell RNA sequencing technology was successfully used to construct an ovarian map of female C57 mice at multiple ages, and the differential expression of Enc1 was discovered. Human ovarian tissue sequencing data in a public database was also used for comparison, and the results showed that the expression of ENC1 in human ovarian tissue decreased with age, and there were significant differences between the old and young groups. These data fully demonstrate that ENC1 has the potential to serve as a specific marker for diagnosing ovarian dysfunction.

[0011] As a further optimization scheme of the present invention, the ovarian function decline includes ovarian damage and / or ovarian aging.

[0012] As a further optimization scheme of the present invention, the ovarian function decline includes ovarian damage and / or ovarian aging caused by one or more factors of age, genetics, immunity, iatrogenicity, infection, environment, behavior, and social psychology.

[0013] As a further optimization scheme of the present invention, the characteristics of ovarian dysfunction include human serum ovarian anti-Müllerian hormone level <1.1 ng / ml, and / or, the number of ovarian antral follicles <6, and / or, ovarian follicle-stimulating hormone level >10 U / L.

[0014] As a further optimization scheme of the present invention, the expression level of the ENC1 gene includes the expression level of RNA, DNA methylation, protein, peptide, or a combination thereof.

[0015] As a further optimization scheme of the present invention, the expression level of the ENC1 gene is negatively correlated with the degree of ovarian function decline.

[0016] As a further optimization scheme of the present invention, the test sample is a human tissue sample.

[0017] In a third aspect, the present invention provides a product for diagnosing ovarian dysfunction, characterized in that the product comprises a reagent for detecting the expression level of the ENC1 gene.

[0018] As a further optimization solution of the present invention, the product includes a chip, a preparation or a kit.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] This study is the first to link the ENC1 gene with ovarian function decline, filling the gap in the study of the role of the ENC1 gene in aging and ovarian aging. This discovery provides a new perspective for a deeper understanding of the molecular mechanism of ovarian aging.

[0021] Through single-cell RNA sequencing technology and bioinformatics analysis, the present invention successfully verified the differential expression of the ENC1 gene in ovarian dysfunction, indicating that ENC1 has the potential to be used as a specific marker for diagnosing ovarian dysfunction. This discovery provides a new tool for early warning and diagnosis of ovarian dysfunction.

[0022] Compared with traditional diagnostic methods, using the ENC1 gene as a biomarker can more accurately assess the ovarian function status, helping to reduce misdiagnosis and missed diagnosis. At the same time, the expression level of the ENC1 gene can also provide a quantitative indicator for the severity of ovarian function decline and provide a basis for the formulation of personalized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The expression changes of Enc1 in mouse ovaries with age, where A is the correlation between the overall Enc1 expression in mouse ovaries and age; B is the correlation between the Enc1 expression in various cell types in mouse ovaries and age; C is the expression location of the Enc1 gene in mouse ovaries using single-cell sequencing results of mouse ovaries; D is the differential expression of the Enc1 gene in various cell types of mice, between the old group and the young group.

[0024] Figure 2 To analyze the expression of ENC1 gene in different age groups using human ovarian tissue transcriptome data from the GTExV8 cohort.

[0025] Figure 3 In order to analyze the expression of each ENC1 gene using the single-cell transcriptome data of human ovarian tissue in the public database, A is the expression location of the ENC1 gene in the ovary using the single-cell sequencing results of human ovary; B is the differential expression and expression level of the ENC1 gene in various cell types of the human ovary, between the old group and the young group.

[0026] Figure 4 Results of immunohistochemical staining of ENC1 protein in the ovaries of naturally aged C57 mice (15 months old) and reproductive-age C57 mice (3 months old) and data analysis diagram; n=5 / group, **p<0.01. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings, embodiments and experimental examples. Of course, the protection scope of the present invention is not limited to the following embodiments. Professional and technical personnel in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiment. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible here. The following examples further illustrate the present invention, rather than limiting the present invention. Any equivalent transformation that is only formal and not substantial based on the concept of the present invention should be regarded as the scope of the technical solution of the present invention.

[0028] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] Example 1

[0031] 1. This example performs single cell analysis and target screening.

[0032] Tissue acquisition and single cell suspension preparation: Fresh C57BL / 6 mouse ovaries were obtained from each group of 3M, 6M, 9M, 12M, and 15M, with 6 mice in each group. Within the first 30 minutes of the procedure, the collected fresh tissues were placed on ice and immersed in tissue preservation solution. The specimens were washed three times with Hanks balanced salt solution and then digested with tissue dissociation solution at 37°C for 15 minutes. Next, red blood cell lysis buffer was added and incubated at 25°C for another 10 minutes to remove red blood cells. Centrifuge at 300×g for 5 minutes in a 4°C centrifuge. After removing the supernatant, the solution was gently suspended in phosphate-buffered saline. Finally, trypan blue staining was performed and cell viability was analyzed using an inverted microscope. Cell suspensions that meet the above standards can be used for single cell library construction if the viability is greater than 90%, the clumping rate is less than 5%, and the nucleation rate is greater than 70%.

[0033] The present embodiment relates to a method for performing age-related analysis on mouse ovarian cells using single-cell transcriptome sequencing technology to identify specific age-related biomarkers. Specifically, the present embodiment first performs an analysis step to identify genes whose expression levels in the ovaries are correlated with the age of the mouse (the absolute value of the correlation coefficient is greater than 0.7). Furthermore, the present embodiment, through the analysis of the composition of ovarian cells, reveals that the number of granulosa cells and their proportion in total cells significantly decrease with age. In view of the importance of granulosa cells in providing nutrition to oocytes and secreting hormones, the present embodiment further extracts and compares the differentially expressed genes between the granulosa cells of the old group and the young group. By cross-comparing the results of the above two analysis steps, the present embodiment ultimately identified a gene Enc1 that is negatively correlated with age. The data show that the expression of Enc1 in multiple cell types of the ovary is negatively correlated with age (see Figure 1 Through detailed analysis of the expression level and cellular localization of Enc1, this example found that Enc1 is mainly expressed in granulosa cells (GCs) and theca stromal cells (T&Ss) of the mouse ovary (see Figure 1 In addition, this example further analyzed the differentially expressed genes of various cells between the old group (15 months old) and the young group (3 months old). The results showed that the expression of Enc1 in mouse granulosa cells was significantly downregulated with age (see Figure 1 D).

[0034] In order to establish the conservation of the differential expression pattern of the ENC1 gene among species, this example uses the human ovarian tissue sequencing data in the GTEx V8 database to evaluate the expression level of the ENC1 gene in human ovaries at different ages (see Figure 2 ). The analysis results revealed that the expression of the ENC1 gene in human ovarian tissue decreased significantly with age, indicating that the differential expression pattern of the ENC1 gene is conserved among different species. According to the analysis results of the human ovarian single-cell sequencing dataset (GSE202601) in the public database, this example found that the ENC1 gene is mainly expressed in granulosa cells and theca cells in human ovarian tissue (see Figure 3 Further analysis showed that the expression of ENC1 gene in granulosa cells, theca cells and epithelial cells in the elderly group showed a significant down-regulation trend compared with the young group (see Figure 3 B).

[0035] In summary, based on the experimental data and analysis results of this example, it can be reasonably inferred that the ectodermal neural cortex 1 (ENC1) gene can be used as a biomarker for evaluating ovarian function decline. This discovery of this example provides a new perspective for in-depth exploration of the pathological mechanism of ovarian aging, and has important scientific and clinical significance for the development of diagnostic methods and treatment strategies for ovarian function decline.

[0036] Example 2

[0037] In this example, immunohistochemical staining of paraffin sections of ovarian tissues of C57 mice with ovarian dysfunction due to natural aging and C57 mice of reproductive age was performed.

[0038] Immunohistochemistry was used to detect the expression of ENC1 (Proteintech, 15007-1-AP) protein in mouse ovaries. The specific steps are as follows:

[0039] (1) Baking: Place the selected ovarian paraffin sections in a 65°C oven and bake for 2 h;

[0040] (2) Dewaxing to hydration: The baked slides were hydrated in the following order: environmental dewaxing solution I for 20 min → environmental dewaxing solution II for 20 min → anhydrous ethanol for 10 min → 95% ethanol for 10 min → 85% ethanol for 5 min → 75% ethanol for 5 min → washing with PBS for 3 × 5 min;

[0041] (3) Wash the tissue with PBS three times for 5 min;

[0042] (4) Antigen retrieval: Boil the antigen retrieval solution at high heat, boil it at medium heat twice for 7 min, and cool it naturally at room temperature;

[0043] (5) Wash the tissue with PBS three times for 5 min;

[0044] (6) Removal of endogenous peroxidase: incubate the tissue with 3% H2O2 at 37°C for 15 min;

[0045] (7) Wash the tissue with PBS three times for 5 min;

[0046] (8) Block with goat serum and incubate the tissue at 37°C for 30 min;

[0047] (9) Remove the blocking solution and incubate the tissue with primary antibody at 4°C overnight;

[0048] (10) The next day, after rewarming at room temperature, the cells were washed three times with PBS for 5 min;

[0049] (11) Incubate sections with secondary antibody at 37°C for 1 h; wash with PBS 3 times for 8 min;

[0050] (12) DAB color development: After 80% to 90% of the tissue has developed color, terminate the color development with tap water, and then rinse with tap water for 30 minutes;

[0051] (13) Counterstain with hematoxylin for 5 min and rinse with tap water three times;

[0052] (14) Soak in differentiation solution for 2 to 3 seconds, then rinse three times with tap water;

[0053] (15) Immerse in bluing solution for 5 seconds and rinse with tap water three times;

[0054] (16) Reverse alcohol gradient: 75% ethanol 5 min → 85% ethanol 5 min → 95% ethanol 5 min → anhydrous ethanol 5 min → xylene II 10 min → xylene I 10 min;

[0055] (17) Air-dry in a fume hood and seal with neutral resin;

[0056] (18) Observe and take photos under a microscope and perform histochemical scoring.

[0057] Data analysis: Figure 4 As shown, optical microscopy showed that ENC1 protein in mouse ovaries was mainly expressed in granulosa cells and interstitial cells of the follicle, and the expression level of ENC1 protein in the ovaries of old mice was significantly decreased compared with that in the ovaries of young mice.

[0058] In summary, this invention provides a new direction for research in related fields by revealing the important role of ENC1 gene in ovarian dysfunction, and also provides new ideas and methods for clinical diagnosis and treatment. This discovery has important scientific value and clinical significance, and is expected to make an important contribution to the prevention and treatment of female reproductive health and aging-related diseases.

[0059] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of ENC1 gene as a biomarker in the preparation of products for diagnosing ovarian dysfunction.

2. Application of reagents for detecting ENC1 gene expression levels in the preparation of products for diagnosing ovarian dysfunction.

3. The use according to claim 1 or 2, characterized in that: The ovarian function decline includes ovarian damage and / or ovarian aging.

4. The use according to claim 3, characterized in that: The ovarian function decline includes ovarian damage and / or ovarian aging caused by one or more factors of age, genetics, immunity, iatrogenicity, infection, environment, behavior, and social psychology.

5. The use according to claim 3, characterized in that: The characteristics of ovarian dysfunction include a human serum ovarian anti-Müllerian hormone level of less than 1.1 ng / ml, and / or an ovarian antral follicle number of less than 6, and / or an ovarian follicle-stimulating hormone level of more than 10 U / L.

6. The use according to claim 2, characterized in that: The expression level of the ENC1 gene includes the expression level of RNA, DNA methylation, protein, peptide, or a combination thereof.

7. The use according to claim 2 or 6, characterized in that: The expression level of the ENC1 gene is negatively correlated with the degree of ovarian dysfunction.

8. The use according to claim 2, characterized in that: The test samples are human tissue samples.

9. A product for diagnosing ovarian dysfunction, characterized in that: The product comprises a reagent for detecting the expression level of the ENC1 gene according to any one of claims 1 to 8.

10. The product according to claim 9, characterized in that The product includes a chip, a preparation or a kit.

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