Application of NR1D1 gene in regulation and control of granular cells

Abstract

The invention relates to application of an NR1D1 gene in regulation and control of granular cells, and belongs to the technical field of biology. The invention provides an application of an NR1D1 gene in regulation and control of granulosa cells, including an application of a sequence shown in siRNA of the NR1D1 gene in preparation of a product for regulation and control of sheep granulosa cells, and the sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2. According to the regulation method and product of the granulosa cells, the proliferation apoptosis and activity of the granulosa cells can be effectively regulated, and the expression of follicular development related genes can be regulated. The method has the beneficial effects that in-vivo or in-vitro regulation and control effects on follicles and / or ovaries are realized, and a method and a product guarantee are provided for research on development and physiological and biochemical functions of granular cells, follicles and ovaries.

Description

Technical Field

[0001] The invention relates to application of NR1D1 gene in regulating granulocytes and belongs to the field of biotechnology. Background Art

[0002] In female animals, successful ovulation in each estrus cycle is crucial to the production of offspring. Follicular development is a continuous process, from the formation of primordial follicles to final ovulation, of which only a few follicles can mature and enter the ovulation stage, while the rest are atretic or even die. Follicular atresia refers to the damage to the follicle structure and the stunted development, a phenomenon that persists in mammals. Studies have shown that the proliferation and apoptosis of granulosa cells play a key role in follicular development and atresia. Although the relationship between follicular development and granulosa cell function has been revealed, the upstream signaling mechanisms regulating cell proliferation and apoptosis remain unclear.

[0003] In mammals, many behaviors and physiological functions are carried out alternately during the day and night to adapt to changes in the external environment. This phenomenon is called "circadian rhythm", which is mediated by the body's internal regulation to adapt to changes in the internal and external environment. From a systemic perspective, circadian rhythm is not only regulated by the brain, but also by reproductive organs such as testes and ovaries. The key factor of circadian rhythm, BMAL1 / CLOCK complex, initiates the transcription of NR1D1, a member of the nuclear receptor family. Core clock proteins (BMAL1, NR1D1 and DBP) regulate the circadian rhythm of organisms through cis regulatory elements such as E-box, RORE and D-box located in the promoter region and exon region, thereby maintaining biological functions. Both male and female mice with NR1D1 gene knockout show infertility, indicating that the circadian rhythm system is essential for the reproductive function of mammals. Studies have shown that NR1D1 is involved in spermatogenesis, inhibits the proliferation of epithelial ovarian cancer cells through the SOCS3 / JAK / STAT3 signaling pathway, and participates in regulating multiple biological processes including metabolism, inflammation, apoptosis and autophagy.

[0004] The secretion of granulosa cells and their related hormones provides a sufficient physical and chemical environment for the development of oocytes, and plays a vital role in the maturation and development of oocytes and the transmission of endocrine signals. Therefore, the development of methods and products for regulating granulosa cells is of great significance for a deeper understanding of the physiological and biochemical functions of granulosa cells and follicles. Summary of the invention

[0005] The purpose of the present invention is to address the defects of the prior art and speculate that NR1D1 may regulate follicle development by affecting granulosa cell function, thereby affecting the reproductive process of livestock. To confirm this speculation, the present invention proposes the application of NR1D1 gene in regulating granulosa cells, downregulating the expression level of NR1D1 by siRNA technology, and using molecular experiments and commercial kits to detect the effect of NR1D1 on granulosa cell function, laying a foundation for the study of NR1D1 in reproductive function.

[0006] The first purpose of the present invention is to provide an application of the NR1D1 gene in the preparation of a product for regulating granulosa cells, so that the prepared product for regulating granulosa cells has at least one of the functions of regulating granulosa cell apoptosis, vitality, cell proliferation and gene expression.

[0007] The second object of the present invention is to provide a product for regulating granulosa cells.

[0008] The third object of the present invention is to provide a method for regulating granulosa cells.

[0009] The fourth object of the present invention is to provide the application of the above-mentioned product for regulating granulosa cells or the above-mentioned method for regulating granulosa cells.

[0010] The present invention achieves the above-mentioned purpose through the following technical scheme: The present invention provides the use of NR1D1 gene in the preparation of a product for regulating granulosa cells; the regulation of granulosa cells includes: regulating granulosa cell proliferation and apoptosis, regulating the expression of granulosa cell genes, and regulating at least one of granulosa cell activity and steroid hormone-related genes.

[0011] The regulating the expression of granulosa cell genes includes regulating at least one of the BAX gene, the BCL2 gene, the HSD17B12 gene, the CYP11A1 gene and the CYP19A1 gene.

[0012] The present invention also provides a product for regulating granulosa cells, the product comprising small interfering RNA (siRNA) for inhibiting the expression of NR1D1 gene, the sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0013] The present invention further provides a method for regulating granulosa cells, which comprises designing a small interfering RNA (siRNA) of NR1D1 according to the CDS region sequence of the sheep NR1D1 gene published in the NCBI database, wherein the sequence is synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., and performing a qPCR experiment using the above sequence.

[0014] The present invention also provides the product for regulating granulosa cells or the method for regulating granulosa cells for use in at least one of the following (1) to (4); 1. Regulate granulosa cell function; 2. Regulate follicle development; 3. Regulate ovarian development; 4. Prepare products for regulating follicle development. Based on the regulatory effect of the NR1D1 gene on granulosa cells discovered in the experiments of the present invention, the present invention provides the use of the NR1D1 gene in preparing a product for regulating granulosa cells, a product for regulating granulosa cells, a method for regulating granulosa cells and its application.

[0015] The present invention discovered through experiments that the NR1D1 gene can promote the proliferation of granulosa cells; the NR1D1 gene can inhibit the apoptosis of granulosa cells; the NR1D1 gene can promote the vitality of granulosa cells; the NR1D1 gene is also related to the expression of the BAX gene, the BCL2 gene, the HSD17B12 gene, the CYP11A1 gene and the CYP19A1 gene in the granulosa cells, and the NR1D1 gene can inhibit the BAX gene and promote the expression of the BCL2 gene, the HSD17B12 gene, the CYP11A1 gene and the CYP19A1 gene.

[0016] In view of the role of NR1D1 gene in granulosa cell regulation, the regulation method of granulosa cells provided by the present invention and its related products can effectively regulate the proliferation, apoptosis and vitality of granulosa cells, and regulate the expression of genes related to follicle development. In view of the regulatory effect of granulosa cells on follicle and ovarian development, the regulation method and product of the above-mentioned granulosa cells are further applied to the regulation of follicle and ovarian development, or for the preparation of products for regulating follicle and ovarian development, and its beneficial effect is to achieve the in vivo or in vitro regulation of follicles and / or ovaries, and provide the guarantee of methods and products for the development and physiological and biochemical functions of granulosa cells, follicles and ovaries. In addition, it is helpful to deepen the understanding of the mechanism of oocyte maturation in the late stage of follicular development, further explore the factors related to follicular development, improve the signal pathways related to follicular development, reveal the mechanism of follicular development, and optimize the in vitro culture system of oocytes. This is of great significance to the research fields such as mammal superovulation, multi-birth variety selection, improvement of abnormal follicular development and treatment of infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the relative expression level of NR1D1 gene after siRNA transfection of granulosa cells in Example 1 of the present invention; Figure 2 The results of CCK8 detection of cell viability after NR1D1 gene interference in Example 2 of the present invention; Figure 3 The results of flow cytometry apoptosis detection after NR1D1 gene interference in Example 2 of the present invention; Figure 4 The expression changes of apoptosis-related genes after NR1D1 gene interference in Example 2 of the present invention; Figure 5 The results of Edu detection of cell proliferation after NR1D1 gene interference in Example 3 of the present invention; Figure 6 This is the change in the expression level of steroid-related genes after NR1D1 gene interference in Example 3 of the present invention. DETAILED DESCRIPTION

[0018] The sheep ovarian follicular fluid granulosa cells used in the following examples were purchased from a slaughterhouse and obtained by centrifuging healthy ovarian follicular fluid, which will not be described in detail.

[0019] Example 1 NR1D1 gene siRNA interference: Based on the CDS region sequence of the sheep NR1D1 gene published in the NCBI database, the NR1D1 small interfering RNA (siRNA) was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. An interference fragment was designed for the sheep NR1D1 gene, and the interference sequence is shown in Table 1: Table 1

[0020] In Table 1, "siNC" represents the untreated control group; "si-NR1D1" represents the NR1D1 gene interference treatment group. Data are expressed as mean ± SEM, and t-test was used for statistical analysis. Different lowercase letters represent significant differences, p < 0.05. The same below.

[0021] Cell transfection: primary GCs were thawed and cultured to a cell density of 60%, and transfected according to the instructions of Lipofectamine®3000. Taking a 6-well plate as an example: Opti-MEM diluted siRNA-NC and siRNA-NR1D1 liposome complexes, where lip3000 was used in each well at a dosage of 4 μL, and 20 mM siRNA was added to each well at 10 μL. After standing at room temperature for 15 min, it was slowly added to the replaced fresh culture medium so that the liquid volume in the 6-well plate was uniformly maintained at 2 mL. After culturing for 24 h or 48 h, the cells were washed 2-3 times with DPBS solution and collected for total RNA extraction. Cell RNA extraction and reverse transcription: refer to the instructions of commercial RNA extraction and reverse transcription kits. The brief steps are as follows: add appropriate amounts of TRIzol, chloroform, isopropanol and 75% ethanol and other reagents, dissolve the RNA precipitate with DEPC water after elution and centrifugation, and use it for subsequent reverse transcription and fluorescence quantitative PCR.

[0022] Example 2 Effects of NR1D1 gene interference on cell viability: The activity of sheep granulosa cells after NR1D1 gene interference was tested by CCK-8 method (Vazyme), and it was found that cell proliferation increased after NR1D1 gene interference. Figure 2 shown.

[0023] Example 3 Effects of NR1D1 gene interference on cell apoptosis: Cell apoptosis detection: The cells were pre-treated according to the cell cycle detection method, and stained according to the instructions of the Annexin V-FITC / PI apoptosis detection kit (Vazyme, A211). The steps are as follows: thaw the primary cells and transfect them according to the test requirements, and set up untreated cell samples for negative control and single staining tubes. Digest the cells with EDTA-free cell digestion solution for about 10 min, centrifuge, discard the supernatant, wash with pre-cooled DPBS, centrifuge, and resuspend. Add 5 μL of Annexin V-FITC and 5 μL of PI to the resuspended sample, stain in the dark for 10 minutes, homogenize and mix well, and detect with a flow cytometer. Flow Jo 10.8.1 software was used for cell apoptosis analysis.

[0024] The apoptosis of sheep granulosa cells after NR1D1 gene interference was detected by flow cytometry. Figure 3 As shown, it was found that the apoptosis rate of granulosa cells in the NR1D1 gene interference group was significantly lower than that in the blank control group.

[0025] Example 4 Effects of NR1D1 gene interference on expression changes of apoptosis-related genes: Cell RNA extraction, reverse transcription and fluorescence quantitative PCR were performed according to the instructions of the commercial kits HiScript®III RTSuperMix (Vazyme R311) and ChamQ SYBR qPCR Master Mix (Vazyme Q711). The primer sequences required for qPCR are shown in Table 2.

[0026] Table 2

[0027] The gene expression of BAX and BCL2 genes related to cell apoptosis in sheep granulosa cells after NR1D1 gene interference was detected. It was found that after NR1D1 gene interference, the expression of BAX gene in granulosa cells was significantly downregulated, while the expression of BCL2 gene was significantly upregulated. This indicates that NR1D1 gene affects the expression of BAX and BCL2 genes, two apoptosis-related genes in sheep granulosa cells. Figure 4 shown.

[0028] Example 5 Effects of NR1D1 gene interference on cell proliferation: EdU detection: refer to the instructions of EdU cell proliferation detection kit (Biyuntian, C0075S), as follows: Thaw the primary GCs in a 24-well plate with cell slides, and perform liposome transfection when the cell density is about 50-60%. After 24 hours of treatment, dilute EdU with culture medium to a working solution concentration of 20 μM. Aspirate the culture medium, wash with DPBS 1-2 times, and incubate with EdU working solution at 37 °C for 2 hours. During the incubation process, EdU replaces thymidine and is incorporated into newly synthesized DNA for labeling. After labeling, fix the cells with 4% paraformaldehyde for 15 minutes. Remove the fixative and wash, and permeabilize with 0.3% Triton-X100 for 15 minutes. Prepare the Click reaction working solution according to the instructions, add 100 μL of the reaction mixture to each well and incubate in the dark for 30 minutes, so that EdU reacts covalently under the catalysis of monovalent copper ions and combines with the fluorescently labeled small molecule azide probe (AzideAlexa Flour 555). After incubation and washing, use 1× Hoechst3342 solution to stain the nucleus at room temperature and avoid light for 10 min. Take out the slide, add anti-fluorescence quenching mounting medium, and invert it on the slide. In the laser confocal imaging detection, the maximum excitation wavelength of Azide555 red fluorescence is 555 nm, and the maximum excitation wavelength of Hoechst3342 blue fluorescence is 346 nm.

[0029] The proliferation level of granulosa cells after interference with NR1D1 was detected by the Edu method. Figure 5 Compared with the control group, the number of Edu-positive cells in granulosa cells increased after treatment, indicating that the cell proliferation ability was promoted.

[0030] Example 6 Effects of NR1D1 gene interference on cellular steroid-related gene expression: The gene expression levels of steroid-related genes HSD17B12, CYP11A1, and CYP19A1 in sheep granulosa cells after NR1D1 gene interference were detected. Figure 6 As shown in the figure. After the NR1D1 gene was interfered, the steroid-related genes in granulosa cells increased significantly, indicating that the NR1D1 gene can function in sheep ovarian granulosa cells.

[0031] In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. The application of NR1D1 gene in regulating granulosa cells is characterized by: The invention comprises the use of the sequence shown by the siRNA for regulating the NR1D1 gene in preparing a product for regulating sheep granulosa cells, wherein the sequence is shown by SEQ ID NO:1 and SEQ ID NO:

2.

2. The use of the NR1D1 gene in regulating granulosa cells according to claim 1, characterized in that: The regulation of sheep granulosa cells includes at least one of regulating granulosa cell proliferation and apoptosis, regulating granulosa cell activity, regulating granulosa cell gene expression and regulating follicular granulosa cell steroid hormone secretion genes.

3. The use of the NR1D1 gene in regulating granulosa cells according to claim 2, characterized in that: The application of the sequence shown by NR1D1 gene siRNA in down-regulating the expression level of NR1D1 gene and inhibiting the gene expression of apoptosis of sheep granulosa cells.

4. The use of the NR1D1 gene in regulating granulosa cells according to claim 3, characterized in that: The gene regulating the proliferation and apoptosis of sheep granulosa cells includes at least one of regulating the expression of BAX and BCL2 genes.

5. The use of the NR1D1 gene in regulating granulosa cells according to claim 2, characterized in that: The regulation of granulosa cell activity includes downregulating the expression of NR1D1 gene in granulosa cells, and increasing cell activity and proliferation levels.

6. The use of the NR1D1 gene in regulating granulosa cells according to claim 2, characterized in that: The genes regulating the secretion of steroid hormones in follicular granulosa cells include at least one of the HSD17B12 gene, the CYP11A1 gene and the CYP19A1 gene.

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