Application of HK1 gene in mouse ovarian granular cells

Abstract

The invention relates to an application of an HK1 gene in mouse ovarian granular cells, in particular to an application in regulating proliferation and apoptosis of the mouse ovarian granular cells and / or steroid hormone synthesis. According to the invention, mouse ovarian granular cells are taken as a research object, and the expression of the HK1 gene is interfered to verify that the HK1 gene is taken as a key target spot for regulating and controlling the functions of the mouse ovarian granular cells so as to influence steroid hormone synthesis and cell proliferation and apoptosis processes. The invention not only provides more reliable technical support for ovarian function regulation, but also provides new thinking and technical guidance for mechanism research of reproductive diseases and preparation and development of related drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to a gene regulation technology, in particular to the application of HK1 gene in mouse ovarian granulosa cells. BACKGROUND

[0002] Hexokinase (HK) is a key enzyme involved in glycolysis, which is essential for glucose utilization. In mammals, five HK isozymes have been identified, including hexokinase 1 (HK1), hexokinase 2 (HK2), hexokinase 3 (HK3), glucokinase (GCK), and hexokinase domain-containing 1 (HKDC1). Studies have shown that interfering with HK1 can significantly inhibit the proliferation, migration and invasion of breast cancer cells. Liver cancer cells with relatively low levels of HK1 can hijack HK1 from large extracellular vesicles to enhance their proliferation through glucose metabolic reprogramming. In addition, targeting HK1 expression can further promote the aging of mesenchymal stem cells. However, there is little research on how HK1 affects ovarian granulosa cells.

[0003] Granulosa cells (GCs) are the most important cells in the ovary, which play an important role in follicular development and oocyte maturation. Their proliferation and apoptosis determine the fate of follicles and oocytes, further affecting ovarian function. Studies have shown that cell cycle genes CDK1, CDC20 and CCND1 have an important influence on cell proliferation. BAK1 and Caspase3 are pro-apoptotic genes that play a key regulatory role in cell apoptosis. In mammals, GnRH secreted by the hypothalamus specifically binds to its receptor GnRHR to promote pituitary secretion of LH. LH specifically binds to its receptor LHR to promote follicular development, while promoting the secretion of progesterone (P4) and estrogen by the ovary, thereby regulating animal reproductive performance.

[0004] At present, it is not clear how HK1 affects the proliferation, apoptosis, steroid hormone synthesis pathway and reproductive-related levels of mouse ovarian granulosa cells and how to regulate it. SUMMARY

[0005] The first technical problem to be solved by the present application is to provide the application of HK1 gene in mouse ovarian granulosa cells.

[0006] The second technical problem to be solved by the present application is to provide the application of HK1 gene as a biomarker in the preparation of products for intervening in the function of mouse ovarian granulosa cells.

[0007] The technical solution adopted by the present application to solve the first technical problem is the application of HK1 gene in mouse ovarian granulosa cells, which includes any one of the following applications:

[0008] 1) Interfering with HK1 gene expression promotes the proliferation of mouse ovarian granulosa cells;

[0009] 2) Interfering with HK1 gene expression inhibits apoptosis of mouse ovarian granulosa cells;

[0010] 3) Interfering with HK1 gene expression inhibits steroid hormone synthesis in mouse ovarian granulosa cells.

[0011] Preferably, the interference with HK1 gene expression is achieved using siRNA technology.

[0012] Preferably, the steroid hormone is estradiol and / or progesterone.

[0013] The technical solution adopted by this invention to solve its second technical problem is the application of the HK1 gene as a biomarker in the preparation of products that regulate the function of mouse ovarian granulosa cells, characterized in that the application includes any one of the following:

[0014] 1) Interfering with HK1 gene expression promotes the proliferation of mouse ovarian granulosa cells;

[0015] 2) Interfering with HK1 gene expression inhibits apoptosis of mouse ovarian granulosa cells;

[0016] 3) Interfering with HK1 gene expression inhibits steroid hormone synthesis in mouse ovarian granulosa cells.

[0017] Preferably, the interference with HK1 gene expression is achieved using siRNA technology.

[0018] Preferably, the steroid hormone is estradiol and / or progesterone.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention is the first to propose and verify that the HK1 gene is a key target for regulating the function of mouse ovarian granulosa cells, thereby affecting steroid hormone synthesis and cell proliferation and apoptosis.

[0021] This invention significantly interferes with HK1 expression, extremely significantly downregulates CDK1 gene expression in mouse ovarian granulosa cells (P<0.01), and significantly downregulates CDC20 and CCND1 gene expression (P<0.05), thereby inhibiting granulosa cell proliferation and achieving precise intervention in cell function.

[0022] By interfering with HK1 expression, the relative expression levels of BAK1 and Caspase3 in mouse ovarian granulosa cells were significantly upregulated, thereby promoting granulosa cell apoptosis; the expression level of CYP11A1 gene was significantly reduced, thereby decreasing steroid hormone synthesis, providing a new strategy and target for improving reproductive performance. This not only provides more reliable technical support for the regulation of ovarian function, but also offers new ideas and technical guidance for the study of reproductive disease mechanisms and the development of related drugs. Attached Figure Description

[0023] Figure 1 The image shows the results of detecting CCK-8 and transfection efficiency in granulosa cells after interfering with HK1.

[0024] Among them, A, the detection of cck-8 in the Si-HK1 group and the Si-NC group; B, the detection of transfection efficiency in the Si-HK1 group and the Si-NC group;

[0025] Note: "**" indicates a highly significant difference from the control group (P<0.01).

[0026] Figure 2 The effect of interference from HK1 on granulocyte proliferation level is shown in the figure.

[0027] The data includes: A) Flow cytometry cell cycle analysis of the Si-HK1 and Si-NC groups; B) Cell cycle distribution of the Si-HK1 and Si-NC groups; C) EdU detection results of granulosa cells (200×), with EdU (red) fluorescence indicating cell proliferation and DAPI (blue) representing the cell nucleus; D) EdU positivity rate; and E) Relative expression levels of cell cycle-related genes (CDK1, CDC20, CCND1) in the Si-HK1 and Si-NC groups.

[0028] Note: "*" indicates a significant difference from the control group (P<0.05), and "**" indicates an extremely significant difference from the control group (P<0.01), the same applies below.

[0029] Figure 3 The effect of interference from HK1 on the level of apoptosis in granulocytes is shown in the figure.

[0030] Among them, A) flow cytometry apoptosis detection images of Si-HK1 group and Si-NC group; B) statistical results of apoptosis in each region of flow cytometry; C) expression level of apoptosis-related genes in granulocytes.

[0031] Figure 4 Figure showing the effects of interference with HK1 on granulosa cell reproduction-related levels and steroid hormone synthesis pathways.

[0032] Among them, A, expression level of genes related to granulosa cell reproduction; B, expression level of genes related to steroid hormone synthesis pathway in granulosa cells; C, expression level of estradiol in granulosa cell culture medium; and D, expression level of progesterone in granulosa cell culture medium. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] The sources of some of the reagents and instruments used in the embodiments of this invention are as follows:

[0036] Reagents: 10% fetal bovine serum (Gibco, MA, USA); 1% penicillin-streptomycin M199 complete medium (Gibco, MA, USA); 0.25% Trypsin-EDTA (Gibco, MA, USA); PrimeScript TM RTreagent Kit (Perfect Real Time) (TAKARA, Japan); CCK-8 reagent (AFExBIO, Houston, TX, USA); Annexin V-FITC apoptosis detection kit (Nanjing Kaiji Biotechnology Co., Ltd., Nanjing, China); EdU cell proliferation kit (Meilun, Dalian, China); TRIzol reagent (Invitrogen, CLD, USA); SYBR Green Kit (TransGen, Beijing, China); estradiol ELISA kit (Aifang, Changsha, Hunan); progesterone ELISA kit (Aifang, Changsha, Hunan).

[0037] Instruments: Microplate reader (Thermo Scientific, MA, USA); Inverted fluorescence microscope (ZEISS, Germany); Ordinary microscope (ZEISS, Germany); CO2 incubator (Eppendorf, Germany); Flow cytometer (BD, NJ, USA); CFXConnect real-time quantitative PCR instrument (BioRad, CA, USA).

[0038] Unless otherwise specified, all other reagents and instruments used in this embodiment are commercially available products.

[0039] In this embodiment of the invention, SPSS 27.0 software was used for statistical analysis. The significance of the comparison between the two groups was calculated using the t-test, and the confidence interval was set at 95%. P<0.05 indicates a significant difference, and P<0.01 indicates a highly significant difference.

[0040] Example 1: Culture and transfection of mouse ovarian granulosa cells

[0041] The granulosa cells used in this embodiment were immortalized mouse ovarian granulosa cells (iCell-0114a) purchased from Shanghai Mirror Cell Technology Co., Ltd. The granulosa cells were cultured in M199 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 cell culture incubator. When the cell confluence reached approximately 80%, the cells were digested with 0.25% Trypsin-EDTA for 2 min, and digestion was terminated by adding an equal proportion of prepared M199 complete medium. The cells were centrifuged at 1300 r·min⁻¹ for 3 min at room temperature. After discarding the supernatant, 3 ml of culture medium was added, the cells were resuspended, and seeded into 12-well or 96-well plates, then incubated at 37°C in a 5% CO2 cell culture incubator. Granulosa cells were isolated and cultured. Once the cells reached approximately 70% confluence and were stably adhered, the original culture medium was removed, and an HK1 interference assay was performed. Transfection was conducted using Lipofectamine 3000 (Invitrogen, CLD, USA). In the HK1 interference assay, the interference group (Si-HK1 group) was transfected with 5 μL of 20 μmol·L⁻¹ Si-HK1, while the blank control group (NC group) received only an equal amount of Lipofectamine 3000. Si-HK1 and Si-NC (Table 1) were designed using DSIR software and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0042] Table 1 siRNA sequences

[0043]

[0044] Example 2: Total RNA Extraction and Quantitative PCR

[0045] RNA was extracted using the traditional TRIzol reagent method and TAKARA's PrimeScript. TMThe RTreagent Kit (Perfect Real Time) was used to reverse transcribe RNA into cDNA. qRT-PCR was performed using the SYBR Green Kit. The mRNA expression levels of HK1, GnRHR, LHR, CYP11A1, CYP19A1, 3β-HSD, CDK1, CDC20, CCND1, BAK1, and Caspase3 in granulosa cells were quantitatively determined. Primers for these genes and the internal control β-actin gene primer were designed using Primer 6 based on reference sequences from NCBI and synthesized by Beijing Qingke Biotechnology Co., Ltd. (Hunan). All primers are listed in Table 2. The qRT-PCR reaction system consisted of: 10 μL Master Mix, 1 μL each of forward and reverse primers, 6 μL ddH2O, and 2 μL cDNA. Reaction conditions were set as follows: 94 °C pre-denaturation for 30 s; 94 °C denaturation for 5 s, Tm annealing / extension for 30 s, for a total of 45 cycles; the β-actin gene was used as the internal control gene, with three replicates per sample. Two-phase flow PCR was employed. –ΔΔCt The relative expression levels of each gene can be calculated.

[0046] Table 2 Primer Sequences

[0047]

[0048] Example 3: Detection of CCK-8 and Transfection Efficiency in Granular Cells

[0049] Granulocytes were divided into 1×10 5 Cells were seeded at a density of 96 wells per well. Transfection was performed when cell confluence reached 70%. After culturing at 37 °C for 0, 48, and 72 hours, cells were transferred to medium containing 10% CCK-8 reagent and incubated at 37 °C for 4 hours. The absorbance of the cells at 450 nm was measured using a microplate reader.

[0050] The results showed that at 48 h, the OD value of the Si-HK1 group at 450 nm was significantly lower than that of the Si-NC group (P<0.01), indicating that interference with HK1 inhibited granulosa cell proliferation; at 72 h, there was no significant difference in the OD value of the Si-HK1 group at 450 nm between the Si-NC group and the Si-HK1 group (P>0.05). Figure 1 A). Subsequently, the transfection efficiency of granulosa cells treated with HK1 interference was detected. The results showed that at 48 h, the relative expression level of HK1 in the Si-HK1 group was significantly lower than that in the Si-NC group (P<0.01). Figure 1 B), with a transfection efficiency of 49%. Therefore, subsequent treatment of HK1 interference was performed for 48 hours.

[0051] Example 4: Cell Proliferation Assay

[0052] Cell cycle detection: Granulocytes from the Si-HK1 group and the Si-NC group were collected separately. Pre-cooled 75% ethanol solution was added to the cell pellet to resuspend the cells. The cells were fixed at 4 ℃, centrifuged at 1000 r·min-1 for 5 min, the supernatant was removed, and the cells were washed with sterile PBS buffer to remove residual fixative. Finally, cell cycle staining solution was added, and the cells were incubated at 4 ℃ in the dark for 30 min. The cell cycle distribution of the two groups was detected and analyzed by flow cytometry.

[0053] The results showed that, compared with the Si-NC control group, the proportion of cells in G1 phase was significantly increased in the Si-HK1 experimental group (P<0.05), while the proportion of cells in S+G2 phase was significantly decreased (P<0.05). Figure 2 A and 2B). EdU detection in granulocytes showed that ( Figure 2 Compared to the Si-NC control group, the EdU positivity rate in the Si-HK1 group was significantly lower (P<0.01). Simultaneously, detection of cell cycle-related genes revealed that interfering with HK1 significantly downregulated CDK1 gene expression (P<0.01) and significantly downregulated CDC20 and CCND1 gene expression (P<0.05). Figure 2 E). The results showed that interfering with HK1 could inhibit the proliferation of granulosa cells.

[0054] Example 5 Apoptosis Assay

[0055] Granulocytes from the Si-HK1 and Si-NC groups were collected separately. Pre-cooled 75% ethanol solution was added to the cell pellet to resuspend the cells. The cells were fixed at 4 °C, centrifuged at 1000 r·min⁻¹ for 5 min, the supernatant was removed, and the cells were washed with sterile PBS buffer to remove residual fixative. Finally, cell cycle staining solution was added, and the cells were incubated at 4 °C in the dark for 30 min. The cell cycle distribution of the two groups was detected and analyzed by flow cytometry.

[0056] After flow cytometry analysis of apoptosis in the Si-HK1 and Si-NC groups of granulocytes, the results showed that ( Figure 3 In both A and 3B groups, the survival rate of cells in the Si-HK1 group (Q4) was significantly lower than that in the Si-NC group (P<0.05), and the early apoptosis rate in the Si-HK1 group (Q3) was significantly lower than that in the Si-NC group (P<0.05). Simultaneously, the expression levels of apoptosis-related genes in granulocytes were detected, revealing that the relative expression levels of BAK1 and Caspase3 in the Si-HK1 group were significantly higher than those in the Si-NC group (P<0.01). Figure 3 C). The results showed that interfering with HK1 could promote apoptosis in granulocytes.

[0057] Example 6: Effects of Cell Reproduction-Related Levels and Steroid Hormone Synthesis

[0058] The expression levels of genes related to granulosa cell reproduction were detected, and the results showed that ( Figure 4 A) The expression level of GnRHR gene in the Si-HK1 group was significantly lower than that in the Si-NC group (P<0.01), and the expression level of LHR gene was significantly lower than that in the Si-NC group (P<0.05). The expression levels of genes related to the steroid hormone synthesis pathway in granulosa cells were detected, and the results showed ( Figure 4 B), the expression level of CYP11A1 gene in the Si-HK1 group was significantly lower than that in the Si-NC group (P<0.05), while the expression levels of CYP19A1 and 3β-HSD genes did not differ significantly (P>0.05). The levels of reproductive hormones in the cell culture medium were measured, and the results showed ( Figure 4 (C and 4D) Interference with HK1 significantly reduced the levels of estradiol (E2) and progesterone (P4) in the cell supernatant (P<0.01).

[0059] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Use of HK1 gene in regulating the function of mouse ovarian granulosa cells, characterized in that, The application includes any one of the following applications: 1) interfering with HK1 gene expression, promoting proliferation of mouse ovarian granulosa cells; 2) interfering with HK1 gene expression, inhibiting apoptosis of mouse ovarian granulosa cells; 3) interfering with HK1 gene expression, inhibiting steroid hormone synthesis in mouse ovarian granulosa cells.

2. Use according to claim 1, characterized in that, The steroid hormone is estradiol and / or progesterone.

3. Use of the HK1 gene as a biomarker in the preparation of a product for regulating the function of mouse ovarian granulosa cells, characterized in that, The application includes any one of the following applications: 1) interfering with HK1 gene expression, promoting proliferation of mouse ovarian granulosa cells; 2) interfering with HK1 gene expression, inhibiting apoptosis of mouse ovarian granulosa cells; 3) interfering with HK1 gene expression, inhibiting steroid hormone synthesis in mouse ovarian granulosa cells.

4. Use according to claim 1, characterized in that, The steroid hormone is estradiol and / or progesterone.

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