Use of pias4 in preparing a product for diagnosing polycystic ovary syndrome

Abstract

The application provides application of PIAS4 in preparation of a product for diagnosing polycystic ovary syndrome, and relates to the technical field of biotechnology.The inventor has found that, compared with healthy subjects, PIAS4 is differentially expressed in polycystic ovary syndrome patients, the expression of PIAS4 is related to polycystic ovary syndrome, the expression amount of PIAS4 can be detected to realize diagnosis of polycystic ovary syndrome, and the diagnosis has high specificity and sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of PIAS4 in the preparation of products for diagnosing polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine disorder and reproductive dysfunction in women of reproductive age. It is characterized by chronic anovulation (disorder or loss of ovulation) and hyperandrogenemia. The main clinical manifestations are menstrual disorders, infertility, hirsutism, and obesity, accompanied by metabolic abnormalities such as insulin resistance, hyperinsulinemia, and hyperlipidemia, which have a great impact on reproductive health.

[0003] The clinical manifestations of PCOS are heterogeneous, and the methods for diagnosing this disease still need to be improved.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide the application of PIAS4 (Sumo E3 ligase protein inhibitor of activated STAT4) in the preparation of products for diagnosing polycystic ovary syndrome, in order to solve at least one of the above-mentioned problems.

[0006] A second objective of this invention is to provide a reagent for diagnosing polycystic ovary syndrome.

[0007] A third objective of this invention is to provide a kit for diagnosing polycystic ovary syndrome.

[0008] In a first aspect, the present invention provides the application of PIAS4 in the preparation of products for diagnosing polycystic ovary syndrome, wherein PIAS4 is the mRNA or protein of the PIAS4 gene.

[0009] As a further technical solution, the product includes reagents or kits for diagnosing polycystic ovary syndrome by detecting the expression level of PIAS4 gene mRNA or protein in the subject.

[0010] As a further technical solution, the mRNA of the PIAS4 gene is downregulated in the oocytes of patients with polycystic ovary syndrome.

[0011] As a further technical solution, the protein of the PIAS4 gene is downregulated in the peripheral blood of patients with polycystic ovary syndrome.

[0012] Secondly, the present invention provides a reagent for diagnosing polycystic ovary syndrome, the reagent comprising primers for detecting PIAS4 gene mRNA or biomolecules for detecting PIAS4 gene protein.

[0013] As a further technical solution, the biomacromolecule includes an antibody or an antibody functional fragment.

[0014] As a further technical solution, the test samples for the reagent include oocytes or peripheral blood.

[0015] Thirdly, the present invention provides a kit for diagnosing polycystic ovary syndrome, the kit comprising the reagents described above.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The inventors' research found that PIAS4 is differentially expressed in patients with polycystic ovary syndrome (PCOS) compared to healthy subjects. PIAS4 expression is associated with PCOS, and PCOS can be diagnosed by detecting the expression level of PIAS4 with high specificity and sensitivity. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The expression levels of the PIAS4 gene in the PCOS group and the control group;

[0020] Figure 2 ROC curve for PIAS4 gene used in the diagnosis of PCOS;

[0021] Figure 3 The WB experimental results provided in Example 2;

[0022] Figure 4 The expression levels of PIAS4 protein in the PCOS group and the control group;

[0023] Figure 5 ROC curve for PIAS4 protein used in the diagnosis of PCOS. Detailed Implementation

[0024] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] In a first aspect, the present invention provides the application of PIAS4 in the preparation of products for diagnosing polycystic ovary syndrome, wherein PIAS4 is the mRNA or protein of the PIAS4 gene.

[0026] The inventors' research found that, compared with healthy subjects, the expression of PIAS4 gene mRNA is downregulated in the oocytes of patients with polycystic ovary syndrome (PCOS), and the expression of PIAS4 gene protein is downregulated in the peripheral blood of patients with PCOS. The expression of PIAS4 gene mRNA or protein is associated with PCOS, and PCOS can be diagnosed by detecting the expression level of PIAS4 gene mRNA or protein.

[0027] In some preferred embodiments, the product includes, but is not limited to, reagents or kits.

[0028] The reagents or kits diagnose polycystic ovary syndrome by detecting the expression levels of the PIAS4 gene mRNA or protein in the subject.

[0029] Secondly, the present invention provides a reagent for diagnosing polycystic ovary syndrome, the reagent comprising primers for detecting PIAS4 gene mRNA or biomolecules for detecting PIAS4 gene protein.

[0030] The reagents provided by this invention detect the expression level of PIAS4 gene mRNA by specifically recognizing primers that target PIAS4 gene mRNA, or detect the expression level of PIAS4 gene protein by specifically recognizing the biomolecule PIAS4 gene protein. The reagents provided by this invention diagnose polycystic ovary syndrome by detecting the expression level of PIAS4 gene mRNA or protein.

[0031] The detection reagent for the protein can be any reagent known to those skilled in the art, such as a fluorescently labeled biomolecule that can react with the protein to produce an antigen-antibody reaction.

[0032] In some preferred embodiments, the biomacromolecule includes an antibody or an antibody functional fragment.

[0033] In some preferred embodiments, the test sample for the reagent includes oocytes or peripheral blood.

[0034] Thirdly, the present invention provides a kit for diagnosing polycystic ovary syndrome, the kit comprising the reagents described above.

[0035] The kit provided by this invention includes the above-mentioned reagents, has all the beneficial effects of the reagents, and can be used to diagnose polycystic ovary syndrome.

[0036] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0037] Example 1

[0038] 1. Clinical specimen inclusion

[0039] Inclusion criteria: All specimens were from the Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences.

[0040] Control group:

[0041] ① Age 20-35;

[0042] ② No PCOS, and no ovarian cysts, ovarian dysfunction, or other diseases that affect ovarian function;

[0043] ③ Use a long protocol for ovulation induction;

[0044] ④BMI stabilized between 18.5 and 26 kg / m² 2 between.

[0045] PCOS Group:

[0046] ① Age 20-35;

[0047] ② Diagnose PCOS according to the Rotterdam criteria;

[0048] ③ Use a long protocol for ovulation induction;

[0049] ④BMI stabilized between 18.5 and 26 kg / m² 2 between.

[0050] Exclusion criteria:

[0051] Infertility may include other factors.

[0052] ① Hyperprolactinemia, thyroid dysfunction;

[0053] ② Type 2 diabetes;

[0054] ③ Serum FSH > 10 mIU / mL;

[0055] ④ Endometriosis.

[0056] PCOS patients underwent ovulation induction treatment using the long protocol. Patients were downregulated with GnRH agonists, followed by ovarian stimulation with FSH or HMG. When three follicles reached a diameter of 18 mm, hCG was administered to promote follicle maturation, and oocytes were retrieved 36 hours later. The control group underwent ovulation induction using the long protocol and oocytes were retrieved. The oocyte source information for the PCOS group and the control group is summarized in Table 1.

[0057] Table 1 Summary of information on the source of human eggs

[0058]

[0059]

[0060] - Abbreviations: BMI, Body Mass Index; D2, Day 2; FSH, Follicle-Stimulating Hormone; LH, Luteinizing Hormone; E2, Estradiol; Testosterone; TSH, Thyroid-Stimulating Hormone; T3, Triiodothyronine; T4, Thyroxine; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; TG, Triglycerides; T-CHO, Total Cholesterol; IVF, In Vitro Fertilization; Prolactin. - P-value < 0.05 indicates statistical significance. Values ​​are expressed as mean ± SD (range).

[0061] 2. Single-cell sequencing

[0062] The obtained oocytes were analyzed using an Illumina HiSeq 4000 to obtain scRNA-seq data.

[0063] Preparation of single-cell transcriptome scRNA-seq libraries includes:

[0064] 1) Cell sample preparation: Use a Pasteur pipette to aspirate germ cells at different stages, resuspend them in 1×PBS, and place them at the bottom of an EP tube. Perform on ice.

[0065] 2) First-strand cDNA synthesis: Prepare Lysis buffer, add it to an EP tube containing a single cell, add Oligo(dT)VN primer and dNTP Mix, mix gently, and then react with a PCR instrument. Reaction conditions: 72℃ extension for 3 min; immediately place on ice for 2 min. Prepare the reverse transcription reaction system and perform the reaction at 42℃ for 90 min; 70℃ for 15 min; cool at 4℃.

[0066] 3) Full-length cDNA amplification and purification: Prepare the PCR amplification system, mix well, and react with a PCR instrument. Reaction conditions: 98℃ denaturation for 10s; 65℃ annealing for 15s; 72℃ extension for 6min (16 cycles); 72℃ extension for 5min; cooling at 4℃. After purification by magnetic bead sorting, the product quality control and identification were performed using an Agilent 2100 Bioanalyzer.

[0067] 4) Library construction and sequencing: After processing the above product fragments according to the DNA library preparation kit, the samples were sequenced on the Illumina Hiseq platform in PE150 mode.

[0068] 3. Results

[0069] The expression levels of the PIAS4 gene in the PCOS group and the control group were as follows: Figure 1 As shown in the figure. The results indicate that, compared with the control group, the expression of the PIAS4 gene was differentially downregulated in the PCOS group, suggesting that the PIAS4 gene has the potential to serve as a diagnostic biomarker for the diagnosis of PCOS.

[0070] Based on the sequencing data of the PIAS4 gene, the inventors established the ROC curve for the PIAS4 gene in the diagnosis of PCOS. Figure 2 The AUC value was 0.778. These results indicate that the PIAS4 gene alone, as a diagnostic biomarker, has high accuracy in the diagnosis of PCOS.

[0071] Example 2

[0072] To verify the above conclusions, the inventors extracted total protein from peripheral blood of PCOS patients and healthy subjects. Blood was collected in anticoagulant tubes, and 2 μL of a protease inhibitor mixture and 2 μL of protein stabilizer were added to every 300 μL of protein extract. The process was performed on ice. 300 μL of whole blood sample was taken, and 300 μL of cooled protein extract was added. The mixture was thoroughly mixed, vortexed at 4°C for 10 minutes, centrifuged at 12000g for 10 minutes, and the supernatant was collected, which was the total protein from whole blood.

[0073] The inclusion criteria for PCOS patients (PCOS group) and healthy subjects (control group) were the same as in Example 1, and the clinical information is shown in Table 2.

[0074] Table 2 Clinical information of the PCOS group and the control group

[0075]

[0076]

[0077] - Abbreviations: BMI, Body Mass Index; D2, Day 2; FSH, Follicle-Stimulating Hormone; LH, Luteinizing Hormone; E2, Estradiol; Testosterone; TSH, Thyroid-Stimulating Hormone; T3, Triiodothyronine; T4, Thyroxine; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; TG, Triglycerides; T-CHO, Total Cholesterol; IVF, In Vitro Fertilization; Prolactin. - P-value < 0.05 indicates statistical significance. Values ​​are expressed as mean ± SD (range).

[0078] The results of the WB experiment are as follows: Figure 3 As shown, the expression levels of PIAS4 protein in the PCOS group and the control group are as follows: Figure 4 As shown in the figure. The results indicate that, compared with the control group, the expression of PIAS4 protein was downregulated in the PCOS group, verifying that the PIAS4 gene can be used as a biomarker for the diagnosis of PCOS.

[0079] Based on the expression level data of PIAS4 protein, the inventors established a ROC curve for the use of PIAS4 protein in the diagnosis of PCOS. Figure 5 The AUC value was 0.873. These results indicate that PIAS4 protein alone, as a diagnostic biomarker, has high accuracy in the diagnosis of PCOS.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a reagent for detecting PIAS4 in the preparation of products for diagnosing polycystic ovary syndrome, characterized in that, The PIAS4 is the mRNA or protein of the PIAS4 gene.

2. The application according to claim 1, characterized in that, The product includes reagents that diagnose polycystic ovary syndrome by detecting the expression levels of PIAS4 gene mRNA or protein in the subject's body.

3. The application according to claim 1, characterized in that, The mRNA of the PIAS4 gene was downregulated in the oocytes of patients with polycystic ovary syndrome.

4. The application according to claim 1, characterized in that, The protein expression of the PIAS4 gene is downregulated in the peripheral blood of patients with polycystic ovary syndrome.

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