Application of UTP6 in the preparation of products for diagnosing polycystic ovary syndrome

Abstract

The present invention provides the use of UTP6 in the preparation of a product for diagnosing polycystic ovary syndrome, relating to the field of biotechnology. Compared with healthy subjects, UTP6 is differentially expressed in patients with polycystic ovary syndrome. The expression of UTP6 is related to polycystic ovary syndrome, and the diagnosis of polycystic ovary syndrome can be achieved by detecting the expression level of UTP6. In addition, compared with healthy subjects, PIAS4 is differentially expressed in patients with polycystic ovary syndrome. The expression of PIAS4 is related to polycystic ovary syndrome, and the combined use of UTP6 and PIAS4 for the diagnosis of polycystic ovary syndrome has higher detection accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of UTP6 in the preparation of products for diagnosing polycystic ovary syndrome. Background Art

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

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

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

[0005] The first object of the present invention is to provide the application of UTP6 (U3 small nucleolar RNA-associated protein 14 homolog) in the preparation of products for diagnosing polycystic ovary syndrome, so as to solve at least one of the above problems.

[0006] The second object of the present invention is to provide the application of UTP6 combined with PIAS4 (protein inhibitor of activated STAT 4, Sumo E3 ligase protein inhibitor of activated STAT 4) in the preparation of products for diagnosing polycystic ovary syndrome.

[0007] The third object of the present invention is to provide a reagent for diagnosing polycystic ovary syndrome.

[0008] The fourth object of the present invention is to provide a kit for diagnosing polycystic ovary syndrome.

[0009] In a first aspect, the present invention provides the application of UTP6 in the preparation of products for diagnosing polycystic ovary syndrome, and the UTP6 is the mRNA or protein of the UTP6 gene.

[0010] As a further technical solution, the product includes a reagent or a kit, and polycystic ovary syndrome is diagnosed by detecting the expression level of the mRNA or protein of the UTP6 gene in a subject.

[0011] As a further technical solution, the expression of the mRNA or protein of the UTP6 gene is down-regulated in patients with polycystic ovary syndrome.

[0012] In a second aspect, the present invention provides the use of UTP6 in combination with PIAS4 in the preparation of a product for diagnosing polycystic ovary syndrome, wherein the UTP6 is the mRNA or protein of the UTP6 gene;

[0013] The PIAS4 is the mRNA or protein of the PIAS4 gene.

[0014] As a further technical solution, the product includes a reagent or a kit, and polycystic ovary syndrome is diagnosed by detecting the expression levels of the mRNA or protein of the UTP6 gene and the PIAS4 gene in a subject.

[0015] As a further technical solution, the mRNA or protein of the UTP6 gene is down-regulated in patients with polycystic ovary syndrome;

[0016] The mRNA or protein of the PIAS4 gene is down-regulated in patients with polycystic ovary syndrome.

[0017] In a third aspect, the present invention provides a reagent for diagnosing polycystic ovary syndrome, and the reagent includes primers for detecting the mRNA of the UTP6 gene or biological macromolecules for detecting the protein of the UTP6 gene.

[0018] As a further technical solution, the reagent further includes primers for detecting the mRNA of the PIAS4 gene or biological macromolecules for detecting the protein of the PIAS4 gene.

[0019] As a further technical solution, the biological macromolecules include antibodies or antibody functional fragments.

[0020] In a fourth aspect, the present invention provides a kit for diagnosing polycystic ovary syndrome, and the kit includes the above-mentioned reagent.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] It has been found by the inventors' research that, compared with healthy subjects, UTP6 is differentially expressed in patients with polycystic ovary syndrome, the expression of UTP6 is related to polycystic ovary syndrome, and the diagnosis of polycystic ovary syndrome can be achieved by detecting the expression level of UTP6. In addition, it has been further found by the inventors' research that, compared with healthy subjects, PIAS4 is differentially expressed in patients with polycystic ovary syndrome, the expression of PIAS4 is related to polycystic ovary syndrome, and the combined use of UTP6 and PIAS4 for the diagnosis of polycystic ovary syndrome has higher detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 are the expression levels of the UTP6 gene in the PCOS group and the control group;

[0025] Figure 2 are the expression levels of the PIAS4 gene in the PCOS group and the control group;

[0026] Figure 3 is the ROC curve of the UTP6 gene for diagnosing PCOS;

[0027] Figure 4 is the ROC curve of the PIAS4 gene for diagnosing PCOS;

[0028] Figure 5 is the ROC curve of the combined use of the UTP6 gene and the PIAS4 gene for diagnosing PCOS;

[0029] Figure 6 are the WB experimental results provided in Example 2;

[0030] Figure 7 are the expression levels of the UTP6 protein in the PCOS group and the control group;

[0031] Figure 8 are the expression levels of the PIAS4 protein in the PCOS group and the control group;

[0032] Figure 9 is the ROC curve of the UTP6 protein for diagnosing PCOS. Specific Embodiments

[0033] The following will describe the implementation schemes of the present invention in detail in combination with the embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0034] In a first aspect, the present invention provides the use of UTP6 in the preparation of a product for diagnosing polycystic ovary syndrome, wherein the UTP6 is the mRNA or protein of the UTP6 gene.

[0035] Through the research of the inventor, it is found that, compared with healthy subjects, the mRNA or protein of the UTP6 gene is differentially expressed and down-regulated in patients with polycystic ovary syndrome. The expression of the mRNA or protein of the UTP6 gene is related to polycystic ovary syndrome, and the diagnosis of polycystic ovary syndrome can be achieved by detecting the expression level of the mRNA or protein of the UTP6 gene.

[0036] In some preferred embodiments, the product includes, but is not limited to, a reagent or a kit.

[0037] The reagent or kit diagnoses polycystic ovary syndrome by detecting the expression level of the mRNA or protein of the UTP6 gene in a subject.

[0038] In a second aspect, the present invention provides the use of UTP6 in combination with PIAS4 in the preparation of a product for diagnosing polycystic ovary syndrome, wherein the UTP6 is the mRNA or protein of the UTP6 gene;

[0039] The PIAS4 is the mRNA or protein of the PIAS4 gene.

[0040] Through further research by the inventor, it is found that, compared with healthy subjects, the mRNA or protein of the PIAS4 gene is differentially expressed and down-regulated in patients with polycystic ovary syndrome. The expression of PIAS4 is related to polycystic ovary syndrome, and the combined use of UTP6 and PIAS4 for the diagnosis of polycystic ovary syndrome has higher detection accuracy.

[0041] In some preferred embodiments, the product includes, but is not limited to, a reagent or a kit. Polycystic ovary syndrome is diagnosed by detecting the expression levels of the mRNA or protein of the UTP6 gene and the PIAS4 gene in a subject.

[0042] In a third aspect, the present invention provides a reagent for diagnosing polycystic ovary syndrome, the reagent including primers for detecting the mRNA of the UTP6 gene or biomacromolecules for detecting the protein of the UTP6 gene.

[0043] The reagent provided by the present invention detects the expression level of the mRNA of the UTP6 gene by specifically recognizing the primers of the UTP6 gene mRNA, or detects the expression level of the protein of the UTP6 gene by specifically recognizing the biomacromolecules of the UTP6 gene protein. The reagent provided by the present invention diagnoses polycystic ovary syndrome by detecting the expression level of the mRNA or protein of the UTP6 gene.

[0044] In some preferred embodiments, the reagent further comprises primers for detecting PIAS4 gene mRNA or biological macromolecules for detecting PIAS4 gene protein.

[0045] By jointly detecting UTP6 and PIAS4, the accuracy of the reagent of the present invention for diagnosing polycystic ovary syndrome can be further improved.

[0046] The detection agent for protein can be a reagent well-known to those skilled in the art, such as a biological macromolecule that can undergo an antigen-antibody reaction with the protein and is labeled with a fluorescent label.

[0047] In some preferred embodiments, the biological macromolecule comprises an antibody or an antibody functional fragment.

[0048] In some preferred embodiments, the detection sample of the reagent comprises oocytes or peripheral blood.

[0049] In a fourth aspect, the present invention provides a kit for diagnosing polycystic ovary syndrome, and the kit comprises the above reagent.

[0050] The kit provided by the present invention comprises the above reagent, has all the beneficial effects of the reagent, and can be used for diagnosing polycystic ovary syndrome.

[0051] The present invention will be further illustrated by specific examples and comparative examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0052] Example 1

[0053] 1. Inclusion of clinical specimens

[0054] Inclusion criteria: The specimens were all from the Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences.

[0055] Control group:

[0056] ① Aged 20 - 35 years old;

[0057] ② Without PCOS, not complicated with diseases affecting ovarian function such as ovarian cysts and ovarian function decline;

[0058] ③ Using the long protocol for ovulation induction;

[0059] ④ BMI stable between 18.5 and 26 kg / m 2 2

[0060] PCOS group:

[0061] ① Aged 20 - 35 years old;

[0062] ②Diagnose PCOS according to the Rotterdam criteria;

[0063] ③Use the long protocol for ovulation induction;

[0064] ④BMI is stable between 18.5 and 26 kg / m 2 .

[0065] Exclusion criteria:

[0066] Including infertility due to other factors;

[0067] ①Hyperprolactinemia, abnormal thyroid function;

[0068] ②Type 2 diabetes;

[0069] ③Serum FSH > 10 mIU / mL;

[0070] ④Endometriosis.

[0071] PCOS patients were treated with ovulation induction according to the long protocol. The patients were down-regulated with GnRH agonist, and then FSH or HMG was given for ovarian stimulation. When the diameter of three follicles reached 18 mm, hCG was given to promote follicle maturation, and eggs were retrieved 36 hours later. The control group used the long protocol for ovulation induction and obtained oocytes. The summary of egg source information for the PCOS group and the control group is shown in Table 1.

[0072] Table 1 Summary of human egg source information

[0073]

[0074]

[0075] - Abbreviations: BMI, body mass index; D2, day 2; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; Testosterone, testosterone; TSH, thyroid-stimulating hormone; T3, triiodothyronine; T4, thyroxine; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; T-CHO, total cholesterol; IVF, in vitro fertilization; Prolactin, prolactin. - A P-value of < 0.05 represents a significant statistical difference. Values are expressed as mean ± SD (range).

[0076] 2. Single-cell sequencing

[0077] The obtained oocytes were measured using Illumina HiSeq 4000 to obtain scRNA-seq data.

[0078] Single-cell transcriptome scRNA-seq library preparation includes:

[0079] 1) Preparation of cell samples: 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. Operate on ice.

[0080] 2) First-strand cDNA synthesis: Prepare Lysis buffer, add it to the EP tube containing a single cell, add Oligo(dT)VN primer and dNTP Mix, gently mix, and react in a PCR instrument. Reaction conditions: Extend at 72°C for 3 min; immediately place on ice for 2 min. Prepare the reverse transcription reaction system, perform at 42°C for 90 min; 70°C for 15 min; cool at 4°C.

[0081] 3) Full-length cDNA amplification and purification: Prepare the PCR amplification system, mix well, and react in a PCR instrument. Reaction conditions: Denature at 98°C for 10 s; anneal at 65°C for 15 s; extend at 72°C for 6 min (16 cycles); extend at 72°C for 5 min; cool at 4°C. Purify by magnetic bead sorting, and use an Agilent 2100 Bioanalyzer for quality control and identification of the product.

[0082] 4) Library construction and loading: Fragment the above product, then operate according to the DNA library preparation kit and load it onto the Illumina Hiseq platform for sequencing in the PE150 mode.

[0083] 3. Results

[0084] The expression levels of the UTP6 gene in the PCOS group and the control group are as Figure 1 shown, and the expression levels of the PIAS4 gene in the PCOS group and the control group are as Figure 2 shown. The results show that compared with the control group, the differential expression of the UTP6 gene and the PIAS4 gene is down-regulated in the PCOS group, and the UTP6 gene or the PIAS4 gene has the potential to be used as a diagnostic marker for PCOS diagnosis.

[0085] The inventor established an ROC curve for the UTP6 gene to diagnose PCOS based on the sequencing data of the UTP6 gene ( Figure 3 ), with an AUC value of 0.769; based on the sequencing data of the PIAS4 gene, an ROC curve for the PIAS4 gene to diagnose PCOS was established ( Figure 4 ), with an AUC value of 0.778; the ROC curve for the combined use of the UTP6 gene and the PIAS4 gene to diagnose PCOS is as Figure 5As shown, the AUC value is 0.815. The results indicate that using the UTP6 gene alone as a diagnostic marker for PCOS diagnosis has a relatively high accuracy. Using the UTP6 gene in combination with the PIAS4 gene for PCOS diagnosis has a higher accuracy compared to using a single marker.

[0086] Example 2

[0087] The inventors extracted total protein from the peripheral blood of PCOS patients and healthy subjects to verify the above conclusion. Blood was collected in anticoagulant tubes. For every 300 μl of protein extraction solution, 2 μl of protease inhibitor mixture and 2 μl of protein stabilizer were added, and the operation was carried out on ice. 300 μl of whole blood sample was taken, 300 μl of cooled protein extraction solution was added, mixed well, shaken at 4°C for 10 minutes, centrifuged at 12,000 g for 10 minutes, and the supernatant was collected, which is the total protein of whole blood.

[0088] 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.

[0089] Table 2 Clinical information of PCOS group and control group

[0090]

[0091] - Abbreviations: BMI, body mass index; D2, day 2; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; Testosterone, testosterone; TSH, thyroid-stimulating hormone; T3, triiodothyronine; T4, thyroxine; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; T-CHO, total cholesterol; IVF, in vitro fertilization; Prolactin, prolactin. - A P-value of <0.05 represents a significant statistical difference. The values are expressed as mean ± SD (range).

[0092] The results of the WB experiment are as Figure 6 shown. The expression levels of UTP6 protein in the PCOS group and the control group are as Figure 7 shown. The expression levels of PIAS4 protein in the PCOS group and the control group are as Figure 8 shown. The results indicate that compared with the control group, the differential expression of UTP6 protein and PIAS4 protein is down-regulated in the PCOS group, verifying that the UTP6 gene or the PIAS4 gene can be used as a marker for PCOS diagnosis.

[0093] Based on the expression level data of UTP6 protein, the inventors established an ROC curve for using UTP6 protein to diagnose PCOS ( Figure 9) The AUC value was 0.910. The results showed that UTP6 protein alone as a diagnostic marker for PCOS diagnosis had high accuracy.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting UTP6 in the preparation of a product for diagnosing polycystic ovary syndrome, characterized in that, The UTP6 is the mRNA or protein of the UTP6 gene.

2. The application according to claim 1, characterized in that, The product includes a kit for diagnosing polycystic ovary syndrome by detecting the expression level of the mRNA or protein of the UTP6 gene in a subject.

3. The application according to claim 1, wherein The mRNA or protein of the UTP6 gene is down-regulated in patients with polycystic ovary syndrome.

4. Use of a reagent for detecting UTP6 in combination with a reagent for detecting PIAS4 in the preparation of a product for diagnosing polycystic ovary syndrome, characterized in that, The UTP6 is the mRNA or protein of the UTP6 gene; The PIAS4 is the mRNA or protein of the PIAS4 gene.

5. The application according to claim 4, wherein The product includes a kit for diagnosing polycystic ovary syndrome by detecting the expression levels of the mRNA or proteins of the UTP6 gene and the PIAS4 gene in a subject.

6. The application according to claim 4, wherein The mRNA or protein of the UTP6 gene is down-regulated in patients with polycystic ovary syndrome; The mRNA or protein of the PIAS4 gene is down-regulated in patients with polycystic ovary syndrome.

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