Diagnostic biomarker and application thereof

A technology of biomarkers and therapeutic drugs, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effect of inhibiting proliferation and inhibiting the production of estrogen

Inactive Publication Date: 2021-05-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on circRNA in diseases has achieved diagnostic and therapeutic results in the fields of tumors and cardiovascular diseases. At the same time, some studies have shown that follicular fluid, granulosa cells,

Method used

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  • Diagnostic biomarker and application thereof
  • Diagnostic biomarker and application thereof
  • Diagnostic biomarker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1.1 Collection of samples

[0023] Ovarian granulosa cells (hGC) from 13 patients with polycystic ovary syndrome and 8 normal women were collected from Peking University Third Hospital.

[0024] 1.2 Extraction and reverse transcription of total cellular RNA

[0025] The total RNA of cells was extracted by Trizol method, and the RNA concentration was determined by Nano Drop.

[0026] The reverse transcription system is as follows:

[0027]

[0028] After mixing the above reagents, react at 37°C for 30 minutes, then add 1 μL EDTA and react at 65°C for 10 minutes.

[0029]

[0030] Add the above reagents, 20 μL of the total system, vortex and mix well, and then carry out the reverse transcription step according to the reaction conditions in the table below:

[0031]

[0032] After the reverse transcription is completed, the product can be stored in a -20°C refrigerator for a short time or carried out for Real-Time PCR reaction.

[0033] 1.3 cDNA dilution

[0...

Embodiment 2

[0049] 2.1 Expression of hsa_circRNA_0000284 in granulosa cells of patients with polycystic ovary syndrome

[0050] The relative expression of hsa_circRNA_0000284 in the collected primary human granulosa cells (hGC) was detected by using SYBRGreen Mixture (GenStar Company) as an internal reference for GAPDH, with 2 -△△Ct Calculation of relative expression ( figure 2 ).

[0051] 2.2 Design of hsa_circRNA_0000284-specific siRNA

[0052]Based on the sequence of hsa_circRNA_0000284, the siRNA sequence si-circ-284 spanning the cleavage point of hsa_circRNA_0000284 and the siRNA sequence si-NC (synthesized by Gemma Gene Corporation) as a control were designed. Its sequence is as follows:

[0053] hsa_circ_0000284siRNA sequence:

[0054] F: 5′-GGUACUACAGGUAUGGCCUTT-3′

[0055] R: 5′-AGGCCAUACCUGUAGUACCTT-3′

[0056] si-NC sequence:

[0057] F: 5′-UUCUCCGAACGUGUCACGUTT-3′

[0058] R: 5′-ACGUGACACGUUCGGAGAATT-3′

[0059] 2.3 siRNA transfected cells

[0060] Using lipofectami...

Embodiment 3

[0067] 3.1 Determination of cytokines

[0068] After the transfected cells were cultured for 48 hours, the cell supernatant was collected and sent to Beijing North Institute of Biotechnology to measure the content of estradiol in the cell supernatant. It was found that the low expression of hsa_circ_0000284 inhibited the secretion of estradiol ( Figure 5 ).

[0069] 3.2 CCK-8 detection of cell proliferation

[0070] Prepare 4 pieces of 96-well plates, add 100 μL of cell suspension to each well (each well contains 2000 cells), set 5 duplicate wells for each group of cells, and the cell adhesion rate is about 70%-80% after 18 hours for cell transfection , the cell transfection steps are the same as above. Use the CCK8 proliferation assay to detect the effect of hsa_circ_0000284 on the proliferation of granulosa cells. The specific operation steps are: use the CCK8 reagent produced by Beyontian Biological Company of China, and operate according to the instructions: take out t...

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Abstract

The invention provides a circRNA biomarker for diagnosing a polycystic ovarian syndrome, wherein the circRNA biomarker is hsa-circ-0000284. The expression conditions of the hsa-circ-0000284 in ovarian granular cells of PCOS patients and normal people are detected, and it is found that the expression level of the hsa-circ-0000284 is significantly reduced; by silencing the expression of hsa-circ-0000284 in human ovarian granular cells, the proliferation of the granular cells can be inhibited, and the generation of estrogen is reduced. The research shows that the hsa-circ-0000284 can be used as a biomarker of the polycystic ovarian syndrome, a target is provided for clinical diagnosis and treatment, and a theoretical basis is provided for pharmacy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to hsa_circ_0000284 as a polycystic ovary syndrome diagnostic biomarker and its application. Background technique [0002] Infertility has become the third major disease seriously affecting human health after cancer and cardiovascular disease worldwide. Among them, female infertility accounts for 40%. Ovulation disorders account for 25%-30% of female infertility. Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder in women of childbearing age, 5% to 10% of women of childbearing age are susceptible to this disease, accounting for about 75% of anovulatory infertility patients. [0003] Polycystic ovary syndrome (PCOS) is clinically characterized by oligoovulation or anovulation, hyperandrogen or insulin resistance, and polycystic ovaries. Excessive androgen levels in the body caused by the imbalance of estrogen and androgen ratio are the main...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/178C12Q2600/136C12Q2600/158
Inventor 李向东刘亚静
Owner CHINA AGRI UNIV
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