CN112626220A - Biomarker and application thereof - Google Patents
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- CN112626220A CN112626220A CN202110064422.5A CN202110064422A CN112626220A CN 112626220 A CN112626220 A CN 112626220A CN 202110064422 A CN202110064422 A CN 202110064422A CN 112626220 A CN112626220 A CN 112626220A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention provides a miRNA biomarker for diagnosis of polycystic ovarian syndrome, wherein the miRNA biomarker is miR-96-5 p. The invention detects the expression of miR-96-5p in PCOS patients and normal human granulosa cells, and finds that the miR-96-5p level in the PCOS patients is obviously reduced. The miR-96-5p is over-expressed in the granular cells to promote the proliferation of the granular cells and promote the production of estrogen. The over-expression of miR-96-5p in the theca of the follicle inhibits the production of testosterone. The research shows that miR-96-5p can be used as a biomarker of polycystic ovarian syndrome and provides a theoretical basis for clinical pharmacy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a miRNA biomarker for diagnosis of polycystic ovarian syndrome and application thereof.
Background
Infertility has become the third largest disease in the world that seriously affects human health following cancer, cardiovascular diseases. Wherein female infertility accounts for 40%. In female infertility, the rate of ovulation failure accounts for 25% -30%. Polycystic ovarian syndrome (PCOS) is the most common disorder of reproductive endocrine disorders in women of reproductive age, and 5-10% of women of reproductive age are susceptible to the disorder, accounting for about 75% of anovulatory infertility patients.
Polycystic ovarian syndrome (PCOS) is a syndrome of endocrine disorders characterized by infrequent or no ovulation, high androgen or insulin resistance, and polycystic ovary. Excessive androgen levels in vivo due to dysregulation of the estrogen/androgen ratio are a major cause of PCOS. miRNA is small non-coding RNA with the endogenous length of about 21-25nt, and plays an important role in regulating and controlling the degradation and inhibition of target gene mRNA. mirnas, as biomarkers of potential tissue-specific diseases, can provide effective measures for many pathological diagnoses. The studies of mirnas in PCOS are currently mainly focused on insulin resistance, obesity, etc., and the pathogenic mechanism of this key factor, hormonal imbalance, is still unclear.
Disclosure of Invention
The invention aims to provide miRNA biomarkers for diagnosis of polycystic ovarian syndrome and application thereof.
The miRNA biomarker for diagnosis of polycystic ovarian syndrome provided by the invention is miR-96-5 p. The mature sequence is as follows: UUUGGCACUAGCACAUUUUUGCU are provided.
The invention also provides application of the miR-96-5p in preparation or screening of a diagnosis and treatment medicament for the human polycystic ovary syndrome.
The invention further provides a kit for diagnosing polycystic ovarian syndrome, which comprises one or more primers for specifically detecting the expression level of miR-96-5 p.
The primer is designed based on the real-time quantitative PCR method for detecting miR-96-5p, and the primer sequence is as follows:
miR-96-5p-RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCAAAAA
miR-96-5p-F:ACACTCCAGCTGGGTTTTGGCACTAGCACATT
miR-96-5p-R:TGGTGTCGTGGAGTCG。
mirnas are widely expressed in female reproductive tract ovaries, uterus, endometrium, oviducts, and during embryonic development, and by specifically binding to their target genes, cause degradation of the target mRNA or inhibit its translation. The research on miRNA perfects and enriches the molecular biology and genetics mechanism of disease occurrence and development. Research shows that the overexpression of miR-96-5p promotes granulosa cells to generate estrogen and inhibits the production of testosterone by theca cells. The low expression of miR-96-5p in granulosa cells causes the dyssecretion of estrogen and androgen in follicular membranes of the granulosa cells, and causes polycystic ovary syndrome.
The invention detects the expression of miR-96-5p in granulosa cells of PCOS patients and normal people, finds that the level of miR-96-5p is obviously reduced, can use miR-96-5p as a biomarker of polycystic ovarian syndrome, and provides a theoretical basis for clinical pharmacy.
Drawings
FIG. 1 is a graph showing the detection of the expression level of miR-96-5p in granulosa cells of patients with polycystic ovarian syndrome and normal humans;
FIG. 2 shows that the designed micic sequence of miR-96-5p can effectively improve the expression level of miR-96-5p in vitro;
FIG. 3 shows that the overexpression of miR-96-5p promotes the increase of estrogen secretion of human primary granulosa cells (hGC);
FIG. 4 is a graph showing that miR-96-5p overexpression promotes estrogen production in mouse primary granulosa cells (mGC);
FIG. 5 shows that testosterone secretion of theca cell is inhibited after miR-96-5p is over-expressed.
Detailed Description
The following examples are given to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1
1.1 Collection of samples
Ovarian granulosa cells (hGC) and 20 normal female ovarian granulosa cells (hGC) were collected from 26 patients with polycystic ovarian syndrome at third hospital, beijing university.
1.2 isolation of mouse Primary granulosa cells and blastocyst cells
Taking 10-15g immature female mice, injecting 5-10IU of PMSG subcutaneously firstly, and injecting 5-10IU of HCG after 40-48 h; killing the mouse by breaking the neck after 15-20h, fixing the mouse, sterilizing with 75% alcohol, quickly taking off the ovary in an aseptic manner, placing the ovary in preheated PBS, quickly removing attachments such as envelopes and tissues around the ovary under a body microscope, and transferring the stripped ovary into a new PBS buffer solution to wash for 2 times; transferring the ovary into a mixed basal culture medium, and incubating for 10-15min at 37 ℃; puncturing the follicle with a 1mL syringe needle under a dissecting mirror, releasing granular cells and oocytes, and collecting the cells into a 15mL centrifuge tube; adding collagenase type I and hyaluronidase mixed enzyme for digestion for 3-5min, filtering with 200 mesh sieve, collecting filtrate, and centrifuging at 1000rpm for 5 min; the cell pellet was resuspended in DMEM/F12 medium containing 10% fetal bovine serum and seeded into a cell flask for culture.
Washing the residual ovarian tissue PBS without the granular cells twice, shearing, adding McCoy's5a culture solution containing collagenase IV (4g/L collagenase is dissolved in McCoy's5a culture solution) in a shaker at 37 ℃ to digest and separate the mesenchymal cells of the theca membranes, digesting for 1h, filtering by a 200-mesh screen, collecting filtrate, and centrifuging for 5min at 1000 rpm; resuspending the cell pellet in McCoy's5a medium containing 10% fetal calf serum, inoculating into a cell flask, and culturing
1.3 extraction and reverse transcription of Total RNA from cells
Extracting total RNA of the cells by using a Trizol method, determining the RNA concentration by using Nano Drop, and storing the extracted RNA in a refrigerator at the temperature of-80 ℃ for later use.
Reverse transcription system: mu.L of 5 × reaction buffer, 1. mu.L of dNTP (10mM), 1. mu.L of specific stem-loop structure reverse transcription primer (5. mu.M), 0.5. mu. L M-MLV reverse transcriptase, 1. mu.gRNA, 0.5. mu.L of RNase inhibitor, DEPC water make-up to 20. mu.L were added to a 0.2mL PCR tube without RNase in this order. Reaction conditions are as follows: 30min at 16 ℃, 30min at 42 ℃ and 5min at 85 ℃. In the invention, a specific stem-loop structure reverse transcription primer is designed for miRNA, and cDNA is synthesized by a single tube by the operation.
The stem-loop refers to a reverse transcription primer of a stem-loop structure used in a reverse transcription process, and consists of the following nucleotide sequences:
miR-96-5p-RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCAAAAA
after the reverse transcription is finished, the product can be stored in a refrigerator at the temperature of-20 ℃ for a short Time or subjected to Real-Time PCR reaction.
1.4 cDNA dilution
After reverse transcription, 80. mu.L of RNase free H2O was added to each well for dilution.
1.5 fluorescent quantitative PCR
Take 2. mu.L of diluted cDNA product, 10. mu.L of SYBR Green I Master (2X), 0.4. mu.L of forward primer (10. mu.M) and 0.4. mu.L of reverse primer (10. mu.M), ddH2O to make up 20. mu.L.Real-time fluorescent quantitative PCR reactions were performed on an Agilent StrataGene Mx3005P instrument with U6 as internal reference. The reaction conditions were 95 ℃ for 10min, then 95 ℃ for 15s, 60 ℃ for 30s, 45 cycles. After the reaction is finished, the CT value in each reaction tube is calculated by matched computer software. By 2-ΔΔCTCalculating the relative expression quantity of miRNA.
The fluorescent quantitative PCR primer sequence is as follows:
miR-96-5p：
F:ACACTCCAGCTGGGTTTTGGCACTAGCACATT
R:TGGTGTCGTGGAGTCG
U6：
F:CTCGCTTCGGCAGCACA
R:AACGCTTCACGAATTTGCGT
example 2
2.1 expression of miR-96-5p in granulosa cells of patients with polycystic ovarian syndrome
Relative expression of miR-96-5p in collected primary human granulosa cells (hGC) was examined for U6 reference using SYBR Green mix (GenStar Inc.) at 2 (FIG. 1)-ΔΔCTCalculating the relative expression quantity of miRNA.
2.2 design of miR-96-5p specific mimic
Based on the sequence of the miR-96-5p, miR-96-5p imic, UUCACAGUGGCUAAGUUCCGC and miR-NC are designed, and the level of miR-96-5p expressed in-vitro cells is detected (figure 2).
miR-NC mimic:
sense:UUCUCCGAACGUGUCACGUTT
antisense:ACGUGACACGUUCGGAGAATT
2.3 siRNA transfected cells
Lipofectamine manufactured by Invitrogen corporation was usedTM3000, operate as described (taking 12-well plates, A, B in total, one well for each group as an example, 3 multiple wells for each treatment group at the time of actual transfection):
cells (1X 10) were seeded 24h before transfection5One/hole), the cell adherence rate is about 70-80% after 24h when the cells are cultured in a serum-containing culture medium; transfection was performed in the following A, B two groups, group C being diluted transfection reagent.
A: 40pmol miR-NC micic +75 mu L serum-free DMEM/F12 culture medium
B: 40pmol miR-96-5p micic +75 mu L serum-free DMEM/F12 culture medium
C：3μL LipofectamineTM3000+75 mu L serum-free DMEM/F12 medium
Mixing the above two premixed solutions, blowing, stirring, and standing at room temperature for 5 min. And (3) mixing the A, B two groups of premixed solutions with the C group of premixed solution in equal volume respectively to obtain about 150 mu L of transfection reagent (small RNA mixed solution), blowing and uniformly mixing, incubating at room temperature for 5min, adding 150 mu L of the prepared transfection reagent into each hole, incubating in an incubator at 37 ℃ for 6h, then changing a normal 10% FBS DMEM/F12 culture medium to continue culturing for 18-48h, and observing the result.
EXAMPLE 3 cytokine assay
In vitro culturing primary human granular cells hGC, primary mouse granular cells mGC and primary mouse theca cells mTC, dividing the cells into an experimental group and a control group, respectively transfecting miR-96-5p imic and miR-NC, after culturing the transfected cells for 48 hours, collecting cell supernatants, sending the cell supernatants to Beijing northern biotechnology research institute, and measuring the contents of estradiol and testosterone in the cell supernatants.
By measurement, miR-96-5p overexpression promotes estradiol secretion (FIGS. 3 and 4).
The influence of miR-96-5p on the secretion of testosterone in theca cells is determined by the method. By measurement, miR-96-5p overexpression inhibits testosterone secretion (figure 5).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (6)
1. The miRNA biomarker for diagnosis of polycystic ovarian syndrome is miR-96-5 p.
2. The use of the miRNA biomarker of claim 1 in the preparation or screening of medicaments for diagnosis and treatment of human polycystic ovarian syndrome.
3. Use of the miRNA biomarker of claim 1 in preparation of a human polycystic ovary syndrome detection reagent.
4. The use according to claim 3, wherein the detection reagent is selected from reagents related to the detection of the biomarker miR-96-5p by southern blotting, northern blotting, PCR, reverse transcriptase PCR, real-time quantitative PCR, nano-array, macro-array, autoradiography or in situ hybridization.
5. The kit for diagnosing the polycystic ovarian syndrome is characterized by comprising one or more primers for specifically detecting the expression level of miR-96-5 p.
6. The kit according to claim 5, wherein the kit comprises primers for detecting miR-96-5p by using a real-time quantitative PCR method, and the primer sequences are as follows:
miR-96-5p-RT:5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCAAAAA-3′
miR-96-5p-F:5′-ACACTCCAGCTGGGTTTTGGCACTAGCACATT-3′
miR-96-5p-R:5′-TGGTGTCGTGGAGTCG-3′。
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| CN113817845A (en) * | 2021-10-27 | 2021-12-21 | 金陵科技学院 | miRNA (micro ribonucleic acid) molecule related to porcine luminal ovarian follicle atresia and application thereof |
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