CN115011596A

CN115011596A - Circular circRNA _ BECN1 gene and application thereof

Info

Publication number: CN115011596A
Application number: CN202210088181.2A
Authority: CN
Inventors: 王丽萍; 崔恒宓; 周罗晶; 孔祥; 张晓梅; 蒋敏; 高玲玲; 周东杰
Original assignee: Northern Jiangsu Peoples Hospital
Current assignee: Northern Jiangsu Peoples Hospital
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-09-06

Abstract

The invention relates to a circular circRNA _ BECN1 gene, belonging to the field of molecular biology. The nucleotide sequence of the circular circRNA _ BECN1 gene is shown as SEQ ID NO:1, the research of the inventor finds that the circular circRNA _ BECN1 is highly expressed in the ovarian follicle of a patient with polycystic ovarian syndrome, and the gene and the expression product thereof can be used as a marker for early diagnosis of the polycystic ovarian syndrome. The invention also provides an expression detection kit for detecting the expression rule of circRNA _ BECN1, provides a research method and a foundation for detecting the expression rule of circRNA _ BECN1, and the kit can be used for detecting the developmental maturation state of oocytes of patients with polycystic ovarian syndrome.

Description

Circular circRNA _ BECN1 gene and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a circular circRNA _ BECN1 gene and application thereof.

Background

Polycystic Ovarian Syndrome (PCOS) is a common reproductive endocrine disease of women in the reproductive age, accounts for 70-80% of patients with ovulation failure, and plays an important role in infertility etiology. PCOS clinical manifestations are dominated by reproductive dysfunction and metabolic abnormalities, increasing the lifelong risk of developing type 2 diabetes, insulin resistance, oxidative stress and cardiovascular disease. The pathological change process of PCOS mainly comprises the following steps: abnormal antral follicular development, granulosa cell hyperproliferation and apoptosis, androgen increase, and oocyte dysgenesis. The cause of the disease of PCOS has not been clarified so far, and the present study is considered to be the result of the combined action of genetic factors and environmental factors. Although the clinical diagnosis has the criterion of Rotterdam, the clinical manifestations of patients have high heterogeneity due to different regions and ethnicities, so that the application of the criterion has certain limitations. Early diagnosis and early treatment have positive significance for PCOS patients. Therefore, screening and identifying specific markers associated with the onset of PCOS is an important scientific problem in the study of PCOS oocyte developmental disorders.

circRNA is a noncoding RNA molecule that differs from traditional linear RNA, does not have a 5 'terminal cap and a 3' terminal poly (a) tail, and forms a circular structure with covalent bonds, has tissue expression specificity, is relatively conserved in biological evolution, while the covalently closed circular structure enhances the stability of circRNA from degradation by exonucleases. It has been found that circRNA is very abundant in mammals and can exert important regulatory effects on gene expression at the transcriptional or post-transcriptional level. Until recently, some circrnas have been shown to be involved not only in human premature ovarian failure regulation, but also in developmental maturation disorders of oocytes.

Disclosure of Invention

The invention aims to provide a circular circRNA _ BECN1 gene, and the gene and an expression product thereof are used as markers for predicting oocyte maturation development, so that a new idea is provided for early diagnosis of a PCOS oocyte maturation barrier patient.

The inventor identifies a circRNA _2003_ BECN1 in human genome by high-throughput sequencing, and the parent gene is named as circRNA _ BECN 1. Research shows that circRNA _ BECN1 can cause oocyte developmental disorder, so that it is important to further understand the expression rule of circRNA _ BECN1 in PCOS oocytes.

A circular circRNA _ BECN1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The application of the circular circRNA _ BECN1 gene in preparing a reagent for diagnosing PCOS oocyte developmental maturation. The inventor finds that the circular circRNA _ BECN1 is highly expressed in the ovarian follicle of the PCOS patient by detecting the expression level of the circular circRNA _ BECN1 in the ovarian follicle of the PCOS patient, so the circular circRNA _ BECN1 and the expression product thereof can be used as a marker for diagnosing the early developmental maturity of the PCOS oocyte.

A kit for detecting the circular circRNA _ BECN1 gene comprises a chain specificity RT primer and a fluorescent quantitative PCR detection primer; the sequence of the chain specificity RT primer is shown as SEQ ID NO.2 and SEQ ID NO. 3; the sequence of the fluorescent quantitative PCR detection primer is shown as SEQ ID NO.4 and SEQ ID NO. 5.

circRNA_BECN1-RT1(SEQ ID NO.2)：5’-GATGGACAGACCGTTGAG-3’

circRNA_BECN1-RT2(SEQ ID NO.3)：5’-CCTCATCCGTCTCGCTTA-3’

circRNA_BECN1-F2(SEQ ID NO.4)：5’-GACTACTGCCATAACTAAGG-3’

circRNA_BECN1-R2(SEQ ID NO.5)：5’-CAGAAGTCACAGCCAGAT-3’。

According to the invention, the research discovers that the circular circRNA _ BECN1 is highly expressed in the follicle of the PCOS patient, the circular circRNA _ BECN1 and the expression product thereof can be used as a marker for early diagnosis of PCOS oocyte developmental maturation, the invention also provides an expression detection kit for detecting the expression rule of the circular circRNA _ BECN1, provides a research method and a basis for detecting the expression rule of the circular RNA _ BECN1, and the kit can be used for detecting the developmental maturation state of the oocyte of the patient with polycystic ovarian syndrome.

Drawings

FIG. 1 shows the results of the expression level test of circRNA _ BECN1 in the PCOS/non-PCOS group;

FIG. 2 is an electrophoretogram after digestion of linear RNA with circRNA _ BECN1 RNaseR;

FIG. 3 is the results of Sanger sequencing of circRNA _ BECN 1;

FIG. 4 shows the results of the circRNAFISH identification of the location of circ _ BECN1 in GV stage oocytes;

FIG. 5 shows the results of the measurement of the expression level of circRNA _ BECN1 at different stages of oocyte maturation;

FIG. 6 shows the results of a test for the effect of interfering with circRNA _ BECN1 on oocyte maturation.

Detailed Description

The present invention will be further described with reference to the following drawings and specific examples, which should not be construed as limiting the invention thereto. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

During the research of follicles of patients suffering from infertility caused by polycystic ovarian syndrome, the inventor finds a circular RNA, the sequence of which is as follows:

aggcggaggttgcagtaagccgagatcatgccattgcacttcagcctgggcaacaagagcgaaactccatttcaaaaaaa aaaaaaacccacaaaaagaaaagaaaaaaaaataaaaatatgccaggcacagtggctcacgtctgtaatcccagcacttt gggaggccaaggcgggtggatcatgaggtcaggagatcaagaccatcatggataacatggtgaaaccccgtcactacca aaaatacaaaaaattagccaggcattgtggtgggcgcctgtaatcccagctactcgggaggagaggaggctgaggcagg agaactgcgtgaacctgggaggcagagcctgcaatgagccgagatcacgccactgcactccagcctgggtgacagagt gagactccgtctcaaaataaataaataaataaaaataataaaaaatgctacaaaacctgaaactttttgaccattgatatgatgc cacaaggggaaaattccatacctgatacctttgcttttttttttttttttttttttttttgagacagaggcttgctttgtcgcccaggctg gagtgcagtggcgcaatctcggcttactgtaagctctgcctcccaggttcacgccattctcctgccactgcctcccgagtag ctgggaccacaggcacctgccaccacgcccagctgattttttttttttttttgtatttttagtagagacggggtttcactgtgttag ccaggatggtctcaatctcctgaccttgtgatccacccgccttggcctcccaaagtgctgggattacaggcgtgagccactg cacccggccctttgctttctgatgattcaatgtatacaagctttgtttcatgcacaaaattatttaaaatattgtatgaaattacctc agactacatgtatcaggtatatataaaacaggccaggcacggtggctcatgcctgtaatcctagaactttgggaggctgagg cgggtggatcacctgaggtcaggagttcgagaccagcctggtccaacatggtaaaaccccatctctactaaaaatacaaaa attagctgggcctggtggtgggcacctgtaatcccagctactccagaggctgaggcaggagactcacttgaacctgggag gcggaggttgcagtgagccgagatcgtgccactgcactccagtctgggcaacagagcaagactctgtctcagaagaaga aacataaatgaattttgtgtttagacttgggtcccattcccaagatatcttagtatctatatgccaatatcccaaaatctgaaaaaa aaaatccaaaatctaaaacacttctggtgatactcaacctgtaaacatctatctcacggtgattcttttcctagcctagtgcacac accctcttctgacctgcccatatcccttctcaacagactgtcaaaacctgatttctgtctacaagacccccaaaactaaacctg aactagacaagatattgaacaatatactggctcttcatatctaggtctaaaaaataaattgaatgccaatatcaaagcaaacca tccattttcttaagagatataagaactctgtattccagtgaaaatggagcttcacagagtaccttagatttgtctgtcagagactc cagatatgaatggtttccgtaaggaacaagtcggtatctaaaatagagatacaaacagagatggatacaggactcctcccaa agtgattattgggaagttacaacccaatgatttcactcttcattctggccaagggcttcttttattatgctcaactccctctcccac ctccccattgaatccatgtttaaaagattttgtttaatgaacaaactacatttattatataattgcctgggggccttccatgaacaa atgttatgttttggtttaaatttaagaaacaaaaactgaaaatatcctgaggaacactaaggctctggggactgtaggctggga actatgctatagtcacgcaactcagtgactgtgtgatcagtggagagagccctgtgatgaggggaaaatcagatgctgaca gctcttcagccacagaaaactcccagtctgtgggcagcaagggctcctgacatggtggacagcagtaccaggttggagat acaaattatatcaatgcacatcactccccaagaaagggctacacttcctac (SEQ ID NO: 1), which was named circRNA _ BECN 1.

Example 2

Material and treatment:

1) collection of follicular fluid specimens

Samples are collected and treated by an assisted reproduction technology in the reproductive medicine center of Subei people hospital in Yangzhou, 7-9 months in 2017, after ovulation promotion, vaginal ultrasound-guided puncture aspiration ovum taking patients are performed according to a scheme, all test objects adopt the same pituitary depression length-regulating scheme to promote ovulation, and the test objects are divided into a PCOS group (25 cases) and a non-PCOS group (25 cases). Puncturing to take out ovum under ultrasonic guidance for 36h after hCG injection, collecting oocyte under a microscope for fertilization, collecting residual follicular fluid, standing at room temperature for 30min, storing at low temperature of 4 ℃, centrifuging for 10min at 3000min, and taking supernatant to store in a refrigerator at-80 ℃ for later use. Patients were informed of the study, signed an informed consent, and submitted for approval by the hospital ethics committee.

2) Preparation and evaluation of samples

2ml of follicular fluid were taken per group and the cells were pipetted several times into the lysate using 1ml of TRIZOL reagent without washing the cells prior to the TRIZOL reagent addition to avoid increasing the likelihood of mRNA degradation. Adding 0.2mL of chloroform into each 1mL of sample homogenized by the TRIzol reagent, and tightly covering a tube cover; manually and violently oscillating the tube body for 15s, and incubating for 2-3min at 15-30 ℃; centrifugation at 12,000 Xg for 15mim at 4 ℃; after centrifugation, the mixed liquid is divided into a lower red phenol chloroform phase and an upper colorless water phase; the RNA was totally partitioned in the aqueous phase; the volume of the aqueous phase was about 60% of the TRIzol reagent added during homogenization. Transferring the aqueous phase to a new centrifuge tube; mixing the aqueous phase with isopropanol to precipitate the RNA therein; mixing, incubating at 15-30 deg.C for 10min, and centrifuging at 4 deg.C for 10min at 12,000 Xg; RNA precipitation will form a gel-like pellet on the bottom and side walls of the tube. Removing the supernatant, adding 1mL of at least 75% ethanol per 1mL of the homogenized sample of LTRIzol reagent for washing RNA precipitate; after shaking, the cells were centrifuged at 7500 Xg for 5min at 4 ℃. Finally, the RNA pellet was not completely dried in air for 5-10min in order not to decrease the solubility of RNA. When dissolving RNA, adding RNase-free water, repeatedly blowing with a gun for several times, and incubating at 55-60 deg.C for 10 min. Following assessment of RNA quantity and quality using NanoDrop, absorbance measurements at 260 and 280mn required a A260/A280 ratio of approximately 2.0 (both 1.8-2.1) and an A260/A230 ratio of greater than 1.8. RNA integrity and purity were assessed by standard denaturing gel electrophoresis.

3) Library construction and quality control

After the total RNA extracted from the sample is qualified through quality inspection, the rRNA truncated RNA is purified by an epicentre Ribo-zeroTM kit of ILLumina company, then is randomly broken into short fragments, the fragmented rRNA truncated RNA is used as a template to obtain cDNA, finally, the cDNA is obtained through PCR amplification, a sequencing library is obtained, then ILLumina Hiseq4000 is used for sequencing, and the sequencing read length is 2 × 150bp with two ends. Qubit2.0 is used for preliminary quantification, the library is diluted to 1ng/ul, then an inserted fragment of a sequencing library is detected by an Agilent 2100 Bioanalyzer, and after the inserted fragment is consistent with the expected value, the effective concentration of the library is accurately quantified by a qPCR method (the effective concentration of the library is more than 2 nmol/L.

Example 3

In the early stage of the research, the follicle of a female with PCOS/non-PCOS reproductive age is subjected to circRNA high-throughput sequencing analysis, and screening and classification are carried out according to the length, the number and the exon number of the circRNA. As a result, it was found that circRNA is abundantly present in all chromosomes except the Y chromosome. Wherein, 412 different circular circRNAs are detected in two groups, wherein 167 circRNAs are up-regulated and 245 circRNAs are down-regulated, and data support is provided for the next experiment.

According to the circRNA expression difference multiple and the p-value, qRT-PCR verification is carried out on 10 different circRNAs obtained by sequencing by subsequently expanding clinical samples, the dissolution curves are all unimodal, the trend of 90% of circRNAs is consistent with high-throughput sequencing, and the reliability of the sequencing result is verified. Wherein the expression of circRNA BECN1 in PCOS follicles was significantly higher than in the non-PCOS group, it was speculated that circRNA BECN1 might be involved in the pathogenesis of polycystic ovarian syndrome.

And (3) performing real-time fluorescent quantitative PCR detection on circRNA _ BECN 1:

1) primer design

The invention screens circRNA _ BECN1 for fluorescent quantitative verification according to high-throughput sequencing results. By 2 ^-ΔΔ And calculating the difference multiple by a Ct method, and detecting by RT-qPCR. The PCR primer sequences were as follows:

circRNA _ BECN1 primer 1:

F：5’-AGAGAGCCCTGTGATGAG-3’(SEQ ID NO.6)

R：5’-CGCCTGTAGGAAGTGTAG-3’(SEQ ID NO.7)

circRNA _ BECN1 primer 2:

F：5’-GGAACACTAAGGCTCTGG-3’(SEQ ID NO.8)

R：5’-ATGATCTCGGCTTACTGC-3’(SEQ ID NO.9)

GAPDH primer:

F：5’-ACCCACTCCTCCACCTTTGAC-3’(SEQ ID NO.10)

R：5’-TGTTGCTGTAGCCAAATTCGTT-3’(SEQ ID NO.11)。

using PrimeScript ^TM The reagent provided in RT reagent kit with gDNA Eraser kit (Nanjing Novovisan Biotech Co., Ltd.) removes the genome. The reaction system comprises: mu.g of the total RNA obtained in step (2) of example 2 as a template, 2. mu.L of 5 XgDNA Eraser Buffer, 1. mu.L of gDNA Eraser, RNase Free dH ₂ The content of O was adjusted to 10. mu.L. The reaction conditions are as follows: 2min at 42 ℃; maintaining the temperature at 4 ℃.

Using PrimeScript ^TM The reagent provided in the RT reagent kit with gDNA Eraser kit carries out reverse transcription reaction to carry out reverse transcription of total RNA into cDNA products. Wherein the nucleotide sequences of the primers circRNA _ BECN1-RT1 and circRNA _ BECN1-RT2 are as follows:

circRNA_BECN1-RT1(SEQ ID NO.2)：5’-GATGGACAGACCGTTGAG-3’

circRNA_BECN1-RT2(SEQ ID NO.3)：5’-CCTCATCCGTCTCGCTTA-3’

the reaction system comprises: example 2 mu.L of the reaction product obtained in step (2) was used as a template, 5. mu.L of primeScript RT 1-RT1 and circRNA _ BECN1-RT2 were used as RT Mix primers, 1. mu.L of primeScript RT Enzyme Mix I, 4. mu.L of 5 XprimeScript Buffer 2 and 4. mu.L of RNase Free dH 2O. The reaction conditions are as follows: 15min at 42 ℃; 5s at 85 ℃; maintaining at 4 ℃.

2) Validation by RT-qPCR

Use of

Premix Ex Taq ^TM The reagents provided in the kit (Nanjing Novodka Biotechnology Co., Ltd.) were used for the fluorescent quantitative PCR reaction. Wherein, the nucleotide sequences of the used specific primers circRNA _ BECN1-F2 and circRNA _ BECN1-R2 are as follows:

circRNA_BECN1-F2(SEQ ID NO.4)：5’-GACTACTGCCATAACTAAGG-3’

circRNA_BECN1-R2(SEQ ID NO.5)：5’-CAGAAGTCACAGCCAGAT-3’；

the reaction system comprises: mu.L of the cDNA product obtained in step (3) of example 2 as a template, 0.4. mu.L (10. mu.M) of the forward primer circRNA _ BECN1-F2 and 0.4. mu.L (10. mu.M) of the circRNA _ BECN1-R2 as amplification primers, respectively, 10. mu.L

Premix Ex Taq ^TM (2X) and 8.2. mu. LddH ₂ And O. The reaction conditions are as follows: 94 ℃, 5min, 94 ℃, 30s, 52 ℃, 30s, 72 ℃, 1min, 72 ℃, 7min, 4 ℃ and infinity; 40 PCR cycles. The housekeeping gene GAPDH (the expression quantity is basically constant among different samples) is used as an internal reference, the value of the gene to be detected of the sample is divided by the value of the internal reference of the sample, and the ratio obtained finally is the relative content of the gene to be detected of the sample.

The result of the fluorescent quantitative PCR detection is shown in FIG. 1, wherein P is a PCOS group, N is a non-PCOS group, and as can be seen from FIG. 1, the expression of circRNA _ BECN1 in the follicles of the PCOS group is significantly higher than that of the non-PCOS group, and it is presumed that circRNA _ BECN1 may be involved in the maturation process of the follicles of the polycystic ovarian syndrome.

Example 4

circular structure identification of circRNA _ BECN 1:

the circRNA _ BECN1 is exon-derived circRNA that is cleaved after transcription of the chr17:74600501-74601413 gene to form a nucleotide fragment of 2003nt in length. As can be seen in FIG. 2, the circRNA _ BECN1 band becomes pale and the linear GAPDH band disappears after RNase R digestion, as a result of digestion of GV stage oocyte RNA with circRNA _ BECN1 RNaseR.

The circRNA _ BECN1 is a new circular RNA, the sequence of the ring-forming site has been verified through the Sanger sequencing result in the early stage, and the Sanger sequencing result of the circRNA _ BECN1 is shown in figure 3, thereby providing theoretical support for the later functional verification.

Example 5

And (3) carrying out positioning identification on circRNA _ BECN1 FISH:

(1) CircRNA _ BECN1 FISH probe design

The circRNA _ BECN1 FISH probe was designed and synthesized by Ribo Co. The probe is designed to cross the backsple junction site area.

(2) Cell fixation and permeabilization

This was mainly done by the circRNA FISH Probe Mix kit supplied by Ribo Co.

First, oocyte crawls were placed at the bottom of the wells of a 24-well plate. Adding 2.5uL of 20uM [ CircRNA FISH Probe Mix stock ] or [ internal reference FISH Probe Mix stock ] into [ hybridization solution ] under the condition of keeping out of light; discarding the [ prehybridization solution ] in each well of cells, adding appropriate amount of [ Probe hybridization solution ] containing probe, keeping out of the sun, and hybridizing at 37 deg.C overnight; washing cells in each well 3 times (5 min each time) at 42 deg.C in the dark to reduce background signal;

(3) probe detection

Finally, keeping out of the sun, and dyeing for 10min in a DAPI dyeing solution; the cell slide was carefully removed from the well, fixed to a slide using a mounting medium, and subjected to fluorescence detection using a confocal microscope. The results are shown in FIG. 4.

As can be seen from FIG. 4, it was confirmed by circRNA FISH experiments that circ _ BECN1 is mainly localized to the cytoplasm in GV-stage oocytes.

Example 6

Identification of the expression level of circRNA _ BECN1 in different stages of oocyte maturation:

selecting healthy 24-28-day-old ICR female mice, injecting Pregnant Mare Serum Gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for superovulation, collecting GV-stage oocytes, culturing for 0h, 3h, 8h and 14h, collecting GV-stage oocytes in The follicular development and maturation process (GV, GVBD, MI and MII), extracting total RNA by using The RNeasy Plus Micro Kit (BWN-Qiagen-74034) Kit, carrying out reverse transcription to obtain cDNA, and using The cDNA

Premix Ex Taq ^TM The reagent provided in the kit (Nanjing Novodka Biotechnology Co., Ltd.) is used for carrying out fluorescence quantitative PCR reaction, and the expression quantity of the circRNA _ BECN1 in different developmental maturity stages of the oocyte is tested. Wherein, the nucleotide sequences of the used specific primers circRNA _ BECN1-F2 and circRNA _ BECN1-R2 are as follows:

circRNA_BECN1-F2(SEQ ID NO.4)：5’-GACTACTGCCATAACTAAGG-3’

circRNA _ BECN1-R2(SEQ ID NO. 5): 5'-CAGAAGTCACAGCCAGAT-3', respectively; the reaction system comprises: mu.L of cDNA product obtained from oocytes at different times was used as template, 0.4. mu.L (10. mu.M) of forward primer circRNA _ BECN1-F2 and 0.4. mu.L (10. mu.M) of circRNA _ BECN1-R2 were used as amplification primers, 10. mu.L

Premix Ex Taq ^TM (2X) and 8.2. mu.L of ddH 2O. The reaction conditions are as follows: 94 ℃, 5min, 94 ℃, 30s, 52 ℃, 30s, 72 ℃, 1min, 72 ℃, 7min, 4 ℃ and infinity; 40 PCR cycles.

The housekeeping gene GAPDH (the expression quantity is basically constant among different samples) is used as an internal reference, the value of the gene to be detected of the sample is divided by the value of the internal reference of the sample, and the ratio obtained finally is the relative content of the gene to be detected of the sample.

FIG. 5 shows the results of the expression level test of circRNA _ BECN1 at different stages of oocyte maturation, showing that circRNA _ BECN1 is expressed at different stages of oocyte maturation (GV, GVBD, MI, MII).

Example 7

The effect on the capacity of follicle development to mature after interference with circRNA _ BECN1 was tested:

(1) design of circRNA _ BECN1 interference sequence

The circRNA _ BECN1 interference sequence was designed and synthesized from the full-length sequence of circRNA _ BECN 1:

circ_BECN1-1:(SEQ ID NO.12)

5’-GGCUACACUUCCUACAGGCTTGCCUGUAGGAAGUGUAGCCTT-3’

circ_BECN1-2:(SEQ ID NO.13)

5’-CACUUCCUACAGGCGGAGGTTCCUCCGCCUGUAGGAAGUGTT-3’；

(2) construction of Lentiviral shRNA interference vector (pCDH-shRNA)

First, the interference sequences circ _ BECN1-1, circ _ BECN1-2 and the control group GFP sequence were constructed to lentiviral shRNA interference vectors (pCDH-shRNA), respectively, according to a conventional method. Then, pCDH-CMV-GFP-Puro and pCDH-CMV-circ _ BECN1 shRNA were mixed with 400ul Opti-MEM using HighGene (ABClonal) transfection reagent, and then co-transfected into 293T cells, in the system shown in Table 1:

TABLE 1

After obtaining the virus solution, the optimal virus titer and infection time of the immature oocyte are searched for by measuring the virus titer according to a fluorescence method.

(3) Effect test on the ability of ovarian follicles to develop maturity following interference with circRNA _ BECN1

The lentivirus overexpression shRNA interference vector is injected into mouse GV stage zona pellucida oocytes in a microinjection way, DMSO is microinjected by a Control group by the same method, and the influence of interference circRNA _ BECN1 on phenotypes such as oocyte development maturation related indexes-GVBD incidence, Pb1 discharge rate, MII formation rate and the like is observed. The results are shown in FIG. 6.

As can be seen from FIG. 6, the incidence of GVBD, the discharge rate of Pb1, and the formation rate of MII in the oocytes after the interference of circRNA _ BECN1 all showed a decrease compared to the Control group, indicating that they affect the maturation of the oocytes.

Sequence listing

SEQ ID NO:1

Nucleotide sequence of circular circRNA _ BECN1 gene

aggcggaggttgcagtaagccgagatcatgccattgcacttcagcctgggcaacaagagcgaaactccatttcaaaaaaa aaaaaaacccacaaaaagaaaagaaaaaaaaataaaaatatgccaggcacagtggctcacgtctgtaatcccagcacttt gggaggccaaggcgggtggatcatgaggtcaggagatcaagaccatcatggataacatggtgaaaccccgtcactacca aaaatacaaaaaattagccaggcattgtggtgggcgcctgtaatcccagctactcgggaggagaggaggctgaggcagg agaactgcgtgaacctgggaggcagagcctgcaatgagccgagatcacgccactgcactccagcctgggtgacagagt gagactccgtctcaaaataaataaataaataaaaataataaaaaatgctacaaaacctgaaactttttgaccattgatatgatgc cacaaggggaaaattccatacctgatacctttgcttttttttttttttttttttttttttgagacagaggcttgctttgtcgcccaggctg gagtgcagtggcgcaatctcggcttactgtaagctctgcctcccaggttcacgccattctcctgccactgcctcccgagtag ctgggaccacaggcacctgccaccacgcccagctgattttttttttttttttgtatttttagtagagacggggtttcactgtgttag ccaggatggtctcaatctcctgaccttgtgatccacccgccttggcctcccaaagtgctgggattacaggcgtgagccactg cacccggccctttgctttctgatgattcaatgtatacaagctttgtttcatgcacaaaattatttaaaatattgtatgaaattacctc agactacatgtatcaggtatatataaaacaggccaggcacggtggctcatgcctgtaatcctagaactttgggaggctgagg cgggtggatcacctgaggtcaggagttcgagaccagcctggtccaacatggtaaaaccccatctctactaaaaatacaaaa attagctgggcctggtggtgggcacctgtaatcccagctactccagaggctgaggcaggagactcacttgaacctgggag gcggaggttgcagtgagccgagatcgtgccactgcactccagtctgggcaacagagcaagactctgtctcagaagaaga aacataaatgaattttgtgtttagacttgggtcccattcccaagatatcttagtatctatatgccaatatcccaaaatctgaaaaaa aaaatccaaaatctaaaacacttctggtgatactcaacctgtaaacatctatctcacggtgattcttttcctagcctagtgcacac accctcttctgacctgcccatatcccttctcaacagactgtcaaaacctgatttctgtctacaagacccccaaaactaaacctg aactagacaagatattgaacaatatactggctcttcatatctaggtctaaaaaataaattgaatgccaatatcaaagcaaacca tccattttcttaagagatataagaactctgtattccagtgaaaatggagcttcacagagtaccttagatttgtctgtcagagactc cagatatgaatggtttccgtaaggaacaagtcggtatctaaaatagagatacaaacagagatggatacaggactcctcccaa agtgattattgggaagttacaacccaatgatttcactcttcattctggccaagggcttcttttattatgctcaactccctctcccac ctccccattgaatccatgtttaaaagattttgtttaatgaacaaactacatttattatataattgcctgggggccttccatgaacaa atgttatgttttggtttaaatttaagaaacaaaaactgaaaatatcctgaggaacactaaggctctggggactgtaggctggga actatgctatagtcacgcaactcagtgactgtgtgatcagtggagagagccctgtgatgaggggaaaatcagatgctgaca gctcttcagccacagaaaactcccagtctgtgggcagcaagggctcctgacatggtggacagcagtaccaggttggagat acaaattatatcaatgcacatcactccccaagaaagggctacacttcctac

SEQ ID NO.2

circRNA_BECN1-RT1

gatggacagaccgttgag

SEQ ID NO.3

circRNA_BECN1-RT2

cctcatccgtctcgctta

SEQ ID NO.4

circRNA_BECN1-F2

gactactgccataactaagg

SEQ ID NO.5

circRNA_BECN1-R2

cagaagtcacagccagat

SEQ ID NO.6

circRNA _ BECN1 primer 1F

agagagccctgtgatgag

SEQ ID NO.7

circRNA _ BECN1 primer 1R

cgcctgtaggaagtgtag

SEQ ID NO.8

circRNA _ BECN1 primer 2F

ggaacactaaggctctgg

SEQ ID NO.9

circRNA _ BECN1 primer 2R

atgatctcggcttactgc

SEQ ID NO.10

GAPDH primer F

acccactcctccacctttgac

SEQ ID NO.11

GAPDH primer R

tgttgctgtagccaaattcgtt

SEQ ID NO.12

circ_BECN1-1

ggcuacacuuccuacaggcttgccuguaggaaguguagcctt

SEQ ID NO.13

circ_BECN1-2

cacuuccuacaggcggaggttccuccgccuguaggaagugtt

Sequence listing

<110> Subei people hospital of Jiangsu province

<120> circular circRNA _ BECN1 gene and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2212

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 1

aggcggaggt tgcagtaagc cgagatcatg ccattgcact tcagcctggg caacaagagc 60

gaaactccat ttcaaaaaaa aaaaaaaccc acaaaaagaa aagaaaaaaa aataaaaata 120

tgccaggcac agtggctcac gtctgtaatc ccagcacttt gggaggccaa ggcgggtgga 180

tcatgaggtc aggagatcaa gaccatcatg gataacatgg tgaaaccccg tcactaccaa 240

aaatacaaaa aattagccag gcattgtggt gggcgcctgt aatcccagct actcgggagg 300

agaggaggct gaggcaggag aactgcgtga acctgggagg cagagcctgc aatgagccga 360

gatcacgcca ctgcactcca gcctgggtga cagagtgaga ctccgtctca aaataaataa 420

ataaataaaa ataataaaaa atgctacaaa acctgaaact ttttgaccat tgatatgatg 480

ccacaagggg aaaattccat acctgatacc tttgcttttt tttttttttt tttttttttt 540

tgagacagag gcttgctttg tcgcccaggc tggagtgcag tggcgcaatc tcggcttact 600

gtaagctctg cctcccaggt tcacgccatt ctcctgccac tgcctcccga gtagctggga 660

ccacaggcac ctgccaccac gcccagctga tttttttttt ttttttgtat ttttagtaga 720

gacggggttt cactgtgtta gccaggatgg tctcaatctc ctgaccttgt gatccacccg 780

ccttggcctc ccaaagtgct gggattacag gcgtgagcca ctgcacccgg ccctttgctt 840

tctgatgatt caatgtatac aagctttgtt tcatgcacaa aattatttaa aatattgtat 900

gaaattacct cagactacat gtatcaggta tatataaaac aggccaggca cggtggctca 960

tgcctgtaat cctagaactt tgggaggctg aggcgggtgg atcacctgag gtcaggagtt 1020

cgagaccagc ctggtccaac atggtaaaac cccatctcta ctaaaaatac aaaaattagc 1080

tgggcctggt ggtgggcacc tgtaatccca gctactccag aggctgaggc aggagactca 1140

cttgaacctg ggaggcggag gttgcagtga gccgagatcg tgccactgca ctccagtctg 1200

ggcaacagag caagactctg tctcagaaga agaaacataa atgaattttg tgtttagact 1260

tgggtcccat tcccaagata tcttagtatc tatatgccaa tatcccaaaa tctgaaaaaa 1320

aaaatccaaa atctaaaaca cttctggtga tactcaacct gtaaacatct atctcacggt 1380

gattcttttc ctagcctagt gcacacaccc tcttctgacc tgcccatatc ccttctcaac 1440

agactgtcaa aacctgattt ctgtctacaa gacccccaaa actaaacctg aactagacaa 1500

gatattgaac aatatactgg ctcttcatat ctaggtctaa aaaataaatt gaatgccaat 1560

atcaaagcaa accatccatt ttcttaagag atataagaac tctgtattcc agtgaaaatg 1620

gagcttcaca gagtacctta gatttgtctg tcagagactc cagatatgaa tggtttccgt 1680

aaggaacaag tcggtatcta aaatagagat acaaacagag atggatacag gactcctccc 1740

aaagtgatta ttgggaagtt acaacccaat gatttcactc ttcattctgg ccaagggctt 1800

cttttattat gctcaactcc ctctcccacc tccccattga atccatgttt aaaagatttt 1860

gtttaatgaa caaactacat ttattatata attgcctggg ggccttccat gaacaaatgt 1920

tatgttttgg tttaaattta agaaacaaaa actgaaaata tcctgaggaa cactaaggct 1980

ctggggactg taggctggga actatgctat agtcacgcaa ctcagtgact gtgtgatcag 2040

tggagagagc cctgtgatga ggggaaaatc agatgctgac agctcttcag ccacagaaaa 2100

ctcccagtct gtgggcagca agggctcctg acatggtgga cagcagtacc aggttggaga 2160

tacaaattat atcaatgcac atcactcccc aagaaagggc tacacttcct ac 2212

<210> 2

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 2

gatggacaga ccgttgag 18

<210> 3

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 3

cctcatccgt ctcgctta 18

<210> 4

<211> 20

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 4

gactactgcc ataactaagg 20

<210> 5

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 5

cagaagtcac agccagat 18

<210> 6

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 6

agagagccct gtgatgag 18

<210> 7

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 7

cgcctgtagg aagtgtag 18

<210> 8

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 8

ggaacactaa ggctctgg 18

<210> 9

<211> 18

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 9

atgatctcgg cttactgc 18

<210> 10

<211> 21

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 10

acccactcct ccacctttga c 21

<210> 11

<211> 22

<212> DNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 11

tgttgctgta gccaaattcg tt 22

<210> 12

<211> 42

<212> DNA/RNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 12

ggcuacacuu ccuacaggct tgccuguagg aaguguagcc tt 42

<210> 13

<211> 42

<212> DNA/RNA

<213> non-coding RNA molecule (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 13

cacuuccuac aggcggaggt tccuccgccu guaggaagug tt 42

Claims

1. A circular circRNA _ BECN1 gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. Use of the circular circRNA BECN1 gene of claim 1 in the preparation of a reagent for diagnosing the developmental maturation of oocytes with polycystic ovary syndrome.

3. A kit for detecting the circular circRNA BECN1 gene of claim 1, comprising a strand specific RT primer and a fluorescent quantitative PCR detection primer; the sequence of the chain specificity RT primer is shown as SEQ ID NO.2 and SEQ ID NO. 3; the sequence of the fluorescent quantitative PCR detection primer is shown as SEQ ID NO.4 and SEQ ID NO. 5.