CN112159811A

CN112159811A - Targeted circRNA capable of competitively binding oar-miR-29b and application thereof

Info

Publication number: CN112159811A
Application number: CN202011060230.9A
Authority: CN
Inventors: 李讨讨; 马友记; 雒瑞瑞; 王慧慧; 张勇; 赵兴绪
Original assignee: Gansu Agricultural University
Current assignee: Gansu Agricultural University
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-01-01

Abstract

The invention discloses a circRNA with target competitive binding of oar-miR-29b and application thereof. The invention takes healthy male Tibetan sheep testis tissues at different development stages as test materials, and circular RNA in the testis is identified and analyzed by an RNA-SEQ technology and a related bioinformatics analysis method to excavate circular RNA related to testis development and function maintenance, wherein the circular RNA is circ _024949 and/or circular RNA circ _026259, and the nucleotide sequence of the circular RNA circ _024949 is shown as SEQ ID No: 1, the nucleotide sequence of the circular RNA circ _026259 is shown as SEQ ID No: 2, respectively. On the basis, the invention discloses application of the circRNA as a marker for detecting the function or the development condition of the Tibetan sheep testis, in preparation of a medicine for regulating the function or the development of the Tibetan sheep testis and in expression analysis or research of a downstream target gene BOLL. The circular RNA circ _029155 can be used for detecting and evaluating the testis development and spermatogenic function of sheep, and lays a foundation for further exploring the molecular action mechanism of sheep testis development and spermatogenesis and subsequent application development.

Description

Targeted circRNA capable of competitively binding oar-miR-29b and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a circRNA with target competitive binding of oar-miR-29b and application thereof.

Background

The testes, an important component of the reproductive and endocrine systems, function primarily to produce male gametes (sperm) and sex hormones, and are the reproductive organs essential for maintaining normal reproductive function in adult males. The most diverse transcriptome within the testis, including coding RNA (mrna) and non-coding RNA (e.g. circRNA, lncRNA and miRNA), is involved in regulating testicular development and spermatogenesis processes through a highly synergistic effect. Circular RNA (circRNA) is taken as endogenous non-coding RNA which has a closed circular structure and is abundantly expressed in eukaryotes, and can be used as a sponge of miRNA to regulate and control the expression and function of downstream genes. However, at present, the application of circRNA in the testis development and function maintenance detection is still in the blank stage in sheep, an important economic livestock species.

The Tibetan sheep serving as one of three major crude sheep varieties in China is mainly distributed in Qinghai-Tibet plateaus with the altitude of more than 3000m and adjacent areas thereof, provides extremely important living materials and economic income for local farmers and herdsmen, and plays an important role in biodiversity and ecosystem functions of alpine meadow grasslands. However, the sheep testicle feed has the characteristics of long development period, late sexual maturity, low fecundity and the like due to the living environment, long-term grazing and lack of supplementary feeding, and is an ideal variety for researching the sheep testicle development and function maintenance regulation and control mechanism.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a circRNA sequence of targeted competitive binding oar-miR-29b and application thereof.

The invention is realized by a circRNA of oar-miR-29b through targeted competitive binding, wherein the circRNA is circular RNA circ _024949 and/or circular RNA circ _026259, and the nucleotide sequence of the circular RNA circ _024949 is shown as SEQ ID No: 1, the nucleotide sequence of the circular RNA circ _026259 is shown as SEQ ID No: 2, respectively.

The invention further discloses application of the circRNA in serving as a marker for detecting the function or the development condition of the Tibetan sheep testis.

The invention further discloses application of the circRNA in preparation of an exogenous medicament for regulating and controlling the function or development of the Tibetan sheep testis.

Preferably, the circRNA regulates the function and development of Tibetan sheep testis by: the circRNA sequences are competitively and targetedly combined with oar-miR-29b to regulate and control the expression of a downstream target gene IGF1, so that the regulation and control of the function or development of the Tibetan sheep testis are realized.

The invention further discloses application of the circRNA in expression analysis or research of downstream target gene IGF 1.

The invention overcomes the defects of the prior art and provides a circRNA with target competitive binding of oar-miR-29b and application thereof. The circRNA of the invention comprises a circular RNAcir _024949 and a circular RNA circ _026259, wherein the nucleotide sequence of the circular RNA circ 024949 is shown as SEQ ID No: 1, the nucleotide sequence of the circular RNA circ _026259 is shown as SEQ ID No: 2, respectively. The identification and screening method process of the circRNA comprises the following steps: (1) circRNA sequencing of 3 developmental stage (3 months, 1 and 3 years) Tibetan sheep testis tissues each with 4 biological replicates; (2) identifying and screening the differentially expressed circRNAs among the 3 age groups based on sequencing data and related bioinformatics analysis; (3) constructing a circRNA-miRNA-mRNA regulation network of different circRNAs based on a cepRNA hypothesis; (4) performing functional annotation and enrichment analysis on a target gene (mRNA) of the circRNA in the network; (5) based on the analysis, circ _024949 and circ _026259 can regulate and control the expression and the function of a downstream target gene IGF1 through targeted combination oar-miR-29b, and further play a role in the development and the function maintenance of the Tibetan sheep testis; (6) the binding sites between circ _024949/circ _026259 and oar-miR-29b and between oar-miR-29b and IGF1 are verified by adopting a dual-luciferase reporter gene detection system; (7) the expression levels of circ _024949, circ _026259, oar-miR-29b and IGF1 in 3 age groups of testis tissues each having 8 biological replicates (containing 4 RNA samples for RNA-seq analysis) were verified by real-time fluorescent quantitative PCR.

On the basis of determining the targeting competitive relationship of the circRNAs, oar-miR-760-3p and BOLL to realize the regulation and control of the function or development of the Tibetan sheep testis, the invention can use the circRNAs as markers for detecting the function or development condition of the Tibetan sheep testis, such as the detection of the expression abundance of the circRNAs or the correlation of expression patterns among the circRNAs, oar-miR-760-3p and BOLL, and judge the function, the spermatogenic function and the development condition of the sheep testis for the markers according to the detection result; or the circRNA can be applied to the preparation of exogenous drugs for regulating and controlling the function or development of the Tibetan sheep testis, and the expression of downstream related target molecules is directionally regulated and controlled in forms of injection and the like so as to regulate the target competition relationship, so that the regulation and control of the function or development of the Tibetan sheep testis are finally realized; or, the invention can use the circRNA in further research on molecular action mechanism of sheep testis development and spermatogenesis to enrich transcriptome information of sheep testis tissue, further understand action mechanism of sheep spermatogenesis and testis function maintenance, and provide theoretical basis for further and deeply disclosing testis development regulation mechanism and breeding of high-fertility stud ram.

Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the circular RNA circ _024949 and circ _026259 can be used for detecting and evaluating the normal testis development and spermatogenic function of sheep, so that the sheep testis transcriptome data are enriched, and a new perspective is provided for further researching the expression regulation and control effect and action mechanism of the stud ram testis function gene.

Drawings

FIG. 1 shows the validation results of dual-luciferase reported target binding sites in the examples of the present invention; wherein, the picture a is the verification of the targeted binding site between circ _024949 and oar-miR-29b, the picture b is the verification of the targeted binding site between circ _026259 and oar-miR-29b, and the picture c is the verification of the targeted binding site between oar-miR-29b and IGF 1;

FIG. 2 is a comparison of the results of the qRT-PCR assay of circ _024949 and circ _026259 with the results of the RNA-seq analysis in an example of the invention; wherein, the graph a is the result of detecting the expression level of circ _024949, and the graph b is the result of detecting the expression level of circ _ 026259;

FIG. 3 is a comparison of the results of qRT-PCR detection of circular RNA downstream target molecules oar-miR-29b and IGF1 with the results of RNA-seq analysis in an example of the invention; wherein, the graph a is the detection result of the expression level of IGF1, and the graph b is the detection result of the expression level of oar-miR-29 b;

FIG. 4 shows validation of circ _024949 and circ _026259 circularized ligation sites based on Sanger sequencing in an example of the invention; wherein, the figure a is the circularized ligation site sequence information of circ _024949, and the figure b is the circularized ligation site sequence information of circ _ 026259;

FIG. 5 is a representation of the cellular localization of circular RNAs circ _024949 and circ _026259 and their downstream target molecules oar-miR-29b and IGF1 in developing Tibetan sheep testis in an example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Test animal and sample collection

The invention takes healthy male tibetan sheep from 3 months old (immature mature), 1 year old (sexual mature) and 3 years old (adult) as test animals, all the test sheep have similar genetic backgrounds (same male parent and different female parents), and are freely fed under the grazing condition of a natural grassland fence with the elevation of 3000-3500m without supplementary feeding. After slaughtering the test sheep, the right testicular tissue of each animal was rapidly harvested and the tunica albuginea was peeled off, cut into small pieces, snap frozen in liquid nitrogen and stored at-80 ℃ for total RNA isolation.

Second, Total RNA isolation

Frozen testis tissues are taken out, rapidly ground into powder by using liquid nitrogen, and total RNA of each tissue is separated by using a Trizol reagent. The mass and concentration of total RNA was then detected using an Agilent 2100 bioanalyzer and a NanoDrop spectrophotometer, respectively. The RNA integrity value (RIN) of all RNA samples to be used was greater than 7.5. For 8 RNA samples from 8 sheep per age group, 4 were randomly selected for RNA sequencing and all 8 testis RNA samples were used to verify sequencing results.

Third, cDNA library construction and sequencing

After removing ribosomal RNA from total RNA using the RiboZero rRNA removal kit, the remaining RNA was fragmented into short fragments by mixing with a fragmentation buffer. The fragmented RNA is used as a template, a random hexamer primer is used for synthesizing first strand cDNA, and buffer solution, dNTPs, RNaseH and DNA polymerase I are used for synthesizing second strand cDNA. And then purifying by a QiaQuick PCR kit, repairing the tail end, adding a base A, adding a sequencing joint, and then digesting by uracil-N-glycosylase (UNG) enzyme to degrade a second chain. Fragment size selection was performed by agarose gel electrophoresis and PCR amplification was performed. The final sequencing library was constructed in Illumina HiSeq^TMSequencing was performed on the 4000 platform.

Fourth, bioinformatics analysis

1. Sequencing data filtering

Raw off-machine sequencing data were processed as follows: (1) removing reads containing the adapter; (2) removing reads containing more N (the N accounts for more than 10 percent, and N represents uncertain base sequence information); (3) low quality reads (the number of bases with a quality value Q < 20 accounts for more than 50% of the total reads) are removed to obtain high quality reads for subsequent bioinformatic analysis.

2. circRNA identification and differential expression analysis

For the high quality reads obtained in example 4, after removing the reads aligned to ribosomal RNA using bowtie2 alignment tool, they were aligned to the sheep reference genome (Oar _ v4.0) using TopHat2(2.1.1) software. Extracting sequences at both ends of the remaining unaligned reads, realigning the sequences on a sheep reference genome by using a bowtie2 alignment tool, and identifying the circRNA by using Find _ Circ software. The identified circRNAs were quantified using the RPM method (number of reads from a circRNA per million reads) followed by identification of differentially expressed circRNAs using the edgeR package based on screening criteria with fold difference in expression >2 between two age groups and P value < 0.05.

4.3 CircRNA-miRNA-mRNA Co-expression network and functional annotation of target genes

The Mireap, miRanda and TargetScan software was used to predict the interaction between miRNA and potential target circRNA or target mRNA in conjunction with previous miRNA and mRNA sequencing data (from the same tibetan sheep testis samples). And further selecting a relation pair that the miRNA and the candidate circRNA/mRNA have a targeting relation and have negative correlation of expression quantity (the spearman grade correlation coefficient is less than-0.7) and have positive correlation of expression quantity (the pearson correlation coefficient is more than 0.9) based on the "cerRNA hypothesis" for constructing the circRNA-miRNA-mRNA network. Further, functional annotation of the target gene of circRNA was performed using the Gene Ontology (GO) database (http:// www.geneontology.org /). Through analysis, it is found that the differential expression of circular RNAs circ _024949 and circ _026259 can target and regulate oar-miR-29b, and further regulate and control the expression of a target gene IGF1 of oar-miR-29 b. IGF1, as a downstream target gene of circ _024949 and circ _026259, is mainly involved in growth and development (GO:0032502// development process; GO:0050793// development process regulation; GO:2000026// regulation of development of multicellular organisms; GO:0048589// developmental growth; GO:0040007// growth; GO:0048513// development of animal organs; GO:0048468// cell development; GO:0048869// cell development process), cell division (GO:0048523// negative regulation of cell process; GO:0051726// cell cycle regulation; GO:0051302// cell division regulation; GO:0007346// mitotic cell cycle regulation), reproduction (GO:0000003// reproduction; GO: 0003006/reproduction-related development process; GO:0061458// reproduction system development; GO:0048608// reproduction structure development; 002214// reproduction/growth of growth/ Reproductive processes) related biological processes. The above analysis shows that circ _024949 and circ _026259 play important roles in normal sheep testicular development and functional maintenance (spermatogenesis and formation).

Five, two luciferase gene report system verification target relation

Wild Type (WT) nucleotide sequences (SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 in this order) of the circ _024949, circ _026259 and IGF 13' UTR containing the putative binding site of oar-miR-29b and their corresponding Mutant (MUT) nucleotide sequences (SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8 in this order) were designed and synthesized. The mutant nucleotide sequence takes the corresponding wild-type sequences of circ _024949, circ _026259 and IGF 13' UTR as templates, and the target site base potentially combined with the oar-miR-29b sequence is mutated into a complementary base. The wild-type and mutant sequences of circ _024949, circ _026259, and IGF 13' UTR described above were loaded into Xho I and Sal I sites of a pmirGLO dual-luciferase miRNA target expression vector (Promega, usa), respectively. oar-miR-29b mimic (mimic) and its negative control (mimic NC) were synthesized by Gilmar pharmaceutical technology, Inc. (Shanghai). HEK-293T cells were recovered and plated in 24-well plates (plating density of about 1.0X 10 per well) in DMEM high-glucose medium containing 10% FBS⁵Single cell), 5% CO₂After static culture in a 37 ℃ incubator for 24 hours (cell growth is 70% -80% fused), Lipofectamine is used^TM2000 transfection kit (Invitrogen, USA), according to the operation instructions, constructed wild type and mutant recombinant dual luciferase reporter vectors were co-transfected with oar-miR-29b mimic or mimic NC to 293T cells. Cells were collected after 48h, and firefly fluorescence and rennin luciferase activities were measured using a dual-luciferase reporter gene assay kit (Promega, USA), with the results expressed as the ratio of firefly luciferase activity to rennin luciferase activity. Each experiment was set up in 3 replicates. As shown in figure 1, oar-miR-29b can significantly inhibit luciferase activities of wild-type circ _024949, circ _026259 and IGF 1-3' UTR (P) compared with control group<0.01) without significant effect on the corresponding mutants (P)<0.05). These results further validate and confirm that circular RNAs circ _024949 and circ _026259 directly target oar-miR-29b, and that IGF1 is the downstream direct target of oar-miR-29 b.

Sixthly, real-time fluorescence quantitative PCR and cyclic RNA cyclization site verification

1. First Strand cDNA Synthesis

The 24 testis total RNA samples extracted from the second part are used as templates for synthesizing first strand cDNA. The method for preparing cDNA by reverse transcription is as follows: for circRNA and mRNA, 1. mu.L of total RNA, 1. mu.L of gDNA Clean Reagent, 2. mu.L of 5 XgDNA Clean Buffer and 6. mu.L of RNase-free Enzyme water were added to the Reaction system, and incubated at 42 ℃ for 2min to remove genomic DNA, followed by addition of 1. mu.L of Evo M-MLV RTase Enzyme Mix, 1. mu.L of RT Primer Mix, 4. mu.L of 5 XRTase Reaction Buffer Mix I and 4. mu.L of RNase-free Enzyme water, incubation at 37 ℃ for 15min, and inactivation of the Enzyme at 85 ℃ for 5 s; for miRNA, 3.75. mu.L of RNA template, 5. mu.L of mRQ Buffer (2X) and 1.25. mu.L of reverse transcriptase mRQ Enzyme (Takara, Japan) were added to the reaction and the Enzyme was inactivated by 5min at 85 ℃ after incubation for 1h at 37 ℃.

2. Real-time fluorescent quantitative PCR (qRT-PCR)

The cDNA synthesized above was used as a template for qRT-PCR amplification on a LightCycler 96 real-time fluorescent quantitative PCR instrument. The involved qRT-PCR reaction system is as follows: mu.L of cDNA, 0.4. mu.L of forward primer (10. mu. mol/L), 0.4. mu.L of reverse primer (10. mu. mol/L), 10. mu.L of 2 XSSYBR Green Pro Taq HS Premix (Excre, Hunan), 8.2. mu.L ddH₂And O. The qRT-PCR amplification procedure employs a two-step process: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s and annealing at 60 ℃ for 30s for 40 cycles. The information of the primers involved in the qRT-PCR reaction system is shown in Table 1.

TABLE 1 primer information for real-time fluorescent quantitative PCR amplification

Beta-actin is used as an internal reference gene to correct the expression level of circ _024949, circ _026259 and IGF1, and U6 is used as an internal reference gene to correct the expression level of oar-miR-29 b. By using 2^-△△CtThe method calculates the relative expression levels of circ _024949, circ _026259, IGF1 and oar-miR-29 b. One-way anova was performed using SPSS 21.0 statistical software, and the Duncan multiplex test determined significance of data difference between groups, and results were expressed as Mean ± standard deviation (Mean ± SD). By ". about" and". X" indicates P respectively<0.05 and P<0.01。

The results are shown in figure 2 and figure 3, the expression change trend of circ _024949, circ _026259 and IGF1 is in positive correlation, and the expression level is gradually reduced with the increase of age; in contrast, the expression level of oar-miR-760-3p increases with age; the patterns of changes in the expression levels of circ _024949, circ _026259, IGF1 and oar-miR-760-3p obtained by real-time fluorescent quantitative PCR are consistent with the results of RNA sequencing. The results described above indicate that the expression of circular RNAs circ _024949 and circ _026259 decreased in an age-dependent manner in developing Tibetan sheep testis.

Considering that the circRNA is reverse-sheared into loops, Sanger sequencing (skyscraper, west ampere) is further performed on qRT-PCR amplification products of the circ _024949 and circ _026259 to verify the accuracy of the sequences of the circularization sites thereof, and the results show that the sequences are completely consistent with the sequence information of the circularization sites obtained from the sequencing results and are reversely looped (see fig. 4), further confirming that the obtained circ RNAs circ _024949 and circ _026259 are reliable.

Seventhly, detecting the location of circ _024949, circ _026259, IGF1 and oar-miR-760-3p by using an RNA fluorescence in situ hybridization technology

Digoxin-labeled RNA specific antisense hybridization probe sequences of circ _024949, circ _026259, oar-miR-29b and IGF1 were designed and synthesized for RNA fluorescence in situ hybridization detection. Dewaxing 5 μm paraffin section to water by conventional method, adding protease K solution dropwise for digestion, and adding 3% methanol-H dropwise₂O₂The solution blocks endogenous peroxidase activity. Adding the prehybridization solution dropwise, incubating at 37 ℃ for 1h, adding digoxin-labeled antisense RNA probe, and hybridizing at 42 ℃ overnight. The hybridization solution was washed with 2 XSSC buffer, 1 XSSC buffer and 0.5 XSSC buffer in this order. After the serum is sealed for 30min at room temperature, the mouse anti-digoxin labeled peroxidase is dripped to incubate for 1h at 37 ℃, and a positive signal of the serum is visualized by using a FITC-TSA reagent. After nuclear staining with DAPI reagent, the anti-fluorescence quencher solution was mounted and placed under a fluorescence microscope (nikon, japan) for observation and photographing. The results are shown in figure 5, and circ _024949, circ _026259, oar-miR-29b and IGF1 are in 3 age groups of Tibetan sheep testis cellsWith a similar RNA distribution pattern: all are widely present in testicular interstitial cells, supporting cells and spermatogenic cells of all levels. Based on the above results, during sheep testicular development, circ _024949/circ _026259 may play multiple roles in the functional maintenance of leydig and supporting cells and in the developmental maturation of germ cells through the oar-miR-29b-IGF1 axis to maintain normal spermatogenic function.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A circRNA for targeted competitive binding of oar-miR-29b, wherein the circRNA is circular RNA circ _024949 and/or circular RNA circ _026259, wherein the nucleotide sequence of the circular RNA circ _024949 is shown as SEQ ID No: 1, the nucleotide sequence of the circular RNA circ _026259 is shown as SEQ ID No: 2, respectively.

2. Use of the circRNA of claim 1 as a marker for detecting function or developmental status of Tibetan sheep testis.

3. Use of the circRNA of claim 1 in the preparation of an exogenous medicament for modulating the function or development of Tibetan sheep testis.

4. The use of claim 3, wherein the circRNA regulates Tibetan sheep testicular function and development by: the circRNA sequences are competitively and targetedly combined with oar-miR-29b to regulate and control the expression of a downstream target gene IGF1, so that the regulation and control of the function or development of the Tibetan sheep testis are realized.

5. Use of the circRNA of claim 1 in expression analysis or study of a downstream target gene IGF 1.