Lncrna mstrg.5970.28, applications thereof, products and methods for regulating ovarian development

Abstract

The application provides a long-chain non-coding RNA MSTRG.5970.28 and application thereof, a product and a method for goose ovary development, relates to the technical field of biology, and provides a new long-chain non-coding RNA MSTRG.5970.28; the lncRNA MSTRG.5970.28 is related to the proliferation and apoptosis of goose follicular granulosa cells; by regulating the expression of the lncRNA MSTRG.5970.28, the related miRNA and gene of the proliferation and apoptosis of the follicular granulosa cells can be correspondingly regulated. By applying the lncRNA MSTRG.5970.28 and the corresponding product, the research on the proliferation and apoptosis of the follicular granulosa cells and the physiological and biochemical functions can be effectively guaranteed. In addition, by regulating the proliferation and apoptosis of the follicular granulosa cells through the lncRNA MSTRG.5970.28, the population reproductive capacity can be improved while the excellent characteristics of the goose germplasm resources are maintained, so that the industrialization development of geese is promoted, and the economic benefits are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and relates to a kind of lncRNA MSTRG.5970.28 and its application, the technical field of product and method for regulating follicular development. BACKGROUND

[0002] Goose belongs to seasonal breeding animal, and the strong nest property and the low individual egg production seriously affect the scale development of goose breeding industry.In actual production, the average annual egg production of goose is 30-60 per hen, and the individual difference is large.The improvement of egg production performance is one of the main breeding objectives in current goose breeding.Because the egg production performance belongs to quantitative trait, the heritability is low, and it is difficult to make significant genetic progress by using traditional quantitative genetics breeding method.

[0003] Follicular development is a key factor determining the egg production performance of female birds.Follicular development is a very complex biological process, and numerous genes and pathways are involved in its proliferation and differentiation.Follicular granulosa cells can provide nutrients for follicular development, and the proliferation and apoptosis of granulosa cells are important factors and necessary conditions for follicular maturation or atresia, and the proliferation of granulosa cells can promote follicular development and maturation, while the apoptosis of granulosa cells can cause follicular atresia.It can be seen that granulosa cells play a decisive role in follicular development.The proliferation and apoptosis of granulosa cells are regulated by various factors, including genetics, nutrition and hormones, etc.With the continuous development of molecular biology technology, revealing the molecular mechanism of granulosa cell proliferation or apoptosis to improve the egg production performance of female birds has become an important research means.

[0004] Long non-coding RNA (lncRNA) is a non-coding RNA of more than 200 nt, which can regulate the transcription and post-transcriptional level by sponging microRNA (miRNA) or other molecules.Studies have shown that lncRNA plays a positive or negative regulatory role in various biological processes.

[0005] However, the current research on goose lncRNA, especially the research on goose ovary lncRNA and its regulatory mechanism is still very insufficient.Therefore, it is urgent to find lncRNA that can regulate the proliferation and apoptosis of goose ovary cells, which can help to clarify the molecular mechanism of goose egg production and lay a theoretical foundation, and greatly accelerate the breeding process of high egg production goose. SUMMARY

[0006] The present application aims to overcome the research gap in the prior art, and provides a kind of lncRNA MSTRG.5970.28 and its application, product and method for regulating ovary development.

[0007] To achieve the above-mentioned purpose, the present application realizes the following scheme:

[0008] The first object of the present application is to provide a new lncRNA MSTRG.5970.28 to at least alleviate one of the technical problems existing in the prior art.

[0009] The second object of the present application is to provide the use of the above-mentioned lncRNA MSTRG.5970.28 in the preparation of a product for regulating the development of ovarian follicles.

[0010] The third object of the present application is to provide a method for regulating the development of ovarian follicles.

[0011] The fourth object of the present application is to provide a product for regulating the development of ovarian follicles.

[0012] To solve the above technical problems, the present application specifically adopts the following technical solutions:

[0013] The lncRNA MSTRG.5970.28 provided by the present application has a nucleotide sequence as shown in SEQ ID NO. 1.

[0014] The present application also provides the use of the above-mentioned lncRNA MSTRG.5970.28 in the preparation of a product for regulating the development of ovarian follicles.

[0015] Further, the development of ovarian follicles is regulated by any one of the following (1)-(4):

[0016] (1) regulating the proliferation of ovarian granulosa cells;

[0017] (2) regulating the apoptosis of ovarian granulosa cells;

[0018] (3) regulating the proliferation or apoptosis of follicular granulosa cells, the miRNA including miR-133a-3p;

[0019] (4) regulating the proliferation or apoptosis of follicular granulosa cells, the miRNA including ANOS1.

[0020] Further,

[0021] (1) overexpressing lncRNA MSTRG.5970.28 to promote the apoptosis of ovarian granulosa cells;

[0022] (2) overexpressing lncRNA MSTRG.5970.28 to inhibit the proliferation of ovarian granulosa cells;

[0023] (3) the proliferation and apoptosis of granulosa cells caused by overexpression of lncRNA MSTRG.5970.28 are weakened by miR-133a-3p;

[0024] (4) overexpressing lncRNA MSTRG.5970.28 to promote the expression of ANOS1 gene.

[0025] Further, overexpression of lncRNA MSTRG.5970.28 is achieved by a recombinant vector of lncRNA MSTRG.5970.28.

[0026] Further, the expression of ANOS1 is affected by lncRNA MSTRG.5970.28 as a ceRNA of miR-133a-3p, to achieve affecting the development of granulosa cells.

[0027] Further, the ovary development is the ovary development of a goose.

[0028] The application also provides a method for regulating ovary development, which comprises regulating the expression of lncRNA MSTRG.5970.28.

[0029] Further,

[0030] (1) overexpressing lncRNA MSTRG.5970.28 to promote the apoptosis of ovary granulosa cells;

[0031] (2) overexpressing lncRNA MSTRG.5970.28 to inhibit the proliferation of ovary granulosa cells;

[0032] (3) the proliferation and apoptosis of granulosa cells caused by overexpression of lncRNA MSTRG.5970.28 are weakened by miR-133a-3p;

[0033] (4) overexpressing lncRNA MSTRG.5970.28 to promote the expression of ANOS1 gene.

[0034] Further, overexpression of lncRNA MSTRG.5970.28 is achieved by a recombinant vector of lncRNA MSTRG.5970.28.

[0035] Preferably, the follicle development is the follicle development of a goose.

[0036] In addition, the application also provides a product for regulating follicle development, comprising a substance for overexpressing lncRNA MSTRG.5970.28 and / or a substance for silencing lncRNA MSTRG.5970.28.

[0037] Compared with the prior art, the application has the following beneficial effects:

[0038] The application provides a new lncRNA MSTRG.5970.28, which is found to be differentially expressed in goose ovaries at each stage before and after egg production. In addition, the inventor of the application finds through experiments that the lncRNA MSTRG.5970.28 is related to the proliferation or apoptosis of granulosa cells, and the expression of the lncRNA MSTRG.5970.28 can also have a corresponding regulatory effect on the related pathways and genes of ovarian development. Therefore, the application of the lncRNA MSTRG.5970.28 and the corresponding product provided by the application can effectively regulate the ovarian development from different angles, and provides a powerful guarantee for the research on the development and physiological and biochemical functions of the ovary. In addition, the regulation of the lncRNA MSTRG.5970.28 on the ovarian development can further carry out high product breeding and the like, so as to maintain the excellent characteristics of the goose germplasm resources, improve the population reproductive capacity, promote the industrialization development of geese, and improve the economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The figure shows the identification, nuclear-cytoplasm separation and hormone treatment results of the ovarian granulosa cells.

[0040] Among them, figure A shows the goose ovarian granulosa cells adhered to the wall; figure B shows the identification of the goose ovarian granulosa cells by cell immunohistochemistry; figure C shows the nuclear-cytoplasmic ratio of the lncRNA MSTRG.5970.28; and figure D shows the influence of FSH or hCG on the expression of the lncRNA MSTRG.5970.28.

[0041] In the figure * P<0.05, ** P<0.01. All the drawings adopt the same kind of mark.

[0042] Figure 2 The figure shows the lncRNA MSTRG.5970.28 and miR-133a-3p target verification result graph.

[0043] Among them, figure A shows the predicted binding information of MSTRG.5970.28 and miR-133a-3p; figure B shows the binding site prediction and vector construction of MSTRG.5970.28 and miR-133a-3p; figure C shows the dual luciferase activity detection of the target relationship between MSTRG.5970.28 and miR-133a-3p; figure D shows the overexpression and interference efficiency detection of miR-133a-3p; and figure E shows the expression change of MSTRG.5970.28 after overexpression / inhibition of miR-133a-3p.

[0044] Figure 3Figure shows lncRNA MSTRG.5970.28 regulates the proliferation and apoptosis of goose granulosa cells through miR-133a-3p.

[0045] Figure A shows the overexpression and interference efficiency detection of lncRNA MSTRG.5970.28; Figure B shows the effect of overexpression / inhibition expression of MSTRG.5970.28 on granulosa cell proliferation; Figure C shows the effect of overexpression / inhibition expression of MSTRG.5970.28 on granulosa cell apoptosis; Figure D shows the effect of overexpression / inhibition expression of miR-133a-3p on granulosa cell proliferation; Figure E shows the effect of overexpression / inhibition expression of miR-133a-3p on granulosa cell apoptosis; Figure F shows the effect of overexpression / inhibition expression of MSTRG.5970.28 and miR-133a-3p on granulosa cell proliferation; Figure G shows the effect of overexpression / inhibition expression of MSTRG.5970.28 and miR-133a-3p on granulosa cell apoptosis.

[0046] Figure 4 Figure shows the targeting verification of miR-133a-3p and ANOS1.

[0047] Figure A shows the predicted binding information of miR-133a-3p and ANOS1; Figure B shows the predicted binding site of miR-133a-3p and ANOS1 and vector construction; Figure C shows the dual luciferase activity detection of the targeting relationship between miR-133a-3p and ANOS1; Figure D shows the mRNA and protein expression changes of ANOS1 after overexpression of miR-133a-3p; Figure E shows the mRNA and protein expression changes of ANOS1 after inhibition of miR-133a-3p.

[0048] Figure 5 Figure shows that lncRNA MSTRG.5970.28 regulates the expression of ANOS1 by acting as a ceRNA of miR-133a-3.

[0049] Figure A shows the mRNA expression changes of ANOS1 after co-transfection of MSTRG.5970.28 and miR-133a-3p overexpression plasmid; Figure B shows the protein expression changes of ANOS1 after co-transfection of MSTRG.5970.28 and miR-133a-3p overexpression plasmid. DETAILED DESCRIPTION

[0050] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application. Modifications or replacements of the method, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0051] Reagents used in this application: PBS buffer, Collagenase type II, M199 complete medium (containing 10% serum), M199 complete medium (containing 10% fetal bovine serum and 1% double antibody), Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Canada), Trizol reagent (Life technogies), Lipofectamine TM 2000 (Thermo Fisher Scientific), 10 IU / mL follicle-stimulating hormone FSH (Solaibao, Beijing, China), 5 IU / mL human chorionic gonadotropin hCG (Solaibao, Beijing, China), Lip3000 reagent (Invitrogen), dual luciferase reporter gene assay kit (RG027, Beyotime Institute of Biotechnology), PrimeScript TM RT reagent Kit with gDNA Eraser kit (TaKaRa), 2x SYBR Green qPCR Master Mix (Servicebio, Wuhan, China), CCK-8 detection kit (Servicebio, Wuhan, China), Annexin V-FITC cell apoptosis (CA1020, Servicebio, China) detection kit, RIPA cell lysis buffer (containing 1 mM PMSF) (Biosharp, Hefei, China), 10% SDS-PAGE gel, BCA kit, 5% skim milk powder, anti-goat anti-mouse IgG, goat anti-rabbit IgG, ANOS1 (1:500), β-actin (1:1000), PBST washing solution, horseradish peroxidase (HRP), ECL hypersensitive luminescent kit (Thermo Scientific)

[0052] Instruments used in this application: constant temperature water bath, centrifuge, vortex, surgical knife, sterile culture dish, surgical scissors, centrifuge tube, pipette, gun head, stainless steel sieve, 6-hole culture dish, cell seed plate, ABI StepOnePlus (ABI, Foster City, CA, USA) instrument, enzyme-linked immunoassay instrument, NovoCyte flow cytometer (Agilent, Santa Clara, CA, USA)

[0053] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0054] Example One:

[0055] The application provides a new lncRNA MSTRG.5970.28, which has a nucleotide sequence as shown in SEQ ID NO. 1.

[0056] The application also provides application of the lncRNA MSTRG.5970.28 in preparation of a product for regulating ovary development.

[0057] Further, the ovary development is regulated by any one of the following (1)-(4):

[0058] (1) regulating proliferation of ovary granulosa cells;

[0059] (2) regulating apoptosis of ovary granulosa cells;

[0060] (3) regulating miRNA related to proliferation or apoptosis of follicular granulosa cells, wherein the miRNA comprises miR-133a-3p;

[0061] (4) regulating genes related to proliferation or apoptosis of follicular granulosa cells, wherein the genes related to proliferation or apoptosis of follicular granulosa cells comprise ANOS1.

[0062] Further,

[0063] (1) overexpressing the lncRNA MSTRG.5970.28 to promote apoptosis of ovary granulosa cells;

[0064] (2) overexpressing the lncRNA MSTRG.5970.28 to inhibit proliferation of ovary granulosa cells;

[0065] (3) the proliferation and apoptosis of granulosa cells caused by overexpression of the lncRNA MSTRG.5970.28 are weakened by miR-133a-3p;

[0066] (4) overexpressing the lncRNA MSTRG.5970.28 to promote expression of an ANOS1 gene.

[0067] Further, overexpression of the lncRNA MSTRG.5970.28 is achieved by a recombinant vector of the lncRNA MSTRG.5970.28.

[0068] Further, by lncRNA MSTRG.5970.28 as a ceRNA of miR-133a-3p, the expression of ANOS1 is affected, and the granulosa cell development is affected.

[0069] Further, the ovary development is the ovary development of a goose.

[0070] The application also provides a method for regulating ovary development, which comprises regulating the expression of lncRNA MSTRG.5970.28.

[0071] Further,

[0072] (1) overexpressing lncRNA MSTRG.5970.28 to promote ovary granulosa cell apoptosis;

[0073] (2) overexpressing lncRNA MSTRG.5970.28 to inhibit ovary granulosa cell proliferation;

[0074] (3) the proliferation and apoptosis of granulosa cells caused by overexpression of lncRNA MSTRG.5970.28 are weakened by miR-133a-3p;

[0075] (4) overexpressing lncRNA MSTRG.5970.28 to promote the expression of ANOS1 gene.

[0076] Further, the overexpression of lncRNA MSTRG.5970.28 is realized by a recombinant vector of lncRNA MSTRG.5970.28.

[0077] Preferably, the follicle development is the follicle development of a goose.

[0078] In addition, the application also provides a product for regulating follicle development, which comprises a substance for overexpressing lncRNA MSTRG.5970.28 and / or a substance for silencing lncRNA MSTRG.5970.28.

[0079] Example II:

[0080] 1. Materials and methods

[0081] 1.1 Goose follicular granulosa cell culture, identification and nuclear-cytoplasmic separation

[0082] All animal experimental procedures were performed in accordance with the relevant guidelines and regulations of the Ministry of Agriculture of the People's Republic of China and were approved by the Animal Ethics Committee of Xinjiang Agricultural University (Approval No. 2019004). The laying geese in the laying period were selected, and the whole ovary tissue was taken after being sacrificed by jugular vein exsanguination. The washed follicle was moved into a sterile culture dish containing pre-cooled PBS buffer, and the follicle membrane, connective tissue and vascular network were stripped. After the follicle was cut, the yolk was released, and the washed follicle membrane was cut as much as possible and placed in a 15 mL centrifuge tube. 4 mL of medium was added and blown for 1 min, and the supernatant was discarded after natural sedimentation for 5 min. 4 mL of 0.2% type II collagenase was added, the precipitate was resuspended, and the digestion was performed in a 37°C constant temperature water bath for 30 min. 4 mL of M199 complete medium (containing 10% serum) was added to terminate the digestion. The filter was collected by passing through a 200-mesh stainless steel sieve, and the cells were collected by centrifugation at 12000 r / min for 5 min. After resuspension in M199 complete medium (containing 10% fetal bovine serum and 1% double antibody), the cell density was determined, and the cells were inoculated in a 6-well culture dish and placed in a 37°C, 5% CO2 incubator for 24 h before being used for subsequent experiments. The follicular granulosa cells were identified by FSHR immunofluorescence method. The cytoplasmic and nuclear RNA were separated according to the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Canada) kit instructions, and the total RNA was extracted from the cell nucleus and cytoplasm using Trizol reagent (Life technogies).

[0083] 1.2 Cell transfection

[0084] The mimics-miR-133a-3p, inhibitor-miR-133a-3p, OE-MSTRG.5970.28, Si-MSTRG.5970.28 and empty vector required for the experiment were synthesized by Shanghai Jimabio Pharmaceutical Technology Co., Ltd. (GenePharm, Shanghai, China). All constructs were verified by sequencing technology. The sequence information is shown in Table 1. The cells were transfected with transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) according to the instructions. The cells were collected 24 h after transfection. TM 2000 (Thermo Fisher Scientific) according to the instructions. The cells were collected 24 h after transfection.

[0085] Table 1: Related primer sequence information table

[0086]

[0087] 1.3 FSH and hCG hormone treatment

[0088] The granulosa cells were treated with 10 IU / mL follicle-stimulating hormone FSH (Solaibao, Beijing, China) or 5 IU / mL human chorionic gonadotropin hCG (Solaibao, Beijing, China) after 48 hours of culture after changing the medium the day after plating the cells, and the control group was added with the same volume of PBS. The cells were collected after 24 h of action, total RNA was extracted, and the expression of lncRNA MSTRG.5970.28 was determined.

[0089] 1.4 Dual-luciferase reporter assay

[0090] After constructing the psiCheck2-MSTRG.5970.28-WT, psiCheck2-MSTRG.5970.28-MUT, psiCheck2-ANOS1-WT, and psiCheck2-ANOS1-MUT vectors by Hefei Yuanen Biotechnology Co., Ltd., 293T cells were seeded in 24-well plates (5 x 10 5 The above vectors were co-transfected into cells with NC mimics or miR-133a-3p mimics using Lip3000 reagent (Invitrogen), and after 48 hours, the relative luciferase activity was measured using a dual-luciferase reporter gene assay kit (RG027, Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The relative luciferase activity was calculated by comparing the ratio of Firefly / Renilla luciferase activity.

[0091] 1.5 Real-time quantitative PCR

[0092] Total RNA of granulosa cells was extracted using Trizol reagent (Life technogies), and PrimeScript TMRT reagent Kit with gDNA Eraser kit (TaKaRa) was used for reverse transcription. qPCR reaction was performed using 2x SYBR Green qPCR Master Mix (Servicebio, Wuhan, China) on ABI StepOnePlus (ABI, Foster City, CA, USA) instrument. The PCR reaction system was 20 μL: cDNA template 3 μL, 2x SYBR Green qPCR Master Mix 10 μL, 0.4 μL of each primer, RNase Free water to 20 μL. The specific primers were designed by Primer Premier (V5.0). The mRNA and lncRNA expression was calculated with β-actin as the internal reference gene, and the miRNA expression was calculated with U6 as the internal reference gene. The primer sequence information is shown in Table 2. The primers were synthesized by General Biosystems Co., Ltd. (Anhui) Co., Ltd. The relative expression level of the target gene was calculated by 2 -ΔΔCT -ΔCt method.

[0093] Table 2: Internal reference gene primer sequence information

[0094]

[0095]

[0096] 1.6 CCK-8 detection

[0097] The isolated goose follicular granulosa cells were inoculated into 96-well plates (1x10 4 -1 ) and transfected for 48 hours. The cell activity detection was performed according to the CCK-8 detection kit (Servicebio, Wuhan, China) instructions. Then the absorbance value of each well was measured at OD 450 nm in the enzyme-linked immunoassay instrument, and the blank hole was set, and the cell proliferation activity was calculated.

[0098] 1.7 Apoptosis detection

[0099] The cells were inoculated in 6-well plates, and when the cell confluence reached about 80%, the transfection was performed. After 24 h of culture, the cells were digested and collected. The granulosa cell apoptosis detection was performed according to the Annexin V-FITC apoptosis (CA1020, Servicebio, China) detection kit instructions. Finally, the NovoCyte flow cytometer (Agilent, Santa Clara, CA, USA) was used for detection and analysis, and 3 replicates were set for each test group.

[0100] 1.8 Western blot detection of protein level ​

[0101] Cell pellets were collected, and 100 μL of RIPA cell lysis buffer (containing 1 mM PMSF) (Biosharp, Hefei, China) was added to each 6-well plate. Lysis was performed on ice for 30 min. The cells were centrifuged at 12000 rpm for 15 min, and the supernatant was collected as total protein. Protein concentration was determined using a BCA assay kit. The total protein extract was separated on a 10% SD-PAGE gel and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder at room temperature for 2 h. After blocking, the membrane was incubated overnight at 4°C with primary antibodies: goat anti-mouse IgG, goat anti-rabbit IgG, ANOS1 (1:500), and β-actin (1:1000). The membrane was then washed with PBST. Horseradish peroxidase (HRP)-labeled secondary antibody (1:10000) was diluted 1:10000 with secondary antibody dilution buffer and incubated at room temperature for 2 h. The membrane was then washed with PBST. Protein bands were observed using an ECL ultrasensor assay kit (Thermo Scientific), and protein grayscale was analyzed using ImageJ software.

[0102] 1.9 Statistical Analysis

[0103] Statistical analysis was performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA), and graphs were generated using GraphPadPrism 8.0. One-way ANOVA was used to compare multiple datasets, and independent t-tests were used to compare two groups. Experiments were repeated at least three times. Results are expressed as mean ± standard error (Mean ± SEM). P < 0.05 indicated a significant difference, and P > 0.05 indicated no significant difference.

[0104] 2 Results and Analysis

[0105] 2.1 Identification, nucleocytoplasmic separation, and hormone treatment of goose ovarian granulosa cells

[0106] Goose ovarian granulosa cells adhered to the culture wall and grew after 24 hours, exhibiting spindle-shaped and irregular triangular forms, as shown in the attached image. Figure 1 As shown in Figure A. Granulosa cell purity was determined by utilizing FSHR specifically expressed in granulosa cells, as shown in the attached figure. Figure 1 As shown in Figure B, after staining, the cytoplasm of granulosa cells showed red fluorescence, while the nucleus was stained blue. Quantitative real-time PCR was used to detect the expression of lncRNA MSTRG.5970.28 in the nucleus and cytoplasm of granulosa cells. The results showed that MSTRG.5970.28 was expressed in both the nucleus and cytoplasm, with high expression in the cytoplasm, as shown in the attached figure. Figure 1Figure 2A shows the expression of MSTRG.5970.28 in goose granulosa cells treated with hCG (5 IU / mL) or FSH (10 IU / mL). Figure 2B shows the expression of MSTRG.5970.28 in goose granulosa cells treated with hCG (5 IU / mL) or FSH (10 IU / mL). Figure 1 Figure 2C shows the expression of MSTRG.5970.28 in goose granulosa cells treated with hCG (5 IU / mL) or FSH (10 IU / mL).

[0107] 2.2 Targeting verification of the relationship between lncRNA MSTRG.5970.28 and miR-133a-3p

[0108] Figure 3A shows the predicted binding sites between MSTRG.5970.28 and miR-133a-3p. Figure 2 Figure 3B shows the construction of MSTRG.5970.28-wt and MSTRG.5970.28-mut plasmids containing the binding sites of miR-133a-3p. Figure 2 Figure 3C shows the co-transfection of wild-type or mutant plasmids with NC mimics or miR-133a-3p mimics into 293T cells. Figure 2 Figure 3D shows the co-transfection of wild-type or mutant plasmids with NC mimics or miR-133a-3p mimics into 293T cells. Figure 2 Figure 4A shows the construction of overexpression vector mimics-miR-133a-3p and inhibition vector inhibitor-miR-133a-3p of miR-133a-3p. Figure 2 Figure 4B shows the expression of MSTRG.5970.28 in goose granulosa cells treated with mimics-miR-133a-3p or inhibitor-miR-133a-3p.

[0109] 2.3 Regulation of the proliferation and apoptosis of goose follicular granulosa cells by lncRNA MSTRG.5970.28 through miR-133a-3p

[0110] Compared with the NC group, the expression of MSTRG.5970.28 in the OE-MSTRG.5970.28 group was extremely significantly increased (P<0.01); the expression of MSTRG.5970.28 in the Si-1, Si-2 and Si-3 groups was extremely significantly decreased (P<0.01), and the decrease in the Si-3 group was the most significant, which indicated that the overexpression and interference plasmid of lncRNA MSTRG.5970.28 were successfully constructed, as shown in FIGS. 1A-1C. Figure 3 A. Subsequently, we analyzed the effect of lncRNA MSTRG.5970.28 on the proliferation and apoptosis of granulosa cells. The results showed that, compared with the NC group, the cell proliferation activity in the OE-MSTRG.5970.28 group was extremely significantly decreased (P<0.01), and the cell apoptosis rate was extremely significantly increased (P<0.01); the cell proliferation activity in the Si-MSTRG.5970.28 group was extremely significantly increased (P<0.01), and the cell apoptosis rate was extremely significantly decreased (P<0.01), as shown in FIGS. 2A-2C. Figure 3 B, FIGS. 2D-2F. Figure 3 C. By analyzing the effect of miR-133a-3p on the proliferation and apoptosis of granulosa cells, it was found that, compared with the NC, the cell proliferation activity in the mimics-miR-133a-3p group was extremely significantly increased (P<0.01), and the cell apoptosis rate was extremely significantly decreased (P<0.01); the cell proliferation activity in the inhibitor-miR-133a-3p group was extremely significantly decreased (P<0.01), and the cell apoptosis rate was extremely significantly increased (P<0.01), as shown in FIGS. 3A-3C. Figure 3 D, 3E. To further analyze the effect of lncRNA MSTRG.5970.28 on the function of goose follicular granulosa cells through miR-133a-3p, the overexpression MSTRG.5970.28 and miR-133a-3p plasmid were transfected into granulosa cells. The results showed that, compared with the NC group, the cell proliferation activity in the mimics-miR-133a-3p+OE-NC group was extremely significantly increased (P<0.01), and the cell apoptosis rate was extremely significantly decreased (P<0.01); the cell proliferation activity in the mimics-NC+OE-MSTRG.5970.28 group was extremely significantly decreased (P<0.01), and the cell apoptosis rate was extremely significantly increased (P<0.01); and the co-transfection of mimics-miR-133a-3p+OE-MSTRG.5970.28 group reduced the inhibition of granulosa cell proliferation and the promotion of cell apoptosis caused by the overexpression of lncRNA MSTRG.5970.28, as shown in FIGS. 4A-4G. Figure 3 F, 3G.

[0111] 2.4 Verification of the targeting relationship between miR-133a-3p and ANOS1

[0112] RNA-hybrid analysis showed that miR-133a-3p has a predicted binding site with ANOS1, as shown in the attached image. Figure 4 As shown in Figure A. To verify the targeting relationship between miR-133a-3p and ANOS1, ANOS1-wt and ANOS1-mut plasmids containing miR-133a-3p binding sites were constructed, as shown in the attached figure. Figure 4 As shown in Figure B. Subsequently, wild-type or mutant plasmids were co-transfected into 293T cells with NC mimics or miR-133a-3p mimics. Dual-luciferase activity analysis showed that co-transfection with miR-133a-3p mimics and ANOS1-wt significantly reduced the fluorescence activity of the wild-type plasmid (P < 0.01), but had no significant effect on the mutant plasmid (P > 0.05), as shown in the attached figure. Figure 4 As shown in Figure C. To further verify whether ANOS1 is a functional target of miR-133a-3p, the overexpression vector mimics-miR-133a-3p or the inhibitor vector inhibitor-miR-133a-3p of miR-133a-3p was transfected into granulosa cells. Compared with the NC group, the mRNA and protein expression of ANOS1 in the mimics-miR-133a-3p group were significantly decreased (P < 0.01), while the mRNA and protein expression of ANOS1 in the inhibitor-miR-133a-3p group were significantly increased (P < 0.01), as shown in the attached figure. Figure 4 As shown in D and 4E.

[0113] 2.5 lncRNA MSTRG.5970.28 regulates ANOS1 expression as a ceRNA acting as miR-133a-3.

[0114] To further analyze how lncRNA MSTRG.5970.28 regulates ANOS1 expression as a ceRNA of miR-133a-3p, MSTRG.5970.28 and miR-133a-3p overexpression plasmids were co-transfected into granulosa cells. The results showed that, compared with the NC group, the mRNA and protein expression of ANOS1 in the Control+mimics-miR-133a-3p+OE-NC group were significantly decreased (P < 0.01), while the mRNA and protein expression of ANOS1 in the Control+mimics-NC+OE-MSTRG.5970.28 group were significantly increased (P < 0.01). In the co-transfected Control+mimics-miR-133a-3p+OE-MSTRG.5970.28 group, the mRNA and protein expression of ANOS1 were significantly higher than those in the NC group (P < 0.01), but significantly lower than those in the Control+mimics-NC+OE-MSTRG.5970.28 group (P < 0.01). (See attached image.) Figure 5 As shown in A and 5B.

[0115] As described above, the present invention can be well implemented. The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all changes and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the present invention.

Claims

1. IncRNA MSTRG.5970.28, characterized in that, The nucleotide sequence of the lncRNA MSTRG.5970.28 is shown as SEQ ID NO.

1.

2. A product for regulating proliferation or apoptosis of goose follicular granulosa cells, characterized in that, The product is siRNA for silencing the lncRNA MSTRG.5970.28 according to claim 1, and the nucleotide sequence of the siRNA is GGCUUAAAUAGCUCUUAAATT and UUUAAGAGCUAUUUAAGCCTT.

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