MiRNA for regulating estrogen production of fish ovary and application thereof

Abstract

The application discloses a kind of miRNA of regulating fish ovary estrogen production and its application, the miRNA of regulating fish ovary estrogen production, the miRNA is miR-143-3p and / or miR-143-5p, the sequence of the miR-143-3p is as shown in SEQ ID NO:1, the sequence of the miR-143-5p is as shown in SEQ ID NO:2, the application of the above-mentioned miRNA in regulating fish ovary estrogen production or in preparation regulating fish ovary estrogen production product, and a kind of method for regulating fish ovary estrogen production.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a miRNA that regulates estrogen production in fish ovaries and its applications. Background Technology

[0002] In aquaculture, gonadal development and sex regulation in fish directly affect their growth rate, sexual maturity time, and economic value. During fish reproduction and gonadal development, ovarian maturation and estrogen synthesis are crucial for maintaining normal reproductive function and the profitability of aquaculture. Estrogen production is mainly regulated by steroid synthesis-related enzymes in the ovary (such as aromatase CYP19A1A and steroid production regulator StAR), and its expression level is influenced by a complex endocrine and molecular regulatory network. In recent years, non-coding small RNAs—especially microRNAs (miRNAs)—have been widely recognized as important factors in gene expression regulation and have been shown to participate in physiological processes such as gonadal development and hormone regulation in animals. miRNAs are a class of non-coding RNAs approximately 22 nucleotides in length that regulate the translation or degradation of mRNAs by targeting them. miRNAs play a vital role in gonadal development by influencing the expression of genes related to gonadal development and controlling the synthesis and secretion of sex hormones. In tilapia, miR-133b may participate in early oogenesis by regulating tagln2 expression, while miR-33, targeting the TGFβ1I1 axis, is involved in tilapia follicle development. In medaka, miR-202-5p has been found to be a key candidate factor for male differentiation and development; female medaka with miR-202 knockout either do not lay eggs or exhibit a sharp reduction in egg number. miR-26 may affect gonadal development in medaka by regulating ddx3x expression. In Japanese flounder, the let-7 family may participate in gonadal development by targeting cbx2. MiR-143 has been less studied in fish; it has been characterized in the gonads of both male and female adult brown toothfish and yellow trevally. To date, the specific regulatory mechanism of miR-143 on gonadal development in fish remains unclear.

[0003] miR-143 is a highly conserved miRNA that has been found to participate in various physiological processes in mammals, including lipid metabolism, cell proliferation, and steroid hormone synthesis. miR-143 can be processed from its precursor miRNA to produce two mature strands: miR-143-3p and miR-143-5p. Existing research has largely focused on the function of a single strand, particularly miR-143-3p in mammals. However, in fish, especially in exploring the regulatory roles of both miR-143-3p and miR-143-5p in estrogen synthesis, remains a gap in our understanding.

[0004] Currently, in the field of ovarian development and sex regulation in fish, some studies have attempted to use miRNA to regulate the expression of gonad-related genes, but few have focused on fish, and the functional differences between the two mature strands of miR-143 have not been systematically analyzed. Summary of the Invention

[0005] The purpose of this invention is to provide a miRNA that regulates the production of estrogen in fish ovaries.

[0006] The present invention also aims to provide the application of the above-mentioned miRNA in regulating estrogen production in fish ovaries or in the preparation of products that regulate estrogen production in fish ovaries.

[0007] The final objective of this invention is to provide a method for regulating estrogen production in fish ovaries.

[0008] The first objective of the present invention can be achieved by the following technical solution: a miRNA that regulates the production of estrogen in the ovaries of fish, wherein the miRNA is miR-143-3p and / or miR-143-5p, the sequence of miR-143-3p is as shown in SEQ ID NO:1, and the sequence of miR-143-5p is as shown in SEQ ID NO:2.

[0009] miRNA sequence:

[0010] miR-143-3p: The mature strand sequence is 5'-UGAGAUGAAGCACUGUAGCUC-3' (SEQ ID NO: 1).

[0011] miR-143-5p: The mature strand sequence is 5'-GGUGCAGUGCUGCAUCUCUGG-3' (SEQ ID NO:2).

[0012] Furthermore, the miRNA targets and regulates the yap1 gene, the binding site of the 3′UTR of the yap1 gene to the miR-143-3p is ACAGTG, and the binding site of the 3′UTR of the yap1 gene to the miR-143-5p is ATGCAG.

[0013] Target gene validation:

[0014] Bioinformatics (TargetScan, miRanda) and dual-luciferase reporter assays confirmed that both miR-143-3p and miR-143-5p target the 3'UTR region of yap1, inhibiting its translation. RNA FISH was used to verify the co-localization of miR-143-3p, miR-143-5p, and yap1 in the gonads.

[0015] The second objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned miRNA in regulating estrogen production in fish ovaries or in the preparation of products that regulate estrogen production in fish ovaries.

[0016] Preferably, the product is an agonist or inhibitor of miR-143-3p and / or miR-143-5p. The agonist or inhibitor can be manufactured by a gene company using conventional methods in the art.

[0017] miRNA overexpression:

[0018] Based on the miR-143-3p and miR-143-5p sequences, a chemically modified miRNA agomir (agonist) was designed and synthesized. Specific high expression was achieved by injection into the ovarian tissue of fish. Real-time quantitative PCR (q-RT PCR) and Western blot were used to verify the mRNA and protein expression of the target gene and downstream related genes after miRNA agomir overexpression. Estrogen concentration in the ovarian tissue was also measured.

[0019] miRNA inhibition:

[0020] A chemically modified antagomir (antisense inhibitor) was designed with a sequence complementary to miR-143-3p / 5p to block its binding to the target gene. Specific inhibition of expression was achieved by injection into the ovarian tissue of fish. Real-time quantitative PCR (q-RTPCR) was used to verify the mRNA expression of the target gene and downstream related genes after antagomir inhibition.

[0021] The results showed that, compared with the control group, the concentration of estradiol in the ovaries of the agomiR-143-3p group and the agomiR-143-3p+5p group was significantly downregulated.

[0022] Studying miRNA function: By analyzing the effects of miRNA overexpression in cell or animal models, we can investigate the function and mechanism of miRNA in regulating biological processes such as gene expression, signal transduction, cell proliferation, differentiation, and apoptosis.

[0023] Expression of yap1 and estrogen-related genes after estrogen and anti-estrogen treatment: Female large yellow croaker were injected with estrogen and anti-estrogen drugs respectively, and the expression of target gene yap1 and estrogen synthesis-related genes was verified by real-time quantitative PCR (q-RT PCR).

[0024] Expression of yap1 and estrogen-related genes after treatment with verteporfen, an inhibitor of the target gene yap1: Verteporfen was injected into female large yellow croaker, and the expression of estrogen synthesis-related genes was verified by real-time quantitative PCR (q-RT PCR), as well as ovarian histology.

[0025] The last objective of the present invention can be achieved by the following technical solution: a method for regulating estrogen production in fish ovaries, which regulates estrogen production in fish ovaries by promoting or inhibiting the expression of miR-143-3p and / or miR-143-5p in fish and targeting the target gene yap1 or cyp19a1a protein.

[0026] Preferably, the fish is the large yellow croaker.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) First discovery: This invention systematically reveals for the first time the dual-stranded synergistic mechanism of miR-143-3p and miR-143-5p in regulating estrogen production in fish ovaries;

[0029] Mechanism clearly defined: This invention establishes the regulatory relationship between target genes and ovarian steroid synthesis genes through target gene prediction and verification;

[0030] The experiments were thorough: combining expression profiling, miRNA intervention experiments (mimetic / inhibitor injection), target gene reporter experiments, and estrogen detection, the regulatory effects of miRNA in vivo were verified.

[0031] Dynamic regulation: By overexpressing or inhibiting miR-143-3p / 5p, estrogen levels can be precisely regulated to adapt to the needs of different reproductive stages.

[0032] (2) Application advantages:

[0033] High theoretical value: It enriches the molecular regulatory network of fish sex determination and gonadal development, and provides a reference for subsequent miRNA function research;

[0034] Environmentally friendly: This technology provides a way to regulate estrogen production in fish ovaries through endogenous miRNA, avoiding pollution and operational risks associated with exogenous hormones and reducing damage to aquatic ecosystems.

[0035] It has broad application prospects: it can be used in aquaculture practices such as molecular marker-assisted breeding, sex control, and reproductive regulation.

[0036] (3) Expanding application potential:

[0037] Cross-species applicability: It has been proven effective in large yellow croaker and can be extended to other economically important fish species;

[0038] Endangered species protection: assisting in the artificial breeding of endangered fish by regulating gonadal development. Attached Figure Description

[0039] Figure 1 The expression profile of small RNAs in the gonads of large yellow croaker in Example 1 is shown in A: A volcano plot shows the differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs between the ovaries and testes of large yellow croaker (p<0.05 and log2FC≥1.0 or log2FC≤-1.0). Red and green dots represent differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs upregulated and downregulated in the testes, respectively. Gray dots represent miRNAs with no significant differential expression. B: Gene analysis of gonadal development-related genes targeting the differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs between the ovaries and testes of large yellow croaker. Ontology (GO) enrichment analysis analyzed target genes of differentially expressed miRNAs, target genes of differentially expressed lncRNAs, and host genes of differentially expressed circRNAs. C: Regulatory network between differentially expressed miRNAs and their target mRNAs, related functions, pathways, and biological processes. Triangular nodes represent miRNAs, circular nodes represent mRNAs, and rectangular nodes represent functions, pathways, and biological processes. Red and green nodes represent RNAs that are upregulated and downregulated in the testes, respectively, and yellow nodes represent mRNAs that are not significantly differentially expressed between the testes and ovaries.

[0040] Figure 2 To verify the differentially expressed RNA between male and female gonads of large yellow croaker in Example 1, A: RNA-Seq expression of mature miRNAs in both 3p and 5p forms in the gonads of large yellow croaker. Pink represents females, and light blue represents males; solid rectangles represent 5p, and rectangles with diagonals represent 3p. The left side of the dashed line indicates 3p-biased expression, and the right side of the dashed line indicates 5p-biased expression. B: Verification of LncRNA and circRNA expression profiles in the gonads of large yellow croaker. Red represents females, and blue represents males. C: Verification of miR-143 and yap1 mRNA expression in the gonads of large yellow croaker. Red represents females, and blue represents males. D: Verification of miR-143-3p, miR-143-5p, and yap1 expression in multiple tissues of large yellow croaker using qRT-PCR. Each experiment was performed in triplicate. Red represents females, blue represents males, and lowercase "ns" indicates no significant difference (p>0.05).

[0041] Figure 3This section illustrates the sequence conservation analysis of miR-143-3p / -5p across different species and its validation of targeting yap1 mRNA in Example 2. A: Sequence conservation analysis of pre-miR-143 and mature miR-143 across different species; yellow highlights indicate conserved sequences in multiple species. Red boxes indicate the precursor arm regions that produce mature miR-143-3p and miR-143-5p. Asterisks indicate the predicted binding sites of miR-143-3p and miR-143-5p in the yap1 3′UTR. B: Cross-species conservation analysis of the binding sites of miR-143-3p / 5p in the yap1 3′UTR. Black highlights indicate conserved sequences in multiple species. C: Schematic diagram of miR-143-3p / 5p targeting the yap1 3′UTR. Dual-luciferase reporter assays were performed using plasmids carrying predicted miR-143-3p and miR-143-5p binding sites in the yap1 3′UTR. Wild-type and mutant vectors were constructed for validation. The D: dual-luciferase assay verified the targeting and regulation of yap1 by miR-143-3p / 5p. pGL3 luciferase vectors carrying the yap1 3′UTR sequence (wild-type or mutant) were co-transfected into HEK-293T cells with miR-143-3p / 5p mimics, inhibitors, or negative controls (NC). The ratio of Firefly to Renilla luciferase activities was normalized relative to the NC group. Data are expressed as mean ± standard deviation (SD) and are from three independent replicates. Statistical significance: p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).

[0042] Figure 4 The fluorescence in situ hybridization (FISH) detection of miR-143-3p, miR-143-5p, and Yap1 in the gonads of large yellow croaker in Example 2 demonstrates the localization and expression of miR-143-3p, miR-143-5p, and yap1 in the ovaries and testes of large yellow croaker. DAPI staining shows that the cell nucleus is blue, yap1 is green, miR-143-3p is red, and miR-143-5p is pink. The figures are labeled as follows: OG represents oocyte primordial germ cells, Nu represents oocyte nucleoli, SG represents spermatogonia, LC represents interstitial cells, and ST represents sperm. The scale bars for the first and fourth columns are 50 μm; the second column is 20 μm; and the third column is 100 μm. Three female and three male large yellow croakers were used for the experiment.

[0043] Figure 5The following figures illustrate the expression of sex-related genes in the gonads of large yellow croaker after injection of miR-143 agonists and inhibitors in Example 3: A: Modeling effect of injecting miR-3p / -5p / 3p+5p agonist (agomiR) into the gonads; BD: mRNA expression levels of yap1, cyp19a1a, foxl2, and esr1 after ovarian injection of agomiR-3p / -5p / 3p+5p and its negative control (agomiR NC); EG: mRNA expression levels of yap1, cyp19a1a, foxl2, and esr1 after testicular injection of agomiR-3p / -5p / 3p+5p and agomiR NC; H: Modeling effect of injecting miR-3p / -5p / 3p+5p inhibitor (antagomiR) into the gonads; IK: ovarian injection of antagomiR-3p / -5p / 3p+5p and its negative control (antagomiR NC). After NC, the mRNA expression levels of yap1, cyp19a1a, foxl2 and esr1 were measured. LN: after testicular injection of antagomiR, the mRNA expression levels of yap1, cyp19a1a, foxl2 and esr1 were measured.

[0044] Figure 6 The expression of related proteins and estradiol concentration in the ovaries of large yellow croaker after injection of miR-143 agonists and inhibitors in Examples 3-4 were analyzed. AC: After ovarian injection of miR-143 agonists and inhibitors, the expression of Cyp19a1 and Yap1 proteins was evaluated by Western blot analysis. Gapdh was used as a loading control. The bands of cyp19a1 and yap1 proteins were statistically analyzed using ImageJ. The gray values ​​were normalized to the corresponding control gray values ​​for each group. D: Estradiol concentration was assessed after ovarian injection of miR-143 agonist. E: qRT-PCR was used to detect the expression levels of yap1, cyp19a1a, foxl2, and esr1 in the ovary after estradiol treatment. F: qRT-PCR was used to detect the expression levels of yap1, cyp19a1a, foxl2, and esr1 in the ovary after tamoxifen treatment. Error bars show the standard error (SEM) of the mean of at least three biological replicates for each group (*p<0.05; **p<0.01).

[0045] Figure 7The following diagram illustrates the changes in estrogen-related gene expression in the ovaries of large yellow croaker treated with Verteporfin in Example 5. A: Schematic diagram of the injection experiment. B: qRT-PCR detection of the expression levels of yap1, cyp19a1a, foxl2, and esr1 in the ovaries after Verteporfin treatment. Error bars represent the standard errors (SEM) obtained from at least three biological replicates for each group. Asterisks indicate statistical significance: *p<0.05, **p<0.01. C: Comparison of ovarian tissue morphology between the Verteporfin-treated group and the control group. a) Ovarian tissue of the control group, HE stained, scale bar = 100 μm; b) Enlarged view of the area within the red box in Figure a, scale bar = 20 μm; c) Verteporfin. Detailed Implementation

[0046] The technical solution of the present invention will be described in detail below through specific embodiments, aiming to help those skilled in the art to more accurately understand and smoothly implement the technical solution of the present invention. It should be noted that the following embodiments and accompanying drawings are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources. Furthermore, unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0047] Example 1: Sequencing to identify the expression profile of small RNAs in the gonads of large yellow croaker

[0048] Gonads were collected from healthy 6-month-old large yellow croaker (3 females and 3 males), total RNA was extracted, and lncRNA and small RNA libraries were constructed. Whole transcriptome sequencing and assembly were performed. Differential expression and enrichment analyses identified key sex-biased miRNAs and their target genes, which were then validated by qPCR.

[0049] result:

[0050] Screening and validation of miR-143-3p / 5p:

[0051] Based on female vs. male comparisons, genes reported to be associated with gonadal development and sex differentiation, including dmrt1, amh, amhr2, gsdf, sox9a, and esr1, showed significantly upregulated expression, while foxl2 and cyp19a1a showed no significant difference in expression, and yap1 showed significantly upregulated expression. Among the reported miRNAs associated with gonadal development, miR-143-3p and miR-143-5p showed significantly upregulated expression, while miR-92a and miR-25 showed significantly downregulated expression. Figure 1(Figure A). KEGG analysis of mRNA, miRNA target genes, and LncRNA target genes based on ovary vs. testis revealed enrichment of several pathways related to gonadal development: MAPK signaling pathway, Neuroactive ligand-receptor interaction, Wnt signaling pathway, TGF-betasignaling pathway, and Progesterone-mediated oocyte maturation. De_CircRNAs were fewer in number, and their host genes were not enriched for related pathways. Figure 1 (Figure B). GO analysis was performed on target genes predicted by DE_miRNA to screen for functions, pathways, and biological processes related to gonadal development and sex differentiation. A network diagram of DE miRNA-target genes-functions, pathways, and biological processes was then constructed. Figure 1 (See Figure C). Based on the ovaries vs. testes of large yellow croaker, the upregulated miRNAs were miR-133-3p, miR-199-5p, miR-143-3p, miR-143-5p, and miR-145-5p, with corresponding downregulated target genes including yap1, det1, and tdrkh; the downregulated miRNAs were miR-140, miR-217, miR-25, and miR-92a, with corresponding upregulated target genes including clockb and setd2. Figure 1 (See Figure C). yap1 is a target gene of miR-143-5p, miR-143-3p, miR-92a, and miR-25. Therefore, miR-143-3p, miR-143-5p, and yap1 were selected as research targets.

[0052] Validation of miR-143-3p / 5p with target genes:

[0053] The expression patterns of 5p and 3p in the gonadal tissue of large yellow croaker are consistent, such as... Figure 2As shown in Figure A, the miRNAs to the left of the dashed line all showed high expression levels of 3p in both testes and ovaries; the miRNAs to the right of the dashed line all showed high expression levels of 5p in both testes and ovaries. Significant differences were found in the expression levels of 3p and 5p of miR-143 and miR-10545 in male and female gonads. DE mRNA (yap1), lncRNAs (Lnc_ptx3a, Lnc_scb4), circRNA (circ_igdcc3), and DE miRNAs (miR-143-5p, miR-143-3p) were analyzed by qRT-PCR to assess the reliability of RNA-seq data expressed as fpkm values. The results of qRT-PCR were consistent with the RNA-seq data. Figure 2 Figures A and B confirm the accuracy of the RNA-seq data, indicating the existence of sexual dimorphic expression profiles in the male and female gonads of the large yellow croaker. Simultaneously, qRT-PCR analysis of the expression of DE mRNA (yap1) and DE miRNAs (miR-143-5p, miR-143-3p) in other male and female tissues of the large yellow croaker showed no significant differences. Figure 2 Figure C shows that the expression pattern of sex dimorphism has a high degree of tissue specificity.

[0054] Example 2: Identification and Function of Core miRNAs

[0055] Experimental Design:

[0056] Bioinformatics analysis confirmed that both miR-143-3p and miR-143-5p can hybridize to the 3'UTR region of yap1. A dual-luciferase reporter assay was performed, in which pGL3 luciferase vectors carrying the yap1 3'UTR sequence (wild-type or mutant) were co-transfected with miR-143-3p / 5p mimics, inhibitors, or negative controls (NC) into HEK-293T cells. Fluorescence intensity was used to confirm whether miR-143-3p and miR-143-5p bound to the 3'UTR region of yap1 and inhibited its translation. RNA FISH was used to verify the co-localization of miR-143-3p, miR-143-5p, and yap1 in the gonads.

[0057] result:

[0058] Target gene validation:

[0059] Cross-species sequence conservation analysis showed that the sequences of precursors miR-143, miR-143-3p, and miR-143-5p were relatively conserved in several fish species (e.g., Astotilapia burtoni, Gadus morhua, Ictalurus punctatus, Oreochromisniloticus, Oryzias latipes, Danio rerio), reptiles (e.g., Xenopustropicalis, Gallus gallus), and mammals (e.g., Mus musculus, Homo sapiens). Figure 3 (Figure A). According to predictions from RNAhybrid tools, the sequences of miR-143-3p and miR-143-5p can hybridize with different regions of the 3′UTR of yap1 mRNA. Notably, the ATGCAG binding site of miR-143-5p in the yap1 3′UTR of large yellow croaker is also present in the yap1 3′UTR of other species, including Oreochromis niloticus (Nile tilapia), Takifugu rubripes (red-finned pufferfish), Oryzias latipes (killifish), Danio rerio (zebrafish), Xenopus laevis (African clawed frog), Gallus gallus (chicken), and Mus. musculus (mouse). Similarly, the miR-143-3p binding site at the yap1 3′UTR in large yellow croaker is also present in the yap1 3′UTR region of other species, such as Seriola dumerili, Oreochromis niloticus, Oryziaslatipes, Danio rerio, Gallus gallus, Mus musculus, and Homo sapiens. Figure 3 (Figure B in the middle)

[0060] Dual-luciferase reporter assay:

[0061] Dual-luciferase reporter assay to verify the direct binding of miR-143-3p and miR-143-5p to the yap1 3′UTR. Figure 3 (Figure C) The results showed that, compared with the NC group, the relative luciferase activity was significantly decreased in the yap1 3′UTR-wt and miR-143-5p / 3p mimcs experimental groups, while there was no significant change in the relative luciferase activity in the yap13′UTR-mut and miR-143-5p / 3p mimcs experimental groups. Figure 3 (Figure D). Compared with the NC group, relative luciferase activity was significantly upregulated in the yap1 3′UTR-wt and miRNA inhibitor groups, while no significant change was observed in the yap1 3′UTR-mut and miRNA inhibitor groups. Figure 3 (Figure D). This demonstrates that miR-143-5p and -3p can bind to the yap1 3′UTR and inhibit its expression.

[0062] RNA FISH experiment:

[0063] RNA FISH results showed that the expression level of miR-143-3p was significantly higher than that of miR-143-5p in both the testes and ovaries. Figure 4 The two strands of miR-143 exhibit different spatial distribution patterns in gonadal tissue: miR-143-3p shows a strong hybridization signal but limited cytoplasmic localization; while miR-143-5p shows a weaker signal and partial nuclear enrichment, with their intracellular localizations not completely overlapping. In the ovary, miR-143-3p and Yap1 are co-expressed in the cytoplasm of oogonia, while miR-143-5p is expressed only in the nucleolus of oogonia. In the testes, miR-143-3p and Yap1 are co-expressed in spermatogonia at all stages, while miR-143-5p and Yap1 are co-expressed in spermatogonia and spermatocytes. Significant differences in the expression levels of miR-143-3p and 5p exist in both the ovary and testes. Their RNA-FISH co-localization in the gonads also shows significant differences in signal strength, and their intracellular localizations do not completely overlap.

[0064] Example 3: In vivo functional verification of miRNA

[0065] Experimental steps:

[0066] One-year-old large yellow croakers were divided into 8 groups. The agonist group included: NC agomir group, agomiR-143-3p group, agomiR-143-5p group, and agomiR-143-3p+5p group. The inhibitor group included: NC antagomir group, antagomiR-143-3p group, antagomiR-143-5p group, and antagomiR-143-3p+5p group. There were 3 males and 3 females in each group.

[0067] Local injections of 1 nmol of NC agomir, agomiR-143-3p, agomiR-143-5p, NCantagomir, antagomiR-143-3p, and antagomiR-143-5p were administered into the gonads. The agomiR-143-3p+5p group received a mixture of 0.5 nmol each of agomiR-143-3p and agomiR-143-5p; the antagomiR-143-3p+5p group received a mixture of 0.5 nmol each of antagomiR-143-3p and antagomiR-143-5p. After 5 hours of rest, gonadal samples were collected.

[0068] The levels of target genes and estrogen-related genes were detected by RT-QPCR, and the levels of target genes and cyp19a1 protein in the ovary were detected by Western blotting. The ovarian estrogen levels in the miRNA overexpression group were detected by ELISA kit.

[0069] result:

[0070] RT-QPCR detection of target gene and estrogen-related gene levels:

[0071] Compared with the control group, injection of agomiR-143-3p into the ovaries of female large yellow croaker significantly downregulated the mRNA levels of yap1 and cyp19a1a, while there was no significant difference in the mRNA levels of foxl2 and esr1. Figure 5 (Figure B). Injection of agomiR-143-5p into the female ovaries and injection of agomiR-143-3p + agomiR-143-5p into the female ovaries resulted in a significant downregulation of yap1 mRNA levels, while there were no significant differences in the mRNA levels of cyp19a1a, foxl2, and esr1. Figure 5 (Figures C-D). Compared with the control group, injection of antagomiR-143-3p into the ovaries of female large yellow croaker significantly upregulated the mRNA levels of yap1 and cyp19a1a, significantly downregulated the mRNA level of esr1, while there was no significant difference in the mRNA level of foxl2. Figure 5 (Figure I). Injection of antagomiR-143-5p into the female ovary significantly upregulated the mRNA levels of yap1 and cyp19a1a, while there was no significant difference in the mRNA levels of foxl2 and esr1. Figure 5 (See Figure J). In the antagomiR-143-3p + antagomiR-143-5p group, the mRNA level of yap1 in the ovary was significantly upregulated, while the mRNA levels of cyp19a1a, foxl2, and esr1 showed no significant difference. Figure 5(See Figure K). Compared with the control group, no significant changes were observed in the mRNA levels of yap1, cyp19a1a, foxl2, and esr1 in the testes of male large yellow croaker injected with either the agonist or inhibitor groups. Figure 5 (E-G diagram, LM diagram).

[0072] Conclusion: In vivo experiments at the transcriptional level confirmed that miR-143-3p and miR-143-5p directly inhibit the expression of the target gene Yap1.

[0073] Western blot analysis of target gene and cyp19a1 protein levels in the ovary:

[0074] Compared with agomiR NC, injection of agomiR-143-3p / 5p / 3p+5p resulted in a downregulation of Yap1 protein levels in the ovaries (e.g., Figure 6 (Figures A-C) Compared to antagomiR NC, injection of antagomiR-143-3p / 5p / 3p+5p upregulated Yap1 protein levels in the ovary. Figure 6 Figures A-C). Compared with the agomiR NC group, injection of agomiR-143-3p downregulated Cyp19a1 protein levels in the ovaries. Figure 6 (Figure A). Compared with anagomiR NC, injection of anagomiR-143-3p and anagomiR-143-5p upregulated Cyp19a1 protein levels in the ovary. Figure 6 (Figures A-C)

[0075] Conclusion: In vivo experiments confirmed at the protein level that miR-143-3p and miR-143-5p directly inhibit the expression of the target gene Yap1.

[0076] ELISA kit for detecting ovarian estrogen levels in miRNA-overexpressing groups:

[0077] Compared with the control group, the concentration of estradiol in the ovaries of the agomiR-143-3p group and the agomiR-143-3p+5p group was significantly downregulated. Figure 6 (D diagram).

[0078] Conclusion: In vivo experiments demonstrated that miR-143-3p and miR-143-5p directly inhibit the expression of the target gene Yap1, thereby downregulating the expression of cyp19a1a and the concentration of estrogen in the gonads, especially the ovaries.

[0079] Example 4: Experimental design for the expression of yap1 and estrogen-related genes after treatment with estrogen and tamoxifen:

[0080] One-year-old large yellow croakers were divided into three groups: a control group, an estrogen injection group, and a tamoxifen injection group, with three female fish in each group.

[0081] Control group: 250 μL corn oil, single intraperitoneal injection;

[0082] Estradiol group: 4 mg / kg (250 μL), single intraperitoneal injection;

[0083] Tamoxifen group: 4 mg / kg (250 μL), single intraperitoneal injection;

[0084] After 5 hours of temporary rearing, ovaries were collected. The mRNA levels of target genes and estrogen-related genes were detected by RT-qPCR.

[0085] result:

[0086] Compared with the control group, there were no significant differences in the mRNA levels of yap1, cyp19a1a, foxl2, and esr1 in the ovaries of the estrogen injection group. Figure 6 (Figure E). In the tamoxifen injection group, the mRNA levels of yap1, cyp19a1a, and foxl2 in the ovaries were significantly upregulated compared to the control group, while the expression level of esr1 showed no significant difference. Figure 6 (China E diagram).

[0087] Conclusion: Exogenous estrogen injection does not affect the concentrations of yap1, cyp19a1a, foxl2, and esr1; however, exogenous tamoxifen injection upregulates the transcriptional levels of yap1, cyp19a1a, and foxl2.

[0088] Example 5: Expression of yap1 and estrogen-related genes after treatment with verteporfin, a target gene inhibitor of yap1.

[0089] Experimental Design:

[0090] The experimental large yellow croakers were divided into two groups: a control group and a verteporfin injection group, with three female fish in each group.

[0091] Control group: 250 μL corn oil, single intraperitoneal injection ( Figure 7 (Figure A in the middle);

[0092] Verteporfen group: 10 mg / kg (250 μL), single intraperitoneal injection ( Figure 7 (Figure A in the middle);

[0093] After 5 hours of temporary rearing, gonadal samples were taken. RT-qPCR was used to detect the levels of target genes and estrogen-related genes, and HE staining was used to observe histological changes in the ovaries.

[0094] result:

[0095] Compared with the control group, intraperitoneal injection of verteporfen inhibited the mRNA level of yap1 in the ovary and led to a significant downregulation of the mRNA level of cyp19a1a. Figure 7 (Figure B) No significant difference was observed in the mRNA levels of foxl2 and esr1. Figure 7 Figure B in the middle section). HE staining analysis showed that the ovarian tissue in the verteporfen treatment group exhibited significant morphological changes (Figure B in the middle section). Figure 7 (See Figure C). Compared with the control group, the treatment group showed an abnormally high proliferation of primary oocytes and an increased cell density, resulting in dense oocyte clusters occupying the follicular cavity. High-power microscopy revealed abnormal nuclear morphology in the proliferating cells, with enlarged nucleoli and increased mitotic activity. Figure 7 (Figure C in the middle)

[0096] Conclusion: When the mRNA level of yap1 is inhibited, the transcriptional level of cyp19a1a is subsequently downregulated.

[0097] Therefore, by inhibiting the target gene yap1, yap1 will downregulate cyp19a1a (estrogen synthase), thereby reducing estrogen concentration.

[0098] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of miRNAs that regulate estrogen production in fish ovaries in the preparation of products that regulate estrogen production in fish ovaries; The miRNA is miR-143-3p and / or miR-143-5p, the sequence of miR-143-3p is shown in SEQ ID NO:1, and the sequence of miR-143-5p is shown in SEQ ID NO:2; The miRNA targets and regulates the yap1 gene, and the binding site between the 3′UTR of the yap1 gene and the miR-143-3p is ACAGTG, while the binding site between the 3′UTR of the yap1 gene and the miR-143-5p is ATGCAG. The product is an agonist or inhibitor of miR-143-3p and / or miR-143-5p; The agonists of miR-143-3p and / or miR-143-5p are miRNA overexpression; The inhibitors of miR-143-3p and / or miR-143-5p are: chemically modified antagomir with sequences complementary to miR-143-3p / 5p, which block their binding to the target gene; The fish in question is the large yellow croaker.

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