A sheep small RNA miR-433-3p and its applications

Abstract

This invention discloses a sheep small RNA miR-433-3p and its applications, belonging to the field of animal molecular breeding and reproductive biology technology. The mature nucleotide sequence of miR-433-3p is shown in SEQ ID NO.1. Studies have shown that after transfection of miR-433-3p mimic into sheep follicular granulosa cells, the expression level of miR-433-3p significantly increased, the cellular EdU positivity rate decreased, the cell cycle G0 / G1 phase ratio increased while the S and G2 phase ratios decreased, the apoptosis rate increased, and the expression of proliferation-related genes / proteins and apoptosis-related genes / proteins changed accordingly; transfection of miR-433-3p inhibitor showed the opposite changes. Based on the above results, miR-433-3p can be used to regulate the proliferation and apoptosis of sheep follicular granulosa cells, and can serve as a candidate molecular marker for constructing an evaluation system related to sheep high fertility screening / breeding, providing experimental basis for the subsequent establishment of molecular evaluation indicators for fertility; its mimics, inhibitors, expression vectors or kits containing it can also be used to screen candidate nutritional factors, bioactive factors or reproductive regulatory factors that affect granulosa cell function.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding and reproductive biology technology, specifically relating to sheep small RNA miR-433-3p and its applications. Background Technology

[0002] Sheep fertility is an important economic trait affecting the efficiency of herd expansion, the speed of breeding, and the profitability of farming. Especially in large-scale livestock production, early identification and rational retention of ewes with high fertility can help improve the utilization efficiency of foundation ewes, shorten the breeding cycle, and enhance the overall production level of the herd.

[0003] Currently, sheep fertility is often assessed using phenotypic indicators such as lambing number, conception rate, and ovulation count. However, these indicators typically require one or more reproductive cycles to be consistently obtained, resulting in significant phenotypic lag, long screening cycles, and difficulties in early breeding decisions. Meanwhile, while methods such as ultrasound monitoring and hormone testing can reflect ovarian status to some extent, they still face challenges in large-scale production conditions, including high costs, limited sampling time windows, and difficulties in standardized implementation.

[0004] Granulosa cells are key somatic cells in the follicular microenvironment, and their proliferation capacity, cell cycle progression, and apoptosis level directly affect follicular growth, atresia, and the formation of dominant follicles. Granulosa cell functional status is closely related to follicular developmental activity; therefore, molecular indicators that can stably reflect granulosa cell functional status can provide an important biological basis for establishing subsequent evaluation indicators related to reproductive potential.

[0005] miRNAs are a class of endogenous non-coding small RNAs, typically 18-24 nt in length, that can regulate target gene expression at the post-transcriptional level and participate extensively in biological processes such as cell proliferation, differentiation, apoptosis, and metabolism. Current technology lacks a systematic research protocol on the function of miRNAs in sheep follicular granulosa cells and their application value in high-fertility screening and breeding, and also lacks standardized descriptions of miRNA candidate molecular marker research. Summary of the Invention

[0006] The purpose of this invention is to provide a sheep small RNA miR-433-3p.

[0007] Another object of the present invention is to provide an application of the above-mentioned sheep small RNA miR-433-3p.

[0008] The mature nucleotide sequence of the sheep small RNA miR-433-3p according to a specific embodiment of the present invention is shown in SEQ ID NO.1: AUCAUGAUGGGCUCCUCGGUGU.

[0009] This invention provides a sheep small RNA miR-433-3p and its application in regulating granulosa cell function, constructing a high fertility evaluation system, and developing a detection kit. The overall technical solution, as described in the embodiments of this application, mainly includes the following core technical elements: a miR-433-3p molecular entity with a clearly defined mature sequence (SEQ ID NO.1), a delivery vector or liposome system constructed based on this sequence, its functional application in the bidirectional regulation of granulosa cell proliferation / apoptosis, its use as a biomarker in the construction of a high fertility screening and evaluation system, and a kit using this miRNA as the detection target. These technical elements work together to constitute the overall technical solution of this invention.

[0010] The recombinant vector or liposome containing the encoding gene of the above-mentioned sheep small RNA miR-433-3p according to specific embodiments of the present invention.

[0011] The DNA sequence encoding sheep small RNA miR-433-3p can be inserted into a suitable vector. The expression vector is preferably one capable of expressing miR-3p in eukaryotic cells (such as sheep follicular granulosa cells), such as, but not limited to, lentiviral vectors, adenovirus vectors, and plasmid vectors (such as pcDNA series vectors, pCDNA3.0, pSilencer series, etc.). The vector may contain a suitable promoter, such as the U6 promoter or CMV promoter, to drive efficient transcription of sheep small RNA miR-433-3p.

[0012] Liposomes are artificially prepared phospholipid bilayer vesicles that can be used to encapsulate mimics, inhibitors, or expression vectors of the sheep small RNA miR-433-3p to improve its transfection efficiency or in vivo delivery stability. The liposomes can be selected from, but are not limited to, cationic liposomes (such as Lipofectamine 2000, Lipofectamine 3000, DOTAP, etc.) or neutral liposomes. In this invention, an overexpression plasmid is constructed by cloning miR-433-3p into a vector and then encapsulated in liposomes to achieve efficient introduction and sustained expression of miR-433-3p in sheep follicular granulosa cells. The recombinant vector ensures long-term stable transcription, while the liposomes enhance transient transfection efficiency and reduce cytotoxicity. The resulting delivery system can effectively alter the abundance of endogenous miRNAs at physiologically relevant concentrations, supporting its application in functional regulation and mechanistic studies.

[0013] The application of sheep small RNA miR-433-3p according to specific embodiments of the present invention in the preparation of products for regulating the proliferation or apoptosis of sheep follicular granulosa cells.

[0014] In this invention, miR-433-3p mimic or inhibitor is introduced exogenously to dynamically intervene in its expression level in sheep follicular granulosa cells, thereby regulating the transcription and translation of downstream target genes PCNA and BAX. Specifically, the product upregulates miR-433-3p expression to inhibit sheep follicular granulosa cell proliferation and promote apoptosis; or the product downregulates miR-433-3p expression to promote sheep follicular granulosa cell proliferation and inhibit apoptosis.

[0015] The product of this invention achieves its function by downregulating (i.e., reducing or inhibiting) the expression level of endogenous miR-433-3p in sheep follicular granulosa cells. For example, the product may contain a miR-3p inhibitor (also known as anti-miR), antagomir, or a miR-3p gene knockout / knockdown system (such as the CRISPR / Cas9 system, RNA interference vector, etc.). These products can specifically bind to endogenous mature miR-3p or disrupt the miR-3p encoding gene at the genomic level, thereby relieving the inhibitory effect of miR-3p on its downstream target genes, restoring the proliferative capacity of granulosa cells, reducing unnecessary apoptosis, and promoting follicular dominance selection and normal development.

[0016] This invention also provides an application of sheep small RNA miR-433-3p as a biomarker in screening highly fertile sheep.

[0017] Preferably, by collecting blood exosomes or ovarian tissue samples from ewes with different reproductive performance, total RNA is extracted, and the expression level of miR-433-3p is quantified by qRT-PCR. The average expression level of miRNA in individuals is detected. The expression level of miR-433-3p is directly correlated with the functional status of granulosa cells in follicles: the low expression group has a high EdU positivity rate and a low apoptosis rate in granulosa cells, reflecting stronger follicle development activity. It can be used as an early screening indicator to identify potential high-fertility candidates in the young, non-lambing stage, overcoming the shortcomings of traditional phenotypic selection, which has strong lag and long cycle.

[0018] Specifically, by detecting the expression level of miR-433-3p in sheep samples (such as blood, ovarian tissue, follicular fluid, granulosa cells, etc.) and comparing it with a normal control or a predetermined threshold, if the expression level is significantly lower than the normal control or threshold, it indicates that the sheep may have high fertility; conversely, if the expression level is significantly higher than the normal control or threshold, it indicates that the sheep may have low fertility. This application can assist breeders in early, rapid, and non-invasive screening of breeding sheep with excellent reproductive traits, shortening the breeding cycle and reducing breeding costs.

[0019] This invention also provides an application of sheep small RNA miR-433-3p as a biomarker in constructing an evaluation system for screening highly fertile sheep.

[0020] This evaluation system can include: a sample processing module (for extracting total RNA from sheep test samples), a detection module (for quantitatively detecting miR-3p expression levels, such as through real-time quantitative PCR, Northern blot, gene chip, or high-throughput sequencing), and a data analysis module (for comparing the measured miR-433-3p expression level with a preset reference range and outputting evaluation results of high or low fertility based on the comparison results). This evaluation system can further integrate other known fertility-related molecular markers (such as polymorphisms of genes like BMPR-IB and FecB) to construct a multi-index comprehensive evaluation model, improving the accuracy and reliability of screening.

[0021] In this invention, miR-433-3p serves as an endogenous molecular indicator reflecting the functional state of the follicular microenvironment, overcoming the limitations of imaging and hormone testing in reflecting cell activity in real time. This makes the evaluation system both in-depth in terms of molecular mechanisms and feasible in clinical operation, supporting large-scale, precise seed preservation decisions.

[0022] This invention also provides a kit for detecting the proliferation and apoptosis of sheep follicular granulosa cells, using sheep small RNA miR-433-3p as the analyte.

[0023] In this invention, by setting miR-433-3p as the detection target, the kit can measure the functional status of granulocytes, which can be completed using a conventional qRT-PCR platform.

[0024] The kit includes one or more of the following: primers and / or probes for detecting miR-433-3p expression levels, a negative control, an internal control reagent, and reagents for RNA extraction, reverse transcription, or quantitative real-time PCR. According to the example kit, the kit includes one or more of the following: primers and / or probes for detecting miR-433-3p expression levels, a negative control, an internal control reagent, and reagents for RNA extraction, reverse transcription, or quantitative real-time PCR.

[0025] The beneficial effects of this invention: This invention provides a sheep small RNA miR-433-3p and its applications. The invention found that transfection of miR-433-3p mimic into sheep follicular granulosa cells significantly increased miR-433-3p expression, decreased EdU positivity, increased G0 / G1 phase ratio and decreased S phase ratio, and increased apoptosis rate. Corresponding changes were observed in the expression of proliferation-related genes / proteins and apoptosis-related genes / proteins. Transfection with miR-433-3p inhibitor showed the opposite results. Therefore, this invention demonstrates that miR-433-3p can precisely regulate the proliferation and apoptosis processes of sheep follicular granulosa cells, providing a novel drug target and molecular tool for artificial intervention in follicular development, treatment of ovarian diseases, and improvement of sheep fertility.

[0026] Based on the above results, miR-433-3p can be used to regulate the proliferation and apoptosis of sheep follicular granulosa cells, and can serve as a candidate molecular marker for constructing an evaluation system related to sheep high fertility screening / breeding, providing experimental basis for the subsequent establishment of molecular evaluation indicators for fertility; its mimics, inhibitors, expression vectors or kits containing it can also be used to screen candidate nutritional factors, bioactive factors or reproductive regulatory factors that affect granulosa cell function. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Bar chart showing the relative expression levels of miR-433-3p after transfection of sheep follicular granulosa cells with miR-433-3p mimic and miR-433-3p inhibitor; (A) overexpression efficiency of miR-433-3p mimic; (B) interference efficiency of miR-433-3p inhibitor.

[0029] Figure 2 Figure 1 shows the results of EdU proliferation detection in sheep follicular granulosa cells after overexpression and interference with miR-433-3p; (A) Percentage of positive cells after transfection with miR-433-3p mimic; (B) Percentage of positive cells after transfection with miR-433-3p inhibitor.

[0030] Figure 3Flow cytometry results of sheep follicular granulosa cells after overexpression and interference with miR-433-3p; (A) Cell cycle distribution after transfection with miR-433-3p mimic; (B) Cell cycle distribution after transfection with miR-433-3p inhibitor.

[0031] Figure 4 Flow cytometry results of apoptosis in sheep follicular granulosa cells after overexpression and interference with miR-433-3p; (A) Distribution of apoptosis after transfection with miR-433-3p mimic; (B) Distribution of apoptosis after transfection with miR-433-3p inhibitor.

[0032] Figure 5 Figure 1 shows the qRT-PCR results of the expression levels of proliferation and apoptosis-related genes in sheep follicular granulosa cells after overexpression and interference with miR-433-3p; (A) Expression of proliferation and apoptosis-related genes after transfection with miR-433-3p mimic; (B) Expression of proliferation and apoptosis-related genes after transfection with miR-433-3p inhibitor.

[0033] Figure 6 Western blot results for quantitative analysis of proliferation and apoptosis-related protein expression in sheep follicular granulosa cells after overexpression and interference with miR-433-3p; (A) Expression of proliferation and apoptosis-related genes and proteins after transfection with miR-433-3p mimic; (B) Expression of proliferation and apoptosis-related genes and proteins after transfection with miR-433-3p inhibitor. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] The main reagents used in the embodiments of this invention are from the following sources: (1) The EdU cell proliferation assay kit was Cell-Light™ EdU Apollo567 In Vitro Kit, purchased from RIBOBIO (Guangzhou, China).

[0036] (2) The liposome transfection reagent was Lipofectamine™ 3000, purchased from Thermo Fisher Scientific (Hercules, CA, USA).

[0037] (3) The miRNA reverse transcription kit was the miRNA 1st Strand cDNA Synthesis Kit, purchased from Vazyme (Nanjing, China).

[0038] (4) The cell cycle detection kit containing RNase was the Cell Cycle Detection Kit, purchased from Bioss (Beijing, China).

[0039] (5) The Annexin V-FITC / PI apoptosis detection kit was purchased from Bioss (Beijing, China).

[0040] (6) The reverse transcription kit was the PrimeScript™ FAST RT Reagent Kit with gDNA Eraser, purchased from TaKaRa Bio (Kusatsu, Shiga, Japan). Unless otherwise specified, all reagents were used in accordance with the manufacturer's instructions.

[0041] In this embodiment of the invention, all experiments were performed with at least three biological replicates and each sample was performed with three technical replicates. Data are expressed as Mean ± SEM. The Student t-test or one-way ANOVA was used for comparisons between groups, and P < 0.05 was considered to be significant.

[0042] Example 1: Isolation, culture and identification of sheep follicular granulosa cells Healthy ewe ovaries were selected as experimental materials. The ovaries were placed in PBS containing 1% penicillin-dextrose antibodies, stored at 4 °C, and transported back to the laboratory as soon as possible. Follicles larger than 5 mm in diameter were punctured, follicular fluid was aspirated, and granulosa cells were collected by centrifugation and cultured in DMEM / F12 medium. After reaching an appropriate density at 37 °C and 5% CO2, subsequent experiments were conducted. Cells were identified using the FSHR granulosa cell marker.

[0043] Example 2 miR-433-3p transfection and efficiency verification The obtained granulocytes were seeded into 6-well plates, and transfection was performed when the cell density reached 50%-70%. The experimental groups that underwent transfection included the miR-433-3p mimic group, the mimic NC group, the miR-433-3p inhibitor group, and the inhibitor NC group.

[0044] Among them, miR-433-3p mimic is designed according to SEQ ID NO.1, which is AUCAUGAUGGGCUCCUCGGUGU.

[0045] miR-433-3p inhibitor is a functional repressive oligonucleotide designed specifically for this mature sequence.

[0046] The mimic NC group served as the control group transfected with a negative control mimic, which was a random oligonucleotide that matched the length and chemical modification of the miR-433-3p mimic but did not target known sheep miRNA and mRNA sequences. The miR-433-3p inhibitor group consisted of an antisense inhibitory oligonucleotide designed to specifically inhibit endogenous miR-433-3p, transfected with the mature sequence shown in SEQ ID NO. 1. The inhibitor NC group served as the control group transfected with a negative control inhibitor, which was a random oligonucleotide that matched the length and chemical modification of the miR-433-3p inhibitor but did not target known sheep miRNA and mRNA sequences. All of the miR-433-3p mimic, mimic NC, miR-433-3p inhibitor, and inhibitor NC were synthesized by GenScript (Shanghai, China) and their sequences were validated.

[0047] The final concentration of miRNA was 50 nM. The transfection was performed using liposome transfection reagent according to the instructions. After 6 h of transfection, the medium was replaced with complete medium, and the culture was continued for 48 h.

[0048] Total RNA was extracted after transfection, and cDNA was synthesized using a miRNA reverse transcription kit. The relative expression level of miR-433-3p was detected by qRT-PCR, with U6 or other stable small RNAs used as internal controls.

[0049] The results are as follows Figure 1 As shown, compared with their respective negative controls, the expression level of miR-433-3p in the mimic group was significantly increased, while the expression level of miR-433-3p in the inhibitor group was significantly decreased, indicating that both the overexpression and interference systems were successfully constructed and can be used for subsequent functional studies.

[0050] Example 3: Effect of miR-433-3p on the proliferation of sheep follicular granulosa cells After 48 h of transfection with miR-433-3p in each experimental group, cell proliferation capacity was detected using the EdU cell proliferation assay kit.

[0051]

[0052] Note: A, B; C, D: p < 0.01.

[0053] The results are shown in Table 1 and Figure 2 As shown, the proportion of EdU-positive cells in the miR-433-3p mimic group was significantly lower than that in the mimicNC group, while the proportion of EdU-positive cells in the miR-433-3p inhibitor group was significantly higher than that in the inhibitor NC group. This indicates that overexpression of miR-433-3p inhibits granulocyte proliferation, while interference with miR-433-3p promotes granulocyte proliferation.

[0054] Example 4: Effect of miR-433-3p on the cell cycle of sheep follicular granulosa cells Cells were collected 48 h after transfection of each miR-433-3p experimental group. PI staining was performed using a cell cycle detection kit containing RNase, and cell cycle distribution was detected by flow cytometry.

[0055] The results are as follows Figure 3 As shown, overexpression of miR-433-3p increases the proportion of cells in the G0 / G1 phase and decreases the proportion of cells in the S and G2 phases, indicating that cell cycle progression is inhibited; while interference with miR-433-3p decreases the proportion of cells in the G0 / G1 phase and increases the proportion of cells in the S and G2 phases, indicating that the transition of cells from the quiescent / preparation phase to the DNA replication phase is enhanced.

[0056] Example 5: Effect of miR-433-3p on apoptosis in sheep follicular granulosa cells After 48 h of transfection with miR-433-3p, each experimental group was stained using the Annexin V-FITC / PI apoptosis detection kit, and the apoptosis rate was analyzed by flow cytometry.

[0057] The results are as follows Figure 4 As shown, the total apoptosis rate of the miR-433-3p mimic group was higher than that of the mimic NC group, while the total apoptosis rate of the miR-433-3p inhibitor group was lower than that of the inhibitor NC group. This indicates that overexpression of miR-433-3p can promote apoptosis of granulocytes, while downregulation of miR-433-3p helps maintain the survival of granulocytes.

[0058] Example 6: Effects of miR-433-3p on the mRNA expression of proliferation and apoptosis-related genes in granulosa cells Forty-eight hours after transfection with miR-433-3p in each experimental group, total RNA was extracted and reverse transcribed using a reverse transcription kit. GAPDH was used as an internal control. The expression of proliferation-related genes and apoptosis-related genes was detected by qRT-PCR. The detection indicators included PCNA, CDK4, and BAX.

[0059]

[0060] Note: a, b: P < 0.05; A, B: p <0.01.

[0061] The results are shown in Table 2 and Figure 5 As shown, overexpression of miR-433-3p can downregulate the expression of the proliferation-related gene PCNA and cause the apoptosis-related gene BAX to change in a pro-apoptotic direction; while interference with miR-433-3p shows the opposite trend.

[0062] Example 7: Effects of miR-433-3p on the expression of related proteins in granulosa cells Forty-eight hours after transfection with miR-433-3p in each experimental group, total protein was extracted using RIPA lysis buffer and analyzed by SDS-PAGE electrophoresis and Western blot, with α-Tubulin as an internal control. The assay markers included PCNA protein, BAX protein, and CDK4 protein.

[0063]

[0064] Note: A, B: p <0.01.

[0065] The results are shown in Table 3 and Figure 6 As shown, overexpression of miR-433-3p can downregulate the proliferation-related protein PCNA and induce the apoptosis-related protein BAX to change in a pro-apoptotic direction; interference with miR-433-3p causes the protein expression to change in the opposite direction, further verifying its regulatory role on the biological behavior of granulocytes at the protein level.

[0066] Therefore, miR-433-3p, as shown in SEQ ID NO.1, has a clear bidirectional regulatory function in sheep follicular granulosa cells: upregulation of its expression inhibits granulosa cell proliferation and promotes apoptosis, while downregulation of its expression promotes granulosa cell proliferation and reduces apoptosis. Thus, miR-433-3p can serve as a molecular tool for regulating granulosa cell function and as a candidate molecular marker in the construction of evaluation systems related to high fertility screening / breeding; existing experimental results support its candidate marker properties and research application value.

[0067] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A sheep small RNA miR-433-3p, characterized in that... Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant vector or liposome containing the encoding gene of the sheep small RNA miR-433-3p as described in claim 1.

3. The use of the sheep small RNA miR-433-3p according to claim 1 in the preparation of products for regulating the proliferation or apoptosis of sheep follicular granulosa cells.

4. The application according to claim 3, characterized in that... The product upregulates miR-433-3p expression to inhibit the proliferation of sheep follicular granulosa cells and promote apoptosis of sheep follicular granulosa cells; or The product downregulates miR-433-3p expression to promote the proliferation of sheep follicular granulosa cells and inhibit sheep follicular granulosa cell apoptosis.

5. Application of sheep small RNA miR-433-3p as a biomarker in screening high-fertility sheep.

6. Application of sheep small RNA miR-433-3p as a biomarker in constructing an evaluation system for screening high-fertility sheep.

7. A kit for detecting the proliferation and apoptosis of sheep follicular granulosa cells, characterized in that... The kit uses the sheep small RNA miR-433-3p described in claim 1 as the analyte.

8. The detection kit according to claim 7, characterized in that... The kit includes one or more of the following: primers and / or probes for detecting miR-433-3p expression levels, negative controls, internal control reagents, and RNA extraction, reverse transcription, or quantitative real-time PCR reagents.

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