Molecular marker for promoting proliferation of sheep endometrial cells and application of molecular marker

Abstract

The invention discloses a molecular marker for promoting proliferation of sheep endometrial cells and application of the molecular marker, and relates to the field of gene agriculture, the molecular marker is oar-miR-493-3p, and the sequence of the oar-miR-493-3p is 5 '-UGAAGGUCUACUGUGUGCCAGG-3' SEQ ID No: 01; the invention finds that the molecular marker oar-miR-493-3p can increase the expression quantity of receptivity related genes AREG and LTF, and promote the proliferation of sheep endometrial cells; the method is widely applied to the field of sheep reproduction diagnostic kits, and can be used for early pregnancy diagnostic kits or embryo implantation diagnostic kits and the like.

Description

Technical Field

[0001] The invention relates to the field of gene agriculture, in particular to a molecular marker for promoting the proliferation of sheep endometrial cells and an application thereof. Background Art

[0002] Poor endometrial receptivity is closely related to embryo implantation failure, embryo loss, and embryo death, and endometrial epithelial cell apoptosis is an important process for establishing uterine receptivity and embryo implantation. Therefore, studying the mechanism of endometrial epithelial cell apoptosis is closely related to improving endometrial receptivity and embryo implantation rate.

[0003] Studies have found that exosomes secreted by the embryo can change the morphology of the endometrium and its gene expression profile, thereby regulating the positioning of the embryo and promoting embryo implantation.

[0004] In the study of embryonic exosomes, it was found that miRNAs can regulate the biological processes of early embryonic development. In addition, the abnormal expression of miRNAs has been shown to be associated with reproductive system diseases such as polycystic ovary syndrome and endometriosis. In summary, miRNAs play a major role in the regulation of endometrial function (epithelial-mesenchymal transition, migration, and endometrial decidualization, etc.) and the differentiation, migration, and invasion of embryonic trophoblast cells, and play an important regulatory role in the communication between the uterus and the embryo.

[0005] The applicant previously used exosomes derived from sheep trophoblast cells to treat Hu sheep endometrial cells, and confirmed its promoting effect on the proliferation and migration of Hu sheep endometrial cells, and speculated that it could promote the formation of sheep endometrial receptive state. By sequencing endometrial cells treated with exosomes derived from sheep trophoblast cells and untreated endometrial cells, multiple differentially expressed miRNAs were found, and oar-miR-493-3p, which was upregulated in the exosome treatment group, was selected for subsequent experiments.

[0006] Related studies have shown that miR-493-3p is closely related to the proliferation, apoptosis, and invasion of human ovarian cancer cells. In addition, studies on sheep have found that miR-493-3p can affect the proliferation of granulosa cells and testicular cells. Therefore, it is speculated that miR-493-3p is related to the proliferation and apoptosis of sheep endometrial cells. The present invention has experimentally verified for the first time that the molecular marker oar-miR-493-3p promotes the proliferation of sheep endometrial cells, broadening its application in the field of sheep reproductive diagnostic kits. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a molecular marker for promoting the proliferation of sheep endometrial cells and its application. The present invention finds that the molecular marker oar-miR-493-3p can increase the expression of receptivity-related genes AREG and LTF, and promote the proliferation of sheep endometrial cells; it has a wide range of applications in the field of sheep reproductive diagnostic kits.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A molecular marker for promoting the proliferation of sheep endometrial cells, the molecular marker is oar-miR-493-3p, and the sequence of oar-miR-493-3p is: 5'-UGAAGGUCUACUGUGUGCCAGG-3'SEQ ID No: 01.

[0010] The aforementioned molecular marker that promotes the proliferation of sheep endometrial cells, oar-miR-493-3p miRNAminics, treated Hu sheep endometrial cells, and the expression levels of uterine receptivity-related genes AREG and LTF increased.

[0011] The aforementioned molecular marker that promotes the proliferation of sheep endometrial cells, oar-miR-493-3p, can promote the formation of the endometrial receptive state of Hu sheep.

[0012] The aforementioned molecular marker related to the body shape traits of Hu sheep, oar-miR-493-3p, increases cell proliferation and cell activity.

[0013] The aforementioned application of a molecular marker related to the body shape trait of Hu sheep is applied to an early sheep pregnancy diagnostic kit.

[0014] The aforementioned application of a molecular marker for promoting the proliferation of sheep endometrial cells is applied to an embryo implantation diagnosis kit.

[0015] Proper Noun:

[0016] EECs:endometrial epithelial cells;

[0017] minics: miRNA mimics, miRNA mimics are a type of synthetic small RNA molecules, usually designed as a double-stranded structure that mimics the function of endogenous miRNA.

[0018] The present invention is beneficial in that:

[0019] The experiment of the present invention confirmed for the first time that oar-miR-493-3p can increase the expression of receptivity-related genes AREG and LTF, and promote the proliferation of sheep endometrial cells;

[0020] The molecular markers of the present invention can be applied to early pregnancy diagnostic kits to improve the reproduction success rate, reduce breeding costs, and promote the healthy development of the sheep industry; the molecular markers of the present invention can be applied to diagnostic kits for embryo implantation, and in production to improve the implantation success rate of technologies such as embryo transplantation, bring significant economic benefits and improve reproduction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the expression results of oar-miR-493-3p after the exosomes of the present invention are treated in sheep EECs;

[0022] Figure 2 Schematic diagram of the expression levels of genes related to the receptivity state of EECs after transfection with oar-miR-493-3p minics of the present invention;

[0023] Figure 3 Schematic diagram of the experiment of detecting the proliferation ability of EECs treated with oar-miR-493-3p by EdU and CCK-8 of the present invention (ab, schematic diagram of cell proliferation number observed by Nikon Eclipse Ts2R inverted microscope; c, schematic diagram of oar-miR-493-3p cell activity). DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Molecular marker screening method:

[0026] ①Small RNA sequencing and differential miRNAs screening:

[0027] After the samples were tested and qualified, the library was constructed. The special structure of the 3' and 5' ends of Small RNA (complete phosphate groups at the 5' end and hydroxyl groups at the 3' end) was used. The total RNA was used as the starting sample, and the adapters were directly added to both ends of the Small RNA, and then reverse transcribed to synthesize cDNA. Subsequently, PCR amplification, PAGE gel electrophoresis to separate the target DNA fragments, and the gel was cut and recovered to obtain the cDNA library. The sequencing results were analyzed by bioinformatics technology, and the Edge R differential analysis software was used to standardize the data. The differential analysis obtained the significantly differentially expressed miRNAs in the exosome-treated group and the untreated group, and the differential screening condition was p-Value≤0.05. Among them, there were 20 upregulated miRNAs in the exosome-treated group. In the validation experiment, it was found that oar-miR-493-3p was the most significantly upregulated, so this miRNA was selected for subsequent experiments.

[0028] The sequence of Oar-miR-493-3p is: 5'-UGAAGGUCUACUGUGUGCCAGG-3'

[0029] ②Real-time fluorescence quantitative PCR:

[0030] Total RNA was extracted from cells and reverse transcribed into cDNA. qPCR was performed using the procedure of qPCR SYBR Green Master Mix (No Rox) (11201ES08).

[0031] The qPCR reaction system is as follows:

[0032] (1) Add to each well of the PCR plate 5 μL of qPCR SYBR Green Master Mix (No Rox), 0.5 μL of upstream and downstream primers (10 μmol / L), 1 μL of cDNA, and then add ddH2O to make up to 10 μL.

[0033] (2) The qPCR reaction procedure was as follows: pre-denaturation at 95°C for 10 min; followed by 40 cycles of 95°C for 15 s; 55-58°C for 20 s; and then 72°C for 20 s.

[0034] After the reaction, U6 and β-Actin (ACTB) were selected as internal reference genes, and 2 -△△Ct The relative expression levels of target genes were analyzed.

[0035] ③EdU proliferation assay

[0036] Huyang endometrial cells were passaged into 96-well plates and transfected when the cell density reached 50%. After 48 hours of transfection, the preheated EdU working solution was added to the 96-well plates in the form of replacement solution and incubated in a 37°C, 5% CO2 incubator for 2 hours. TM The results were processed using Image J.

[0037] ④CCK-8 proliferation assay

[0038] Hu sheep endometrial cells were passaged into 96-well plates and transfected when the cell density reached 50%. CCK-8 working solution was added to the 96-well plates in the form of replacement solution at 12, 24, 48, and 72 hours after cell transfection, respectively, and incubated in a 37°C, 5% CO2 incubator for 2 hours. The absorbance at 450 nm was then measured using an ELISA reader.

[0039] 3. Experimental Results

[0040] 1. Expression of oar-miR-493-3p in sheep EECs after exosome treatment Figure 1 As shown, Figure 1 qPCR results showed that the expression level of oar-miR-493-3p was significantly increased after Hu sheep endometrial cells were treated with exosomes derived from sheep trophoblast cells.

[0041] 2. The expression levels of genes related to receptivity in EECs after transfection with oar-miR-493-3p minics Figure 2 As shown, Figure 2 qPCR results showed that after oar-miR-493-3p minics treated Hu sheep endometrial cells, the expression levels of uterine receptivity-related genes AREG and LTF were significantly increased. Therefore, it is speculated that oar-miR-493-3p can promote the formation of Hu sheep endometrial receptivity.

[0042] 3. EdU and CCK-8 detection of the proliferation ability of EECs treated with oar-miR-493-3p minics Figure 3 As shown, EdU results showed that the number of cell proliferation increased significantly after oar-miR-493-3p treatment, and CCK-8 results showed that the cell activity increased after oar-miR-493-3p treatment.

[0043] In summary:

[0044] The experiment of the present invention confirmed for the first time that oar-miR-493-3p can increase the expression of receptivity-related genes AREG and LTF, and promote the proliferation of sheep endometrial cells. The molecular marker of the present invention can be applied to early pregnancy diagnosis kits to improve the success rate of reproduction, reduce breeding costs, and promote the healthy development of the sheep industry; the molecular marker of the present invention can be applied to the diagnosis kit of embryo implantation, and in production to improve the implantation success rate of embryo transplantation and other technologies, it brings significant economic benefits and reproductive efficiency improvement.

[0045] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A molecular marker for promoting the proliferation of sheep endometrial cells, characterized in that: The molecular marker is oar-miR-493-3p, and the sequence of oar-miR-493-3p is: 5'-UGAAGGUCUACUGUGUGCCAGG-3'SEQ ID No:

01.

2. A molecular marker for promoting the proliferation of sheep endometrial cells according to claim 1, characterized in that: After the oar-miR-493-3p miRNA minics were used to treat Hu sheep endometrial cells, the expression levels of uterine receptivity-related genes AREG and LTF increased.

3. A molecular marker for promoting the proliferation of sheep endometrial cells according to claim 2, characterized in that: The oar-miR-493-3p can promote the formation of the endometrial receptive state of Hu sheep.

4. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: oar-miR-493-3p increases cell proliferation and cell activity.

5. The use of a molecular marker related to the body shape traits of Hu sheep according to claim 1, characterized in that: Applied in early sheep pregnancy diagnostic kit.

6. The use of a molecular marker for promoting the proliferation of sheep endometrial cells according to claim 1, characterized in that: Applied to embryo implantation diagnosis kit.

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