A nucleotide sequence targeting PRDM1 to improve bovine endometritis and its application

Abstract

The present invention provides a nucleotide sequence targeting PRDM1 and its use, belonging to the field of gene engineering technology. The nucleotide targeting PRDM1 is shown in SEQ ID NO.2. The nucleotide targeting PRDM1 and reducing the expression of PRDM1 by siRNA, thereby increasing the vitality and proliferation number of endometrial epithelial cells in the process of cow endometrial inflammation, can be used in the treatment process of improving cow endometrial inflammation, such as preparing a nucleotide drug for treating cow endometritis.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a nucleotide sequence targeting PRDM1 and an application thereof in improving bovine endometritis. Background Art

[0002] The uterus is an important organ in the reproductive system of dairy cows (Bos taurus). The health of the uterus directly affects the reproductive performance of dairy cows, and reproductive performance is a key factor in determining the profitability of dairy farms. Endometritis is a type of uterine inflammation in dairy cows, which seriously endangers the health of the reproductive system of dairy cows and leads to reproductive disorders in dairy cows. It is one of the most common reproductive disorders in my country's dairy farming industry. The incidence of endometritis in dairy cows in different regions at home and abroad varies, but it is basically between 14% and 40%. The main reason for endometritis to cause reproductive disorders in dairy cows is that a large number of white blood cells and bacteria produced in the uterus inhibit the combination of sperm and eggs, leading to infertility in dairy cows, accounting for 70% of infertility in dairy cows. Endometritis is more common after dairy cows give birth. Damage to the birth canal during delivery, retained placenta, miscarriage, uterine infection, etc. can all cause endometritis. The main symptoms of sick cows are fever, decreased appetite, listlessness, discharge of odorous lochia, and delayed estrus. If treated in a timely and effective manner, dairy cows can quickly restore their reproductive performance. If improperly treated, it may induce sepsis, leading to long-term infertility or secondary miscarriage in dairy cows, which will eventually be forced to be culled, damaging the breeding benefits.

[0003] Histologically, endometritis is characterized by the disintegration of endometrial epithelial cells, the aggregation of lymphocytes and plasma cells to varying degrees on the surface of the endometrium, inflammatory cell infiltration, vascular congestion, and matrix edema. The endometrium is the first barrier to pathogenic microbial infection in the reproductive system of female livestock. It is a mucosal layer containing a single layer of columnar epithelial cells covering a matrix rich in blood vessels, immune cells, and endometrial stromal cells. Endometrial epithelial cells are present on the inner surface of the uterus and are in direct contact with the uterine cavity. The endometrial epithelium accepts embryo implantation and comes into contact with the reproductive tract flora, and is part of the innate immunity of the uterus. Endometrial epithelial cells play a sentinel role through multiple pathways and have secretory and immune response functions. Endometrial epithelial cells form a physical barrier through their own tight junctions between cells and mucin secreted into the uterine cavity. The integrity of endometrial epithelial cells is of great significance to maintaining the normal reproductive function of the body, such as the implantation of fertilized eggs and the maintenance of pregnancy. When the integrity of the endometrial epithelium is destroyed, the further invasion of pathogenic microorganisms and harmful substances will endanger the health of the entire body. Many pathogens such as Escherichia coli cause uterine diseases by destroying the integrity of the endometrial epithelium. As the outer surface of the uterine cavity, epithelial cells first come into contact with pathogens that enter the uterine cavity. Therefore, communication between epithelial cells and immune cells is particularly important for the body to maintain normal reproductive function and avoid infection. Studies have shown that although cow endometrial epithelial cells cannot directly phagocytize pathogens like immune cells, they can secrete a variety of cytokines such as tumor necrosis factor α, interleukin IL-1, IL-6, IL-8, etc., as the main components of communication between cells in the uterus. In addition, epithelial cells also play an important role in the initiation of inflammatory responses. By expressing pattern recognition receptors such as Toll-like receptors and nucleic acid binding oligomerization domain protein family NOD-like receptors, they participate in the recognition of Escherichia coli and lipopolysaccharide and promote the synthesis and release of inflammatory cytokines. Therefore, in the treatment of endometritis in dairy cows, it is very important to promote the proliferation of endometrial epithelial cells and maintain the activity and integrity of endometrial epithelial cells. PRDM1 protein, whose full name is PR-domain containing protein 1, is also often called Blimp-1 (B lymphocyte-induced maturation protein-1). It is an important transcription factor that plays a key role in cell differentiation and immune regulation. At present, the role and application of nucleotide sequences targeting PRDM1 in the treatment of endometritis in dairy cows have not been reported. Summary of the invention

[0004] Based on the problems existing in the background technology, the purpose of the present invention is to provide a nucleotide sequence targeting PRDM1 and its application in improving bovine endometritis.

[0005] In one aspect, the present invention provides a nucleotide sequence targeting PRDM1, wherein the nucleotide sequence is shown as SEQ ID NO.2.

[0006] Preferably, the nucleotide sequence targeting PRDM1 is siRNA.

[0007] On the other hand, the present invention provides a composition for treating bovine endometritis, characterized in that the composition comprises the aforementioned nucleotide sequence.

[0008] On the other hand, the present invention provides a composition for promoting LPS-induced proliferation of bovine endometrial epithelial cells, characterized in that the composition comprises the aforementioned nucleotide sequence.

[0009] In another aspect, the present invention provides use of any one of the aforementioned nucleotide sequences or compositions in the preparation of a medicament for treating bovine endometritis.

[0010] On the other hand, the present invention provides the use of any one of the aforementioned nucleotide sequences or compositions in the preparation of a drug for promoting LPS-induced proliferation of bovine endometrial epithelial cells.

[0011] On the other hand, the present invention provides a non-disease treatment method for promoting LPS-induced proliferation of bovine endometrial epithelial cells, characterized in that the method is achieved by reducing PRDM1 expression.

[0012] Preferably, the reduction of PRDM1 expression is achieved by interfering with PRDM1 expression.

[0013] Preferably, the interference with PRDM1 expression is achieved by using the nucleotide sequence of claim 1 to perform siRNA interference.

[0014] On the other hand, the present invention provides the use of any of the aforementioned methods as a positive control for evaluating the effectiveness of a method for treating bovine endometritis, wherein the method reduces PRDM1 expression and promotes LPS-induced proliferation of bovine endometrial epithelial cells.

[0015] Preferably, the reduction of PRDM1 expression is achieved by interfering with PRDM1 expression.

[0016] Preferably, the interference with PRDM1 expression is achieved by using the nucleotide sequence of claim 1 to perform siRNA interference.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1) The present invention provides a new siRNA targeting PRDM1, which is a reagent for studying the function of PRDM1 and can be used in any biological function research related to PRDM1.

[0019] 2) The siRNA provided by the present invention significantly promotes the proliferation of bovine endometrial epithelial cells in bovine endometrial inflammation, and has a certain effect on improving bovine endometrial inflammation.

[0020] 3) The method of the present invention can also be used as a positive control in basic scientific research to evaluate the effectiveness of methods for treating bovine endometritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The beneficial effects of the present invention on improving cow endometrial inflammation are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] Figure 1 RT-qPCR was used to detect the interference efficiency of different siRNAs targeting PRDM1.

[0023] Figure 2 RT-qPCR was used to detect the expression levels of CDK2, CCND2, CCNE2 and PCNA mRNA in si-PRDM1_1 samples.

[0024] Figure 3 CCK8 was used to detect cell viability.

[0025] Figure 4 EdU detection of cell proliferation (200×) blue indicates all cells, and red indicates EdU positive cells.

[0026] Figure 5 Cell cycle was detected by flow cytometry. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0029] Example 1: Vector construction and cell transfection

[0030] 1. Cell culture and transfection

[0031] Take out the frozen endometrial epithelial cells from liquid nitrogen, thaw quickly in a water bath at 37°C, discard the supernatant after centrifugation at 1000rpm, add culture medium (DMEM+10% FBS) to resuspend the cells, inoculate in a 10mm culture dish, and culture in a cell culture incubator at 37°C and 5% CO2. When the cells grow to about 80%, pass the cells for subsequent experiments. According to the treatment and biological replication required by the experiment, cells with good growth status are inoculated in a 6-well cell culture plate, and the Opti culture medium is replaced when the density reaches about 50% to 60%. Dilute the siRNA of the gene to a final concentration of 50nM for standby use according to the instructions, and premix according to the transfection system of 110μL Opti+3.5μL Lip 3000, 110μL Opti+5μL siRNA, let stand for 5min, mix the system and let stand for 20min, and evenly drop it into a 6-well plate. 6 h after cell transfection, complete culture medium was replaced, and LPS (final concentration 50 ng / μL) was added at 42 h to induce for 6 h before subsequent detection.

[0032] 2. Gene expression analysis

[0033] Total RNA from endometrial epithelial cells was extracted using the Trizol method. The integrity and concentration of RNA samples were determined by agarose gel electrophoresis and NanoDrop2000c. cDNA was prepared according to the instructions of the TaKaRa reverse transcription kit. RT-qPCR was performed using the 2×M5HiPer SYBR Premix EsTaq instruction system. GAPDH was selected as the internal reference. Finally, 2 -ΔΔCt Methods The relative expression levels of mRNAs were calculated. The primers for RT-qPCR detection are listed in Table 1, and the gene interference sequences are shown in Table 2.

[0034] Table 1 RT-qPCR detection primers

[0035]

[0036] Table 2 Gene interference sequence siRNA

[0037]

[0038]

[0039] Results Figure 1 and Figure 2 Among them, the si-PRDM1_1 interference sequence had the best effect, and the expression of PRDM1 was significantly reduced compared with the control. In the si-PRDM1_1 sample, the expression of CDK2, CCND2, CCNE2, and PCNA genes were significantly upregulated. These genes are proliferation marker genes, reflecting the level of cell proliferation.

[0040] Example 2: Detection of cell proliferation and apoptosis

[0041] The cell proliferation was detected by CCK-8 kit. The well-growing endometrial epithelial cells were inoculated in 96-well plates, and each treatment group had 6 independent replicates. When the density reached about 50%, the Control group was retained, and then si-NC and si-PRDM1_1 were transfected into the endometrial epithelial cells, and the cells were cultured for 6h, 12h, 24h and 48h. LPS was added 6h before the detection to induce inflammation. 10μL CCK-8 reagent was added to the wells, incubated in a cell culture incubator at 37℃ and 5% CO2 for 1h, and the OD value at a wavelength of 450nm was detected using a multifunctional fully automatic microplate reader.

[0042] EdU kit was used to detect cell proliferation. 2× EdU working solution (20 μM) was prepared and preheated, and then added to the culture medium of endometrial epithelial cells transfected for 48 h (LPS induced for 6 h) (final concentration was 10 μM). After incubating the cells for 2 h, the cells were labeled with BeyoClick. TM EdU Cell Proliferation Kit with Alexa Fluor 555 was used for staining and photographed under a fluorescent inverted microscope (each treatment group had 3 biological replicates). Image J was used to count the number of cells, and the EdU labeling index (%) was obtained by calculating the ratio of EdU-positive cells to the total number of DAPI-stained cells in each field of view.

[0043] The cell cycle and apoptosis detection kit was used to detect the cell cycle. The endometrial epithelial cells that were transfected for 48 hours (LPS induced for 6 hours) were digested with trypsin until they could be gently blown down with a gun tip. After neutralization with the medium, they were centrifuged at 1000rpm for 5 minutes, 1mL of pre-cooled PBS was added to resuspend the cells, and the cells were centrifuged again to precipitate the cells. The supernatant was carefully removed, and 1mL of pre-cooled 70% ethanol was added to resuspend the cells, and the cells were fixed at 4℃ for 12 hours. Centrifuge the precipitated cells, add 1mL of pre-cooled PBS to resuspend the cells again, and then continue to centrifuge the precipitated cells. In the dark, 0.5mL of propidium iodide staining solution was added to each tube of cells to slowly and fully resuspend the cell pellet, incubate at 37℃ in the dark for 30 minutes, and detect with a flow cytometer.

[0044] The results are as follows Figure 3 to Figure 5 As shown, the cell viability of the si-PRDM1_1+LPS sample was significantly improved compared with that of the si-NC+LPS sample at 48h ( Figure 3 ), the number of cell proliferation in the si-PRDM1_1+LPS sample was significantly higher than that in the si-NC+LPS sample ( Figure 4), and the si-PRDM1_1+LPS sample had more cells in the active cell cycle compared with si-NC+LPS ( Figure 5 ). In summary, interfering with PRDM1 expression can promote LPS-induced proliferation of bovine endometrial epithelial cells, indicating that this method has potential application value in improving bovine endometritis.

[0045] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nucleotide sequence targeting PRDM1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

2. A composition for treating bovine endometritis, characterized in that: The composition comprises the nucleotide sequence of claim 1.

3. A composition for promoting LPS-induced proliferation of bovine endometrial epithelial cells, characterized in that: The composition comprises the nucleotide sequence of claim 1.

4. Use of the nucleotide sequence or composition according to any one of claims 1 to 3 in the preparation of a medicament for treating bovine endometritis.

5. Use of the nucleotide sequence or composition according to any one of claims 1 to 3 in the preparation of a drug for promoting LPS-induced proliferation of bovine endometrial epithelial cells.

6. A method for promoting LPS-induced proliferation of bovine endometrial epithelial cells for non-diagnostic and non-therapeutic purposes, characterized in that: The method is achieved by reducing the expression of PRDM1, and the reduction of PRDM1 expression is achieved by using the nucleotide sequence of claim 1 to perform siRNA interference.

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