CN118667815B - A nucleotide sequence targeting PRDM1 to improve bovine endometritis and its application - Google Patents
A nucleotide sequence targeting PRDM1 to improve bovine endometritis and its application Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
本发明提供了一种靶向PRDM1的核苷酸序列及其用途，属于基因工程技术领域。靶向PRDM1的核苷酸如SEQ ID NO.2所示。靶向PRDM1的核苷酸和通过siRNA降低PRDM1的表达，从而提高牛子宫内膜炎症过程中的子宫内膜上皮细胞的活力和增殖数量，可在改善牛子宫内膜炎症的治疗过程中进行应用，如制备治疗牛子宫内膜炎的核苷酸药物等。
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
The invention belongs to the technical field of genetic engineering, and particularly relates to a nucleotide sequence of a target PRDM1 and application thereof in improving bovine endometritis.
Background
Uterus is an important organ in the reproductive system of cows (Bos taurus), and whether uterus is healthy directly affects reproductive performance of cows, which is a key factor in determining the profitability of a dairy. Endometritis is one of the uterine inflammations of cows, seriously harms the reproductive system health of cows, causes cow reproductive disorders, and is one of the most common reproductive disorder diseases in cow breeding industry in China. The incidence rate of the endometritis of the dairy cows in different areas at home and abroad is different, but basically is 14% -40%. The main reason for the reproduction disorder of the dairy cows caused by endometritis is that a large number of white blood cells and bacteria generated in the uterus inhibit the combination of sperms and ova, so that the dairy cows are infertility, and the infertility of the dairy cows accounts for 70%. Endometritis is high after delivery of cows. The birth canal of the milk cow is damaged during delivery, and the endometritis can be caused by retained fetal membranes, abortion, uterine infection and the like. The sick cows mainly show symptoms of body temperature rise, appetite reduction, listlessness, bad smell discharge, lochia, delayed estrus and the like. If the treatment can be effectively performed in time, the milk cows can quickly recover reproductive performance. If the improper treatment can cause septicemia, the cows are caused to be sterile for a long time or have secondary abortion, and finally can only be forced to be eliminated, so that the breeding benefit is damaged.
Histologically, endometritis is characterized by disintegration of endometrial epithelial cells, different degrees of lymphocyte and plasma cell aggregation on the surface layer of endometrium, inflammatory cell infiltration, vascular congestion, and stromal edema. Endometrium is the first barrier against infection by pathogenic microorganisms in the female reproductive system, and is a mucosal layer containing a monolayer of columnar epithelial cells, covering a matrix rich in vascular, immune 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. Endometrial epithelium receives embryo attachment and contacts the genital flora and is part of the innate immunity of the uterus. Endometrial epithelial cells function as a guard through a variety of pathways, with secretory and immune response functions. Endometrial epithelial cells constitute a physical barrier by their own, intercellular tight junctions and mucins secreted into the uterine cavity. The integrity of endometrial epithelial cells is of great importance in maintaining normal reproductive functions of the body, such as implantation of fertilized eggs, maintenance of pregnancy. When the integrity of the endometrial epithelium is compromised, further invasion of pathogenic microorganisms and harmful substances will compromise the health of the whole body. An important way for many pathogenic bacteria such as E.coli to cause uterine diseases is to destroy the integrity of the endometrial epithelium. The epithelial cells act as the outer surface of the uterine cavity and are first contacted by pathogenic bacteria that enter the uterine cavity. Thus, communication between epithelial cells and immune cells is particularly important for the body to maintain normal reproductive function and to be protected from infection. Studies have shown that, although cow endometrial epithelial cells cannot directly phagocytose pathogenic bacteria like immune cells, they can be used as a main component of communication between intrauterine cells by secreting various cytokines such as tumor necrosis factor α, interleukin IL-1, IL-6, IL-8, etc. In addition, epithelial cells play an important role in the initiation of inflammatory responses, and participate in the recognition of E.coli and lipopolysaccharide by expressing Toll-like receptors and pattern recognition receptors such as the nucleic acid binding oligomerization domain protein family NOD-like receptors, thereby promoting the synthesis and release of inflammatory cytokines. Therefore, in the course of treatment of endometritis in cows, it is important to promote proliferation of endometrial epithelial cells and maintain the activity and integrity of endometrial epithelial cells. PRDM1 protein, which is commonly referred to as PR domain-containing protein 1 (PR-domain containing protein 1), also commonly referred to as Blimp-1 (B-lymphocyte-induced maturation protein-1), is an important transcription factor, playing a key role in cell differentiation and immunomodulation. At present, the effect and application of the nucleotide sequence of the target PRDM1 in treating the endometritis of the dairy cows have not been reported yet.
Disclosure of Invention
Based on the problems in the background art, the invention aims to provide a PRDM1 targeting nucleotide sequence and application thereof in improving bovine endometritis.
In one aspect, the invention provides a nucleotide sequence of targeting PRDM1, wherein the nucleotide sequence is shown as SEQ ID NO. 2.
Preferably, the nucleotide sequence of the targeting PRDM1 is an siRNA.
In another aspect, the invention provides a composition for treating bovine endometritis, characterized in that the composition comprises the aforementioned nucleotide sequence.
In another aspect, the invention provides a composition for promoting LPS-induced proliferation of bovine endometrium epithelial cells, characterized in that the composition comprises the nucleotide sequence as described above.
In another aspect, the invention provides the use of any one of the nucleotide sequences or compositions described above in the manufacture of a medicament for the treatment of bovine endometritis.
In another aspect, the invention provides the use of any one of the nucleotide sequences or compositions described above in the manufacture of a medicament for promoting LPS-induced proliferation of bovine endometrial epithelial cells.
In another aspect, the invention provides a method of non-disease treatment for promoting LPS-induced proliferation of bovine endometrium epithelial cells, characterized in that said method is effected by reducing PRDM1 expression.
Preferably, said reducing PRDM1 expression is achieved by interfering with PRDM1 expression.
Preferably, said interfering PRDM1 expression is achieved by siRNA interference using the nucleotide sequence of claim 1.
In another aspect, the invention provides the use of any of the methods described above as a positive control for assessing the efficacy of a method of treating bovine endometritis, characterized in that the method reduces PRDM1 expression and promotes LPS-induced proliferation of bovine endometrium epithelial cells.
Preferably, said reducing PRDM1 expression is achieved by interfering with PRDM1 expression.
Preferably, said interfering PRDM1 expression is achieved by siRNA interference using the nucleotide sequence of claim 1.
Compared with the prior art, the invention has the following advantages:
1) The invention provides a brand new siRNA targeting PRDM1, which can be used for researching the function of PRDM1 and is applicable to any biological function research related to PRDM 1.
2) The siRNA provided by the invention can obviously promote proliferation of the bovine endometrial epithelial cells in the bovine endometrial inflammation, and has a certain effect on improving the bovine endometrial inflammation.
3) The method of the invention can also be used as a positive control for evaluating the effect of the method for treating bovine endometritis in basic scientific research.
Drawings
The beneficial effects of the present invention for improving endometritis in cattle are described in detail below with reference to the accompanying drawings and detailed description.
FIG. 1RT-qPCR detection of interference efficiency of siRNA targeting different siRNAs of PRDM 1.
FIG. 2RT-qPCR detection of expression levels of CDK2, CCND2, CCNE2 and PCNA mRNA in si-PRDM1_1 samples.
FIG. 3CCK8 detects cell viability.
FIG. 4EdU detection cell proliferation (200×) blue for all cells and red for EdU positive cells.
FIG. 5 flow cytometry detects cell cycle.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Example 1 vector construction and cell transfection
1. Cell culture and transfection
Frozen endometrial epithelial cells are taken out in liquid nitrogen, melted in a 37 ℃ rapid water bath, centrifuged at 1000rpm, and the supernatant is discarded, added with a culture medium (DMEM+10% FBS) to resuspend the cells, inoculated in a 10mm culture dish, cultured in a 37 ℃ and 5% CO2 cell incubator, and the cells are subjected to cell passage until the cells grow to about 80% for subsequent experiments. According to the treatment and biological repetition of the test requirements, inoculating the cells with good growth state into a 6-hole cell culture plate, and changing the Opti culture medium when the density reaches about 50% -60%. The siRNA of the gene was diluted to a final concentration of 50nM for use according to the instructions, and premixed according to a transfection system of 110. Mu.L Opti+3.5. Mu.L Lip 3000,110. Mu.L Opti+5. Mu.L siRNA, left standing for 5min, the system was mixed well and left standing for 20min, and then dropped into 6 well plates uniformly. After 6h of cell transfection, the complete medium was changed and after 42h of induction with LPS (final concentration 50 ng/. Mu.L) for 6h, the subsequent detection was performed.
2. Gene expression analysis
Total RNA of endometrial epithelial cells is extracted by a Trizol method, the integrity and concentration of RNA samples are determined by agarose gel electrophoresis and NanoDrop 2000c, and cDNA is prepared by referring to TaKaRa reverse transcription kit instructions. RT-qPCR detection was performed by the 2 XM 5 HiPer SYBR Premix EsTaq instruction system, GAPDH was chosen as an internal control, and finally the relative expression levels of mRNAs were calculated using the 2 XM -ΔΔCt method. The RT-qPCR detection primers are shown in Table 1, and the gene interference sequences are shown in Table 2.
TABLE 1RT-qPCR detection primers
TABLE 2 Gene interference sequence siRNA
The results are shown in FIGS. 1 and 2, where the si-PRDM 1-1 interference sequence works best and the PRDM1 expression level is significantly reduced compared to the control. In the si-PRDM1_1 sample, the expression of CDK2, CCND2, CCNE2 and PCNA genes are all obviously up-regulated, and the genes are proliferation marker genes and represent the proliferation level of cells.
Example 2 detection of cell proliferation and apoptosis
Cell proliferation was examined using CCK-8 kit, and endometrial epithelial cells with good growth status were seeded in 96-well plates with 6 independent replicates per treatment group. When the density reached about 50%, the Control group was maintained, and then si-NC and si-PRDM 1-1 were transfected into endometrial epithelial cells, respectively, and culturing was continued for 6h, 12h, 24h and 48h, followed by the addition of LPS to induce inflammation 6h before detection. 10. Mu.L of CCK-8 reagent is added into the well, incubated for 1h in a 37 ℃ and 5% CO 2 cell incubator, and the OD value of 450nm wavelength is detected by using a multifunctional full-automatic enzyme-labeled instrument.
Cell proliferation was examined using the EdU kit, 2 XEdU working solution (20. Mu.M) was prepared and pre-heated, and added to a culture solution of endometrial epithelial cells transfected for 48 hours (LPS-induced 6 hours) (final concentration: 10. Mu.M), and after incubation of the cells for 2 hours, staining was performed according to the BeyoClick TM EdU Cell Proliferation Kit with Alexa Fluor 555 protocol, and photographic observation was performed under a fluorescent inverted microscope (3 biological replicates per treatment group). The number of cells was counted using image J, and the EdU labeling index (%) was obtained by calculating the ratio of EdU-staining positive cells to the total number of DAPI-staining cells in each field.
The cell cycle and apoptosis detection kit is adopted to detect the cell cycle condition, endometrium epithelial cells transfected for 48 hours (LPS induction for 6 hours) are digested by pancreatin until the cells can be gently blown down by a gun head, after the cells are neutralized by adding culture medium, the cells are centrifuged for 5 minutes at 1000rpm, 1mL of precooled PBS is added to resuspend the cells, the sediment cells are centrifuged again, the supernatant is carefully sucked, 1mL of precooled 70% ethanol is added to resuspend the cells, and the cells are fixed for 12 hours at 4 ℃. The pelleted cells were centrifuged, resuspended in 1mL of pre-chilled PBS again, and then the pelleted cells were continued to be centrifuged. In the dark, 0.5mL propidium iodide staining solution was added to each tube of cells to slowly and fully resuspend the cell pellet, and the cells were incubated at 37℃for 30min in the dark and detected by flow cytometry.
As shown in fig. 3-5, the cell viability of the si-PRDM 1+ LPS sample was significantly improved at 48h relative to the si-NC + LPS (fig. 3), the number of cell proliferation of the si-PRDM 1+ LPS sample was significantly improved relative to the si-NC + LPS (fig. 4), and the si-PRDM 1+ LPS sample had more cells in the dividing active cell cycle relative to the si-NC + LPS (fig. 5). In conclusion, the interference of PRDM1 expression can promote the proliferation of bovine endometrium epithelial cells induced by LPS, which shows that the method has potential application value in improving bovine endometritis.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A nucleotide sequence of a target PRDM1, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.
2. A composition for treating bovine endometritis, said composition comprising the nucleotide sequence of claim 1.
3. A composition for promoting LPS-induced proliferation of bovine endometrium epithelial cells, said composition comprising the nucleotide sequence of claim 1.
4. Use of the nucleotide sequence or composition of any one of claims 1-3 in the preparation of a medicament for treating bovine endometritis.
5. Use of the nucleotide sequence or composition of any one of claims 1-3 in the preparation of a medicament for promoting LPS-induced proliferation of bovine endometrium epithelial cells.
6. A method for promoting LPS-induced proliferation of bovine endometrium epithelial cells for non-diagnostic and non-therapeutic purposes, said method being effected by reducing PRDM1 expression by siRNA interference using the nucleotide sequence of claim 1.
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