SiRNA for specifically inhibiting expression of CX3CL1 gene of dairy cow and application of siRNA

Abstract

The invention discloses siRNA for specifically inhibiting the expression of a dairy cow CX3CL1 gene and application of the siRNA, and relates to the technical field of biology. The problems existing in the prior art are solved. The siRNA specifically comprises a positive-sense strand and an antisense strand of the siRNA molecule, and the positive-sense strand is as shown in SEQ ID NO. 1; the antisense strand is as shown in SEQ ID NO. 2; and the mRNA target sequence of the siRNA acting on the CX3CL1 gene is as shown in SEQ ID NO. 3. The expression of the dairy cow CX3CL1 gene can be specifically inhibited on the transcriptional level, the siRNA has the advantages of high interference efficiency, good stability, high specificity and the like, and compared with a traditional gene knockout technology, the siRNA is high in sensitivity, easy and convenient to operate, short in experimental period, low in price and suitable for popularization and application. The method has an important application value for researching the functions of the dairy cow CX3CL1 gene and a regulation mechanism for embryo implantation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a siRNA for specifically inhibiting expression of a CX3CL1 gene of a dairy cow and application thereof. BACKGROUND

[0002] Successful pregnancy is essential for the profitability and sustainability of the dairy industry, and biological factors such as oocyte quality, embryo development capacity, endometrial functional status, and crosstalk between the embryo and the endometrium affect the likelihood of successful pregnancy. Studies have shown that 40% of embryos die within the first 45 days of pregnancy in cattle, and complex biochemical interactions occur between the embryo and the endometrium before and during implantation, and disruption of these interactions can lead to embryo loss and thus implantation failure.

[0003] CX3CL1 is a unique chemokine, and during the process of embryo implantation, CX3CL1 is closely related to cell adhesion, vascular remodeling, cell proliferation and differentiation, migration, etc. Studies have shown that elevated CX3CL1 can exacerbate uterine inflammation, thereby disrupting endometrial receptivity and leading to embryo implantation failure or miscarriage; in addition, abnormal expression of CX3CL1 is associated with various biological processes, such as apoptosis, cell proliferation and differentiation, and repeated implantation failure. Therefore, studying the role of CX3CL1 in embryo implantation, embryo development, and the reproductive system of dairy cows has important value for genetic improvement, disease research, and the biomedical field of dairy cows.

[0004] There is no siRNA for specifically inhibiting expression of a CX3CL1 gene of a dairy cow in the prior art, which hinders researchers from studying the function and regulation mechanism of the CX3CL1 gene of a dairy cow, and thus the function and regulation mechanism of the CX3CL1 gene of a dairy cow are not yet clear. Therefore, developing a siRNA for specifically interfering with expression of the CX3CL1 gene of a dairy cow can provide an important experimental tool for studying the function of a dairy cow gene, disease mechanisms, and gene therapy. SUMMARY

[0005] The present application aims to solve the problems in the prior art and provides a siRNA for specifically inhibiting expression of a CX3CL1 gene of a dairy cow and application thereof.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A siRNA for specifically inhibiting expression of a CX3CL1 gene of a dairy cow, comprising a sense strand and an antisense strand of a siRNA molecule, wherein the sense strand is shown as SEQ ID NO. 1;

[0008] the antisense strand is shown as SEQ ID NO. 2;

[0009] The siRNA acts on the mRNA target sequence of the CX3CL1 gene, as shown in SEQ ID NO. 3.

[0010] Preferably, the sequence of the sense strand is: 5'-GGGCGAAAGCAGUAGUCUGTT-3'.

[0011] Preferably, the sequence of the antisense strand is: 5'-CAGACUACUGCUUUCGCCCTT-3'.

[0012] Preferably, the mRNA target sequence of the CX3CL1 gene on which the siRNA acts is: 5'-GGGCGAAAGCAGUAGUCUG-3'.

[0013] The application of a siRNA that specifically inhibits the expression of the CX3CL1 gene in cows, the siRNA interference sequence is used for biological research and gene therapy of cows, the siRNA can significantly reduce the expression level of the CX3CL1 gene in cows by binding to the target gene mRNA and promoting its degradation, thereby affecting the expression of the endometrial receptivity-related genes in cows, and further affecting the embryo implantation process.

[0014] The beneficial effects of the present application are:

[0015] The sense and antisense strands of the siRNA molecule proposed in the present application can specifically bind to the target gene mRNA and promote its degradation, promote the expression of the receptivity-related genes in cows, thereby significantly reducing the expression level of the CX3CL1 gene in cows, affecting the expression of the endometrial receptivity-related genes in cows, and further affecting the embryo implantation process; the siRNA interference fragment proposed in the present application can specifically inhibit the expression of the CX3CL1 gene in cows at the transcription level, and this siRNA has the advantages of high interference efficiency, good stability, and strong specificity, and compared with traditional gene knockout technology, it has high sensitivity, simple operation, short experimental period, and low price, and has important application value for studying the function of the CX3CL1 gene in cows and the regulation mechanism of embryo implantation, and provides a new tool for biological research and gene therapy of cows. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The ordinary transcriptome sequencing graph after LPS treatment of the endometrial epithelial cells of cows proposed in the present application;

[0017] Figure 2 The mass spectrum of siCX3CL1-153, siCX3CL1-459, siCX3CL1-962, and siCX3CL1-1085 proposed in the present application;

[0018] Figure 3Figure of mRNA and protein expression map determined by Real-time PCR method and Western Blot method of the present application for siRNA interference;

[0019] Figure 4 Figure of mRNA of the influence of interfering CX3CL1 gene on the receptivity genes of dairy cow endometrial epithelial cells detected by Real-time PCR method of the present application;

[0020] Figure 5 Figure of protein of the influence of interfering CX3CL1 gene on the receptivity genes of dairy cow endometrial epithelial cells detected by Western blot method of the present application;

[0021] Figure 6 Figure of mRNA and protein expression map determined by Real-time PCR method and Western Blot method of the present application for overexpression of CX3CL1 gene;

[0022] Figure 7 Figure of mRNA of the influence of overexpression of CX3CL1 gene on the receptivity genes of dairy cow endometrial epithelial cells detected by Real-time PCR method of the present application;

[0023] Figure 8 Figure of protein of the influence of overexpression of CX3CL1 gene on the receptivity genes of dairy cow endometrial epithelial cells detected by Western blot method of the present application. DETAILED DESCRIPTION

[0024] The technical solutions of the present patent will be further described in detail below in combination with specific embodiments.

[0025] The embodiments of the present patent will be described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present patent, and cannot be understood as a limitation of the present patent.

[0026] Embodiment 1:

[0027] A siRNA specifically inhibiting the expression of CX3CL1 gene of dairy cow, comprising a sense strand and an antisense strand of siRNA molecule, the sequence of the sense strand is: 5'-GGGCGAAAGCAGUAGUCUGTT-3'(as shown in SEQ ID NO. 1);

[0028] the sequence of the antisense strand is: 5'-CAGACUACUGCUUUCGCCCTT-3'(as shown in SEQ ID NO. 2);

[0029] The mRNA target sequence of the siRNA acting on the CX3CL1 gene is 5'-GGGCGAAAGCAGUAGUCUG-3' (as shown in SEQ ID NO. 3);

[0030] In use, the sense and antisense strands of the siRNA molecule can specifically bind to the target gene mRNA and promote its degradation, promote the expression of the cow receptivity-related gene, thereby significantly reducing the expression level of the cow CX3CL1, affecting the expression of the cow endometrial receptivity-related gene, and further affecting the embryo implantation process.

[0031] Example 2:

[0032] The application of the siRNA for specifically inhibiting the expression of the cow CX3CL1 gene can be applied to biological research and gene therapy of cows.

[0033] The siRNA interference fragment provided by the application can specifically inhibit the expression of the cow CX3CL1 gene at the transcription level, and has the advantages of high interference efficiency, good stability, strong specificity, high sensitivity, simple operation, short experimental period, low price, important application value for studying the function of the cow CX3CL1 gene and the regulation mechanism of embryo implantation, and provides a new tool for biological research and gene therapy of cows.

[0034] Test Example 1:

[0035] 1. Cell culture: Thaw the laboratory-preserved bovine endometrial epithelial cells (BEECs). Culture the cells in DMEM / F12 medium (Sigma, China, Cat. no.: D8437) supplemented with 10% fetal bovine serum (PAN-Biotech, Germany, Cat. no.: P30-3306) and 1% double antibody, and maintain at 37°C, 5% CO2, and saturated humidity.

[0036] 2. General transcriptome sequencing of cells after LPS treatment:

[0037] ①BEECs were inoculated in 60mm culture dishes, and when the cells grew to 70% confluence, the cells were randomly divided into two groups, namely the control group and the LPS (Solarbio, China, Cat. no.: IL2020) group;

[0038] ②Each group of cells was treated as follows: the control group was cultured using DMEM / F12 + 10% FBS + 1% double-antibody basic culture solution; the LPS group was cultured using basic culture solution + 100 μg / mL LPS.

[0039] ③After 24 h of treatment, the cells were collected using TRNzol (TIANGEN, China, Cat. no.: DP424) and sent to Novogene Bioinformatics Technology Co., Ltd. for ordinary transcriptome sequencing.

[0040] Results are shown in Figure 1 The results show that the differential genes after LPS treatment were analyzed, and it was found that the chemokine family (GRO1, CX3CL1, CXCL3, CXCL5, etc.) was significantly up-regulated, and then GO and KEGG enrichment analysis was performed, and the subsequent experiment mainly focused on the regulation of CX3CL1 on the receptive gene. Table 1 below is the differential expression of the chemokine family genes.

[0041] Table 1:

[0042]

[0043]

[0044] 3、si-CX3CL1 design and synthesis:

[0045] The CDS sequence of CX3CL1 was obtained on NCBI, and siRNA targeting CX3CL1 was designed and synthesized based on this, for reducing the expression level of CX3CL1, and the sequence shown in Table 2 (CX3CL1 target sequence and siRNA sequence) was obtained after screening, and the mass spectrometry results are shown in Figure 2 (The interference fragments of the bovine CX3CL1 gene and the negative control were synthesized by Jimabio Technology Co., Ltd.).

[0046] Table 2:

[0047]

[0048]

[0049] 4、si-CX3CL1 transfection of bovine endometrial epithelial cells, the specific operation is as follows:

[0050] 1) One day before transfection, the bovine endometrial epithelial cells were evenly plated in a six-well plate, and the DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% double-antibody was added, and placed in a 37℃, 5% CO2 incubator for 12 h;

[0051] 2) On the transfection day, replace the DMEM medium (containing 10% FBS) without double-antibody;

[0052] 3) Take 125 μL DMEM to dilute siRNA, and take another 125 μL DMEM to dilute 3000 (American Invitrogen Corporation), mix well, then stand at room temperature for 5 min, then mix the two, stand at room temperature for 15 min;

[0053] 4) Add 250 μL of the above mixture to each well, the final transfection volume is 2 mL, and the working concentration of siRNA is 50 nM;

[0054] 5) After transfection for 48 h, the cells were collected.

[0055] 5, Overexpression of CX3CL1 transfected bovine endometrial epithelial cells, the specific operation as follows:

[0056] 1) One day before transfection, the bovine endometrial epithelial cells were evenly plated in a six-well plate, and the DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% double antibody was placed in a 37℃, 5% CO2 incubator for 12 h;

[0057] 2) On the day of transfection, replace the DMEM medium (containing 10% FBS) without double antibody;

[0058] 3) Configuration of transfection complex: add 125 μL DMEM, 5 μL P3000, 5 μL overexpression plasmid to a 1.5 mL EP tube, take another 1.5 mL EP tube, add 125 μL DMEM, 5 μL 3000, mix well, then stand at room temperature for 5 min, then mix the two, stand at room temperature for 15 min;

[0059] 4) Add 250 μL of the above transfection complex to each well, the final transfection volume is 2 mL, mix well, and incubate in the incubator for 6 h;

[0060] 5) After 6 h of transfection, discard the old medium, and replace it with complete medium for 24 h, and collect the cells.

[0061] 6, total RNA extraction and Real-time PCR:

[0062] Among them, the method for extracting total RNA is as follows:

[0063] 1) Take out the six-well plate, discard the old medium, and wash with pre-cooled PBS for 3 times;

[0064] 2) Add 1 mL of pre-cooled TRNzol to each well of the six-well plate, shake well, and stand on ice for 1 min, then transfer to a 1.5 mL centrifuge tube and label;

[0065] 3) Add 200 μL pre-cooled chloroform to each centrifuge tube, shake well, and let stand on ice for 15 s, then centrifuge at 4°C, 12000 rpm / min for 15 min;

[0066] 4) Centrifuge at 4°C, 12000 rpm / min for 15 min;

[0067] 5) Take about 400 μL supernatant (as much as possible), and transfer to a new centrifuge tube;

[0068] 6) Add an equal volume of pre-cooled isopropanol, shake well, and let stand on ice for 10 min;

[0069] 7) Centrifuge at 4°C, 12000 rpm / min for 10 min to obtain a clear precipitate;

[0070] 8) Carefully remove the supernatant, and add 1 mL pre-cooled 75% ethanol solution to each tube, and gently invert to wash the tube wall and precipitate;

[0071] 9) Centrifuge at 4°C, 7500 rpm / min for 5 min;

[0072] 10) Open the centrifuge tube and invert it on a clean filter paper, and place it in a clean bench to dry the precipitate for 1-2 min to remove ethanol;

[0073] 11) According to the size of the precipitate, add about 20-50 μL DEPC water to dissolve the precipitate, and mix well to make the RNA solution uniform in concentration;

[0074] 12) Measure the concentration of RNA using a microspectrophotometer, and then immediately use it for reverse transcription or store it at -80°C for later use.

[0075] The reverse transcription was performed according to the instructions of the Jiangsu Yugong Biological Reverse Transcription Kit, and the reaction conditions for removing genomic DNA are shown in Table 3, and the reverse transcription reaction system is shown in Table 4.

[0076] Table 3:

[0077]

[0078] Table 4:

[0079]

[0080]

[0081] Among them, Real-time PCR:

[0082] The mRNA expression of the target gene was detected by real-time fluorescent quantitative RT-qPCR, and the reaction system is shown in Table 5.

[0083] Table 5:

[0084] Reagent Amount used ROX 5.0 μL Forward Primer (10 μM) 0.3 μL Reverse Primer (10 μM) 0.3 μL Template DNA 1.0 μL DEPC water 3.4 μL

[0085] Mix the reagents well and add to 96-well plates (10 μL / well) respectively. The reaction conditions are 95℃, 10 min; 95℃, 15 s; 60℃, 1 min, repeated for 40 cycles. The melting curve is collected by the default program of the instrument.

[0086] All primers were synthesized by JESGene Co. Ltd. The sequences of the primers for Real-time PCR are shown in Table 6. The relative expression of the target genes was calculated by the 2-ΔΔCT method using β-actin as the internal reference.

[0087] Table 6:

[0088]

[0089]

[0090] 7. Extraction of total protein and Western Blot:

[0091] The method for extracting total protein is as follows:

[0092] 1) Take out the six-well plate, discard the old culture medium, wash with pre-cooled PBS for 3 times, add 1 mL of trypsin to each well, then put it in a 37℃ incubator for 3-5 min, and terminate the digestion with DMEM / F12 culture medium containing 10% fetal bovine serum;

[0093] 2) Centrifuge at 1500 rpm for 5 min, add 1 mL of pre-cooled PBS, blow the cell pellet to disperse, repeat the blowing and washing, then move to a new EP tube, centrifuge at 1500 rpm for 5 min, and discard all the residual PBS for the next protein extraction or store at -80℃;

[0094] 3) Prepare the protein lysis solution: mix RIPA protein strong lysis solution and PMSF (proteinase inhibitor) at a volume ratio of 200:1 according to the cell amount, mix well, and store at 4℃;

[0095] 4) Add 150-300 μL of protein lysis solution according to the cell sample amount, repeat the blowing to ensure complete lysis, and if necessary, use an ultrasonic cell disruptor, and place it in an ice box for complete lysis for 10 min;

[0096] 5) After complete lysis, centrifuge at 4℃, 13000 g, for 5 min, take the supernatant and move it to a new EP tube, and wait for the concentration determination.

[0097] The method for determining the protein concentration is as follows:

[0098] 1) Refer to the BCA protein concentration assay kit instructions to determine the protein concentration of the sample;

[0099] 2) Adjust the measured protein to the same concentration, add an appropriate amount of 5x Loading Buffer (1 / 4 of the protein volume), mix well, and heat at 100°C for 10 min to denature the protein.

[0100] Western Blot:

[0101] 1) After the protein sample cools to room temperature, use a pipette to add marker and protein sample to the sample well, and control the protein sample in each well to be between 20-40 μg / μL by calculation;

[0102] 2) The first stage runs at 100V constant voltage for 20 min to compress the sample and align the bands; the second stage runs at 140V constant voltage for 50 min to complete the separation process;

[0103] 3) After electrophoresis, assemble the "filter paper→PVDF membrane→gel→filter paper" in order in the membrane transfer device according to the "sandwich" assembly method, connect the power supply, set the constant current condition to 100 mA, and transfer the membrane for 70 min. The actual time is adjusted according to the molecular weight of the target protein;

[0104] 4) Immerse the PVDF membrane in the pre-prepared 5% skim milk blocking solution, place it in a shaker, and block it at room temperature for 2 h at 40 r / min;

[0105] 5) Wash the blocking solution off the PVDF membrane with TBST at room temperature on a shaker at 80 r / min for 8 min each time, for a total of 3 times;

[0106] 6) Immerse the washed PVDF membrane in the diluent containing the primary antibody, and place it in the refrigerator at 4°C overnight;

[0107] 7) Wash the membrane with TBST 3 times for 8 min each time, then incubate the membrane in the secondary antibody-containing incubation solution, and place it in a shaker at room temperature at 40 r / min for 2 h;

[0108] 8) Wash the membrane with TBST 3 times for 8 min each time, then use the Super Sensitive ECL chemiluminescence kit for chemiluminescence imaging detection;

[0109] 9) Use Image J software to measure the gray value of the protein band.

[0110] 8. Detection of si-CX3CL1 transfection efficiency:

[0111] To detect the interference effect of the interfering fragments, when the cell confluence reached about 60%, si-NC (negative control group) and siCX3CL1-153, siCX3CL1-459, siCX3CL1-962, and siCX3CL1-1085 (experimental group) were transfected into the cells. 48 hours after transfection, RNA and protein were extracted from the cells, and the interference effect was detected by Real-time PCR and Western Blot.

[0112] The results are as follows Figure 3 As shown, siCX3CL1-459 (denoted as si-CX3CL1) has a good interference effect and can be used as a direct and effective experimental tool for studying the mechanism of CX3CL1 regulation of receptivity genes in dairy cows.

[0113] 9. Detection of mRNA of receptivity marker gene in bovine endometrial epithelial cells by interfering with the CX3CL1 gene:

[0114] When the cell confluence reached about 60%, si-NC (negative control group) and si-CX3CL1 (experimental group) were transfected into the cells, respectively. 48 h after transfection, the expression level of receptivity marker gene mRNA in bovine endometrial epithelial cells was detected by Real-time PCR.

[0115] The results are as follows Figure 4 As shown, after interfering with CX3CL1, the expression level of VEGF mRNA did not change significantly compared with the negative control group (the same letter indicates P>0.05), while the expression levels of HOXA10 and LIF mRNA were significantly higher than those of the negative control group (different letters indicate P<0.05).

[0116] 10. Detection of the receptivity marker gene protein of bovine endometrial epithelial cells by interfering with the CX3CL1 gene:

[0117] When the cell confluence reached about 60%, si-NC (negative control group) and si-CX3CL1 (experimental group) were transfected into the cells, respectively. 48 h after transfection, the expression level of receptivity marker gene protein in bovine endometrial epithelial cells was detected by Western blot.

[0118] The results are as follows Figure 5 As shown, after interfering with CX3CL1, the expression level of VEGF protein did not change significantly compared with the negative control group (the same letter indicates P>0.05), while the expression levels of HOXA10 and LIF proteins were significantly higher than those of the negative control group (different letters indicate P<0.05).

[0119] 11. Detection of CX3CL1 overexpression transfection efficiency:

[0120] To detect the overexpression effect of CX3CL1, when the cell confluence reached about 60%, NC (negative control group) and OE-CX3CL1 (experimental group) were transfected into the cells, respectively. After 24 h of transfection, the RNA and protein in the cells were extracted, and the overexpression effect was detected by Real-time PCR and Western Blot.

[0121] As shown in the results Figure 6 , the overexpression effect of OE-CX3CL1 was significant (marked as OE), which could be used for the subsequent research on the mechanism of CX3CL1 regulating receptivity genes.

[0122] 12. Detection of mRNA of receptivity marker genes of bovine endometrial epithelial cells by overexpression of CX3CL1 gene:

[0123] When the cell confluence reached about 60%, NC (negative control group) and OE-CX3CL1 (experimental group) were transfected into the cells, respectively. After 24 h of transfection, the expression level of mRNA of receptivity marker genes in bovine endometrial epithelial cells was detected by Real-time PCR.

[0124] As shown in the results Figure 7 , after overexpression of CX3CL1, the mRNA expression level of VEGF was significantly higher than that of the negative control group (different letters represent P<0.05), and the mRNA expression levels of HOXA10 and LIF were significantly lower than those of the negative control group (different letters represent P<0.05).

[0125] 13. Detection of protein of receptivity marker genes of bovine endometrial epithelial cells by overexpression of CX3CL1 gene:

[0126] When the cell confluence reached about 60%, NC (negative control group) and OE-CX3CL1 (experimental group) were transfected into the cells, respectively. After 24 h of transfection, the expression level of protein of receptivity marker genes in bovine endometrial epithelial cells was detected by Western Blot.

[0127] As shown in the results Figure 8 , after overexpression of CX3CL1, the protein expression level of VEGF was significantly higher than that of the negative control group (different letters represent P<0.05), and the protein expression levels of HOXA10 and LIF were significantly lower than those of the negative control group (different letters represent P<0.05).

[0128] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A siRNA that specifically inhibits the expression of a CX3CL1 gene of a dairy cow, comprising a sense strand and an antisense strand of a siRNA molecule, characterized in that, The sense strand is shown as SEQ ID NO.

1. The antisense strand is shown as SEQ ID NO.

2. The siRNA acts on the mRNA target sequence of the CX3CL1 gene, which is shown as SEQ ID NO.

3.

2. The siRNA for specifically inhibiting the expression of CX3CL1 gene in dairy cows according to claim 1, characterized in that, The sequence of the sense strand is: 5'-GGGCGAAAGCAGUAGUCUGTT-3'.

3. The siRNA of claim 1, wherein the siRNA specifically inhibits the expression of CX3CL1 gene in dairy cows. The sequence of the antisense strand is: 5'-CAGACUACUGCUUUCGCCCTT-3'.

4. The siRNA of claim 1, wherein the siRNA specifically inhibits the expression of CX3CL1 gene in dairy cows. The mRNA target sequence of the siRNA acting on the CX3CL1 gene is: 5'-GGGCGAAAGCAGUAGUCUG-3'.

5. Use of a siRNA according to any one of claims 1 to 4, characterized in that, The siRNA interference sequence is used for dairy cow biological research and gene therapy. By binding to the target gene mRNA and promoting its degradation, the siRNA can significantly reduce the expression level of dairy cow CX3CL1, affect the expression of dairy cow endometrial receptivity related genes, and then affect the embryo implantation process.

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