SiRNA for specifically inhibiting expression of HOXA10 gene of dairy cow and application of siRNA

Abstract

The invention discloses siRNA for specifically inhibiting HOXA10 gene expression of a dairy cow and application of the siRNA. The invention designs a siRNA interference sequence of a target dairy cow HOXA10 gene. A positive-sense strand sequence and an antisense strand sequence are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The target sequence of the HOXA10 interfered by the siRNA is as shown in SEQ ID NO. 3. The siRNA can be combined with a target gene mRNA and promote the target gene mRNA to be degraded, so that the expression level of HOXA10 of the dairy cow is remarkably reduced, and the embryo implantation process of the dairy cow is influenced. The siRNA provided by the invention is high in interference efficiency on the transcriptional level, good in specificity, high in sensitivity, simple to operate, short in experimental period and low in price compared with a gene knockout technology, has an important application value in the aspects of exploring HOXA10 gene functions and regulatory pathways thereof, and provides a new tool for biological research and gene therapy of dairy cows.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a siRNA for specifically inhibiting the expression of cow HOXA10 gene and an application thereof. Background Art

[0002] The HOXA10 gene is an important member of the HOX gene family and plays a key role in embryo implantation and development, cell proliferation and differentiation, tissue repair and gene regulation. The abnormal expression of this gene is closely related to the disorder of various biological processes, such as tissue regeneration, cell proliferation, cell migration and differentiation. In particular, the role of HOXA10 in embryo implantation, embryo development and reproductive system of dairy cows has important research value for genetic improvement, disease research and biomedical fields of dairy cows.

[0003] However, the current research on the HOXA10 gene in dairy cows is not in-depth enough, especially in terms of its functional regulation and its mechanism of action at different developmental stages and physiological conditions. In order to better study the specific function of the HOXA10 gene in dairy cows and explore its interaction with other genes or signaling pathways, researchers need to use an efficient and specific technical means to inhibit the expression of the HOXA10 gene.

[0004] Currently, RNA interference (RNAi) technology has become an important tool for studying gene function. By using small interfering RNA (siRNA), the expression of the target gene can be specifically targeted and inhibited. siRNA molecules complementarily pair with the target mRNA, activate the RNA-induced silencing complex (RISC), thereby promoting the degradation of the target mRNA and inhibiting the expression of the target gene.

[0005] There is still a lack of efficient and specific siRNA interference tools for the study of the cow HOXA10 gene. In the prior art, although there are interference methods for the HOXA10 gene of other species, the design of specific siRNA sequences for the cow HOXA10 gene has not been reported. Therefore, the development of a siRNA that specifically interferes with the expression of the cow HOXA10 gene can provide an important experimental tool for the study of cow gene function, disease mechanism research and gene therapy. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a siRNA specifically inhibiting the expression of cow HOXA10 gene and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A siRNA that specifically inhibits the expression of the cow HOXA10 gene. The siRNA binds to the target gene mRNA and promotes its degradation, and has high interference efficiency and good specificity at the transcription level, thereby affecting the embryo implantation of the cow. The sequences of the sense chain and the antisense chain are:

[0009] Sense strand: 5'-GCAGGAUGAAACUGAAGAATT-3' (SEQ ID NO. 1),

[0010] Antisense strand: 5'-UUCUUCAGUUUCAUCCUGCTT-3' (SEQ ID NO. 2).

[0011] Preferably, the mRNA target sequence of the siRNA acting on the HOXA10 gene is: 5'-GCAGGAUGAAACUGAAGAA-3' (SEQ ID NO. 3).

[0012] The invention discloses an application of siRNA for specifically inhibiting the expression of cow HOXA10 gene, which is applied to inhibit gene expression during transcription.

[0013] Preferably, the gene is the HOXA10 gene of dairy cow.

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

[0015] 1. The RNA interference fragment provided by the present invention can specifically inhibit the expression of the cow HOXA10 gene at the post-transcriptional level. This siRNA has the advantages of high efficiency, strong specificity, and simple operation. Compared with traditional gene knockout technology, it has better flexibility and applicability, especially in the study of complex gene regulatory networks. It has important application prospects and provides an effective experimental tool for studying the regulatory mechanism of the cow HOXA10 gene on embryo implantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Cell visual field at different time points after si-HOXA10 transfection.

[0017] Figure 2 Real-time PCR and Western Blot methods were used to determine the mRNA and protein expression of siRNA interference

[0018] Figure 3 This is a graph showing the effect of interfering with the HOXA10 gene on the cell viability of bovine endometrial epithelial cells detected by the CCK8 method.

[0019] Figure 4 This is the common transcriptome sequencing diagram of bovine endometrial epithelial cells after interference with the HOXA10 gene.

[0020] Figure 5 Flow cytometric analysis of the effect of HOXA10 gene interference on the cell cycle of bovine endometrial epithelial cells.

[0021] Figure 6 This is a graph showing the effect of Real-time PCR on the expression of cell cycle genes in bovine endometrial epithelial cells by interfering with the HOXA10 gene.

[0022] Figure 7 This is a graph showing the effect of Real-time PCR on the expression of apoptosis genes in cow endometrial epithelial cells by interfering with the HOXA10 gene.

[0023] Figure 8 This is a diagram of Real-time PCR method used to detect the expression of adhesion factors of cow endometrial epithelial cells by interfering with the HOXA10 gene. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0025] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0026] Example 1: Cell culture

[0027] Bovine endometrial epithelial cells (bEECs) stored in the laboratory were thawed and cultured 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 maintained at 37°C, 5% CO2, and saturated humidity.

[0028] Example 2: si-HOXA10 transfection into cow endometrial epithelial cells

[0029] The interference fragment of the bovine HOXA10 gene (si-HOXA10) and the negative control sequence (si-NC) were synthesized by Jima Biotechnology Co., Ltd. bEECs were inoculated in a 6-well plate, and when the cells grew to 40% confluence, 3000 (Invitrogen, USA) was used for transfection of si-HOXA10 and si-NC. The specific operation is as follows 3000 kit instructions.

[0030] Table 1 siRNA nucleotide sequences

[0031]

[0032] Example 3: Total RNA extraction and Real-time PCR

[0033] (1) Total RNA extraction

[0034] 1) Take out the six-well plate, discard the culture medium, and wash three times with PBS.

[0035] 2) Add 1 mL of Trizol to each well of the six-well plate, shake well, place on ice for 1 min, and pipette the cells.

[0036] 3) Transfer the Trizol in the culture plate into a 1.5 mL EP tube, add 0.2 mL of chloroform to every 1 mL of Trizol, shake vigorously for 15 seconds, and place on ice for 5 minutes.

[0037] 4) Centrifuge at 4°C, 12,000 rpm for 15 min.

[0038] 5) Transfer the upper aqueous phase to a new 1.5 mL EP tube, add an equal volume of isopropanol, mix by inverting, and place on ice for 10 min.

[0039] 6) Centrifuge at 4°C, 12,000 rpm for 10 min.

[0040] 7) Discard the supernatant, add 1 mL of freshly prepared 75% ethanol to each tube, gently invert upside down, and wash the RNA precipitate once.

[0041] 8) Centrifuge at 4°C, 7500 rpm for 5 min.

[0042] 9) Discard the supernatant, place the 1.5 mL EP tube containing RNA precipitate in a clean bench, and dry it with a fan for 5 minutes.

[0043] 10) Add 20 μL DEPC water to the RNA precipitate to dissolve it.

[0044] 11) Measure the concentration of RNA by UV spectrophotometer and then use it for reverse transcription immediately or store it at -80℃.

[0045] (2) Reverse transcription

[0046] The two-step operation was performed according to the instructions of the PrimeScriptTMRTreagentKitwithgDNAEraser (TAKARA) kit. The reaction conditions for removing genomic DNA are shown in Table 2.

[0047] Table 2 Reaction system for removing genomic DNA

[0048]

[0049] The reverse transcription reaction system is shown in Table 3.

[0050] Table 3 TBGreen qPCR reverse transcription system

[0051]

[0052]

[0053] (3) Real-time PCR

[0054] The FastStart Universal SYBR Green Master (Rox) kit was used as described in the instructions. The Real-time PCR reaction system is shown in Table 4.

[0055] Table 4 Real-time PCR reaction system

[0056]

[0057] The above reagents were mixed thoroughly and added to 96-well plates (10 μL / well). The reaction conditions were 95°C, 10 min; 40 cycles 95°C, 15 s; 60°C, 1 min, 40 cycles.

[0058] With β-actin as the internal reference, the relative expression of the target gene was calculated using the 2-ΔΔCT method, where ΔCT=CT(target gene)-CT(β-actin). The Real-time PCR primer sequences of each gene are shown in Table 5.

[0059] Table 5 Real-time PCR primer sequences

[0060]

[0061] Example 4: Total protein extraction and Western Blot

[0062] (1) Extraction of total protein

[0063] 1) Taking a six-well plate as an example, take 1 mL of lysis buffer (RIPA) and add 10 μL of 100 nM PMSF within a few minutes before use to make the final concentration of PMSF 1 mM.

[0064] 2) Collect the cells, wash them three times with PBS, add 1 mL of 0.25% Trypsin-EDTA to each well, place them in a 37°C incubator for digestion for 3-5 minutes, and terminate the digestion with DMEM / F12 culture medium containing 10% fetal bovine serum.

[0065] 3) Centrifuge at 1500 rpm for 5 min; add 1 mL PBS to resuspend the cells, transfer to a new EP tube, centrifuge at 1500 rpm for 5 min, aspirate the PBS and set aside.

[0066] 4) Add 150 μL of cell lysis solution (containing PMSF) to each EP tube, pipette several times to allow the lysis solution to fully contact the cells, and place on ice for 10 minutes.

[0067] 5) After sufficient lysis, centrifuge at 4°C, 12,000 rpm for 5 min, and transfer the supernatant to a new EP tube, which is the total protein of the cells.

[0068] (2) Western Blot

[0069] 1) The concentration of the collected protein was determined using the BCA protein concentration assay kit (enhanced), and the determined protein was adjusted to the same concentration. An appropriate amount of 6× Loading Buffer was added and heated to boil at 100°C for 5 min to fully denature the protein.

[0070] 2) After the protein samples have cooled to room temperature, load 20-40 μg / μL of each sample and perform conventional electrophoresis at 100 V for 20 min; 140 V for 50 min.

[0071] 3) After the electrophoresis is completed, the sponge, transfer filter paper, gel and PVDF membrane are placed in the semi-dry transfer apparatus in order, the power is turned on, and the membrane is transferred at a constant current of 100 mA for 70 minutes.

[0072] 4) Place the PVDF membrane in PBST (blocking solution) containing 5% skimmed milk powder and shake on a shaker at room temperature for 2 h.

[0073] 5) Wash off the blocking solution on the PVDF membrane with PBST, and incubate the PVDF membrane in the dilution solution containing the primary antibody and place it in a 4°C refrigerator overnight.

[0074] 6) Wash the membrane three times with PBST, 10 min each time, and then incubate the membrane in blocking solution containing secondary antibody and shake on a shaker for 2 h at room temperature.

[0075] 7) The membrane was washed three times with PBST, each time for 10 min, and then detected by chemiluminescence imaging using an ultra-sensitive ECL chemiluminescence kit.

[0076] Example 5: Detection of si-HOXA10 transfection efficiency

[0077] In order to detect the interference effect of the interference fragment, when the cell confluence reached about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) were transfected into the cells respectively. After 48 hours of transfection, RNA and protein were extracted from the cells, and the interference effect of si-HOXA10 was detected by Real-time PCR and Western Blot. Figure 2 The results showed that si-HOXA10 had a good interference effect and could be used as a direct and effective experimental tool to study the function and regulatory mechanism of the cow HOXA10 gene.

[0078] Example 6: CCK-8 detection of cell viability after si-HOXA10 interference

[0079] bEECs were seeded in 96-well plates and when the cells grew to 40% confluence, 3000 for transfection of si-HOXA10 and si-NC. Add 10 μL CCK-8 reagent to each well at 0h, 12h, 24h and 48h of transfection, mix gently, and continue to culture for 2-3 hours at 37°C, 5% CO2 and saturated humidity. Measure the absorbance value (OD) at 450nm by an ELISA reader, and calculate the cell viability based on the absorbance value (OD). Repeat the experiment three times for each treatment group. The absorbance value (OD) is between 0.8-2.0. Figure 3 It was shown that interfering with the HOXA10 gene affected cell viability.

[0080] Example 7: Sequencing of normal transcriptome of cells after si-HOXA10 interference

[0081] Taking a 6-well plate as an example, when the cell confluence reaches about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) are transfected into the cells respectively. After 48 hours of transfection, 1 mL of Trizol is added to each well, placed on ice for 1 min, and the cells are blown. The Trizol in the culture plate is transferred to a 1.5 mL EP tube and sent to Novogene Technology Co., Ltd. for conventional transcriptome sequencing. Figure 4 The results of KEGG and GO enrichment analysis of differential mRNA induced by interfering HOXA10 were shown, and subsequent experiments can focus on the regulation of cell cycle, DNA repair, autophagy, cell senescence and apoptosis. Table 6 shows the differential expression of the target genes to be detected.

[0082] Table 6 Differential expression of target genes

[0083]

[0084] Example 8: Flow cytometry detection of the effect of HOXA10 gene interference on the cycle expression of cow endometrial epithelial cells

[0085] Taking a 6-well plate as an example, when the cell confluence reached about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) were transfected into the cells respectively. 48 hours after transfection, the expression levels of each stage of the cell cycle in bovine endometrial epithelial cells were detected by flow cytometry. Figure 5 As shown, interference with the HOXA10 gene blocked the expression of the G1 phase of cow endometrial epithelial cells.

[0086] Example 9: Interference with HOXA10 gene in detecting the mRNA of cycle marker genes in cow endometrial epithelial cells

[0087] Taking a 6-well plate as an example, when the cell confluence reached about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) were transfected into the cells respectively. After 48 hours of transfection, the expression levels of marker genes in the G1 and G2 phases of the cell cycle in bovine endometrial epithelial cells were detected by Real-time PCR. Figure 6 As shown, after HOXA10 gene group interference, the expression levels of CCNE1, CCNB1, and CDK2 were significantly lower than those in the negative control group (** indicates P<0.01), and the expression levels of CDKN1A, CCND1, and CDK4 were significantly higher than those in the negative control group (* indicates P<0.05, ** indicates P<0.01).

[0088] Example 10: Interference with HOXA10 gene to detect apoptosis marker gene mRNA in cow endometrial epithelial cells

[0089] Taking a 6-well plate as an example, when the cell confluence reached about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) were transfected into the cells respectively. After 48 hours of transfection, the expression level of apoptosis marker genes in bovine endometrial epithelial cells was detected by Real-time PCR. Figure 8 As shown, after interfering with the HOXA10 gene set, the expression level of BAX was significantly higher than that of the negative control group (** indicates P<0.01), and the expression level of BCL-2 was significantly lower than that of the negative control group (* indicates P<0.05).

[0090] Example 11: Interference with HOXA10 gene to detect adhesion-related gene mRNA in cow endometrial epithelial cells

[0091] Taking a 6-well plate as an example, when the cell confluence reached about 40%, si-NC (negative control group) and si-HOXA10 (experimental group) were transfected into the cells, and 48 hours after transfection, the expression level of apoptosis marker genes in bovine endometrial epithelial cells was detected by Real-time PCR. Interfering with the HOXA10 genome, the expression of MUC1 was significantly higher than that of the negative control group (** indicates P<0.01), and the expression of ITGB3, ITGB6, and ITGAV was significantly lower than that of the negative control group (* indicates P<0.05, ** indicates P<0.01).

[0092] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A siRNA that specifically inhibits the expression of cow HOXA10 gene, characterized in that: The siRNA binds to the target gene mRNA and promotes its degradation, and has high interference efficiency and good specificity at the transcription level, thereby affecting the embryo implantation of dairy cows. The sequences of the sense chain and antisense chain are: The positive strand is shown in SEQ ID NO.1, The antisense strand is shown in SEQ ID NO.

2.

2. The siRNA for specifically inhibiting the expression of cow HOXA10 gene and its application according to claim 1, characterized in that: The mRNA target sequence of the siRNA acting on the HOXA10 gene is shown in SEQ ID NO.

3.

3. An application of siRNA for specifically inhibiting the expression of cow HOXA10 gene, characterized in that: It is used to inhibit gene expression during transcription.

4. The use of a siRNA for specifically inhibiting the expression of cow HOXA10 gene according to claim 3, characterized in that: The gene is the HOXA10 gene of dairy cows.

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