SiRNA for inhibiting chicken RASGRP3 gene expression and application thereof

Abstract

The invention discloses siRNA (small interfering Ribonucleic Acid) for inhibiting the expression of a chicken RASGRP3 (Reduced Amplification of Sequence Growth Protein 3) gene and application of the siRNA, the siRNA comprises a positive-sense strand and an antisense strand, and is any one of RASGRP3-Anas-214, RASGRP3-Anas-846 and RASGRP3-Anas-1187. According to the specific siRNA aiming at the chicken RASGRP3 mRNA provided by the invention, in a chicken liver cancer cell line (LMH), after liposome-mediated transfection culture is performed for 48 hours, RT-qPCR is adopted to evaluate the transcript level, and a result shows that the relative expression quantity of the chicken RASGRP3 mRNA is remarkably reduced compared with that of negative control, and the repeatability is good. The polypeptide can be further used for inhibiting ALV-J replication, provides a new molecular intervention target for prevention and control of avian leukosis, and has important scientific research and potential economic values.

Description

Technical Field

[0001] This invention belongs to the field of animal and poultry biomedicine, specifically relating to an siRNA that inhibits the expression of the chicken RASGRP3 gene and its application. Background Technology

[0002] The RASGRP3 (RAS guanyl releasing protein 3) gene encodes the Ras small GTPase guanine nucleotide exchange factor (GEF), which promotes the transition of Ras protein from a GDP-binding inactive state to a GTP-binding activated state, thereby activating downstream signaling pathways such as Ras-MAPK. RASGRP3 is highly conserved in vertebrates and participates in regulating key biological processes such as cell proliferation, survival, and metabolic state. Ras-MAPK signaling is closely related to the remodeling of the host cellular environment during various viral infections, potentially providing favorable conditions for viral replication. Existing research mainly focuses on the role of RASGRP3 in tumorigenesis and immune signaling regulation. However, in the field of avian viral infections, especially in the context of Avian leukosis virus subgroup J (ALV-J) infection, there is limited publicly available research on the expression characteristics and functions of RASGRP3, as well as targeted interventions. There is a lack of publicly available information on specific inhibitory nucleic acid molecules (such as siRNA) designed to target the characteristics of the avian RASGRP3 gene sequence and their validation studies on their ability to inhibit ALV-J replication in avian cells or tissues. The relevant mechanisms still need to be further elucidated.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA approximately 21 nt in length, often with 2 nt 3′ overhangs at both ends. After entering the cytoplasm, it is loaded into the RNA-induced silencing complex (RISC), where AGO2 catalyzes the cleavage of complementary mRNA between 10-11 bases at the antisense strand pairing site, thereby inhibiting gene expression at the posttranscriptional level. Compared to vector-expressed shRNA, synthetic siRNA offers advantages such as controllable dosage, rapid onset of action, no risk of genome integration, and ease of standardization in in vitro systems, and has been widely used in gene function studies in cell models. To improve stability and specificity, chemical modifications are often introduced at the sugar ring and backbone levels, and the efficiency of antisense strand entry into the RISC is enhanced by regulating the thermodynamic asymmetry of the double strand and strand loading preference. Sequence design targeting chicken RASGRP3 typically follows these principles: prioritizing accessible fragments located in the RASGRP3 coding region (CDS) or 3′UTR, controlling GC content to approximately 30-55%; performing whole transcriptome alignment in the antisense strand "seed region" (positions 2-8) to exclude potential complementarity with non-target mRNA 3′UTRs; and avoiding known polymorphic sites while incorporating secondary structure prediction to reduce the risk of target site masking. In poultry cells, liposome-mediated transfection is commonly used. With blank and non-target sequence negative controls, the degree of target gene mRNA inhibition is evaluated by RT-qPCR 24-48 h post-transfection. Existing research primarily focuses on human / mouse species or other gene targets; studies targeting chicken RASGRP3 are scarce, and there are no publicly available reports of "using siRNA to interfere with chicken RASGRP3 gene expression." Summary of the Invention

[0004] Objective of the invention: To address the shortcomings of existing technologies, this invention provides an siRNA that inhibits the expression of the chicken RASGRP3 gene. This specific siRNA can reduce the relative expression level of chicken RASGRP3 mRNA and exhibits good reproducibility.

[0005] The present invention also provides the application of the siRNA that inhibits the expression of the chicken RASGRP3 gene.

[0006] Technical solution: In order to achieve the above objective, the present invention provides an siRNA for inhibiting the expression of the chicken RASGRP3 gene, wherein the siRNA comprises a sense strand and an antisense strand, and is any one of the following: RASGRP3-Anas-214, RASGRP3-Anas-846 and RASGRP3-Anas-1187;

[0007] RASGRP3-Anas-214 Justice Chain: GAAGGCUGCAACAUUAGAUTT (SEQ ID NO. 1)

[0008] RASGRP3-Anas-214 antisense chain: AUCUAAUGUUGCAGCCUUCTT (SEQ ID NO. 2)

[0009] RASGRP3-Anas-846 Justice Chain: GGGCAGAAGYGAUCACAAATT (SEQ ID NO. 3)

[0010] RASGRP3-Anas-846 antisense chain: UUUGUGAUCACUUCUGCCCTT (SEQ ID NO. 4)

[0011] RASGRP3-Anas-1187 Justice Chain: CAGCUUUCCAUCACCUUGATT (SEQ ID NO. 5)

[0012] RASGRP3-Anas-1187 antisense chain: UCAAGGUGAUGGAAAGCUGTT (SEQ ID NO. 6).

[0013] The siRNA is RASGRP3-Anas-846.

[0014] The siRNA is synthesized chemically.

[0015] The present invention relates to the application of the siRNA used to inhibit chicken RASGRP3 gene expression in the preparation of reagents that reduce RASGRP3 mRNA levels in chicken-derived cells.

[0016] The application of the siRNA for inhibiting chicken RASGRP3 gene expression described in this invention in the preparation of anti-ALV-J virus drugs.

[0017] The siRNA is used in the preparation of anti-ALV-J virus drugs by reducing the levels of pol mRNA and p27 mRNA.

[0018] The pharmaceutical composition of the present invention contains the siRNA for inhibiting chicken RASGRP3 gene expression and a pharmaceutically acceptable carrier or excipient.

[0019] The carrier is a liposome, nanoparticle, or viral vector.

[0020] The nucleic acid transfection composition for reducing RASGRP3 mRNA levels in chicken cells according to the present invention comprises an effective amount of the siRNA and a transfection reagent.

[0021] The transfection reagent is selected from any one of cationic liposomes or cationic polymer transfection reagents or their equivalents.

[0022] This invention provides a set of small interfering RNAs (siRNAs) for inhibiting chicken RASGRP3 gene expression, wherein the siRNA is any one of the following: RASGRP3-Anas-214, RASGRP3-Anas-846, and RASGRP3-Anas-1187. Each siRNA is a double-stranded RNA, preferably 19-23 nt in length, more preferably about 21 nt, with optional 2 nt 3′ overhangs at both ends of the double strand; wherein the antisense strand is complementary to the target site of the chicken RASGRP3 transcript, and the sense strand is the corresponding complementary strand. Specifically:

[0023] The RASGRP3-Anas-214 is a double-stranded siRNA formed by annealing SEQ ID NO.1 (sense strand) and SEQ ID NO.2 (antisense strand);

[0024] The RASGRP3-Anas-846 is a double-stranded siRNA formed by annealing SEQ ID NO.3 (sense strand) and SEQ ID NO.4 (antisense strand);

[0025] The RASGRP3-Anas-1187 is a double-stranded siRNA formed by annealing SEQ ID NO.5 (sense strand) and SEQ ID NO.6 (antisense strand).

[0026] This invention provides a nucleic acid transfection composition for reducing RASGRP3 mRNA levels in chicken cells, comprising: an effective amount of siRNA (double-stranded, preferably about 21 nt, with 2 nt 3' hangers at both ends of the double strand) as described in any one of claims 1-3; and a transfection reagent selected from any one of cationic liposomes or cationic polymer transfection reagents or equivalents thereof.

[0027] The transfection reagent is a commercially available cationic liposome / polymer transfection reagent, such as TransIntro® EL Transfection Reagent (or its equivalent). The composition may further contain a buffer (such as RNase-free water or Opti-MEM medium), and the siRNA may be in its physiologically acceptable salt or hydrated form.

[0028] This invention provides the use and method of the siRNA in reducing RASGRP3 mRNA levels in chicken cells: The siRNA is transfected into a chicken hepatocellular carcinoma (LMH) cell line or other chicken cells, with a preferred final concentration of 20 nM. After culturing for 48 h, the relative expression level of RASGRP3 mRNA is detected by RT-qPCR; a blank control and a non-target sequence negative control are set up. Under the above conditions, the siRNA can effectively downregulate chicken RASGRP3 at the transcript level.

[0029] This invention proposes an siRNA that can effectively inhibit the expression of the RASGRP3 gene in birds and demonstrates that it can inhibit ALV-J replication, providing a new molecular intervention target for the prevention and control of avian leukosis, which has important scientific research and potential economic value.

[0030] This invention presents a set of specific siRNA oligonucleotides (21–23 nt in length, double-stranded) targeting chicken RASGRP3 mRNA. After transfection into chicken hepatocellular carcinoma cell lines for 48 h, these siRNAs effectively reduced RASGRP3 gene expression, achieving an inhibition efficiency of over 50% and demonstrating improved knockdown efficiency. This siRNA achieves stable and efficient downregulation of the chicken RASGRP3 gene and can be used as an in vitro molecular inhibition tool for targeted intervention of RASGRP3 expression and for in vitro studies of its association with ALV-J infection / replication pathways and disease resistance mechanisms.

[0031] This invention employs a unique technical approach of "sequence design—screening—application verification": three sets of double-stranded siRNA candidate sequences with lengths of 21–23 nt are designed and provided for chicken RASGRP3 mRNA. First, their knockdown efficiency against RASGRP3 is verified by transfection in in vitro cells. Then, ALV-J replication indicators (such as the relative expression levels of pol and p27 transcripts) are used for evaluation and screening to obtain a set of effective siRNAs with stable and reproducible inhibition efficiency. This forms a closed-loop scheme from target to application effect, ensuring that this invention has clear technical effects and feasibility.

[0032] The unique features of the siRNA of this invention lie in its differentiated target, approach, and application: First, the siRNA targets the host gene RASGRP3 rather than the viral sequence, belonging to a host-targeted intervention approach, which differs from the common inhibitory strategies of directly targeting viral genes in existing technologies; Second, this invention does not propose a single sequence, but provides and screens multiple candidate siRNAs, and determines the effective sequence with high knockdown efficiency through in vitro transfection comparison, thereby improving the success rate and reproducibility; Third, the siRNA can not only downregulate RASGRP3 expression, but also further demonstrates a reduction in ALV-J replication indicators, thus clarifying its application value in inhibiting ALV-J replication.

[0033] Based on the omics difference analysis of cell samples from the challenge group and the control group, this invention screened RASGRP3 as a candidate key gene and further verified its function in a cell model. The results showed that RASGRP3 was differentially highly expressed under ALV-J infection conditions, and that inhibiting RASGRP3 expression by siRNA could significantly reduce the replication level of ALV-J, thus establishing RASGRP3 as a host-dependent factor and a new molecular intervention target in the ALV-J infection / replication process.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] This invention provides an siRNA capable of inhibiting chicken RASGRP3 gene expression. In chicken hepatocellular carcinoma (LMH) cells, transfection with siRNA via cationic liposomes (e.g., TransIntro EL) followed by RT-qPCR 48 h post-transfection showed a significant reduction in RASGRP3 mRNA expression level compared to the negative control (n=3, P < 0.05). Furthermore, 24 h after siRNA transfection, a further 24 h challenge followed by RT-qPCR showed a significant reduction in the relative expression levels of p27 and p27 compared to the negative controls (n=3, P < 0.05). This demonstrates that the siRNA not only downregulates RASGRP3 expression but also further reduces ALV-J replication markers (decreased p27 and p27 gene expression). Therefore, this siRNA can be used as an in vitro molecular tool to elucidate the association between host signaling pathways such as RASGRP3-Ras / MAPK and ALV-J replication levels. It can also serve as a tool for verifying the function of candidate host factors and exploring molecular markers in studies of ALV-J disease resistance traits in poultry, demonstrating clear methodological feasibility and promotional value. Attached Figure Description

[0036] Figure 1 Transcriptome sequencing analysis of RASGRP3 expression changes (log) after LMH cell challenge with ALV-J. 2 (fc)).

[0037] Figure 2 The relative expression change of RASGRP3 24 h after ALV-J infection (2) -ΔΔCt ). The difference was statistically significant (P < 0.05). This indicates a highly significant difference (P < 0.01).

[0038] Figure 3 The relative expression level of RASGRP3 mRNA in LMH cells 48 h after transfection with the siRNA was determined. The difference was statistically significant (P < 0.05). This indicates a highly significant difference (P < 0.01).

[0039] Figure 4 The relative expression level of the ALV-J related gene pol was changed 48 h after transfection of LMH cells with the siRNA.

[0040] Figure 5 The relative expression level of the ALV-J related gene p27 was changed 48 h after transfection of LMH cells with the siRNA. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0043] LMH cells and ALV-J virus were both provided by Yangzhou University.

[0044] Example 1

[0045] Relative expression levels of RASGRP3 in ALV-J infected cells and control cells (RT-qPCR)

[0046] 1. Cell grouping

[0047] LMH cells with approximately 70% confluence were selected from each generation, with four biological replicates per group. Cells were collected, flash-frozen in liquid nitrogen, and stored at −80°C for analysis.

[0048] 2. Expression of RASGRP3 in ALV-J infected and control cells

[0049] RT-qPCR and Statistical Analysis: Real-time quantitative PCR was performed using Applied Biosystems' PowerUp™ SYBR™ GreenMaster Mix reagent, with three replicates per sample. The q-PCR reaction program was: 50℃ for 2 min, 95℃ for 2 min; 40 cycles (95℃ for 15 s, 60℃ for 1 min); 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. Relative expression levels were calculated using the 2^-ΔΔCt method: ΔCt = Ct(target gene) − Ct(GAPDH), ΔΔCt = ΔCt(treatment group) − ΔCt(control group). Differences between different siRNA treatment groups and the control group were analyzed using one-way ANOVA, followed by Dunnett's multiple comparison test for post-hoc comparisons; P < 0.05 was considered statistically significant.

[0050] Cell challenge: LMH cells with a confluence of approximately 70% were selected. Each group was biologically replicated four times. Cells were washed twice with PBS, adsorbed with ALV-J virus at MOI=0.2 for 2 hours, washed twice with PBS, and cultured in complete culture medium for 22 hours.

[0051] The expression of RASGRP3 in challenged and control cells was detected using qPCR. RASGRP3 quantitative PCR primers (SEQ ID NO. 7-8) and internal control primer GAPDH (SEQ ID NO. 9-10) were used, as follows: Figure 1 As shown, there was a significant difference in RASGRP3 levels between the challenge group and the control group. (P<0.05) suggests that RASGRP3 may be related to viral replication pathways.

[0052] RASGRP3

[0053] Upstream primer (SEQ ID NO. 7):

[0054] 5'-ATGGGGTCAAACACACTTGG-3'

[0055] Downstream primer (SEQ ID NO.8):

[0056] 5'-GCTATCCTCAGATTCCTGTCT-3'

[0057] GAPDH

[0058] Upstream primer (SEQ ID NO. 9):

[0059] 5'-GGTTGTCTCCTGCGACTTCA-3'

[0060] Downstream primer (SEQ ID NO. 10):

[0061] 5'-TCCTTGGATGCCATGTGGAC-3'

[0062] Example 2

[0063] Design and preparation of siRNA targeting chicken RASGRP3

[0064] Based on the chicken RASGRP3 reference transcript (NCBI, NM_001006401.1:194-2269), three pairs of siRNAs targeting the CDS region of the chicken RASGRP3 gene were designed, with each siRNA sequence suffixed with TT bases. The designed siRNAs were synthesized by Suzhou Jima Biotechnology Co., Ltd. Each 1 OD siRNA was diluted with 125 μL of DEPC water to a final concentration of 20 μM and stored at -20°C until use. The sequences of the three pairs of siRNAs are shown in Table 1.

[0065] Table 1. Sequences of three pairs of siRNAs targeting the chicken RASGRP3 gene.

[0066]

[0067] Example 3

[0068] RT-qPCR detection of RASGRP3 and ALV-J related genes

[0069] 1. Cells and Culture

[0070] Chicken hepatoma cell line (LMH) was routinely cultured in complete medium (DMEM + 10% FBS) in culture dishes pretreated with 0.1% gelatin at 37°C and 5% CO2. Cells were seeded into 12-well plates at a density of approximately 1.5 × 10⁶ cells / well. 5 / hole, wait for adhesion to the wall and transfection at a density of about 70%.

[0071] 2. Transfection conditions

[0072] Following the manufacturer's instructions, the three pairs of siRNAs prepared in Example 1 were transfected into LMH using the cationic liposome transfection aid TransIntro EL (TransGen Biotech, FT201-01). The specific transfection systems were as follows: Experimental group: 100 μL opti-MEM medium + 5 μL siRNA (final concentration 20 μM) + 4 μL TransIntro EL; Control group: 100 μL opti-MEM medium + 5 μL Negative NC (SEQ ID NO. 11-12) (final concentration 20 μM) + 4 μL TransIntro EL. The cells were incubated for 15-20 min to form a transfection complex, which was then transfected into the culture medium. Four hours after transfection, the culture medium was replaced with serum-containing medium, and the cells were cultured for another 24 hours. Cells (sample A) were then collected to detect the siRNA knockout efficiency.

[0073] 3. Infectious Disease Treatment

[0074] Following the transfection method described above, ALV-J challenge (MOI=0.2, same as Example 1) was performed 24 hours after transfection. Cells were then cultured for 24 hours and collected (sample B) for subsequent detection of virus-related gene expression levels.

[0075] 4. Sample collection

[0076] Samples A and B were collected separately. After 48 h, the culture medium was discarded, the samples were washed gently with PBS, and total RNA was extracted by adding lysis buffer and then reverse transcribed to prepare cDNA.

[0077] 5. RT-qPCR and statistics

[0078] Evaluation of RASGRP3 inhibition efficiency: Using the same RASGRP3 quantitative fluorescence primers (SEQ ID NO. 7-8) and internal control primer GAPDH (SEQ ID NO. 9-10) as in Example 1:

[0079] ALV-J related gene detection: Select two representative genes, such as specific primers for p1 and p27 (denoted as SEQ ID NO.13-16), and use GAPDH as the internal reference (same as above) to detect the relative expression level according to the same system.

[0080] pol

[0081] Upstream primer (SEQ ID NO. 13):

[0082] 5'-AGACCTGCCCGCATTGTA-3'

[0083] Downstream primer (SEQ ID NO.14):

[0084] 5'-CACGGCCATGCTGAGTTA-3'

[0085] p27

[0086] Upstream primer (SEQ ID NO. 15):

[0087] 5'-CCGGGGAATTGGTTGCTAT-3'

[0088] Downstream primer (SEQ ID NO.16):

[0089] 5'-AGTCAATGATCACCGGAGCC-3'

[0090] The statistical methods were the same as in Example 1; P < 0.05 was considered statistically significant.

[0091] 6. Results

[0092] like Figure 1 and Figure 2 The results showed that RASGRP3 exhibited an upregulated transcriptomic response in the challenged LMH group (**P<0.01), indicating that viral replication can affect the expression of the RASGRP3 gene, which may be involved in the viral replication process and is likely to promote viral replication. Figure 3 As shown, compared with the negative control, the siRNA significantly downregulated RASGRP3 mRNA in LMH at 48 h ( P<0.05, (P<0.01) Among them, RASGRP3-846 showed the best effect, with an inhibition efficiency of over 50%; under the same inhibition conditions, ALV-J related genes showed a downregulation response at the transcript level. After inhibiting RASGRP3 expression with RASGRP3-846, p27 mRNA was downregulated by about 50%, and pol mRNA was downregulated by more than 50%. Therefore, RASGRP3-846 is the preferred siRNA for targeting RASGRP3, which can not only effectively inhibit RASGRP3 mRNA expression, but also significantly inhibit ALV-J replication (see...). Figure 4 and 5 , P<0.05, P<0.01).

Claims

1. A siRNA for inhibiting the expression of the chicken RASGRP3 gene, said siRNA comprising a sense strand and an antisense strand, which is any one of the following: RASGRP3-Anas-214, RASGRP3-Anas-846 and RASGRP3-Anas-1187; RASGRP3-Anas-214 Justice Chain: GAAGGCUGCAACAUUAGAUTT (SEQ ID NO. 1) RASGRP3-Anas-214 antisense chain: AUCUAAUGUUGCAGCCUUCTT (SEQ ID NO. 2) RASGRP3-Anas-846 Justice Chain: GGGCAGAAGYGAUCACAAATT (SEQ ID NO. 3) RASGRP3-Anas-846 antisense chain: UUUGUGAUCACUUCUGCCCTT (SEQ ID NO. 4) RASGRP3-Anas-1187 Justice Chain: CAGCUUUCCAUCACCUUGATT (SEQ ID NO. 5) RASGRP3-Anas-1187 antisense chain: UCAAGGUGAUGGAAAGCUGTT (SEQ ID NO. 6).

2. The siRNA for inhibiting chicken RASGRP3 gene expression according to claim 1, characterized in that, The preferred siRNA is RASGRP3-Anas-846.

3. The siRNA for inhibiting chicken RASGRP3 gene expression according to claim 1, characterized in that, The siRNA is synthesized chemically.

4. The use of the siRNA of claim 1 for inhibiting chicken RASGRP3 gene expression in the preparation of a reagent that reduces the level of RASGRP3 mRNA in chicken-derived cells.

5. The use of the siRNA for inhibiting chicken RASGRP3 gene expression as described in claim 1 in the preparation of an anti-ALV-J virus drug.

6. The application according to claim 5, characterized in that, The siRNA is used in the preparation of anti-ALV-J virus drugs by reducing pol mRNA and p27 mRNA levels.

7. A pharmaceutical composition, characterized in that, It contains the siRNA for inhibiting chicken RASGRP3 gene expression as described in claim 1, and a pharmaceutically acceptable vector or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The carrier is a liposome, nanoparticle, or viral vector.

9. A nucleic acid transfection composition for reducing RASGRP3 mRNA levels in chicken cells, characterized in that, It contains an effective amount of the siRNA as described in claim 1 and the transfection reagent.

10. The nucleic acid transfection composition according to claim 1, characterized in that, The transfection reagent is selected from any one of cationic liposomes or cationic polymer transfection reagents or their equivalents.

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