Application of REXO2 and siRNA thereof in inhibiting replication of PRRSV

Abstract

This invention belongs to the field of genetic engineering technology and relates to... REXO2 This invention relates to the application of its siRNA in inhibiting PRRSV replication. By establishing a cell model and using RNA interference technology and a live attenuated PRRSV vaccine strain for challenge, this invention detects… REXO2 The effect of silencing on PRRSV replication in cells was found. REXO2 Silencing can significantly inhibit PRRSV replication. This invention provides a new approach for the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS).

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of REXO2 and its siRNA in inhibiting PRRSV replication. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease, is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). It is one of the most important infectious diseases of pigs worldwide, causing huge economic losses to the global pig industry.

[0003] In practice, the primary strategy for preventing PRRS is vaccination. However, current commercially available PRRSV vaccines have efficacy and safety issues, making effective PRRS prevention difficult to achieve through vaccination alone.

[0004] REXO2 encodes RNA exonuclease 2, an oligonucleotide enzyme active in mitochondria. REXO2 is involved in DNA repair, replication, and recombination. Studies have shown that REXO2 degradation of dinucleotides maintains promoter specificity in mammalian mitochondria. Laitman et al. performed whole-exome sequencing on a family with four patients affected by pheochromocytoma across two consecutive generations and identified a novel candidate pathogenic variant (c.531-1G>T(NM 015523.3)) in the REXO2 gene that potentially affects splicing; this variant co-segregates with the phenotype within the family. Lack of REXO2 protein also leads to a significant decrease in mitochondrial nucleic acid content and de novo disruption of mitochondrial protein synthesis. Currently, research on REXO2 is limited, and its role in PRRSV remains unclear. Summary of the Invention

[0005] The main objective of this invention is to provide the application of REXO2 and its siRNA in inhibiting PRRSV replication. This invention discovers that REXO2 silencing can significantly inhibit PRRSV replication, providing a new approach for the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS).

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the application of REXO2 in suppressing PRRSV replication, wherein silencing REXO2 can suppress PRRSV replication.

[0008] A second aspect of the present invention provides siRNA for silencing REXO2, the siRNA sequence of which is as follows:

[0009] The forward sequence is: 5'-GGAAGUCUGGUCUAACCAATT-3';

[0010] The reverse sequence is: 5'-UUGGUUAGACCAGACUUCCTT-3'.

[0011] A third aspect of the present invention provides a kit comprising the siRNA sequence for silencing REXO2 as described in the second aspect.

[0012] The fourth aspect of this invention provides the use of the siRNA for silencing REXO2 described in the second aspect above or the kit described in the third aspect in the preparation of PRRSV inhibition.

[0013] The fifth aspect of this invention provides the use of the siRNA for silencing REXO2 described in the second aspect above or the kit described in the third aspect in the preparation of drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome (PRRS).

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention discovers that silencing REXO2 can significantly inhibit PRRSV replication, and the siRNA or kits used to silence REXO2 can be used to develop drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome (PRRS), thus providing a new approach for the prevention and treatment of PRRS. Attached Figure Description

[0016] Figure 1 The diagram shows the silencing efficiency of the REXO2 gene: A represents the relative expression of REXO2 gene mRNA, and B represents the expression of REXO2 protein.

[0017] Figure 2 The effect of REXO2 gene silencing on PRRSV ORF7 gene expression;

[0018] Figure 3 GO and KEGG enrichment analysis for DEGs: A and B are the significant GO entries (top 30) and KEGG pathways enriched for DEGs of REXO2-siRNA vs. NC, respectively;

[0019] Figure 4 This refers to the expression of GO entries involved by REXO2 and DEGs in the KEGG pathway. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. Furthermore, it should be understood that when the terms "comprising" or "including" are used in this specification, they indicate the presence of features, steps, operations, and combinations thereof.

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0023] The cell line used was porcine renal epithelial PK15. -CD163 Cell lines that stably express CD163 and are susceptible to PRRSV; cell lines that can be used in this application include, but are not limited to, PK15. -CD163 Cell line; the virus used was a highly pathogenic porcine reproductive and respiratory syndrome live vaccine strain (PRRSV-MLV, live strain TJM-F92, Pfizer).

[0024] Example

[0025] (I) Experimental Methods

[0026] (1) siRNA transfection and PRRSV challenge: The REXO2 gene siRNA sequence and NC sequence are shown in Table 1.

[0027] Table 1 siRNA sequences

[0028]

[0029] The transfection reagent Lipofectamine 2000 was used to transfect PK15 cells in pigs. -CD163 Cells were transfected with siRNA at a ratio of 20 μL transfection reagent to siRNA of 200 pmol. Silencing efficiency of REXO2 was assessed at the transcriptional and protein levels using quantitative real-time PCR and Western blotting, respectively, 24 h after siRNA transfection. Cells were challenged with the PRRSV vaccine strain 24 h after siRNA transfection. PK15 levels were detected using quantitative relative PCR 24 h after challenge. -CD163 The expression of the ORF7 gene of PRRSV in cells and the amplification primers are shown in Table 2.

[0030] Table 2 qRT-PCR primer sequences

[0031]

[0032] (2) RNA-seq and sequencing data analysis: Total RNA extracted 24 h after cell challenge was sequenced using transcriptome sequencing, which was performed by BioMed Biotechnology Co., Ltd. After sequencing, the raw data was filtered using FastQC v0.11.9 and Trimmomatic-0.39 to remove reads with adapters and low quality, obtaining high-quality clean reads. The Q30 and GC content of the quality-controlled data were calculated. The quality-controlled clean reads were aligned with the porcine reference genome (Sus scrofa11.1) using hisat2 v2.1.0 software, and the alignment efficiency was statistically analyzed. The aligned transcripts were quantified using HTseq v0.13.5 software to obtain read counts and calculate gene expression levels. The gene expression levels of the interference group and the negative control group, as well as the negative control group and the blank control group, were compared and analyzed using Rpackage DEseq2 v1.30.1. The significance thresholds for differential gene expression in this study were P < 0.05 and |Fold change (FC)| ≥ 2. The differentially expressed genes (DEGs) obtained were analyzed using the online software KOBAS 3.0 (http: / / kobas.cbi.pku.edu.cn / kobas3) via Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The significance level for both GO and KEGG enrichment analyses was set at P < 0.05.

[0033] (II) Experimental Results

[0034] (1) Interference with the REXO2 gene significantly silenced the REXO2 gene at both the transcriptional and translational levels. Twenty-four hours after siRNA interference, the REXO2 gene was challenged with a PRRSV attenuated live vaccine strain. The relative expression level of the REXO2 gene 24 hours after siRNA interference was detected using qRT-PCR. The results are as follows: Figure 1 As shown in Figure A, the transcriptional silencing efficiency of the REXO2 gene was 91.7%. After 48 hours of siRNA interference (24 hours after challenge), the relative expression level of REXO2 protein was detected using Western blotting, and the results are as follows: Figure 1As shown in Figure B, compared with the negative control group, the expression of REXO2 protein in the REXO2 gene interference group decreased by 63.1%, indicating that the REXO2 gene was significantly silenced at the protein translation level through RNA interference.

[0035] (2) REXO2 gene silencing versus PK15 -CD163 The effect of PRRSV replication in cells was measured using relative quantification to detect PK15 levels 24 h after challenge. -CD163 The expression level of PRRSV ORF7 in cells was investigated. The REXO2 gene silencing group showed significantly lower PRRSV ORF7 expression compared to the negative control group, indicating that REXO2 gene silencing has a significant effect on PK15 expression. -CD163 Cell infection with PRRSV has an inhibitory effect. Figure 2 ).

[0036] (3) Biological function analysis of DEGs

[0037] DEGs of REXO2-siRNA vs. NC were enriched in plasma membrane-related GO entries, Rab protein signaling, positive regulation of protein targeting of the membrane, hydrolytic enzyme activity, cholesterol metabolism, neuronal migration, and inflammatory responses—GO entries related to immune responses. Figure 3 A) KEGG enrichment analysis was performed on DEGs using the online software KOBAS. The top 30 most significant KEGG pathways were selected, and a bubble chart was plotted using the R package ggplot2 v3.3.3 (P<0.05). The results are as follows: Figure 3 As shown in B, the DEGs of REXO2-siRNA vs. NC were enriched in important immune-related biological pathways such as the Toll-like receptor signaling pathway, the TNF (tumor necrosis factor) signaling pathway, the MAPK signaling pathway, the interaction between viral proteins and cytokines and cytokine receptors, the IL-17 signaling pathway, antigen processing and presentation, the interaction between cytokines and cytokine receptors, and the PI3K-Akt signaling pathway.

[0038] DEG enrichment analysis of the REXO2-siRNA vs. NC comparison group revealed that REXO2 is involved in four GO pathways: 3'-5'-ribonuclease activity, RNA phosphodiester bond hydrolysis, nucleic acid exonucleation, mitochondrial and nucleic acid binding, as well as the ribosome biogenesis pathway in eukaryotes, but none were statistically significant. Among these REXO2-involved GO pathways and KEGG pathways, five genes—ABCG1, GCKR, CA5A, DDX25, and ABAT—were significantly upregulated in the REXO2-siRNA vs. NC comparison group (P<0.01 and |FC|>2.0), while the expression levels of REXO2 and CYP24A1 were significantly downregulated in the REXO2-siRNA vs. NC comparison group (P<0.01 and |FC|>2.0). Figure 4 The results showed that REXO2 negatively regulates the expression of ABCG1, GCKR, CA5A, DDX25, and ABAT, while positively regulating the expression of the CYP24A1 gene. ABCG1 is an important lipid transporter gene, and ABCG1 protein is one of the main factors inducing cholesterol efflux from cells. Numerous studies have shown that increased cholesterol efflux can enhance resistance to viral infections in vitro.

[0039] The above results indicate that REXO2 gene silencing can inhibit PRRSV replication, and the mechanism is that REXO2 silencing induces a significant upregulation of ABCG1 expression, which in turn increases the efflux of cellular cholesterol. This restricts the virus's ability to replicate using intracellular cholesterol, thereby reducing the ability of PRRSV to infect cells.

[0040] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A siRNA for silencing REXO2, characterized in that, The siRNA sequence is as follows:

2. A reagent kit, characterized in that, It contains the siRNA sequence for silencing REXO2 as described in claim 1.

3. The use of the siRNA for silencing REXO2 as described in claim 1 or the kit as described in claim 2 in the preparation of drugs that inhibit PRRSV replication.

4. The use of the siRNA for silencing REXO2 as described in claim 1 or the kit as described in claim 2 in the preparation of a drug for treating porcine reproductive and respiratory syndrome (PRRS).

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