Use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a medicament for preventing and / or treating porcine reproductive and respiratory syndrome virus infection

Abstract

The present invention relates to the field of biomedical technologies, in particular to the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a medicament for preventing and / or treating porcine reproductive and respiratory syndrome virus (PRRSV) infection. The nucleotide sequence of miR-1386-z according to the present invention is as shown in SEQ ID NO.1. The present invention discovers that a reagent for inhibiting the expression of miR-1386-z can effectively alleviate the increased expression of PRRSV-N protein and E-cadherin protein caused by PRRSV infection, and can effectively alleviate the change in the distribution of ZO-1 and the damage to cell morphology. miR-1386-z can be used as a new molecular target for the treatment of porcine reproductive and respiratory syndrome virus infection, guiding the screening and research and development of related medicaments.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a medicament for preventing and / or treating porcine reproductive and respiratory syndrome virus infection. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen with an enveloped positive-sense RNA genome approximately 15 kb in length, containing 11 open reading frames. PRRSV can inhibit immunity by regulating the secretion of inflammatory cytokines and chemokines, dominating the activities of some immune cells, and inhibiting the production of interferons. Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by viral infection, which can be transmitted through multiple routes such as the respiratory tract, digestive tract, mucosal contact, blood, and placenta. Its main manifestations are disorders of reproductive and respiratory system functions, causing serious economic losses to pig farming. Exploring the regulatory relationship and pattern of differentially expressed RNAs for PRRSV invasion and host immunity will contribute to the analysis of the molecular mechanism of PRRSV infection and the search for new ideas for inhibiting PRRSV infection.

[0003] Currently, research on porcine reproductive and respiratory syndrome (PRRS) mainly focuses on mRNA, and there is less research on the regulatory mechanism of non-coding RNAs (lncRNAs, miRNAs, and circRNAs) after PRRS infection. miRNAs are a class of non-coding RNAs that regulate many intracellular processes by directly binding to mRNAs and affecting translation efficiency or mRNA abundance. miRNAs can affect the replication and infection of RNA viruses by directly binding to the RNA virus genome or through virus-mediated changes in the host transcriptome. Host miRNAs play an important regulatory role in PRRSV infection and can serve as molecular targets for controlling PRRSV infection. However, there is currently a lack of miRNA molecular targets for controlling PRRSV infection. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus (PRRSV) infection. The present invention discovers that the reagent for inhibiting the expression of miR-1386-z can effectively alleviate the increased expression of porcine reproductive and respiratory syndrome virus N protein (PRRSV-N) and E-cadherin caused by PRRSV infection, and can effectively alleviate the altered distribution of Zona Occludens 1 (ZO-1) and the damaged cell morphology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus infection, wherein the nucleotide sequence of miR-1386-z is as shown in SEQ ID NO.1.

[0007] Preferably, the reagent for inhibiting the expression of miR-1386-z includes small interfering RNA targeting miR-1386-z.

[0008] Preferably, the nucleotide sequence of the small interfering RNA targeting miR-1386-z is as shown in SEQ ID NO.6.

[0009] Preferably, the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection includes: alleviating one or more of the increased expression of porcine reproductive and respiratory syndrome virus N protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the increased expression of E-cadherin caused by porcine reproductive and respiratory syndrome virus infection, alleviating the increased expression of Zona Occludens protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the altered distribution of Zona Occludens protein caused by porcine reproductive and respiratory syndrome virus infection, and alleviating the damaged cell morphology caused by porcine reproductive and respiratory syndrome virus infection.

[0010] The present invention provides a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus infection, wherein the active ingredient of the drug includes small interfering RNA; the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO.6.

[0011] Preferably, the unit dose concentration of the small interfering RNA in the drug is 80-120 pmol / L.

[0012] Preferably, the unit dose concentration of the small interfering RNA in the drug is 100 pmol / L.

[0013] Beneficial effects:

[0014] The present invention provides the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, and the nucleotide sequence of the miR-1386-z is as shown in SEQ ID NO.1. The present invention discovers that the reagent for inhibiting the expression of miR-1386-z can resist PRRSV infection. The results of the examples show that miR-1386-z is significantly down-regulated in the lung tissues of 45-day-old Large White pigs infected with PRRSV; ZO-1, E-cadherin and PRRSV-N proteins are significantly up-regulated in ZMAC-4 cells infected with HP-PRRSV; the reagent for inhibiting the expression of miR-1386-z can effectively alleviate the increase in the expression of PRRSV-N protein and E-cadherin protein caused by PRRSV infection, and effectively alleviate the change in ZO-1 distribution and the damage of cell morphology. miR-1386-z can be used as a new molecular target for the treatment of PRRSV infection to guide the screening and research and development of related drugs. Brief Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0016] Figure 1 For the expression of miR-1386-z in lung tissues identified by small RNA sequencing (B) and the qPCR verification results of miR-1386-z (A);

[0017] Figure 2 For the expression results of ZO-1, E-cadherin and PRRSV-N proteins in different groups of ZMAC-4 cells; among them, A is the western blot result diagram; B is the gray value analysis result;

[0018] Figure 3 For the immunofluorescence experiment results of different groups of ZMAC-4 cells;

[0019] Figure 4 For the cell morphology change results of different groups of ZMAC-4 cells. Detailed Embodiments

[0020] The present invention provides the use of a reagent for inhibiting the expression of miR-1386-z in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, and the nucleotide sequence of the miR-1386-z is as shown in SEQ ID NO.1.

[0021] In the present invention, the reagent for inhibiting the expression of miR-1386-z preferably comprises small interfering RNA targeting miR-1386-z. As a specific embodiment, the nucleotide sequence of the small interfering RNA targeting miR-1386-z is as shown in SEQ ID NO.6.

[0022] In the present invention, the prevention and / or treatment of porcine reproductive and respiratory syndrome virus (PRRSV) infection preferably comprises alleviating one or more of the following: the increased expression of PRRSV-N protein caused by PRRSV infection, the increased expression of E-cadherin caused by PRRSV infection, the increased expression of zonula occludens protein caused by PRRSV infection, the altered distribution of zonula occludens protein caused by PRRSV infection, and the damaged cell morphology caused by PRRSV infection. As a specific embodiment, the strain of PRRSV used in the examples of the present invention is WUH3; the strain WUH3 is a highly pathogenic PRRSV virus strain isolated from a "high fever" diseased pig in China, with the GenBank accession number: HM853673, and is disclosed in the literature [Li B, Fang L, Liu S, et al. The genomic diversity of Chinese porcine reproductive and respiratory syndrome virus isolates from 1996 to 2009 [J]. Vet Microbiol, 2010, 146(3-4):226-237.].

[0023] The present invention discovers that the reagent for inhibiting the expression of miR-1386-z can effectively alleviate the increased expression of PRRSV-N protein and E-cadherin protein caused by PRRSV infection, and effectively alleviate the altered distribution of ZO-1 and the damaged cell morphology. miR-1386-z can be used as a new molecular target for the treatment of porcine reproductive and respiratory syndrome virus infection, guiding the screening and development of related drugs.

[0024] Based on the above advantages, the present invention provides a drug for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection, and the active ingredient of the drug comprises small interfering RNA; the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO.6. In the present invention, the unit dose concentration of the small interfering RNA in the drug is preferably 80-120 pmol / L. As a specific embodiment, the unit dose concentration of the small interfering RNA in the drug is 100 pmol / L.

[0025] To further illustrate the present invention, the application of the reagent for inhibiting miR-1386-z expression in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus infection will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 miR-1386-z is Lowly Expressed in Lung Tissues of 45-Day-Old Large White Pigs Infected with PRRSV

[0027] Lung tissue samples were collected from 3 45-day-old large white pigs infected with the PRRSV strain WUH3 and 3 45-day-old large white pigs not infected with the PRRSV strain WUH3, respectively.

[0028] Total RNA of the 6 lung tissue samples was extracted by the Trizol (Invitrogen) method. The quality of the total RNA was evaluated by agarose gel electrophoresis and the micro nucleic acid protein analyzer Quawell UV-Vis Spectrophotometer Q5000. It was required that the three bands of 28S, 18S and 5S were clear and complete after agarose gel electrophoresis; the OD 260 / OD 280 value was between 1.8 and 2.1, the OD 260 / OD 230 ratio was greater than 2.0; the RNA integrity number (RIN) was greater than 8.0.

[0029] 4 μg of total RNA was extracted from each sample. Bands in the range of 18 - 30 nt were selected by polyacrylamide gel (PAGE) electrophoresis and the gel was cut to recover small RNAs; adapters were ligated respectively to construct transcript information; reverse transcription and PCR amplification were carried out; finally, bands of about 140 bp were recovered and purified using PAGE gel, and Elution buffer was added to complete the library construction. Quality inspection was performed using Agilent 2100. After the sequencing library passed the quality inspection, it was sequenced using Illumina HiSeq TM 4000.

[0030] After obtaining the raw data (raw reads) by sequencing, the fastp software was used for filtering, and the filtering criteria were as follows: (1) the quality value Q of the reads < 20, or containing N; (2) the reads containing the 5' adapter and not having the 3' adapter; (3) the reads not containing the insert fragment and the insert fragment being less than 18 nt; (4) the reads containing polyA. The small RNA sequences were aligned to the GenBank and Rfam databases using the blastn software, and the aligned rRNA, scRNA, snoRNA, snRNA, tRNA were removed. The small RNA sequences were aligned to the pig reference genome, and the aligned repetitive sequences were removed.

[0031] The small RNAs were aligned with the miRNA sequences of pigs in the authoritative miRNA database miRBase. The software bowtie was used to align the miRNA precursor sequences to obtain the known miRNAs of pigs. The remaining small RNAs without any annotated information were used for the prediction of novel miRNAs.

[0032] The miRNAs identified from each sample were summarized, and the TPM (Transcripts per million reads) expression levels of the miRNAs were calculated using the formula TPM = T × 10^6 / N, where T represents the number of reads of a certain miRNA input, and N represents the total number of miRNA reads (the sum of the counts of known miRNAs + novel miRNAs). The edgeR software was used for differential analysis of the miRNAs. According to the screening criteria of log2(fc) > 2 and P < 0.05, significantly differentially expressed miRNAs were identified. Among them, miR-1386-z was significantly downregulated in the lung tissues of 45-day-old Large White pigs infected with PRRSV. The results of the differential analysis of miR-1386-z are shown in Figure 1 B in, where **** is P < 0.0001, RNA-SEQ-CTR is the lung tissue sample infected with the PRRSV virus strain, and RNA-SEQ-INF is the non-infected lung tissue sample. The nucleotide sequence of miR-1386-z is as follows: 5'-CUCCUGGCUGGCUCGCCA-3', SEQ ID NO.1.

[0033] Example 2 qPCR verification of miR-1386-z expression

[0034] Total RNA was extracted from the lung tissues of 3 45-day-old Large White pigs infected with the PRRSV virus strain WUH3 (the virus strain described in Example 1) and 3 45-day-old Large White pigs not infected with the PRRSV virus strain WUH3 using the Trizol (Invitrogen) method. Reverse transcription was carried out according to the instructions of the TAKARA (RR047A) reagent.

[0035] The RT-qPCR reaction was carried out according to the instructions of the TaKaRa SYBR Premix EX Taq kit. The reaction was performed on a fluorescence quantitative instrument, the QuantStudio5 real-time PCR system (Applied biosystems). β-actin was used as the internal reference gene. The primer sequences are as follows:

[0036] miR-1386-z-F: 5'-CGCGCTCCTGGCTGGC-3', SEQ ID NO.2;

[0037] miR-1386-z-R: 5'-GTGCAGGGTCCGAGGT-3', SEQ ID NO.3;

[0038] β-actin-F: 5'-GGACTTCGAGCAGGAGATGG-3', SEQ ID NO.4;

[0039] β-actin-R: 5'-AGGTAGTTTCGTGGATGCCG-3', SEQ ID NO.5;

[0040] The reaction system for RT-qPCR was as follows: 0.8 μL of cDNA, 6.1 μL of DEPC water, 7.2 μL of TB Green nucleic acid dye, 0.3 μL of DyeⅡ reference dye, 0.3 μL of upstream primer, and 0.3 μL of downstream primer; The reaction program for RT-qPCR was: 95°C for 5 min; 95°C for 15 s, 6°C for 45 s, 95°C for 15 s, for 40 cycles; 60°C for 1 min, 95°C for 15 s.

[0041] The results are shown in Figure 1 A in, where *, P < 0.05, CTR is the lung tissue sample infected with the PRRSV virus strain, and INF is the uninfected lung tissue sample.

[0042] The results showed that miR-1386-z was significantly downregulated in the lung tissues of 45-day-old Yorkshire pigs infected with PRRSV, which was consistent with the small RNA sequencing results.

[0043] Example 3 Inhibiting the expression of miRNA-1386-z can significantly alleviate the expression of ZO-1, E-cadherin, and PRRSV-N proteins induced by HP-PRRSV infection

[0044] The treatment method of miRNA-1386-z inhibitor was as follows: Small interfering RNAs (siRNAs) targeting miR-1386-z were synthesized by GenePharma and transfected according to the instructions of Lipofectamine 3000. The nucleotide sequence of the miR-1386-z inhibitor was as follows: 5'-UGGCGAGCCAGCCAGGAG-3', SEQ ID NO.6.

[0045] The experiment was divided into four groups, namely the NC group, the miRNA-1386-z group, the PRRSV-NC group, and the PRRSV-miRNA-1386-z group. Among them, the NC group was ZMAC-4 cells (porcine macrophages) without treatment; the miRNA-1386-z group was the treatment group with miRNA-1386-z inhibitor. Lipofectamine 3000 was used to transfect 100 pM miRNA-1386-z inhibitor for 48 hours, and the transfection operation was carried out according to the reagent instructions; the PRRSV-NC group was co-incubated with the XH-GD strain and cells for 24 hours, and the virus titer was 106.7; the PRRSV-miRNA-1386-z group was added with the XH-GD strain after transfecting cells with miRNA-1386-z inhibitor for 48 hours and incubated for 24 hours, and the virus titer was 106.7; the XH-GD strain was kindly provided by Foshan University of Science and Technology and was published in the literature [Cao Z X, Jiao P R, Huang Y M, et al. Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China[J]. Acta Vet Hung, 2012, 60(1):157-164.].

[0046] Proteins of the four groups of samples were extracted using a protein lysate (RIPA lysate), and the protein concentration was detected by the BCA method. By the method of Western blot, namely electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and development, the expression levels of zonula occludens protein 1 (ZO-1), E-cadherin, porcine reproductive and respiratory syndrome virus N protein (PRRSV-N), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins in each group of samples were detected. The results are shown in Figure 2 , where *, ** represent P < 0.05, P < 0.001 respectively, GAPDH is the internal reference protein, PRRSV is the XH-GD strain, and miRNA-1386-z inhibitor is the miRNA-1386-z inhibitor.

[0047] The results showed that PRRSV infection induced an increase in the expression levels of ZO-1, E-cadherin, and PRRSV-N proteins in cells, and the treatment with miRNA-1386-z inhibitor could inhibit the occurrence of this phenomenon.

[0048] Example 4 Inhibiting the expression of miRNA-1386-z can significantly alleviate the changes in the distribution of ZO-1 and cell morphology induced by HP-PRRSV infection

[0049] The experimental groups were divided as described in Example 3. By immunofluorescence method, the distribution of ZO-1 in ZMAC-4 cells was observed under a microscope. The immunofluorescence experiment was operated as follows:

[0050] (1) Culture the cells in a 24-well plate to 80%. Wash the cells 3 times with PBS, fix the cells with 4% paraformaldehyde for 15 min, and then wash the cells 3 times with PBS.

[0051] (2) Permeabilize with 0.5% TritonX-100 at room temperature for 15 min, wash the cells 3 times with PBS, then add 1% BSA blocking solution and block at room temperature for 60 min. Remove the blocking solution, and then add a sufficient amount of diluted primary antibody to the 24-well plate and incubate overnight at 4°C.

[0052] (3) After taking out the 24-well plate, warm it up for 30 min. Wash the 24-well plate 3 times with PBS, add the diluted fluorescent secondary antibody, and place the plate in a wet box and incubate at 20-37°C for 1 h. After the incubation, wash the plate 3 times with PBS again.

[0053] (4) Counterstain the nucleus: Add DAPI and incubate in the dark for 10 min, wash 3 times with PBS.

[0054] (5) Observe and collect images under a fluorescence microscope.

[0055] The results are shown in Figure 3 , HP-PRRSV infection induced changes in the distribution of ZO-1 protein in cells, and inhibiting the expression of miRNA-1386-z partially restored the localization of ZO-1 protein.

[0056] Take cell morphology images, and the results are shown in Figure 4 , HP-PRRSV infection caused damage to cell morphology, and treatment with miRNA-1386-z inhibitor partially restored cell morphology.

[0057] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of small interfering RNA targeting miR-1386-z in the preparation of a medicament for treating porcine reproductive and respiratory syndrome virus infection, wherein the nucleotide sequence of miR-1386-z is as shown in SEQ ID NO.1; and the nucleotide sequence of the small interfering RNA targeting miR-1386-z is as shown in SEQ ID NO.

6.

2. The application according to claim 1, wherein The treatment of porcine reproductive and respiratory syndrome virus infection includes: alleviating one or more of the increased expression of porcine reproductive and respiratory syndrome virus N protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the increased expression of E-cadherin caused by porcine reproductive and respiratory syndrome virus infection, alleviating the increased expression of occludin caused by porcine reproductive and respiratory syndrome virus infection, alleviating the change in the distribution of occludin caused by porcine reproductive and respiratory syndrome virus infection, and alleviating the damage to cell morphology caused by porcine reproductive and respiratory syndrome virus infection.

3. A drug for treating porcine reproductive and respiratory syndrome virus infection, characterized in that, The active ingredient of the medicament includes small interfering RNA; the nucleotide sequence of the small interfering RNA is as shown in SEQ ID NO.

6.

4. The drug according to claim 3, characterized in that, The unit dose concentration of small interfering RNA in the medicament is 80-120 pmol / L.

5. The drug according to claim 4, characterized in that, The unit dose concentration of small interfering RNA in the medicament is 100 pmol / L.

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