Application of reagent for inhibiting expression of miR-186-z in preparation of medicine for preventing and / or treating porcine reproductive and respiratory syndrome virus infection

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a reagent for inhibiting miR-186-z expression in preparation of a medicine for preventing and / or treating porcine reproductive and respiratory syndrome virus infection. The nucleotide sequence of the miR-186-z disclosed by the invention is as shown in SEQ ID NO. 1. The invention finds that the reagent for inhibiting the expression of miR-186-z can effectively relieve the expression increase of PRRSV-N protein and E-cadherin protein caused by PRRSV infection, and can effectively relieve the ZO-1 distribution change and cellular morphology damage. The miR-186-z can be used as a new molecular target for treating porcine reproductive and respiratory syndrome virus infection and is used for guiding screening, research and development of related drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an agent 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. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen with an enveloped positive-sense RNA genome of approximately 15 kb in length, containing 11 open reading frames. PRRSV can suppress immunity by regulating the secretion of inflammatory cytokines and chemokines, dominating the activity of some immune cells, and inhibiting the production of interferon. Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by viral infection that can be transmitted through multiple pathways such as the respiratory tract, digestive tract, mucosal contact, blood, and placenta. It is mainly manifested by dysfunction of the reproductive and respiratory systems, causing serious economic losses to pig farming. Exploring the regulatory relationship and rules of differentially expressed RNA for PRRSV invasion and host immunity will help to analyze the molecular mechanism of PRRSV infection and find new ideas to inhibit PRRSV infection.

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

[0004] In order to solve the above problems, the present invention provides the use of an agent that inhibits the expression of miR-1386-z in the preparation of a drug for preventing and / or treating porcine reproductive and respiratory syndrome virus infection. The present invention found that the agent that inhibits 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 (E-cadherin) caused by PRRSV infection, and effectively alleviate the changes in the distribution of Zona Occludens 1 (ZO-1) and damaged cell morphology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of an agent 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. The nucleotide sequence of the miR-1386-z is shown in SEQ ID NO.1.

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

[0008] Preferably, the nucleotide sequence of the small interfering RNA targeting miR-1386-z is 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 following: alleviating 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 occludens junction protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the change in the distribution of occludens junction protein 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.

[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 comprises small interfering RNA; the nucleotide sequence of the small interfering RNA is 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 an agent 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 shown in SEQ ID NO.1. The present invention finds that an agent for inhibiting the expression of miR-1386-z can resist porcine reproductive and respiratory syndrome virus infection. The results of the embodiment show that miR-1386-z is significantly low-expressed in the lung tissue of 45-day-old large white pigs infected with PRRSV; ZO-1, E-cadherin and PRRSV-N proteins are significantly highly expressed in HP-PRRSV-infected ZMAC-4 cells; the agent 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 change in ZO-1 distribution and 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 to guide the screening and development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0016] Figure 1 The expression of miR-1386-z in lung tissue was identified by small RNA sequencing (B) and the qPCR validation results of miR-1386-z (A);

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

[0018] Figure 3 The immunofluorescence results of ZMAC-4 cells in different groups;

[0019] Figure 4 The results of cell morphological changes of ZMAC-4 cells in different groups. DETAILED DESCRIPTION

[0020] The present invention provides the use of an agent 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. The nucleotide sequence of the miR-1386-z is shown in SEQ ID NO.1.

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

[0022] In the present invention, the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection preferably includes: alleviating 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 occludens junction protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the change in the distribution of occludens junction protein caused by porcine reproductive and respiratory syndrome virus infection and alleviating one or more of the damaged cell morphology caused by porcine reproductive and respiratory syndrome virus infection. As a specific embodiment, the strain of porcine reproductive and respiratory syndrome virus used in the embodiment of the present invention is WUH3; the strain WUH3 is a highly pathogenic PRRSV virus strain isolated from a sick pig with a "high fever" in China, GenBank accession number: HM853673, disclosed in the document [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 found that the reagent that inhibits 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 change in ZO-1 distribution and cell morphological damage. MiR-1386-z can be used as a new molecular target for the treatment of porcine reproductive and respiratory syndrome virus infection to guide the screening and development of related drugs.

[0024] Based on the above advantages, 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 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 to 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 provided by the present invention in the preparation of drugs for preventing and / or treating porcine reproductive and respiratory syndrome virus infection is described in detail below in combination with the accompanying drawings and examples, 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 the lung tissue of 45-day-old Large White pigs infected with PRRSV

[0027] Lung tissue samples were collected from three 45-day-old Large White pigs infected with PRRSV virus strain WUH3 and three 45-day-old Large White pigs not infected with PRRSV virus strain WUH3.

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

[0029] 4 μg of total RNA was extracted from each sample, and the bands in the range of 18 to 30 nt were selected by polyacrylamide gel (PAGE) electrophoresis to recover small RNA; the adapters were connected to construct transcript information; reverse transcription and PCR amplification were performed; finally, the bands of about 140 bp were recovered and purified using PAGE gel, and Elution buffer was added to complete the library construction. Agilent2100 was used for quality inspection. If the sequencing library passed the quality inspection, it was used for Illumina HiSeq TM 4000 were sequenced.

[0030] After sequencing to obtain raw data (rawreads), fastp software was used for filtering. The filtering criteria were as follows: (1) reads with a quality value Q < 20 or containing N; (2) reads containing 5' adapters and no 3' adapters; (3) reads without inserts and inserts less than 18nt; (4) reads containing polyA. The small RNA sequences were aligned to the GenBank and Rfam databases using blastn software, and rRNA, scRNA, snoRNA, snRNA, and tRNA in the alignment were removed. The small RNA sequences were aligned to the pig reference genome, and duplicate sequences in the alignment were removed.

[0031] The small RNA was compared with the pig miRNA sequences in the miRNA authority database miRBase. The software bowtie was used to compare the miRNA precursor sequences and obtain the known pig miRNAs. The remaining small RNAs that were not annotated with any information were used to predict new miRNAs.

[0032] The miRNAs identified in each sample were summarized and the TPM (transcripts per million reads) expression of miRNA was calculated. The formula was TPM = T × 10^6 / N, where T represents the fragment input of a certain miRNA and N represents the total number of miRNA fragments (the sum of known miRNA + new miRNA counts). The edgeR software was used to perform differential analysis of miRNAs. Based on the screening criteria of log2(fc) > 2 and P < 0.05, significantly differentially expressed miRNAs were identified, among which miR-1386-z was significantly underexpressed in the lung tissue of 45-day-old Large White pigs infected with PRRSV. The results of miRNA differential analysis of miR-1386-z are shown in Figure 1 B, where **** means P < 0.0001, RNA-SEQ-CTR is a lung tissue sample infected with PRRSV virus strain, and RNA-SEQ-INF is an uninfected 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 from lung tissues of three 45-day-old Large White pigs infected with PRRSV virus strain WUH3 (the strain described in Example 1) and three 45-day-old Large White pigs not infected with PRRSV virus strain WUH3 was extracted by Trizol (Invitrogen) method. Reverse transcription was performed according to the instructions of TAKARA (RR047A) reagent.

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

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

[0037] miR-1386-zR: 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 of RT-qPCR was: 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 of RT-qPCR was: 95°C for 5 min; 95°C for 15 s, 60°C for 45 s, 95°C for 15 s, 40 cycles; 60°C for 1 min, 95°C for 15 s.

[0041] Results Figure 1 A in the figure, where * means P < 0.05, CTR means lung tissue sample infected with PRRSV virus strain, and INF means uninfected lung tissue sample.

[0042] The results showed that miR-1386-z was significantly lowly expressed in the lung tissue of 45-day-old Large White pigs infected with PRRSV, which was consistent with the results of small RNA sequencing.

[0043] Example 3 Inhibition of miRNA-1386-z expression 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 is as follows: small interfering RNA (siRNAs) targeting miR-1386-z was synthesized by GenePharma and transfected according to the instructions of Lipofectamine 3000. The nucleotide sequence of miR-1386-z inhibitor is as follows: 5'-UGGCGAGCCAGCCAGGAG-3', SEQ ID NO.6.

[0045] The experiment was divided into four groups, namely NC group, miRNA-1386-z group, PRRSV-NC group, and PRRSV-miRNA-1386-z group. Among them, the NC group was ZMAC-4 cells (porcine macrophages) without treatment; the miRNA-1386-z group was a miRNA-1386-z inhibitor treatment group, which was transfected with Lipofectamine 3000, 100pM miRNA-1386-z inhibitor for 48 hours, and the transfection operation was carried out according to the reagent instructions; the PRRSV-NC group was XH-GD strain and cells were co-incubated for 24 hours, and the virus titer was 106.7; the PRRSV-miRNA-1386-z group was miRNA-1386-z inhibitor transfected cells 48 hours later and then added XH-GD strain for 24 hours, and the virus titer was 106.7; the XH-GD strain was donated by Foshan University of Science and Technology and published in the literature [Cao ZX, Jiao PR, Huang YM, et al. Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China[J].ActaVetHung,2012,60(1):157-164.].

[0046] Proteins of the four groups of samples were extracted using protein lysis buffer (RIPA lysis buffer), and the protein concentration was detected by the BCA method. The expression of Zona Occludens 1 (ZO-1), E-cadherin (E-cadherin), porcine reproductive and respiratory syndrome virus N protein (PRRSV-N) and glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) proteins in each group of samples was detected by protein immunoblotting, i.e., electrophoresis, transfer, blocking, primary antibody incubation, secondary antibody incubation and development. The results are shown in Figure 2 , where * is P<0.05, ** is P<0.001, 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 of ZO-1, E-cadherin and PRRSV-N proteins in cells, and treatment with miRNA-1386-z inhibitor could inhibit the occurrence of this phenomenon.

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

[0049] The experimental grouping was as described in Example 3. The distribution of ZO-1 in ZMAC-4 cells was observed under a microscope by immunofluorescence. The immunofluorescence experiment was performed as follows:

[0050] (1) Cells were cultured in a 24-well plate until 80% of the cells were incubated, washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS.

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

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

[0053] (4) Counterstaining of nuclei: Add DAPI and incubate in the dark for 10 min, then wash three times with PBS.

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

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

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

[0057] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of an agent 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 shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The agent for inhibiting the expression of miR-1386-z includes small interfering RNA targeting miR-1386-z.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the small interfering RNA targeting miR-1386-z is shown in SEQ ID NO.

6.

4. The use according to any one of claims 1 to 3, characterized in that: 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 occludens junction protein caused by porcine reproductive and respiratory syndrome virus infection, alleviating the change in the distribution of occludens junction protein 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.

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

6.

6. The drug according to claim 5, characterized in that The unit dosage concentration of the small interfering RNA in the medicine is 80-120 pmol / L.

7. The drug according to claim 6, characterized in that The unit dosage concentration of the small interfering RNA in the drug is 100 pmol / L.

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