Micropeptide miPEP-71 coded by long-chain non-coding RNA NEAT1 and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a micropeptide miPEP-71 coded by long-chain non-coding RNA NEAT1 and application of the micropeptide miPEP-71. The micropeptide is human-derived micropeptide and is named as miPEP-71; tests prove that the micropeptide miPEP-71 coded by the human NEAT1 gene can inhibit the replication of influenza viruses. The invention reveals that the micropeptide miPEP-71 coded by the human NEAT1 gene can significantly inhibit influenza virus replication at the cellular level, can be used as a replication inhibitor of influenza virus, and provides a new choice for research and development of anti-influenza virus drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a micropeptide miPEP-71 encoded by the long non-coding RNA NEAT1 and its applications. Background Technology

[0002] Influenza virus is a common respiratory virus, commonly known as the flu virus. It belongs to the genus *Influenza* of the family Orthomyxoviridae and is an enveloped, negative-sense RNA virus. Based on the antigenicity of its nucleoprotein (NP) and matrix protein (M), it can be classified into four types: A, B, C, and D. Type A influenza virus can cause pandemics; type B influenza virus is currently only found in humans and seals and can cause localized epidemics; type C influenza virus causes rare, mild illness in humans; and type D influenza virus primarily infects livestock.

[0003] Influenza A virus (IAV) is an important zoonotic disease with a very wide host range, infecting birds and various mammals, including humans, pigs, and marine animals. IAV can be further classified into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) based on differences in the viral surface protein antigens hemagglutinin (HA) and neuraminidase (NA). Historically, circulating influenza virus strains include H1N1, H3N2, and H2N2. While H5N1, a highly pathogenic strain, did not cause a global pandemic, its extremely high pathogenicity has attracted widespread attention. IAV can be widely transmitted through birds, and the H1-H16 subtypes have been isolated from waterfowl; therefore, waterfowl are considered natural hosts of IAV. Some influenza virus (IAV) subtypes have also established stable transmission in mammalian species, including the H1N1 and H3N2 subtypes, which are transmissible in both humans and pigs; the H3N8 and H7N7 subtypes in horses; and the H17N10 and H18N11 subtypes found in bats. Because influenza viruses possess antigenic shift and antigenic drift characteristics, they exhibit extremely high variability. Bird viruses can recombine with human and swine viruses to create new pandemic viruses in humans. These factors undoubtedly increase the difficulty of combating influenza viruses.

[0004] Throughout human history, influenza A viruses have caused four major pandemics, resulting not only in enormous losses to the global economy but also in the deaths of millions. The origin of a major human influenza pandemic is the 1918 Spanish flu, which alone caused three major outbreaks in 1918, leading to the loss of 50 million lives worldwide. Although neuraminidase inhibitors (such as oseltamivir) and cap-dependent endonuclease inhibitors (such as mabaloxavir) have been successfully used clinically, forming the cornerstone of current anti-influenza treatment, the continuous emergence and spread of viral resistance, as well as the insufficient efficacy in some patients, highlight the limitations and potential risks of existing treatments. Current mainstream antiviral drug development focuses primarily on highly conserved enzyme active sites of the virus itself (such as neuraminidase and RNA polymerase). However, viruses, through their inherently high mutation rate, can produce amino acid substitutions at these key targets, leading to decreased drug binding or even complete ineffectiveness, resulting in clinical drug resistance. For example, oseltamivir-resistant strains of the H1N1 virus have been reported multiple times. These issues highlight the necessity of developing new antiviral drugs.

[0005] Therefore, there is an urgent need in this field for more novel anti-influenza virus compounds. These compounds should effectively inhibit influenza virus replication while exhibiting high host cell safety to compensate for the shortcomings of existing treatments and provide new weapons against potential future influenza pandemics.

[0006] With continuous innovation in biotechnology and the advancement of high-throughput sequencing, researchers have discovered that small open reading frames (sORFs) on long non-coding RNA gene sequences can encode tiny proteins of less than 100 amino acids, termed micropeptides. Numerous experiments have confirmed that modified micropeptides can perform corresponding functions, and some micropeptides processed from long non-coding RNAs can act on themselves, promoting or inhibiting the expression of their own long non-coding RNAs. The micropeptide miPEP-71 is a human-derived micropeptide encoded by the long non-coding RNA NEAT1. However, the inventors have found that the specific function of miPEP-71 in the host's antiviral innate immune response, particularly during influenza virus infection, has not yet been reported in research. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a micropeptide miPEP-71 encoded by the long non-coding RNA NEAT1 and its applications.

[0008] To achieve the above objectives, the present invention provides a micropeptide miPEP-71, wherein the micropeptide miPEP-71 is a human micropeptide encoded by long non-coding RNA NEAT1, and the amino acid sequence of the miPEP-71 is shown in SEQ ID No. 2.

[0009] Furthermore, the nucleotide sequence encoding the micropeptide miPEP-71 is shown in SEQ ID No. 1.

[0010] This invention also proposes the use of the micropeptide miPEP-71 in any of the following: (1) Application in the preparation of drugs for treating influenza viruses; (2) Use in the preparation of medicines for the treatment and / or prevention of influenza virus infection; (3) Application in the preparation of influenza virus inhibitors; (4) Application in the preparation of drugs that inhibit the expression of influenza virus proteins NP and PB2.

[0011] Furthermore, the influenza virus is an influenza A virus; the influenza A virus is influenza A virus strain PR8.

[0012] Furthermore, the influenza virus inhibitor is used to inhibit the replication of the influenza virus; the influenza virus inhibitor is a drug or a common test reagent for non-medical purposes.

[0013] The beneficial effects of this invention are as follows: Experiments have shown that after cells are transfected with the miPEP-71 plasmid, compared with the control group (EV), miPEP-71 can inhibit influenza virus replication and reduce the viral load, thus serving as an influenza virus replication inhibitor. In summary, this invention provides a micropeptide miPEP-71 capable of inhibiting influenza virus replication, offering a new option for the preparation of anti-influenza virus drugs. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0015] Figure 1 The effect of the micropeptide miPEP-71 on influenza virus replication at the cellular level is shown in Figure A. * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01). Figure A shows the expression of PR8 NP in 293T cells after miPEP-71 overexpression, detected by RT-PCR and Western blotting. Figure B shows the hemagglutination assay for influenza virus replication in 293T cells. Figure C shows the plaque assay for viral load in 293T cells. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] It should also be noted that, in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solution of the present invention are shown in the embodiments, while other details that are not closely related are omitted.

[0018] 293T cells (human embryonic kidney cell line): American Type Culture Collection (ATCC), catalog number CRL-3216. MDCK cells (canine kidney cell line): American Type Culture Collection (ATCC), catalog number CCL-34.

[0019] Example 1. The micropeptide miPEP-71 can reduce the viral load of influenza virus replication. 1. Obtaining the miPEP-71 gene fragment Using the miPEP-71 nucleotide sequence (as shown in SEQ ID No. 1) from human NEAT1 (GenBank accession number: NR_131012.1) as a template, the micropeptide miPEP-71 (amino acid sequence as shown in SEQ ID No. 2) was synthesized by Shanghai Sangon Biotech Co., Ltd. After the micropeptide was synthesized, it was verified by mass spectrometry results to be correct before subsequent cell experiments were carried out.

[0020] 2. The micropeptide miPE-71 inhibits the replication of influenza virus. 293T cells were cultured in DMEM (Gibco) medium supplemented with 100 units / mL penicillin, 100 units / mL streptomycin, and 10% fetal bovine serum (FeBS) (Gibco). The culture was placed in a 37°C, 5% CO2 incubator. When the cell confluence in the 6-well plates reached 70-80%, the culture medium was replaced with 2 mL of complete medium. 2.5 μg of EV plasmid (control group) and miPEP-71 overexpression plasmid (experimental group) were added to 125 μL of transfection buffer (antibiotic-free and serum-free DMEM), followed by 4 μL of lipo8000. TM(Beyotime) The mixture was gently pipetted and added to 293T cells. After 24 hours of transfection, the complete culture medium was discarded, and the cells were infected with influenza virus PR8 (100 HAU / ml) for 2 hours. The cells were then washed three times with PBS and virus maintenance medium was added. Different samples were collected according to different subsequent detection requirements.

[0021] (1) RT-PCR assay to detect PR8 NP expression. After 16 h of culture, cells were removed from the cell culture incubator, washed with PBS, and then NucleoZOL (brand: MNG, catalog number: 740404.200) was added. Total RNA was extracted from the cells according to the NucleoZOL instructions, and the total RNA was reverse transcribed into cDNA. RT-PCR was performed using the cDNA as a template to detect PR8 NP expression, as shown in the attached figure. Figure 1 As shown in Figure A, overexpression of miPEP-71 can reduce the expression of influenza virus NP.

[0022] (2) Western blotting was used to detect the expression of PR8 NP. After culturing for 16 h, cells were removed from the cell culture incubator, washed with PBS, and then collected in 1.5 mL centrifuge tubes using a cell scraper. The cells were centrifuged at 5000 rpm and 4 °C for 5 min, and the PBS supernatant was discarded to obtain the cells. 80 μL of cell lysis buffer containing PMSF (final PMSF concentration was 2 mM) was added to each sample, and the cells were vortexed and placed on ice for lysis for 30 min (vortexing for 10 s every 10 min). After lysis, the cells were centrifuged at 12000 rpm and 4 °C for 10 min, and the supernatant was transferred to a new centrifuge tube. The appropriate loading buffer was added according to the amount of supernatant collected, and the cells were boiled in boiling water for 8 min to denature the protein. Then, SDS-PAGE gel was run, the cells were transferred to a membrane, blocked, and incubated with antibody. Finally, chemiluminescence was used to detect the expression of Flag and the internal control β-actin. Flag antibody and β-actin were purchased from Beijing TransGen Biotech Co., Ltd., and secondary antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. Results are as follows: Figure 1 A showed that overexpression of miPEP-71 reduced the expression of influenza virus PB2.

[0023] (3) Hemagglutination assay to detect influenza virus replication in cells. Supernatants were collected at 14h, 16h, 18h, 20h, and 22h for hemagglutination assay. First, blood was collected from healthy chickens, washed, and the red blood cells were prepared into a 1% suspension with PBS and placed on ice for later use or stored in a 4°C refrigerator. A 96-well V-shaped hemagglutination plate was taken out and labeled, and 25 μL of PBS solution was added to each well. 25 μL of virus solution was added to each well in the first column from the left, mixed with PBS, and then 25 μL of the mixture was serially diluted to the second to last column using a pipette. The rightmost column served as the control group. 1% red blood cell suspension was taken out, gently inverted and mixed, and then 25 μL of 1% chicken red blood cell suspension was added to each well using a pipette.

[0024] After gently agitating the hemagglutination plate, incubate it at 37°C for 15 minutes. Then, observe the agglutination of red blood cells obliquely to determine the viral titer. (See attached image) Figure 1 As shown in B, compared with control cells transfected with empty plasmid (EV), transfection with the micropeptide miPEP-71 plasmid, resulting in overexpression of the micropeptide, significantly inhibited influenza virus replication.

[0025] (4) Plaque assay to detect the viral load of influenza virus in cells. Collect the supernatant after 18 hours for plaque assay. Spread MDCK cells evenly into a six-well plate. When the cell density reaches 90%, the viral plaque assay can be carried out. First, remove the old culture medium, wash three times with 1×PBS, and then add 900 μL of virus maintenance medium to each well and place it in a cell culture incubator. Take 100 μL of the supernatant to be tested and perform a 10-fold serial dilution. Take 100 μL of the diluted virus solution and add it to each well, with three parallel wells for each dilution. Place the six-well plate in a cell culture incubator and incubate for 1 hour for infection and adsorption, gently shaking the cells once every 15 minutes. At the same time, preheat the DMEM medium without phenol red in the incubator. When the infection is about to end, melt 3% low melting point agarose in a microwave oven and mix it with the preheated DMEM without phenol red at a ratio of 1:4. Add 1 μg / mL TPCK-trypsin and mix well. Place the mixture in the incubator for later use. After infection, remove the six-well plate, discard the supernatant, wash three times with 1×PBS, discard the PBS, and quickly add 3% low-melting-point agarose mixture to each well along the sidewall. Place the six-well plate horizontally in a 4°C refrigerator and let it stand for 30 minutes until the mixture solidifies. Then, invert the six-well plate and incubate it in a cell culture incubator for approximately 72 hours to observe the experimental results. When obvious plaques appear in the six-well plate, plaque counting can be performed directly, or counting can be performed after crystal violet staining, and the viral titer can be calculated. Based on the plaque experiment results, see attached... Figure 1 C showed that the addition of the micropeptide miPEP-71 reduced the viral load of influenza virus replication.

[0026] The experimental results of the above embodiments demonstrate that the micropeptide miPEP-71 has the function of inhibiting influenza virus replication and reducing influenza virus replication load, and can be used as an inhibitor of influenza virus replication. Moreover, miPEP-71 is a human micropeptide with high host cell safety, providing a new option for the development of anti-influenza virus drugs.

[0027] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A micropeptide miPEP-71, characterized in that, The micropeptide miPEP-71 is encoded by the long non-coding RNA NEAT1, and the amino acid sequence of the micropeptide miPEP-71 is shown in SEQ ID No.

2.

2. The micropeptide miPEP-71 according to claim 1, characterized in that, The nucleotide sequence encoding the micropeptide miPEP-71 is shown in SEQ ID No.

1.

3. The use of the micropeptide miPEP-71 as described in claim 1 in any of the following: (1) Application in the preparation of drugs for treating influenza viruses; (2) Use in the preparation of medicines for the treatment and / or prevention of influenza virus infection; (3) Application in the preparation of influenza virus inhibitors; (4) Application in the preparation of drugs that inhibit the expression of influenza virus proteins NP and PB2.

4. The application according to claim 3, characterized in that, The influenza virus in question is influenza A virus.

5. The application according to claim 4, characterized in that, The influenza A virus in question is influenza A virus strain PR8.

6. The application according to claim 3, characterized in that, The influenza virus inhibitor is used to inhibit the replication of the influenza virus; the influenza virus inhibitor is a drug or a common test reagent for non-medical purposes.

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