A micropeptide encoded by long-chain non-coding RNA GAS5 and application thereof

Abstract

The application discloses a micropeptide encoded by long-chain non-coding RNA GAS5 and application thereof, and the micropeptide is a micropeptide containing 50 amino acids and encoded by long-chain non-coding RNA GAS5, and is named as GAS5-P50. It is found through blood clotting test and plaque test detection that, compared with a control group, overexpression of the micropeptide GAS5-P50 can significantly increase influenza virus HA titer and virus load, and knockdown of the expression of the micropeptide GAS5-P50 can significantly reduce the influenza virus HA titer and virus load. It is found that the micropeptide GAS5-P50 encoded by long-chain non-coding RNA plays a role in promoting virus replication in the process of influenza virus infection, and a molecule targeting the micropeptide GAS5-P50 can be developed as an anti-influenza virus drug, thereby providing a new option for prevention and treatment of influenza virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a micropeptide encoded by long-chain non-coding RNA GAS5 and applications thereof. Background Art

[0002] Long non-coding RNA (lncRNA) is a type of non-coding RNA greater than 200 bases in length that is abundant in the human body. Recent data indicate that lncRNA-related transcripts account for approximately a quarter of all human gene transcripts. LncRNAs possess a rich array of high-order structures, enabling them to perform diverse functions. With the advancement of sequencing technology, researchers have discovered that many lncRNAs contain small open reading frames (sORFs) that can encode small molecular weight proteins, known as micropeptides.

[0003] Accumulating evidence indicates that sORFs hidden within lncRNAs can be translated to produce novel micropeptides with important physiological and biochemical functions. These micropeptides function in various regulatory networks within the body, such as individual growth and development, tumorigenesis and progression, and cellular metabolism. With in-depth research, more and more functional micropeptides are being discovered, and their diverse regulatory mechanisms are being revealed. For example, Anderson et al. discovered that the lncRNA AFAP1-AS1 encodes a 90-amino acid micropeptide, named ATMLP, located on mitochondria. Although ATMLP is a small protein, it can promote the development and progression of lung cancer. Researchers have found that high serum levels of ATMLP in patients with non-small cell lung cancer are associated with a poor prognosis. FORCP, a conserved small protein encoded by LINC00675, is abundant in well-differentiated colorectal cancer (CRC) cells and functions to inhibit proliferation, clonogenicity, and tumorigenesis. GT3-INCP, a novel micropeptide encoded by LINC00992, has a tumor-promoting function, and its expression is associated with a poor prognosis in luminal tumors. GT3-INCP interacts with GATA3, a key transcription factor in mammary gland development and breast cancer cell proliferation, regulating the expression of multiple breast cancer susceptibility genes and influencing the estrogen response and cell proliferation of tumor cells. These studies suggest that many previously unknown proteins encoded by lncRNAs play multiple roles in the development and progression of diseases, potentially representing new therapeutic targets and holding significant research significance.

[0004] A wide variety of antiviral drugs are currently available on the market, each with a distinct mechanism of action. Many work by interfering with various stages of viral replication. Drugs such as amantadine, rimantadine, enfuvirtide, and maraviroc primarily inhibit viral entry and uncoating, offering broad-spectrum antiviral activity but are prone to side effects. Nucleoside drugs, such as lamivudine, zidovudine, emtricitabine, tenofovir, and adefovir dipivoxil, target the viral reverse transcriptase and interfere with the transcription of viral genetic material. Non-nucleoside drugs such as efavirenz and nevirapine are convenient to administer and play a crucial role in both specific treatments and broad-spectrum antiviral treatments, but prolonged use can easily lead to drug resistance. Neuraminidase inhibitors such as oseltamivir and zanamivir primarily target influenza viruses, inhibiting the release of the virus by inhibiting neuraminidase activity. However, the high mutability of influenza viruses leads to constant changes in neuraminidase antigenicity, which gradually diminishes the effectiveness of these drugs. Biological antiviral drugs primarily include transfer factor, interferon, and interleukin-2, which achieve their antiviral effects by inhibiting viral proliferation, growth, and replication and enhancing immunity. In addition to single-ingredient chemical drugs, many traditional Chinese medicines with complex ingredients also have antiviral effects. These herbs primarily inhibit viruses directly by blocking certain steps in viral reproduction, or indirectly by inducing interferon production and boosting immunity. While the antiviral effects of many traditional Chinese medicines have been clinically tested, their specific mechanisms of action remain elusive.

[0005] Current antiviral drugs have their own advantages and disadvantages in terms of efficacy, cost, and side effects. Traditional chemical drugs have a well-defined mechanism of action, exerting their antiviral effects by directly targeting viral replication. They are fast-acting and have a broad spectrum of efficacy. However, these drugs are often prone to adverse reactions, and as viruses evolve and adapt, drug resistance becomes increasingly prominent. Biological drugs primarily inhibit viral proliferation by modulating the human immune system. However, these drugs cannot be used long-term and are prone to severe side effects. Traditional Chinese medicines offer good efficacy and few adverse reactions, but their development is slow. Despite the emergence of a wide range of antiviral drugs, viruses are also constantly evolving. New strains emerge during transmission, and antigenic shifts lead to increasingly severe drug resistance. During viral infection, many host factors, such as lncRNAs, are involved in regulating viral replication. LncRNAs can directly participate in viral replication and influence viral proliferation by modulating host immune processes. Furthermore, lncRNAs can exert their effects by encoding functional micropeptides, which play a key role in the pathogenesis of various diseases and hold enormous potential for drug development. These micropeptides can also be used as therapeutic targets or viral antagonists in the development of antiviral drugs. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to propose a micropeptide encoded by long non-coding RNA and its application, which can promote the replication of influenza virus and can be used as a target for the design of anti-influenza virus drugs.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] The micropeptide encoded by the long non-coding RNA GAS5 is designated as GAS5-P50, and the amino acid sequence of the micropeptide GAS5-P50 is shown in SEQ ID No.1.

[0009] The nucleic acid molecule sequence encoding the above-mentioned micropeptide, wherein the nucleic acid sequence encoding the micropeptide GAS5-P50 is shown as SEQ ID No.2.

[0010] The use of the micropeptide GAS5-P50 expression inhibitor as described above in one or more of the following (a), (b), and (c):

[0011] (a) preparing anti-influenza virus drugs;

[0012] (b) preparing a drug for inhibiting influenza virus replication in cells;

[0013] (c) preparing a medicament for treating and / or preventing influenza.

[0014] As used above, the expression inhibitor of the micropeptide GAS5-P50 is an antibody that targets and binds to the micropeptide GAS5-P50 or an RNA interference reagent that targets the micropeptide GAS5-P50.

[0015] The influenza viruses mentioned above include (but are not limited to) influenza A viruses.

[0016] In the present invention, the influenza A virus is an H1 subtype influenza virus, specifically an H1N1 subtype influenza virus; more specifically, an H1N1 subtype influenza virus A / PR / 8 / 34 strain (ATCC VR-1469).

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: the present solution found through hemagglutination test and plaque test that overexpression of the micropeptide GAS5-P50 can significantly increase the influenza virus HA titer and viral load compared with the control group, while knocking down the expression of GAS5-P50 significantly reduced the influenza virus HA titer and viral load. In summary, the present invention has identified a micropeptide encoded by long non-coding RNA and having the function of promoting influenza virus replication, which can be used as a new target for anti-influenza virus drugs, providing a new option for the preparation of anti-influenza virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only represent some embodiments of the present application, and all other drawings obtained by those of ordinary skill in the art without creative effort based on these drawings belong to the protection scope of the present application.

[0019] Figure 1 In order to detect that long-chain non-coding RNA GAS5 contains an ORF with coding ability.

[0020] Figure 2 A is to detect that GAS5-P50 has coding ability and can drive the re-expression of mutated GFP and emit green fluorescence; B is that the cells transfected with GAS5-P50-GFP-mut plasmid can fuse to express GAS5-P50 and GFP.

[0021] Figure 3 In order to detect that endogenous micropeptide GAS5-P50 exists in A549 cells by mass spectrometry.

[0022] Figure 4 A is to detect the hemagglutination titer of virus in control cells and overexpression GAS5-P50 cells after infection with influenza virus; B is to detect the viral load in control cells and overexpression GAS5-P50 cells after infection with influenza virus; ** indicates a very significant difference (P<0.01).

[0023] Figure 5 A is to detect the expression of GAS5-P50, B is to detect the hemagglutination titer of virus in control cells and GAS5-P50 knockdown expression cells after infection with influenza virus; C is to detect the viral load in control cells and GAS5-P50 knockdown expression cells after infection with influenza virus; ** indicates a very significant difference (P<0.01). DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in combination with the accompanying drawings and embodiments. It is particularly pointed out that the following embodiments are only used to illustrate the present application, but do not limit the scope of the present application. Similarly, the following embodiments are only some embodiments of the present application, not all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0025] The materials used in the embodiments of the present application and their sources are as follows:

[0026] 293T cells (human embryonic kidney cell line): American Type Culture Collection (ATCC), number CRL-3216.

[0027] A549 cells (human lung cancer cell line): American Type Culture Collection (ATCC), number CCL-185.

[0028] MDCK cells (canine kidney cell line): American Type Culture Collection (ATCC), number CCL-34.

[0029] Example 1. Identification of long non-coding RNA GAS5 encoding micropeptide

[0030] Through the combined analysis of RNA-seq and RIBO-seq, long non-coding RNA GAS5 with potential coding ability ORF was found. The ORF was constructed into pLVX3 vector and a flag tag was added at the C terminal. The plasmid was named pLVX3-GAS5-P50 and sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. After verification, subsequent cell experiments were carried out.

[0031] The successfully constructed plasmid was transfected into 293T cells to detect whether the ORF has the ability to encode proteins by exogenous expression. The specific steps are as follows:

[0032] 293T cells were cultured in DMEM (Gibco) medium supplemented with a final concentration of 100 units / mL penicillin, 100 units / mL streptomycin, and 10% fetal bovine serum (Gibco) in a 37°C, 5% CO2 cell culture incubator. When the cells reached 80-90% confluency in a 6-well plate, 4 μg of pLVX3 empty vector and 4 μg of pLVX3-GAS5-P50 were transfected into the 293T cells according to the instructions of Lip8000 (Biyuntian Biotechnology Co., Ltd.). After 24 h of culture, the cells were removed from the cell culture incubator, washed with PBS, and then collected using a cell scraper in a 1.5 mL centrifuge tube. The cells were centrifuged at 4500 rpm at 4°C for 5 min, and the PBS supernatant was discarded to obtain the cells. Add 60 μL of cell lysis solution containing PMSF (the final concentration of PMSF is 2 mM) to each aliquot, vortex and place on ice for 30 minutes to lyse (vortex and oscillate for 10 seconds every 10 minutes). After the lysis is completed, centrifuge at 12000 rpm and 4°C for 10 minutes, and transfer the supernatant to a new centrifuge tube. Add the corresponding loading buffer according to the amount of supernatant collected, and boil in boiling water for 5 minutes to denature the protein. Then run SDS-PAGE gel, transfer the membrane, block, incubate the antibody, and finally use chemiluminescence to detect the expression of Flag and internal reference β-actin. Among them, Flag antibody and β-actin were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and secondary antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd. Western blot results are as follows. Figure 1 As shown, the ORF can encode a 50-amino acid micropeptide, which is named GAS5-P50, and its amino acid sequence is shown below (SEQ ID No. 1):

[0033] Met Val Leu Gly Ala Asp Ala Val Trp Leu Trp Ile Ala Pro Tyr Gly GlnLeu Cys Pro Gln Gly Arg Met Arg Ile Ala Thr Glu Val Leu Lys Ser Lys Pro AsnSer Ser His Trp His Thr Gly Ile Arg Gln Lys Ala Gly Ser

[0034] Among them, the nucleic acid sequence encoding GAS5-P50 is shown as SEQ ID No.2.

[0035] In addition, the sequence of GAS5-P50 was constructed on the pNL-GFP vector pNL-GFP-mut with the start codon of GFP mutated, and the cells were transfected again. Whether the ORF can drive the mutated GFP to be re-expressed was determined by observing whether the GFP green fluorescence and the expression of GFP protein, so as to further determine whether the ORF has the translation ability. The specific steps are as follows: the sequence of GAS5-P50 was constructed on the pNL-GFP-mut vector, and the insertion position was at the front end of the GFP sequence. The plasmid was named GAS5-P50-GFP-mut. Then the start codon of the ORF fragment in this plasmid was mutated (ATG was mutated into ATT), and the plasmid was named GAS5-P50-mut-GFP-mut. After sequencing by Shanghai Superbioengineering Co., Ltd. and verification, the subsequent cell test was carried out.

[0036] When the confluence of the 293T cells in the 6-well plate reached 80-90%, 4 μg of pNL-GFP, 4 μg of pNL-GFP-mut, 4 μg of GAS5-P50-GFP-mut and 4 μg of GAS5-P50-mut-GFP-mut were transfected into the 293T cells according to the instructions of Lip8000 (Biunten Biotechnology Co., Ltd.). After 24 h of culture, whether the cells had fluorescence after transfection of the plasmid was observed by an inverted fluorescence microscope. Figure 2 As shown in FIG. A, green fluorescence can be observed in the cells transfected with the pNL-GFP plasmid and the cells transfected with the GAS5-P50-GFP-mut plasmid, but no fluorescence can be observed in the cells transfected with the pNL-GFP-mut and GAS5-P50-mut-GFP-mut plasmids, which indicates that the GAS5-P50 can encode the micropeptide and drive the mutated GFP behind the micropeptide to express, and finally emit green fluorescence. Subsequently, the cells were collected, and the protein sample was prepared. The expression of GFP was detected by Western blot experiment. The GFP antibody and the secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd. The Western blot result is shown in FIG. B. Figure 2 As shown in FIG. B, the size of the GFP incubated from the cell sample transfected with the GAS5-P50-GFP-mut plasmid is about 5 KD larger than that of the GFP incubated from the cell sample transfected with the pNL-GFP plasmid, which shows that the GAS5-P50 can successfully fuse GFP to express.

[0037] Subsequently, the endogenous expression of the micropeptide GAS5-P50 in the cells was detected by mass spectrometry analysis. The A549 cells cultured in a 10 cm culture dish were collected, the cells were lysed to obtain total protein lysate, and SDS-PAGE was performed. The gel block below 10 KD was cut, and LC-MS / MS mass spectrometry analysis was performed after enzymolysis. The result is shown in FIG. C. Figure 3As shown, mass spectrometry identified a peptide segment (SEQ ID No. 3) that matched the micropeptide GAS5-P50: MVLGADAVWLWIAPYGQLCPQGRMR, confirming that GAS5-P50 actually exists in endogenous cells.

[0038] Example 2. The long non-coding GAS5-encoded micropeptide GAS5-P50 can promote the replication of influenza virus

[0039] When the confluence of 293T cells in a 6-well plate reaches 80-90%, 4 μg pLVX3 (control group) and 4 μg pLVX3-GAS5-P50 (experimental group) are transfected into 293T cells according to the instructions of Lip8000 (Biyuntian Biotechnology Co., Ltd.). After culturing for 24 hours, wash the cells 2-3 times with PBS, and then add 1 mL of virus maintenance solution (2 μg / mL trypsin in serum-free DMEM) and 5 μL H1N1 influenza virus A / PR / 8 / 34 strain (MOI=1). Then place it in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration for 1 hour, shaking the 6-well plate every 15 minutes during this period. After 1 hour, discard the virus maintenance solution, wash the cells again with PBS 2-3 times, add 2 mL of fresh virus maintenance solution to each well, and then continue to culture in a cell culture incubator. Different samples are collected according to different subsequent detection requirements:

[0040] 1. Hemagglutination test was used to detect the replication of influenza virus in cells. 200 μL of cell supernatant was collected at 16h, 18h, 20h, 22h, and 24h after virus infection and hemagglutination test was performed. Figure 4 As shown in Figure A, the hemagglutination titer of the experimental group was higher than that of the control group, indicating that GAS5-P50 can promote the replication of influenza virus in cells.

[0041] 2. Plaque assay to detect the viral load of influenza virus in cells. Plaque assay is the gold standard for virus titration, and the results are stable and reliable. After 16 hours of influenza virus infection, the cell supernatant was collected and the plaque assay was performed. The specific steps are as follows: First, MDCK cells were plated in a 6-well plate for later use (cell density was 5×10 5 / well is appropriate). The cell supernatant collected above is then diluted 10-fold with virus maintenance solution. Reprocess the MDCK cells, wash the cells 3 times with PBS, take 0.1 mL of the diluted virus solution and add it to each well of the 6-well plate, then add 0.9 mL of virus maintenance solution to each well, and set up 3 parallel wells for each dilution. The cells are cultured in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration to allow the virus to adsorb for 1 hour (shake the 6-well plate once every 15 minutes). During the virus adsorption process, 3% low-melting point agarose (Promega, product number V2111-25g) and phenol red-free DMEM (HYCLONE, product number SH30284.01) incubated at 37°C can be melted in advance by microwave, and then the two are mixed in a ratio of 1:4 (volume ratio), and TPCK-trypsin (Sigma, product number T8802-50MG) with a final concentration of 1 μg / mL is added and set aside. After the virus has been adsorbed for 1 hour, discard the supernatant and wash the cells 3 times with PBS. After absorbing as much PBS as possible, quickly add 2 mL of the pre-prepared mixture to each well. Finally, place the 6-well plate in a 4°C refrigerator for 1 hour. After the gel solidifies, invert it and culture it in a 37°C cell culture incubator for 2-3 days. When white spots appear, count the plaques (you can also count them after staining with neutral red). The results are as follows Figure 4 As shown in Figure B, the influenza virus load in the experimental group was higher than that in the control group, which once again shows that the micropeptide GAS5-P50 can significantly promote the replication of influenza virus.

[0042] Example 3. Knockdown of GAS5-P50 expression inhibits influenza virus replication

[0043] Two siRNAs were designed targeting the nucleotide sequence of GAS5-P50 and are labeled as follows:

[0044] si-GAS5-P50-1 (SEQ ID No. 4), sequence GGCTCTGGATAGCACCTTA;

[0045] si-GAS5-P50-2 (SEQ ID No. 5), sequence GGATGAGAATAGCTACTGA.

[0046] When the confluence of A549 cells in the 6-well plate reached 50-60%, 100 pmol si-NC, 100 pmol si-GAS5-P50-1, and 100 pmol si-GAS5-P50-2 were respectively transfected into A549 cells according to the instructions of Lip3000 (Lipofectamine 3000, Thermo Fisher Scientific). After 36 h of culture, the cells were washed with PBS for 2-3 times, and then 1 mL of virus maintenance liquid (trypsin 2 μg / mL in serum-free DMEM) and 5 μL of H1N1 influenza virus A / PR / 8 / 34 strain (MOI = 1) were added. Then, the 6-well plate was placed in a cell incubator at 37°C, saturated humidity, and 5% CO2concentration for 1 h, and was shaken every 15 min during this period. After 1 h, the virus maintenance liquid was discarded, the cells were washed with PBS again for 2-3 times, 2 mL of fresh virus maintenance liquid was added to each well, and then the plate was placed in the cell incubator for continuous culture. Different samples were collected according to different subsequent detection requirements:

[0047] 1. qRT-PCR detection of the relative expression of GAS5-P50. After 16 h of influenza virus infection, 500 μL of NucleoZOL (brand: MNG, catalog number: 740404.200) was added to each well. Total RNA was extracted according to the instructions of NucleoZOL, and the total RNA was reverse transcribed into cDNA. qRT-PCR was performed using the cDNA as a template to detect the expression of GAS5-P50. As shown in FIG. A of Figure 5 , both siRNAs had good knockdown effect on GAS5-P50.

[0048] 2. Hemagglutination test for detecting the replication of influenza virus in cells. At 16 h, 18 h, 20 h, 22 h, and 24 h after virus infection, 200 μL of cell supernatant was collected for hemagglutination test. The results are shown in FIG. B of Figure 5 . The hemagglutination titers of the si-GAS5-P50-1 or si-GAS5-P50-2 transfected groups were lower than those of the control group, indicating that knockdown of GAS5-P50 can inhibit the replication of influenza virus in cells.

[0049] 3. Plaque assay to detect the viral load of influenza virus in cells. 16 hours after the cells were infected with influenza virus, the cell supernatant was collected and subjected to a plaque assay. The specific steps are as follows: First, MDCK cells were plated in a 6-well plate and set aside. Then, the collected cell supernatant was diluted 10-fold with virus maintenance solution. After the MDCK cells were treated, the cells were washed three times with PBS. 0.1 mL of the diluted virus solution was added to each well of the 6-well plate. Then, 0.9 mL of virus maintenance solution was added to each well. Three parallel wells were set up for each dilution. The cells were cultured in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration to allow virus adsorption for 1 hour (shaking the 6-well plate once every 15 minutes). During the virus adsorption process, 3% low-melting point agarose and phenol red-free DMEM incubated at 37°C were melted in a microwave beforehand. The two were then mixed in a 1:4 (volume ratio) and TPCK-trypsin was added to a final concentration of 1 μg / mL and set aside. After the virus has been adsorbed for 1 hour, discard the supernatant and wash the cells 3 times with PBS. After absorbing as much PBS as possible, quickly add 2 mL of the pre-prepared mixture to each well. Finally, place the 6-well plate in a 4°C refrigerator for 1 hour. After the gel solidifies, invert it and culture it in a 37°C cell culture incubator for 2-3 days. When white spots appear, count the plaques (you can also count them after staining with neutral red). The results are as follows Figure 5 As shown in Figure C, the viral titers of the si-GAS5-P50-1 or si-GAS5-P50-2 transfection groups were lower than those of the control group, again indicating that knocking down the expression of GAS5-P50 can significantly inhibit the replication of influenza virus.

[0050] The above descriptions are only some embodiments of the present invention and do not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of micropeptide GAS5-P50 expression inhibitors in the following (a) or (b): (a) Preparation of medicaments for use against influenza A virus; (b) preparing medicaments for treating and / or preventing influenza A virus infection; The amino acid sequence of the micropeptide GAS5-P50 is shown in SEQ ID No. 1; The expression inhibitor of the micropeptide GAS5-P50 is siRNA targeting the micropeptide GAS5-P50.

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