Micropeptide coded by long-chain non-coding RNA GAS5 and application thereof

Abstract

The invention discloses a micropeptide coded by long-chain non-coding RNA (Ribonucleic Acid) GAS5 and application thereof, the micropeptide is a micropeptide which is coded by long-chain non-coding RNA GAS5 and contains 50 amino acids, and the micropeptide is named as GAS5-P50. According to the scheme, a hemagglutination test and a plaque test detect that compared with a control group, the overexpressed micropeptide GAS5-P50 can obviously increase the HA titer and the virus load of the influenza virus, and the HA titer and the virus load of the influenza virus are obviously reduced by knocking down the expression of the GAS5-P50. The invention finds that the micropeptide GAS5-P50 coded by the long-chain non-coding RNA plays a role in promoting virus replication in the influenza virus infection process, molecules of the targeted micropeptide GAS5-P50 can be developed to serve as anti-influenza virus drugs, and a new choice is provided for prevention and treatment of influenza viruses.

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 an application thereof. Background Art

[0002] Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 bases. It is abundant in the human body. The latest data show that lncRNA-related transcription products account for about a quarter of all human gene transcription products. LncRNA has a rich high-level structure, which enables it to perform a variety of different functions. With the development of sequencing technology, researchers have found that many small open reading frames (sORFs) contained in lncRNA have coding capabilities and can encode small molecular weight proteins, called micropeptides.

[0003] There is increasing evidence that sORFs hidden in lncRNA can be translated to produce novel micropeptides with important physiological and biochemical functions. These micropeptides play a role in various regulatory networks of the body, such as individual growth and development, tumor development, cell metabolism and other life activities. With the deepening of research, more and more functional micropeptides are gradually discovered, and their rich and diverse regulatory mechanisms are constantly revealed. For example, Anderson, DM and others found that lncRNA AFAP1-AS1 encodes a 90-amino acid micropeptide located on the mitochondria, named ATMLP. Although ATMLP is a protein with a very small molecular weight, it can promote the occurrence and development of lung cancer tumors. Researchers have found that patients with non-small cell lung cancer with high levels of ATMLP in the serum of patients have a poor prognosis. FORCP, a conserved small protein encoded by LINC00675, is abundant in well-differentiated colorectal cancer (CRC) cells, and its function is to inhibit proliferation, clonogenicity and tumorigenesis. GT3-INCP, a novel micropeptide encoded by LINC00992, has the function of promoting tumor growth, and the expression of this protein is associated with poor prognosis of luminal tumors. GT3-INCP interacts with GATA3, a key transcription factor in breast development / breast cancer cell proliferation, regulates the expression of multiple breast cancer susceptibility genes, and affects the estrogen response / cell proliferation of tumor cells. These studies show that many unknown proteins encoded by lncRNA play multiple roles in the occurrence and development of diseases, and therefore may become new therapeutic targets, which is of great research significance.

[0004] There are many types of antiviral drugs on the market, and their mechanisms of action are also different. Many antiviral drugs work mainly by interfering with various stages of viral replication. Such as amantadine, rimantadine, enfuvirtide, maraviroc, etc., these drugs mainly inhibit the virus's entry and uncoating process, have a broad-spectrum antiviral effect, but are prone to side effects. Nucleoside drugs target viral reverse transcriptase and can interfere with the transcription process of viral genetic material, such as lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, etc. Non-nucleoside drugs such as efavirenz and nevirapine are easy to use and play a vital role in specific treatment and broad-spectrum antiviral fields, but long-term use of drugs can easily cause drug resistance. Neuraminidase inhibitors such as oseltamivir and zanamivir are mainly targeted at influenza viruses, and inhibit the release process of influenza viruses by inhibiting the activity of influenza virus neuraminidase. However, the high mutability of influenza viruses causes the antigenicity of neuraminidase to change continuously, which gradually weakens the effectiveness of such drugs. Biological antiviral drugs mainly include transfer factor, interferon and interleukin-2, etc., which mainly achieve antiviral effects by inhibiting the proliferation, growth, replication of viruses and enhancing immunity. In addition to single-ingredient chemical drugs, many Chinese herbal medicines with complex ingredients also have antiviral effects. Chinese herbal medicines mainly directly inhibit viruses by blocking a certain link in the reproduction of viruses, or indirectly inhibit virus replication by inducing the body to produce interferon and improving the body's immunity. Although the antiviral effects of many Chinese herbal medicines have passed clinical tests, their specific mechanisms of action are difficult to clarify.

[0005] At present, various types of antiviral drugs have their own advantages and disadvantages in terms of efficacy, cost and side effects. Traditional chemical drugs have a clear mechanism of action, exert antiviral effects by directly targeting the virus replication process, and have a fast effect and a high spectrum. However, these drugs are often prone to adverse reactions, and as the virus evolves adaptively, its drug resistance problem has gradually become prominent. Biological drugs mainly inhibit the proliferation of viruses in the body by regulating human immunity. These drugs cannot be used for a long time and are prone to strong side effects. Chinese herbal medicines have good efficacy and fewer adverse reactions, but the development speed is slow. Although various types of antiviral drugs emerge in an endless stream, viruses are also constantly evolving. During the transmission process, new strains continue to appear, and antigenic changes lead to increasingly serious drug resistance problems. In the process of viral infection of the host, many host factors such as lncRNA are also involved in regulating the process of viral replication. LncRNA can directly participate in the process of viral replication and can also affect the proliferation of the virus by regulating the host immune process. In addition, lncRNA can also play a role by encoding functional micropeptides. These micropeptides play a key role in the occurrence of many diseases and have great 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, wherein the micropeptide 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 set 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 SEQID 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 scheme, the present invention has the following beneficial effects compared with the prior art: the present scheme detected by hemagglutination test and plaque test found that overexpression of micropeptide GAS5-P50 can significantly increase the HA titer and viral load of influenza virus compared with the control group, while knocking down the expression of GAS5-P50 significantly reduced the HA titer and viral load of influenza virus. In summary, the present invention identifies 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 To detect the long non-coding RNA GAS5 contains an ORF with coding ability.

[0020] Figure 2 A is the detection of GAS5-P50's coding ability, which can drive the re-expression of the mutated GFP and emit green fluorescence; B is the cells transfected with GAS5-P50-GFP-mut plasmid can fusion-express GAS5-P50 and GFP.

[0021] Figure 3 Mass spectrometry was used to detect the endogenous presence of the micropeptide GAS5-P50 in A549 cells.

[0022] Figure 4 The results of the hemagglutination experiment of this protocol. In the figure, A is the hemagglutination titer of the virus in the control cells and the cells overexpressing GAS5-P50 after being infected with influenza virus; B is the viral load in the control cells and the cells overexpressing GAS5-P50 after being infected with influenza virus; ** indicates that the difference is extremely significant (P<0.01).

[0023] Figure 5 The results of detecting the expression of GAS5-P50 in this protocol, where A is the detection of GAS5-P50 expression, B is the detection of the hemagglutination titer of the virus in the control cells and the GAS5-P50 knockdown cells after infection with influenza virus; C is the detection of the viral load in the control cells and the GAS5-P50 knockdown cells after infection with influenza virus; ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0025] The materials used in this embodiment 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), numbered CCL-34.

[0029] Example 1. Identification of micropeptides encoded by long noncoding RNA GAS5

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

[0031] The successfully constructed plasmid was transfected into 293T cells, and the ORF was tested for its ability to encode protein by exogenous expression. The specific steps are as follows:

[0032] 293T cells were cultured in DMEM (Gibco) medium, and the medium was supplemented with a final concentration of 100 units / mL penicillin, 100 units / mL streptomycin and 10% fetal bovine serum (Gibco), and placed in a cell culture incubator at 37°C and 5% CO2 concentration. When the confluence of the cells in the 6-well plate reached 80-90%, 4 μg of pLVX3 empty vector and 4 μg of pLVX3-GAS5-P50 were transfected into 293T cells according to the instructions of Lip8000 (Biyuntian Biotechnology Co., Ltd.). After culturing for 24 hours, the cells were removed from the cell culture incubator, washed with PBS, and then collected with a cell scraper in a 1.5 mL centrifuge tube, centrifuged at 4500 rpm, 4°C, for 5 minutes, 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 portion, vortex and place on ice for 30 minutes to lyse (vortex for 10 seconds every 10 minutes). After the lysis is completed, centrifuge at 12000rpm, 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 the secondary antibody was purchased from Wuhan Mitaka Biotechnology Co., Ltd. Western blot results are shown as follows. Figure 1 As shown, the ORF can encode a micropeptide of 50 amino acids, 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 a mutated GFP start codon, and then the cells were transfected. By observing the presence of GFP green fluorescence and the expression of GFP protein, it was determined whether the ORF would drive the mutated GFP to be re-expressed, so as to further determine whether the ORF had 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, and then the start codon of the ORF fragment in this plasmid was mutated (ATG mutated to ATT), and the plasmid was named GAS5-P50-mut-GFP-mut, and sent to Shanghai Biotech Co., Ltd. for sequencing. After verification, subsequent cell experiments were carried out.

[0036] When the confluence of 293T cells in the 6-well plate reached 80-90%, 4 μg pNL-GFP, 4 μg pNL-GFP-mut, 4 μg GAS5-P50-GFP-mut, and 4 μg GAS5-P50-mut-GFP-mut were transfected into 293T cells according to the instructions of Lip8000 (Biyuntian Biotechnology Co., Ltd.). After culturing for 24 hours, the cells were observed for fluorescence after plasmid transfection under an inverted fluorescence microscope. Figure 2 As shown in A, green fluorescence can be observed in cells transfected with pNL-GFP plasmid and cells transfected with GAS5-P50-GFP-mut plasmid, but there is no fluorescence in cells transfected with pNL-GFP-mut and GAS5-P50-mut-GFP-mut plasmids, indicating that GAS5-P50 can encode micropeptides and drive the expression of GFP that has mutated behind the micropeptides, and finally emit green fluorescence. Subsequently, the cells were collected, protein samples were prepared, and the expression of GFP was detected by Western blot experiment. Among them, GFP antibody and secondary antibody were purchased from Wuhan Mitaka Biotechnology Co., Ltd. Western blot results are shown in Figure 2 As shown in B, the size of GFP hatched from the cell sample transfected with GAS5-P50-GFP-mut plasmid is about 5KD larger than that of GFP hatched from the cell sample transfected with pNL-GFP plasmid, which shows that GAS5-P50 can be successfully fused with GFP expression.

[0037] Subsequently, the endogenous expression of the micropeptide GAS5-P50 in cells was detected by mass spectrometry. A549 cells cultured in a 10 cm culture dish were collected, lysed, and the total protein lysate was obtained. SDS-PAGE was performed, and the gel blocks below 10 KD were cut. After enzymatic hydrolysis, LC-MS / MS mass spectrometry analysis was performed. The results are shown in Figure 2. 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 micropeptide GAS5-P50 encoded by the long non-coding GAS5 can promote the replication of influenza virus

[0039] When the confluence of 293T cells in the 6-well plate reaches 80-90%, according to the instructions of Lip8000 (Biyuntian Biotechnology Co., Ltd.), 4μg pLVX3 (control group) and 4μg pLVX3-GAS5-P50 (experimental group) were transfected into 293T cells. After 24h of culture, the cells were washed 2-3 times with PBS, and then 1mL of virus maintenance solution (serum-free DMEM containing 2μg / mL of pancreatin) and 5μL H1N1 influenza virus A / PR / 8 / 34 strain (MOI=1) were added. Then placed in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration for 1h, and the 6-well plate was shaken every 15min during the period. After 1h, the virus maintenance solution was discarded, and the cells were washed 2-3 times with PBS again, and 2mL of fresh virus maintenance solution was added to each well, and then continued to be placed in a cell culture incubator for culture. Different samples were 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 golden 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 (the cell density was 5×10 5 / well is appropriate). Then use the virus maintenance solution to dilute the cell supernatant collected above by 10 times. Re-treat the MDCK cells, wash the cells with PBS 3 times, take 0.1mL of diluted virus solution and add it to each well of the 6-well plate, then add 0.9mL of virus maintenance solution to each well, and set 3 parallel wells for each dilution. Culture the cells in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration to allow the virus to adsorb for 1h (shake the 6-well plate once every 15min). During the virus adsorption process, 3% low-melting point agarose (Promega, product number V2111-25g) and 37°C incubated phenol red-free DMEM (HYCLONE, product number SH30284.01) 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 1μg / mL is added for standby use. 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 in a 37°C cell culture incubator and culture it 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 marked 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 transfected into A549 cells according to the instructions of Lip3000 (Lipofectamine3000, Thermo Fisher Scientific). After 36 h of culture, the cells were washed 2-3 times with PBS, and then 1 mL of virus maintenance solution (serum-free DMEM containing 2 μg / mL of pancreatin) and 5 μL of H1N1 influenza virus A / PR / 8 / 34 strain (MOI=1) were added. Then, the cells were placed in a cell culture incubator at 37°C, saturated humidity, and 5% CO2 concentration for 1 h, and the 6-well plate was shaken every 15 min. After 1 hour, discard the virus maintenance solution, wash the cells with PBS 2-3 times, add 2 mL of fresh virus maintenance solution to each well, and then continue to culture in the cell culture incubator. Collect different samples according to different subsequent detection requirements:

[0047] 1. Detect the relative expression of GAS5-P50 by qRT-PCR. 16 hours after influenza virus infection, add 500 μL of NucleoZOL (brand: MNG, catalog number: 740404.200) to each well. According to the instructions of NucleoZOL, extract total RNA from cells, reverse transcribe total RNA into cDNA, and perform qRT-PCR with cDNA as template to detect the expression of GAS5-P50. Figure 5 As shown in Figure A, both siRNAs have a good effect of knocking down GAS5-P50.

[0048] 2. 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 5 As shown in Figure B, the hemagglutination titers of the groups transfected with si-GAS5-P50-1 or si-GAS5-P50-2 were lower than those of the control group, indicating that knocking down 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. When the cells were treated with influenza virus for 16 hours, the cell supernatant was collected and the plaque assay was performed. The specific steps are as follows: First, the MDCK cells were spread in a 6-well plate for use. Then the cell supernatant collected above was diluted 10 times with the virus maintenance solution. After the MDCK cells were treated again, the cells were washed with PBS 3 times, and 0.1 mL of the diluted virus solution was added to each well of the 6-well plate, and then 0.9 mL of the virus maintenance solution was added to each well, and 3 parallel wells were set for each dilution. The cells were 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 and phenol red-free DMEM 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 with a final concentration of 1 μg / mL is added for use. 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 in a 37°C cell culture incubator and culture it 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 virus titers of the groups transfected with si-GAS5-P50-1 or si-GAS5-P50-2 were lower than those of the control group, which once again indicated that knocking down the expression of GAS5-P50 could significantly inhibit the replication of influenza virus.

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

Claims

1. A micropeptide encoded by long non-coding RNA GAS5, denoted as GAS5-P50, characterized in that: The amino acid sequence of micropeptide GAS5-P50 is shown in SEQ ID No.

1.

2. The nucleic acid molecule sequence encoding the micropeptide according to claim 1, characterized in that: The nucleic acid sequence encoding the micropeptide GAS5-P50 is shown in SEQ ID No.

2.

3. Use of the micropeptide GAS5-P50 expression inhibitor as claimed in claim 1 in one or more of the following (a), (b), and (c): (a) preparing anti-influenza virus drugs; (b) preparing a drug for inhibiting influenza virus replication in cells; (c) preparing a medicament for treating and / or preventing influenza.

4. The use according to claim 3, characterized in that: 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.

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