CN115894658A - Long-chain non-coding RNA ZFAS1 coded micro-peptide and application thereof - Google Patents

Abstract

The invention discloses application of a novel micro-peptide, and belongs to the field of biological medicine research and development. The application provided by the invention is specifically the application of the long-chain non-coding RNA ZFAS1 coded micro-peptide ZFAS1-P56 in the preparation of anti-influenza virus drugs. Experiments prove that the long-chain non-coding RNA ZFAS1 can code a micro-peptide containing 56 amino acids. RT-PCR, hemagglutination test and plaque test tests show that compared with a control group, the overexpression of the micro-peptide ZFAS1-P56 can obviously reduce the mRNA expression quantity of the NP protein of the influenza virus, the HA titer of the virus is obviously reduced, the virus carrying capacity is also obviously reduced, and the micro-peptide ZFAS1-P56 can inhibit the proliferation of the influenza virus in cells. The invention finds the effective application of the long-chain non-coding RNA coded micro-peptide ZFAS1-P56 in anti-influenza virus, has potential new drug development value, and provides a new choice for preparing anti-influenza virus drugs.

Description

Long-chain non-coding RNA ZFAS1 coded micro-peptide and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a long-chain non-coding RNA ZFAS1 coded micro-peptide and application thereof in preparation of anti-influenza virus medicines.

Background

Long non-coding RNAs (lncrnas) are a class of non-coding RNA molecules with a length greater than 200 nucleotides (nt). Over the past decade, due to the rapid development of advanced high-throughput RNA sequencing technologies, tens of thousands of novel lncrnas have been annotated in animal and plant genomes. In addition, many studies now reveal that some long non-coding RNAs are also likely to be translated into stable, functional micro-or small peptides. A micro-peptide is generally defined as a polypeptide having less than 100 amino acids. They are present in eukaryotic and prokaryotic cells and are also produced by upstream ORF (uORF) or larger polypeptide divisions of mRNA. The micro-proteins translated from the uORF may be referred to as uORF-encoding peptides or uPEPs.

Although many putative micro-peptides have been identified in different organisms with advanced technological advances, only a limited number of them have been revealed to have biological functions. Most identified dipeptides function by binding to or modulating larger proteins. In 2016, researchers at the southwestern medical center of texas university identified a small peptide (DWORF) encoded by a non-coding RNA in human heart cells and confirmed that it plays an important role in myocardial contraction. Although DWORF contains only 34 amino acids and has no enzymatic activity, it is able to function as a regulator of the macromolecular complex. Another investigator found that lncRNA LINC00961 encodes a Small Peptide (SPAR) containing 90 amino acids. SPAR is involved in multiple cellular processes such as translation, metabolism and cell growth by regulating the activity of mTORC1 protein complex. Since dysregulation of mTORC1 protein complexes is often present in cancer, researchers are analyzing whether SPAR can affect other disease-associated cellular functions of mTORC 1. In addition, the non-coding RNA MIR155HG can also code a polypeptide (miPEP 155), the miPEP155 influences the antigen presenting function of dendritic cells by combining HSP70, and the polypeptide can play a better treatment effect on a mouse psoriasis-like disease model and an experimental encephalomyelitis model. In conclusion, the discovery and identification of unknown micro-or small peptides encoded by lncRNA and the exploration of new application fields are of great significance for opening the coding door of non-coding RNA and searching for new disease treatment strategies.

Currently known commonly used antiviral drugs mainly include two main classes of chemical drugs and Chinese herbal medicines. The chemical medicine has the advantages of fast effect, good antiviral effect, definite pharmacological mechanism and the like, plays a great role in preventing and treating viruses, and mainly contains nucleosides, non-nucleosides, organisms and polysaccharides. The antiviral drugs of nucleoside include ribavirin, lamivudine, vidarabine, idoxuridine, zidovudine and the like, are convenient to take, play a vital role in the fields of specific treatment and broad-spectrum antiviral, but have obvious side effects, and are easy to cause drug resistance due to long administration time. The non-nucleoside drugs mainly comprise amantadine, peptides, non-peptides, foscarnet and the like, and the drugs have the disadvantages of high cost, short half life, drug resistance caused by repeated medication and the like. The biological antiviral drugs mainly comprise transfer factors, interferon, interleukin-2 and the like, and achieve the antiviral effect mainly by inhibiting the proliferation, growth and replication of viruses, enhancing the immunity and the like. Mannan, dextran sulfate and lentinan belong to polysaccharide antiviral drugs, and the drugs mainly are host immunopotentiators, and can also directly inhibit viruses to lose activity or inhibit replication of the viruses, and the drugs can generate adverse reactions after being used for a long time. The Chinese herbal medicine has the advantages of low toxicity, difficult generation of drug resistance, less drug residue and the like, and is increasingly paid attention to prevention and treatment of viral diseases. The Chinese herbal medicines comprise 2 kinds of single Chinese herbal medicines and compound Chinese herbal medicines, and the virus is directly inhibited by blocking a certain link of virus propagation or indirectly inhibited by inducing an organism to generate interferon and improving the immunity of the organism. A single Chinese herbal medicine has wide variety and obvious effect, and a plurality of Chinese herbal medicines are found to have the effect of inhibiting viruses, but the specific antiviral mechanism needs to be further researched. The compound Chinese herbal medicine is a prescription compatible for definite diseases, and has the advantages of wide antiviral spectrum, wide action mechanism and good antiviral effect.

It is worth mentioning that the research development of polypeptide drugs is very rapid, according to QYResearch statistics: the global polypeptide drug market size is about $ 152 billion in 2010, and reaches $ 285 billion in 2018, and the composite annual average growth rate reaches 8.17%. The polypeptide medicine has obvious activity, strong specificity, good affinity with receptors, weak toxicity and difficult accumulation in vivo. Compared with protein macromolecular drugs, except for the polypeptide vaccine, the polypeptide drug has relatively low immunogenicity, small dosage, higher unit activity, easy synthesis, modification and optimization, high product purity and controllable quality, and can quickly determine the medicinal value. At present, polypeptide drugs are gradually applied and developed in the treatment of cancer, cardiovascular diseases, immune-related diseases, metabolic diseases and infectious diseases. However, the research on the micro-peptide in the treatment of various diseases is less, and the patent utilizes bioinformatics analysis and experimental verification to identify a novel micro-peptide coded by long-chain non-coding RNA in human cells, discusses the application of the novel micro-peptide in the preparation of anti-influenza virus medicines and provides a new scheme for the design of antiviral medicines.

Disclosure of Invention

The invention provides a novel micro peptide coded by long-chain non-coding RNA and application thereof in preparing anti-influenza virus drugs.

In order to achieve the purpose, the invention provides the following technical scheme:

the long-chain non-coding RNA ZPAS 1-coded micro-peptide ZPAS 1-P56 is characterized in that the amino acid sequence of the micro-peptide ZPAS 1-P56 is shown as SEQ ID No. 1.

The application of the micro-peptide ZPAS 1-P56 coded by the ZPAS 1 in the following (a), (b) and (c):

(a) Preparing an anti-influenza virus medicament;

(b) Preparing a medicament for inhibiting the replication of influenza virus in cells;

(c) Preparing the medicine for treating and/or preventing influenza.

All of the above influenza viruses include, but are not limited to, influenza a viruses.

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

The beneficial effects of the invention are:

experiments prove that the long-chain non-coding RNA ZFAS1 can code a micro-peptide containing 56 amino acids. RT-PCR, hemagglutination test and plaque test tests show that compared with a control group, the expression quantity of mRNA of the NP protein of the influenza virus can be obviously reduced by overexpression of the long-chain non-coding RNA ZFAS1 coded micro peptide, the HA titer of the virus is obviously reduced, the virus capacity is also obviously reduced, and the micro peptide ZFAS1-P56 can obviously inhibit the replication level of the influenza virus in cells. In conclusion, the invention identifies the oligopeptide which is coded by the long-chain non-coding RNA and has the function of inhibiting influenza virus replication, and provides a new choice for preparing anti-influenza virus medicaments.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the exogenous expression of a predicted long non-coding RNA ZFAS1 ORF with coding potential in 293T cells.

FIG. 2A shows that ZPAS 1-P56 has coding ability, can bring mutated GFP to re-express, and emit green fluorescence; b is a cell transfected with PEGFP-N1-MUT-ZFAS1-P56 plasmid and can fuse and express ZFAS1-P56 and GFP.

FIG. 3 shows the endogenous presence of the mini-peptide ZFAS1-P56 in A549 cells.

FIG. 4A is a graph showing the effect of overexpression of ZFEAS 1-P56 on the mRNA expression level of influenza NP protein; b is the effect of overexpression of ZFAS1-P56 on influenza replication in cells, indicating very significant differences (P < 0.01); c is the effect of overexpression of ZFAS1-P56 on the viral load of influenza virus in cells, indicates that the difference is very significant (P < 0.01).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

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

A549 cells (human lung cancer cell line): american Type Culture Collection (ATCC) with number CCL-185.

MDCK cells (canine kidney cell line): american Type Culture Collection (ATCC) with the number CCL-34.

The present invention is illustrated in detail below with specific examples, which should not be construed as limiting the scope of the invention.

Example 1 identification of Long non-coding RNA ZFAS 1-encoding Micropeptides

The RNA-seq and RIBO-seq are jointly analyzed, the long-chain non-coding RNA with potential coding capacity is preliminarily verified, and the ORF that the long-chain non-coding RNA ZFAS1 has potential coding capacity is found. The ORF is constructed on a pLVX3 vector, a flag tag is added at the C end, the plasmid is named as pLVX3-ZFAS1-P56, the pLVX3-ZFAS1-P56 is sent to a company Limited in the biological engineering (Shanghai) to carry out sequencing, and after the sequencing is verified to be correct, the subsequent cell test is carried out.

The successfully constructed plasmid is transfected into 293T cells, and whether the ORF has the capability of encoding protein is detected by means of exogenous expression. The method comprises the following specific steps: 293T cells were cultured in DMEM (Gibco) medium supplemented with penicillin 100units/mL, streptomycin 100units/mL and fetal bovine serum 10% (Gibco) at final concentrations, placed at 37 ℃ and 5% CO ₂ In the cell culture box with the concentration, when the confluence of the cells in the 6-well plate reaches 80-90%, 4 mu g of pLVX3 empty vector and 4 mu g of pLVX3-ZFAS1-P56 are transfected into 293T cells respectively according to the specification of Lip8000 (Biyuntian biotechnology Co., ltd.). After 24h of culture, the cells were taken out from the cell incubator, washed with PBS, scraped with a cell scraper, collected in a 1.5ml centrifuge tube at 4500rpm,4 ℃, centrifuged for 5min, and the PBS supernatant was discarded to obtain the cells. mu.L of PMSF-containing cell lysate (PMSF final concentration of 2 mM) was added to each portion, vortexed and then placed on ice for lysis for 30min (vortexed for 10 seconds every 10 min). After the cleavage was completed, the mixture was centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was transferred to a new centrifuge tube. Adding corresponding loading buffer according to the amount of the collected supernatant, and boiling in boiling water for 5min to denature the protein. Then running SDS-PAGE gel, transferring membrane, sealing, incubating antibody, and finally detecting the expression conditions of Flag and internal reference beta-actin by chemiluminescence. The Flag antibody and the beta-actin are purchased from Beijing Quanjin biotechnology limited, and the secondary antibody is purchased from Wuhan Sanying biotechnology limited. The Western blot result is shown in FIG. 1, the ORF can encode 56 amino acid micro-peptide, which is named as ZFAS1-P56, and the amino acid sequence is shown as follows:

Met Thr Ala Pro Lys Ala Phe Ala Gly Leu Ser Arg Pro Gln Arg Lys Gly Asn Pro Ser Ser Gly Leu Val Arg Ala Ala Ser Gly Tyr Arg Met Asp Phe Gly Arg Gly Ser His His Trp Thr Ser Lys Glu Ala Thr Cys Arg His Leu Gln Pro Ser Ile Ser

in addition, the sequence of ZFAS1-P56 is constructed on a PEGFP-N1 vector PEGFP-N1-MUT with GFP initiation codon mutation, cells are transfected, and whether the ORF drives the expression of GFP or not is judged by observing the existence of GFP green fluorescence and the expression condition of GFP protein, so as to further determine whether the ORF has translation capability or not. The method comprises the following specific steps: the sequence of ZFAS1-P56 is constructed in a PEGFP-N1-MUT vector, the inserted position is at the front end of the GFP sequence, the used enzyme cutting is XhoI (upstream) and KpnI (downstream), the plasmid is named PEGFP-N1-MUT-ZFAS1-P56, the plasmid is sent to the company Limited in the biological engineering (Shanghai) to carry out sequencing, and after the verification, the subsequent cell test is carried out.

When the confluence of 293T cells in 6-well plates reached 80-90%, 4. Mu.g of PEGFP-N1-MUT, and 4. Mu.g of PEGFP-N1-MUT-ZFAS1-P56 were transfected into 293T cells, respectively, according to the instruction of Lip8000 (Biyuntian Biotechnology Co., ltd.). After 24h of culture, cells were observed for fluorescence by inverted fluorescence microscopy after transfection of the plasmid. As shown in FIG. 2A, green fluorescence was observed in both PEGFP-N1 plasmid transfected cells and PEGFP-N1-MUT-ZFAS1-P56 plasmid transfected cells, but no fluorescence was observed in PEGFP-N1-MUT plasmid transfected cells, indicating that ZFAS1-P56 was able to encode a small peptide and drive expression of a GFP, which has been mutated behind the small peptide, to emit green fluorescence. Subsequently, cells were harvested, protein samples were prepared, and expression of GFP was detected by Western blot assay. Among them, both the GFP antibody and the secondary antibody were purchased from Wuhan Sanying Biotechnology Ltd. The Western blot result is shown in figure 2B, and the size of GFP hatched from the cell sample transfected with the PEGFP-N1-MUT-ZFAS1-P56 plasmid is about 6KD slightly larger than that of GFP hatched from the cell sample transfected with the PEGFP-N1 plasmid, so that ZFAS1-P56 can achieve achievement fusion of GFP expression.

Subsequently, the endogenous expression of the micro-peptide ZFAS1-P56 in the cells was again detected by mass spectrometry. Collecting A549 cells cultured in a 10cm culture dish, lysing the cells to obtain a total protein lysate, performing SDS-PAGE, cutting gel blocks within 10-15KD, performing LC-MS/MS mass spectrometry after enzymolysis. The results are shown in fig. 3, and mass spectrometry identified 1 peptide fragment matching with the micropeptide ZFAS 1-P56: PQRKGNPSSGLVR, the presence of ZFAS1-P56 in the endogenous source of the cell was determined.

Example 2 Long non-coding RNA ZFAS 1-encoded mini-peptide ZFAS1-P56 inhibits replication of influenza viruses

When the confluence of 293T cells in 6-well plates reached 80-90%, 4. Mu.g of PEGFP-N1 (control group) and 4. Mu.g of PEGFP-N1-MUT-ZFAS1-P56 (experimental group) were transfected into 293T cells according to the specification of Lip8000 (Biyuntian Biotechnology Co., ltd.). After 24H incubation, the cells were washed 2-3 times with PBS and 1ml of virus maintenance medium (2. Mu.g/ml pancreatin in serum-free DMEM) and 5. Mu.l of H1N1 influenza A/PR/8/34 strain (MOI = 1) were added. Then, standing at 37 deg.C, saturated humidity, 5% CO ₂ Culturing in cell culture box with concentration for 1 hr while shaking every 15min6-well plate. After 1h, abandoning the virus maintenance liquid, washing the cells with PBS for 2-3 times again, adding 2ml of fresh virus maintenance liquid into each hole, then continuously placing the fresh virus maintenance liquid into a cell culture box for culture, and collecting different samples according to different subsequent detection requirements:

1. RT-PCR detects the expression of the NP protein of influenza virus at the mRNA level. 12h after viral infection, 1ml of NucleoZOL (brand: MNG, cat # 740404.200) was added per well. The total RNA of the cells is extracted according to the instruction of the nucleoZOL, the total RNA is reversely transcribed into cDNA, the cDNA is used as a template to carry out RT-PCR, the mRNA expression levels of NP protein, ZFAS1-P56 and internal reference GAPDH of the influenza virus are detected, and as shown in figure 4A, after the ZFAS1-P56 is over-expressed, the mRNA expression level of the NP is obviously reduced.

2. The hemagglutination assay detects the replication of influenza virus in cells. At 12h, 14h, 16h, 18h, 20h, 22h, and 24h of virus infection, 200. Mu.L of cell supernatant was collected and subjected to hemagglutination assay. The results are shown in fig. 4B, the hemagglutination price of the control group is higher than that of the experimental group, and the difference is significant, which indicates that ZFAS1-P56 can inhibit the replication of influenza virus in cells.

3. The plaque assay detects the viral load of influenza virus in cells. The plaque assay is the gold standard for virus titration, and the results are stable and reliable. At 16h after infection of the treated cells with influenza virus, cell supernatants were harvested for plaque assay. The method comprises the following specific steps: MDCK cells were first plated in 6-well plates (cell density 5X 10) ⁵ Preferably per hole). The cell supernatants collected above were then diluted 10-fold with virus maintenance solution. And then treating the MDCK cells, washing the cells for 3 times by PBS, respectively taking 0.1ml of diluted virus solution, correspondingly adding the diluted virus solution into each well of a 6-well plate, adding 0.9ml of virus maintenance solution into each well, and setting 3 parallel wells for each dilution degree. Placing the cells at 37 deg.C, saturation humidity, 5% ₂ The cells were incubated at a concentration in an incubator to allow the virus to adsorb for 1h (shaking the 6-well plate once every 15 min). In the virus adsorption process, 3% low melting point agarose (Promega, product No. V2111-25 g) was previously microwaved and incubated with phenol red-free DMEM (HYCLONE, product No. SH 30284.01) at 37 ℃ and then mixed at a ratio of 1,TPCK-pancreatin (Sigma, product No. T8802-50 MG) was added to a final concentration of 1. Mu.g/ml for further use. After 1h of virus adsorption, the supernatant was discarded, the cells were washed 3 times with PBS, and after the PBS was removed as much as possible, 2ml of the prepared mixture was added to each well. And finally, placing the 6-hole plate in a 4 ℃ refrigerator for 1h, and after the gel is solidified, inversely placing the plate in a 37 ℃ cell incubator for culturing for 2-3 days. When white spots appeared, plaques were counted (they were also counted after staining with neutral red). The results are shown in fig. 4C, the influenza virus load of the control group is higher than that of the experimental group, again demonstrating that the mini-peptide ZFAS1-P56 can significantly inhibit the replication of influenza virus.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. The long-chain non-coding RNA ZFAS1 coded micro-peptide ZFAS1-P56 is characterized in that the amino acid sequence of the micro-peptide ZFAS1-P56 is shown as SEQ ID No. 1.

2.use of the mini-peptide ZFAS1-P56 encoded by zfas1 in (a), (b), (c) as follows:

(a) Preparing an anti-influenza virus medicament;

(b) Preparing a medicament for inhibiting the replication of influenza virus in cells;

(c) Preparing the medicine for treating and/or preventing influenza.

Images