CN116355904A

CN116355904A - Long-chain non-coding RNA for broad-spectrum anti-influenza virus and application thereof

Info

Publication number: CN116355904A
Application number: CN202310147990.0A
Authority: CN
Inventors: 胡娇; 胡增垒; 刘秀梵; 张蕾; 蔡欣欣; 郑新新; 刘晓文; 王晓泉; 胡顺林; 顾敏
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-06-30

Abstract

The invention discloses a long-chain non-coding RNA (LncRNA#61) of broad-spectrum anti-influenza virus and application thereof. LncRNA #61 significantly inhibited replication of various subtypes of influenza virus, including human H1N1 virus and various highly pathogenic and less pathogenic avian influenza viruses. The invention constructs lncRNA lipid nanoparticles for delivering LncRNA#61 in vivo based on the lipid nanoparticles, and the LncRNA#61 delivered based on the lncRNA lipid nanoparticles plays a good in vivo antiviral effect. In addition, the antiviral mechanism of lncrna#61 was analyzed, and its antiviral key functional region was identified, and it was found to exert antiviral effects by decreasing the viral polymerase activity and inhibiting the aggregation of polymerase protein nuclei.

Description

Long-chain non-coding RNA for broad-spectrum anti-influenza virus and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to long-chain non-coding RNA for broad-spectrum anti-influenza virus and application thereof.

Background

Currently, influenza a viruses (influenza A virus, IAV) still pose a significant threat to public health safety worldwide. The historically exploded IAV pandemic has had its viral internal genes derived from avian influenza virus (Avian Influenza Virus, AIVs). AIVs belong to the orthomyxoviridae family, influenza A viruses, single-stranded segmented RNA viruses, whose natural host is mainly wild waterfowl. However, AIVs may also infect other hosts, including wild birds, land birds and a wide variety of mammals. In addition, some AIVs subtypes, such as H5N1/N6/N8, H6N1, H7N2/N3/N7/N9, H9N2, H10N7/N8, etc., can also cross-species spread directly to infect humans, causing serious respiratory disease, even death. In addition, it has been reported that some variant H5 subtype AIVs can gain the ability to be aerosol transmitted in ferrets by accumulating few genetic mutations. The recent discovery in spain of H5N1 mutant viruses that can be transmitted with high efficiency in mink has led to a large outbreak of IAV in humans and wild animals.

The main target point of the current anti-influenza virus drugs is influenza virus protein. Influenza viruses are very frequently mutated in their viral proteins due to the nature of RNA viruses and their genomic segments. Therefore, drugs targeting viral proteins can exhibit drug resistance. However, the development of drugs against host gene targets can solve this problem.

Long non-coding RNAs (lncRNAs) are RNAs with a length of greater than 200nt that do not have the ability to encode proteins, which have structural features similar to messenger RNAs (mRNA), with or without a cap structure at the 5 'end, ploy-A tail at the 3' end, and are spliced to convert to mature lncRNAs and form a topology. LncRNAs have been considered to be "noise" generated during gene transcription, and do not have a clear biological function. However, with the continuous development of high-throughput sequencing technologies including gene chips and RNA-seq, the studies on lncRNAs on viruses are more and more intensive. Researches find that lncRNAs can regulate IAV infection through various mechanisms, on one hand, differentially expressed lncRNAs can regulate natural antiviral immune response of a host through positive feedback or negative feedback, and indirectly inhibit or promote viral replication; on the other hand, IAVs can also "hijack" certain host lncRNA, either directly regulate viral life cycle or regulate viral infection by altering host cell metabolism. Therefore, the identification of functional host lncRNAs for regulating and controlling various virus infections is beneficial to the development of novel broad-spectrum antiviral drugs. In detail, it is important to analyze the LncRNA of the host which has broad-spectrum avian influenza virus inhibition, and to prevent IAV.

Disclosure of Invention

The invention aims to provide long-chain non-coding RNA (lncRNA) and application thereof in inhibiting various subtype influenza viruses. Still further, the present invention aims to provide a novel use of long non-coding RNA, lncRNA #61, in inhibiting influenza virus replication. In addition, the invention provides a new target for researching anti-influenza virus replication drugs, lays a foundation for further developing new antiviral strategies, and also provides candidate lncRNA for researching host antiviral mechanisms after influenza virus infection.

In a first aspect, the invention provides a lncRNA having a nucleotide sequence as shown in SED ID No. 1.

In a second aspect, the invention provides an expression cassette, a recombinant vector or a recombinant bacterium comprising said lncRNA.

In a third aspect, the invention provides the lncRNA or the use of an expression cassette, recombinant vector or recombinant bacterium of the lncRNA in any of the following:

(1) The application in preparing anti-influenza virus products;

(2) The application of the recombinant DNA serving as a target in preparing medicaments and/or vaccines for treating and/or preventing influenza;

(3) The application of the recombinant DNA as a target in preparing an anti-influenza virus transgenic cell line;

(4) The application of the recombinant DNA serving as a target in preparing transgenic animals resisting influenza viruses.

Further, the influenza viruses include human influenza viruses and avian influenza viruses; the human influenza virus is H1N1 subtype influenza virus or H3N2 subtype influenza virus, and the avian influenza virus is H3N2/N8, H4N6, H5N1, H6N2/N8, H7N9, H8N4, H10N3 or H11N2/N6/N9 subtype influenza virus.

In a fourth aspect, the present invention provides a product comprising as an active ingredient said lncRNA; the product has the effect of inhibiting influenza virus replication.

In a fifth aspect, the present invention provides an lncRNA lipid nanoparticle, the method of preparing the lncRNA lipid nanoparticle comprising:

dissolving lecithin, cholesterol and the lncRNA plasmid according to claim 1 in a dichloromethane organic solvent, removing the organic solvent by rotary evaporation to synthesize liposome, hydrating the liposome in deionized water, performing ultrasonic dispersion, and performing dialysis purification to obtain the lncRNA lipid nanoparticle.

Further, the ultrasonic treatment is ultrasonic treatment at 80W for 10min.

Further, the dialysis purification is performed by dialysis with a dialysis bag with a molecular weight cut-off of 1000 for 16 hours.

In a sixth aspect, the invention provides the use of the lncRNA lipid nanoparticle in any of the following:

(1) The application in preparing anti-influenza virus products;

The invention provides long-chain non-coding RNA, which is named LncRNA#61, and the nucleotide sequence is shown as SEQ ID NO. 1.

The invention discloses a novel method for delivering LncRNA#61 based on lipid nano-particles (LNP), and successfully delivers LncRNA#61 into mice, thereby exerting good in-vivo antiviral effect.

The experimental results show that the overexpression of LncRNA#61 significantly inhibits the replication of various subtype IAVs, and the interference of the expression of LncRNA#61 significantly promotes the replication of various subtype IAVs. LncRNA #61 delivered based on LNP significantly inhibited viral replication in mice. In addition, the invention also partially analyzes the antiviral mechanism of Lnc#61, identifies the key antiviral functional region of LncRNA#61, and finds that the LncRNA#61 plays an antiviral role by reducing the activity of viral polymerase and inhibiting the aggregation of polymerase protein cores. The invention provides a new target for researching anti-influenza virus replication drugs, lays a foundation for further developing new antiviral strategies, and also provides candidate LncRNA#61 for researching host antiviral mechanisms after influenza virus infection.

Compared with the prior art, the invention discloses and provides a long-chain non-coding RNA-LncRNA #61 with broad-spectrum anti-IAV; a novel method for in vivo efficient delivery of lncrna#61 based on LNP is provided; the LncRNA#61 is hopefully developed into a novel drug for preventing and treating influenza virus and the development of genetically modified animals for resisting the influenza virus, and has important significance for preventing and treating influenza.

Drawings

FIG. 1 shows that LncRNA#61 is capable of inducing high expression by a variety of influenza viruses. FIG. 1A shows up-regulated expression of LncRNA #61 24h after infection of RTE-6TN cells with CK10 virus; FIG. 1B shows that LncRNA #61 is expressed at a peak 28h after CK10 infection; FIG. 1C shows that LncRNA #61 peaks in expression at CK10 at 4MOI doses; figure 1D shows that LncRNA #61 was significantly up-regulated 24h after different subtype IAV infection.

FIG. 2 shows LncRNA#61 subcellular localization after viral infection. FIG. 2A is a schematic illustration of subcellular localization of 18S and U6 based on RNA-FISH technology; FIG. 2B is a subcellular localization of LncRNA#61 based on RNA-FISH technology; FIG. 2C illustrates subcellular localization of LncRNA#61 based on qRT-PCR technique without infection with virus; FIG. 2D shows subcellular localization of LncRNA#61 based on qRT-PCR technique in case of CK10 virus infection.

FIG. 3 is a graph showing that overexpression of LncRNA #61 inhibits replication of multiple subtypes of influenza virus in 293T cells. FIGS. 3A and 3D are diagrams showing that overexpression of LncRNA#61 inhibits replication of CK10 (H5N 1) virus; FIGS. 3B and 3E are diagrams showing that overexpression of LncRNA #61 inhibits replication of S8 (H7N 9) virus; FIGS. 3C and 3F show that overexpression of LncRNA #61 inhibits PR8 (H1N 1) viral replication.

FIG. 4 is a graph showing that overexpression of LncRNA #61 inhibits replication of multiple subtypes of influenza virus in HeLa cells. FIGS. 4A and 4D are graphs showing that overexpression of LncRNA#61 inhibits replication of CK10 (H5N 1) virus; FIGS. 4B and 4E are graphs showing that overexpression of LncRNA #61 inhibits replication of the S8 (H7N 9) virus; FIGS. 4C and 4F are schematic representations of the inhibition of PR8 (H1N 1) virus replication by overexpression of LncRNA #61.

FIG. 5 is a graph showing that overexpression of LncRNA #61 inhibits replication of other influenza subtypes in 293T cells. FIG. 5A is H3N2 virus; FIG. 5B is H3N8 virus; FIG. 5C is H4N6 virus; FIG. 5D is H5N1 (TT 3 strain) virus; FIG. 5E is H6N2 virus; FIG. 5F is H6N8 virus; FIG. 5G is H7N9 (GD 15 strain) virus; FIG. 5H is H8N4 virus; FIG. 5I is H10N3 virus; FIG. 5J is H11N2 virus; FIG. 5K is H11N6 virus; FIG. 5L is H11N9 virus.

FIG. 6 is a graph of interference with LncRNA #61 promoting replication (cell pellet) of multiple subtypes of influenza virus in RTE-6TN cells. FIGS. 6A and 6D are diagrams of interference LncRNA#61 promoting replication of CK10 (H5N 1) virus; FIGS. 6B and 6E are diagrams showing that infection with LncRNA #61 promotes replication of the S8 (H7N 9) virus; FIGS. 6C and 6F are diagrams of interference LncRNA#61 promoting replication of PR8 (H1N 1) virus.

FIG. 7 is a graph of interference with LncRNA #61 promoting replication of multiple subtypes of influenza virus in RTE-6TN cells (cell supernatant). FIGS. 7A and 7D are diagrams showing that interfering LncRNA#61 promotes replication of CK10 (H5N 1) virus; FIGS. 7B and 7E are schematic representations of infection with LncRNA #61 to promote replication of the S8 (H7N 9) virus; FIGS. 7C and 7F are diagrams of interference LncRNA#61 promoting replication of PR8 (H1N 1) virus.

Figure 8 is an in vivo antiviral effect of lipid nanoparticle-based delivery of LncRNA #61 in mice. FIG. 8A is a schematic view of LNP-LncRNA #61 particle; FIG. 8B is an in vivo LNP-LncRNA #61 mouse profile based on a small animal in vivo imaging technique; FIG. 8C shows the in vivo distribution of LNP-LncRNA #61 mice based on qRT-PCR technique; FIG. 8D shows antiviral results in LNP-LncRNA #61 mice (day 3); FIG. 8E shows the in vivo antiviral results (day 5) of LNP-LncRNA #61 mice.

Figure 9 shows that LncRNA #61 exerts antiviral effects at various stages of the influenza virus lifecycle. FIG. 9A is the effect of LncRNA #61 on viral entry after 20min, 40min, 1h of viral infection; FIG. 9B effect of LncRNA #61 on early stages of viral replication 4h after viral infection; FIG. 9B effect of LncRNA #61 on late and mid-viral replication 9h after viral infection; FIG. 9D effect of LncRNA #61 on the viral release phase 12h after viral infection.

FIG. 10 shows the effect of LncRNA #61 on influenza virus polymerase activity and apoptosis and necrosis. FIG. 10A is the effect of LncRNA #61 on influenza virus polymerase activity; FIG. 10B is the effect of LncRNA#61 on apoptosis; FIG. 10C is the effect of LncRNA #61 on cell necrosis.

FIG. 11 shows the effect of LncRNA#61 on viral NP and PA protein nuclear entry. FIG. 11A shows the aggregation of NP and PA nuclei under a 40-fold mirror in the control group after 0h, 9h, 12h of virus infection; FIG. 11B shows the aggregation of NP and PA nuclei under 40-fold mirror of the group #61 overexpressing LncRNA after 0h, 9h, 12h of virus infection; FIG. 11C shows NP and PA nuclear aggregation at 63-fold oil level 9h post virus infection; FIG. 11D is a graph showing the percent nuclear aggregation of NP protein 9h after viral infection; FIG. 11E is a graph showing the percent nuclear aggregation of PA protein 9h after viral infection.

FIG. 12 is a graph showing the determination of the antiviral key functional region of LncRNA #61. FIG. 12A is a schematic representation of LncRNA#61 truncation mutant; FIG. 12B is a circle marking the mutation position; FIG. 12C is a graph showing the determination of LncRNA#61 and mutant exogenous expression in 293T cells by RT-PCR; FIG. 12D shows qRT-PCR detection of CK10 viral NP gene mRNA levels in IAV infected 293T cells overexpressing the LncRNA#61 mutant; FIG. 12E is the polymerase activity of the virus in 293T cells overexpressing the LncRNA#61 mutant.

FIG. 13 shows the effect of LncRNA#61 mutant (A, B, C, D, E, G) on viral NP and PA protein nucleation. FIG. 13A shows that after 9h of virus infection, IFA observes the aggregation of viral NP and PA proteins; FIG. 13B virus infection 9h later, qRT-PCR was performed to detect viral NP gene nuclear cytoplasmic distribution.

FIG. 14 shows the effect of LncRNA#61 mutant (F, H, I) on viral NP and PA protein nucleation. FIG. 14A shows that after 9h of virus infection, IFA observes the aggregation of viral NP and PA proteins; FIG. 14B virus infection 9h later, qRT-PCR was performed to detect viral NP gene nuclear cytoplasmic distribution.

Detailed Description

The invention will be further described with reference to specific examples, and advantages and features of the invention will become apparent from the description. These examples are exemplary and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Example 1: lncRNA#61 can induce high expression by various influenza viruses

(1) To investigate the induction of LncRNA#61 expression in host cells by the H5N1 subtype AIVs CK10 strain, CK10 virus was used to infect RLE-6TN cells at an infectious dose of 1. MOI (multiplicity of infection), and cellular RNA was extracted after 24H. The fluorescent quantitative PCR (qRT-PCR) results showed that CK10 virus infection significantly up-regulated expression of LncRNA #61 compared to uninfected cells (fig. 1-a). The qRT-PCR primers of this patent are detailed in Table 1.

TABLE 1 qRT-PCR primers

(2) To determine the expression profile of LncRNA #61, CK10 virus was used to infect RLE-6TN cells at 1MOI dose, cellular RNA was harvested at different time points (0 h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36 h) and gene expression was detected by fluorescent quantitative PCR. The results showed that the expression level of LncRNA #61 started to rise 12h after infection, followed by a peak at 28h (fig. 1B).

(3) To determine the expression pattern of LncRNA #61, CK10 virus was infected with RLE-6TN cells at different doses (0 MOI, 0.5MOI, 2MOI, 3MOI, 4MOI, 5 MOI) and gene expression was detected by qRT-PCR 24h after infection. The results showed that LncRNA #61 can be significantly induced to differentially express at 0.5MOI infection doses and gene expression levels peaked at 4MOI infection doses (fig. 1C).

(4) To further determine if the upregulated expression of lncrna#61 is characteristic of CK10 virus infection, three additional IAVs strains were selected, including H1N1 (PR 8), H7N9 (S8), and H9N2 (SDKD 1). The viruses are respectively infected with RLE-6TN cells, cellular RNA is collected after 24 hours, and the expression of LncRNA#61 is detected by qRT-PCR. As a result, it was found that lncrna#61 induced by S8 virus strain of H7 subtype AIVs was significantly up-regulated 2-fold (fig. 1D); lncRNA #61 induced by subtype H1 IAVs PR8 virus was significantly up-regulated 10-fold.

Example 2: lncRNA#61 subcellular localization after viral infection

(1) To elucidate the potential role of lncrna#61 in influenza virus replication, subcellular localization of lncrna#61 was examined based on the RNA-FISH (RNA fluorescence in situ hybridization) technique. Cytoplasmic and nuclear fractions of RLE-6TN cells were collected, respectively, and GAPDH and 18S were set as reference standards for cytoplasmic components, and U6 was set as reference standard for nuclear components. RNA-FISH results showed that the 18S content was higher in the cytoplasmic portion of RLE-6TN and the U6 content was higher in the nuclear portion (FIG. 2A) compared to the nuclear portion, indicating successful nuclear and cytoplasmic control settings. Next, by observation of the lncrna#61 and lncrna#45 fluorescent probes, lncrna#61 was found to be mainly distributed in the nucleus (fig. 2B, fig. 2C), but lncrna#61 was transported from the nucleus into the cytoplasm after CK10 virus infection (fig. 2D), suggesting that it may play an important role in the course of virus infection. Wherein, the experimental steps of RNA-FISH are as follows:

1. cell culture: the distribution of LncRNA #61, lncRNA #45 in RLE-6TN cells was determined using Fluorescence In Situ Hybridization (FISH) kit (riboBio, guangzhou, china). The climbing sheet is arranged in the 24-hole plate, and no bubbles are generated between the climbing sheet and the bottom. RLE-6TN cells were cultured therein for 18 hours, followed by infection of CK10 virus at 1MOI dose for 24 hours, while non-infected virus was set as a negative control well.

2. Cell fixation and permeabilisation: cells were fixed in 4% paraformaldehyde for 30min at room temperature and then washed three times with PBS. The permeation was then performed with 0.5% Triton x-100 on ice for 10min and washed three times with PBS.

3. And (3) detecting a probe: adding 200 mu L of prehybridization solution into each hole, sealing for 30min at 37 ℃, and preheating the hybridization solution at 37 ℃; the lncRNA FISH probe and the internal reference FISH probe are respectively added into the probe hybridization solution under the light-shielding condition. U6 served as nuclear reference and 18S served as cytoplasmic reference. Discarding prehybridization solution in each hole of cells, adding probe-containing probe hybridization solution, and performing light-shielding treatment to hybridize at 37 ℃ overnight; the cells were washed 3 times, 5min each, in the dark at 42℃C (hybridization solution I, 4 XSSC); light was protected from the light and washed once at 42 ℃ (hybridization solution II, 2 XSSC); light was prevented and the hybridization solution was washed once at 42℃and 1 XSSC; cells were washed in 1 XPBS in the dark and at room temperature for 5min. (sodium citrate buffer, saline sodium citrate, SSC)

4. DNA staining: light-shading and dyeing with DAPI (DAPI) staining solution for 10min; cells were washed 3 times 5min each with 1 XPBS in the absence of light.

5. Sealing piece: the cell slide in the cell plate was removed in a dark room and fixed on a glass slide with clear nail polish and the confocal fluorescence image was analyzed with a Leica TCS SP-E microscope.

Example 3: overexpression or interference of LncRNA #61 inhibits replication of multiple subtype influenza viruses

(1) To investigate the effect of lncrna#61 on replication of influenza virus, the overexpression vector pcdna3.1-lncrna#61 of lncrna#61 was constructed. As a result, it was found that overexpression of LncRNA #61 significantly reduced the viral titers of CK10 (H5N 1) (fig. 3A), S8 (H7N 9) (fig. 3B) and PR8 (H1N 1) (fig. 3C) in 293T cells. In addition, the expression levels of viral NP genes mRNA, cRNA and vRNA were detected by fluorescent quantitative PCR, and it was found that overexpression of LncRNA #61 significantly down-regulated the mRNA, cRNA and vRNA levels of these viral NP genes (fig. 3D-3F). To further verify the antiviral ability of lncrna#61, the effect on CK10, S8 and PR8 viral replication after overexpression of lncrna#61 in Hela cells was also measured. The results showed that LncRNA #61 was equally effective in inhibiting replication of different subtypes of IAVs in Hela cells (fig. 4).

(2) To verify the antiviral effect of LncRNA #61, IAVs of different subtypes were selected for verification. Overexpression of LncRNA #61 was found to significantly inhibit IAV replication of various subtypes, including human H1N1 virus as well as various AIVs: H3N2/N8, H4N6, H5N1, H6N2/N8, H7N9, H8N4, H10N3, H11N2/N6/N9 (FIG. 5).

(3) To further confirm the antiviral effect of lncrna#61, the effect of knockdown lncrna#61 expression on RNA levels of different subtypes of IAVs NP genes was analyzed. After treatment of the cells with LncRNA#61-targeted siRNAs (siRNA-693, siRNA-736, siRNA-693+siRNA-736) (Table 2), the LncRNA#61 levels in RLE-6TN cells were down-regulated by 65%, 70%, 85%, respectively. Thus, the subsequent knockdown LncRNA#61 expression results were all based on siRNA-693+siRNA-736 co-transfection (FIGS. 6A-6C). As a result, knockdown LncRNA #61 expression was found to significantly up-regulate mRNA, cRNA, and vRNA levels of different subtype influenza NP genes in cell pellets, including CK10, S8, and PR8 viruses (fig. 6D-6F). Similar to the results in cell pellet, NP gene RNA levels of different subtypes of IAVs were also significantly up-regulated in lncrna#61 knockdown cell culture supernatants (fig. 7D-6F).

TABLE 2 siRNA sequences (5 '-3')

Example 4: delivery of LncRNA #61 based on lipid nanoparticles significantly inhibited viral replication in mice

(1) To verify the in vivo antiviral effect of lncrna#61, a lipid nanoparticle (lipid nanoparticle, LNP) -based delivery system (LNP-lncrna#61) encapsulating lncrna#61 was constructed. LNP-LncRNA #61 was prepared by thin film hydration (FIG. 8A). The LncRNA #61 plasmid is extracted in a large quantity, the concentration is more than 2 mug/mu L, 24.65mg of lecithin and 4.28mg of cholesterol are dissolved in 5mL of dichloromethane, and the mixture is placed in a rotary steaming round bottom flask of a rotary steaming instrument, and after the liquid is completely evaporated to dryness. The white adherent film was dissolved with 2mL of ultra-pure water (40. Mu.g of DNA) and placed under ultrasound for 10min at 80W until the white film was visible to the naked eye as a pale blue color. The preparation of ICG liposomes is different from the preparation method of the above-mentioned general liposomes. The difference is that after the spin-steaming instrument has evaporated the liquid, 1mL of ultra-pure water (40. Mu.g of DNA+0.1mg of ICG) is required to dissolve the white adherent film, which is then placed under ultrasound for 10min,80W until dissolution of the white film is seen with the naked eye. Then the solution is dialyzed (dialysis bag with molecular weight cut-off 1000) again for 16 hours, and the water is changed once.

(2) The characterization of LNP-LncRNA #61 is detected by a Markov particle size analyzer, the surface of the LNP-LncRNA #61 is negatively charged, the average diameter of the LNP-LncRNA #61 is 238.9nm, and the characterization is stable, so that the LNP-LncRNA #61 can be used for subsequent in vivo experiments. In order to more intuitively detect the in vivo expression efficiency of Lnc#61, lnc#61-LNP labeled with ICG dye is injected into mice in a leg intramuscular injection mode, and then the in vivo distribution condition of LncRNA#61 is observed by means of a small animal in vivo imaging system. After 12h expression in vivo, lncRNA #61 was found to be predominantly distributed in the leg muscle, liver and kidney of the mice (fig. 8B), indicating that LNP can deliver LncRNA #61 into the mice.

(3) To further confirm in vivo distribution of lncrna#61, LNP-lncrna#61 was delivered in mice by point-by-point injection of leg muscle, and then the expression level of lncrna#61 in each organ of mice was detected by qRT-PCR assay. The experimental results show that the lncrna#61 has different distribution in the organs of the mice at different expression times, and reaches an expression peak at 12 hours, wherein the expression amount of the muscle is the highest, and the liver, spleen, kidney and lung are the next (fig. 8C). To further confirm the in vivo antiviral effect of lncrna#61, LNP-lncrna#61 was delivered in mice 12H after H5N1 subtype influenza virus infection by point-split leg muscle injection. The measurement of the viral titers of each organ was then carried out on

days

3 and 5 after viral infection. As a result, LNP-LncRNA #61 was found to significantly impair viral replication in mice (FIGS. 8D-8F).

Example 5: lnc#61 exerts antiviral effects at various stages of the influenza virus lifecycle

To gain insight into the mechanism of antiviral action of lncrna#61, its effect on viral single round replication was studied. The test uses CK10 to infect 293T cells over-expressing LncRNA#61 at 1MOI dose, and performs fluorescent quantitative PCR detection 20min, 40min, 1h, 4h, 9h and 12h after virus infection. As a result, lncRNA #61 significantly inhibited the entry phase (fig. 9A), early phase (fig. 9B) and late middle replication phase (fig. 9C) of the virus; but does not affect the release phase of the virus (fig. 9D).

Example 6: effects of LncRNA#61 on influenza Virus polymerase Activity and apoptosis and necrosis

Next, it was investigated whether lncrn#61 modulates the polymerase activity of CK10 virus, apoptosis and necrosis induced by viral infection. As shown in fig. 10A, it was found that overexpression of LncRNA #61 in 293T cells significantly reduced the polymerase activity of CK10 virus. In addition, apoptosis and necrosis induced by viruses were examined by flow cytometry, and it was found that overexpression of LncRNA #61 significantly inhibited CK10 virus infection-induced apoptosis (fig. 10B); but had no effect on cell necrosis (fig. 10C). The detailed experimental procedure for the polymerase activity and cell death assays were as follows:

(1) Polymerase Activity assay

1. 293T cells were seeded into 12-well plates and plasmid cotransfection was performed when the cell growth density was 70%.

2. Each experimental well was transfected with 200ng of firefly luciferase reporter plasmid, eukaryotic expression vectors (pcDNA3.1-PA, pcDNA3.1-PB1, pcDNA3.1-PB2, pcDNA3.1-NP) expressing viral PA, PB1, PB2, NP proteins, 20ng of sea cucumber luciferase reporter plasmid, and 500ng of LncRNA#61 or LncRNA#45 overexpression plasmid.

3. After 24h of transfection, the cells were lysed using the Dual-Luciferase Reporter Assay System kit (Promega) to detect reporter activation. Results are shown as mean ± standard deviation of three independent experiments.

(2) Cell death assay

1. 293T cells were cultured overnight in 6-well plates and allowed to grow to 70% transfected over-expression vectors.

2. Subsequently 24h, CK10 virus infected cells at 1MOI, while no infection was provided as a negative control well.

3. 24h after infection, the cells were treated with trypsin and washed three times with PBS. The membrane was stained with Fluorescein Isothiocyanate (FITC) -conjugated protein V and Propidium Iodide (PI) (Roche) in the dark at room temperature for 15min.

4. Stained cells were detected by flow cytometry fluorescence sorting (luorescence activated cell sorting, FACS).

5. Principle of: annexin V has a high affinity for Phosphatidylserine (PS), which translocates outward from the inner leaflet of the plasma membrane during apoptosis. Living and apoptotic cells are not permeable to PI, but stain dead cells and bind tightly to nucleic acids within the cell. Thus, apoptotic cells are cells stained for high annexin V and low PI, while necrotic cells are cells stained for coupled membrane protein V and Propidium Iodide (PI). The total cell fraction of apoptosis or necrosis is the average of at least three independent experiments.

Example 7: effect of LncRNA #61 on viral NP and PA protein insertion into the nucleus

Subsequently, it was further investigated whether lncrna#61 affects the nuclear aggregation ability of the polymerase components NP and PA proteins during CK10 virus infection. CK10 was transfected into 293T cells that overexpressed lncrna#61 at 1MOI dose and tested by indirect immunofluorescence (Immunofluorescence assay, IFA) after 4h, 9h, 12h, respectively, of viral infection. As a result, lncRNA #61 was found to significantly inhibit the nuclear aggregation ability of viral polymerase components NP and PA proteins on 293T cells compared to the control group (fig. 11a,11 b). Notably, NP and PA proteins were significantly different in nuclear aggregation in 293T cells overexpressing lncrna#61 as compared to empty cells after 9h of CK10 infection (fig. 11C-11D). Specifically, NP protein was detected in 95% of the nuclei in the empty vector, whereas NP nuclear localization was shown in only 42% of nuclei in the LncRNA #61 group (fig. 11D); PA protein was detected in 80% of the nuclei in empty vector, whereas PA nuclear localization was shown in only 35% of the nuclei (fig. 11E). It was shown that overexpression of LncRNA #61 significantly inhibited the nuclear aggregation efficiency of viral polymerase components NP and PA protein in 293T cells.

(1) The indirect immunofluorescence experiment operation steps are as follows:

1. cells were plated into 24-well plates and transfected after cells had adhered to the walls and grown to 70%.

2. After 24h of cell transfection, CK10 virus infected cells at 1MOI infection dose.

3. 24h after infection, wash 3 times with PBS.

4. Adding 4% paraformaldehyde after rewarming, and fixing at room temperature for 30min.

5. The paraformaldehyde was discarded and washed three times with PBS. 0.5% TritonX-100 was added for cell permeation for 5min.

6. After washing, 1mL of 100mM glycine was added to each dish and incubated at room temperature for 15min.

7. Glycine was discarded and PBS was added to wash twice. 1% BSA was added and blocked for 1h at room temperature.

8. Primary antibody was incubated after blocking was completed, dilution ratio of influenza virus NP protein antibody (murine) = 1:300; PA protein antibody (rabbit source) dilution ratio = 1:900. after 2h at room temperature or overnight incubation at 4 ℃.

9. After the incubation of the primary antibody is finished, the primary antibody is washed 3 times by PBS, and the corresponding secondary fluorescent antibody is added. Secondary antibody dilution ratio = 1:1000. incubate for 1h at 37℃in the absence of light.

10. The secondary antibody was discarded and then washed three times with PBS. DAPI was used for nuclear staining, and light was protected from room temperature for 5min.

11. DAPI was discarded and PBS was added for washing, each time for 10min, and repeated 3 times.

12. Taking out the climbing sheet, taking out the climbing sheet by using tweezers, and cutting and recording the climbing sheet, namely slightly pressing the surface with cells (frosted) downwards to remove bubbles between the climbing sheet and the glass slide, sealing the glass slide, making detailed marks on one side of the glass slide after airing, and placing the glass slide into a loading box for waiting to be observed.

Example 8: identification of LncRNA#61 antiviral Critical functional region

(1) In order to determine the antiviral key functional region of LncRNA#61, PCR amplification was performed using the cDNA of LncRNA#61 as a template, and a truncated mutant of LncRNA#61 was constructed. The truncated mutants were subcloned into the expression plasmid pcDNA3.1 according to the instructions using a seamless cloning kit (Shanghai Biotechnology, china) according to the primers in Table 3. The mutant vectors were designated LncRNA#61-MutA to LncRNA#61-MutI, respectively (FIGS. 12A, 12B).

TABLE 3 LncRNA#61 truncated mutant primer sequences

(2) The successfully constructed truncated mutant plasmid was transfected into 293T cells, and the exogenous expression efficiency was verified by RT-PCR. RT-PCR results showed successful ectopic expression of LncRNA#61 and its corresponding mutants in 293T cells (FIG. 12C). The influence of the mutants on virus replication is further explored, the CK10 virus is transfected into 293T cells which over express the mutants and the maternal plasmid at a MOI infection dose of 1, cellular RNA is collected after 24h infection, and the mRNA level of the virus NP gene is measured through three independent repeated experiments. As a result of qRT-PCR experiments, the inhibition effect of LncRNA#61-MutB, lncRNA#61-MutC, lncRNA#61-MutE and LncRNA#61-MutI mutant corresponding domains deleted and LncRNA#61 on viral NP gene mRNA is lost. Thus, the secondary domain corresponding to the above mutant plays an important role in regulating replication of H5N1 subtype AIVs (CK 10) (fig. 12D).

(3) The effect of the LncRNA #61 truncation mutant on viral polymerase activity was further demonstrated in subsequent studies. As a result, it was found that the polymerase activity in 293T cells of the empty vector was significantly higher than that in 293T cells overexpressing LncRNA #61. Whereas the CK10 viral polymerase activity was significantly up-regulated in 293T cells overexpressing mutants LncRNA#61-MutB, lncRNA#61-MutC, lncRNA#61-MutE and LncRNA#61-MutI compared to the maternal plasmid (FIG. 12E). It was further verified that these functional region deletions correspond to domains that are critical for the antiviral effect of lncrna#61. Specifically, 150bp-230bp, 260bp-310bp, 500bp-670bp and 910bp-1030bp of LncRNA#61 motif portion form a spatial structure critical to its antiviral function.

Example 9: effect of LncRNA#61 antiviral Critical function on viral NP and PA protein Nuclear insertion

(1) To investigate the effect of lncrna#61 truncation mutants on viral polymerase component NP and PA protein nuclear aggregation. The CK10 virus is infected with 293T cells which over express the empty vector, lncRNA #61 and the truncated mutant thereof at a MOI infection dose of 1, samples are collected after 9 hours of infection, and NP (green) and PA (red) nuclear aggregation conditions are detected by an indirect immunofluorescence test; RNA is extracted from the cytoplasm and the components of the cell nucleus, the mRNA level of the virus NP gene in the cell nucleus is detected by qRT-PCR, GAPDH is taken as a cytoplasm reference, U6 is taken as a nucleus reference, and the calculation is carried out according to the ct value of the gene of the cell nucleus and the ct value of the gene of the cell cytoplasm [ delta ct (nuclear-cytoplasms) ].

Subcellular distribution of NP and PA proteins was observed based on IFA and laser confocal microscopy. The results show that the control CK10 viral NP and PA proteins had completed nuclear entry at 9h of infection, whereas the localization of CK10 viral NP and PA proteins in 293T cells overexpressing lncrna#61 was predominantly cytoplasmic (fig. 13), i.e., lncrna#61 inhibited viral NP and PA protein nuclear aggregation. Whereas the truncated mutant of lncrna#61 (B, C, D, E, G) had a significant difference in influencing nuclear aggregation efficiency of CK10 viral NP and PA proteins in 293T cells compared to the parent lncrna#61 plasmid (fig. 13), and the RNA nuclear distribution level of viral NP gene was examined by qRT-PCR, which showed that viral NP gene was distributed mainly in the nucleus after overexpression of the above mutant, whereas viral NP gene was mainly in the cytoplasm after overexpression of the parent lncrna#61 plasmid. Therefore, the combination of IFA and qRT-PCR results further shows that the secondary domain corresponding to the mutant has a remarkable inhibition effect on viral NP and PA protein nuclear aggregation.

(2) Furthermore, the truncated mutant of lncrna#61 (F, H, I) did not differ in affecting nuclear aggregation efficiency of CK10 viral NP and PA proteins in 293T cells compared to the parent lncrna#61 plasmid (fig. 3-13A), and the viral NP gene RNA nuclear distribution levels were detected by qRT-PCR, which indicated that after over-expression of the above mutant and the parent lncrna#61 plasmid, the viral NP gene was distributed in the cytoplasm (fig. 14). It is speculated that the secondary domain corresponding to the mutant does not exert antiviral effect.

In summary, binding viral NP gene RNA level detection (fig. 12D) and polymerase activity assay (fig. 12E) were screened: the secondary domains corresponding to LncRNA#61-MutB (150 bp-230 bp), lncRNA#61-MutC (260 bp-310 bp), lncRNA#61-MutE (500 bp-670 bp) and LncRNA#61-MutI (910 bp-1030 bp) mutants are key functional regions of LncRNA#61 for exerting antiviral effect.

The above description is only of the preferred embodiments of the present invention, but is not intended to limit the embodiments of the present invention. It should be understood by those skilled in the art that any modifications, equivalents, improvements and modifications made on the basis of the present invention are possible without departing from the inventive concept, and fall within the scope of the present invention as claimed.

Claims

1. A lncRNA, wherein the nucleotide sequence of the lncRNA is as set forth in SED ID No. 1.

2. An expression cassette, recombinant vector or recombinant bacterium comprising the lncRNA of claim 1.

3. Use of the lncRNA of claim 1 or the expression cassette, recombinant vector or recombinant bacterium of the lncRNA of claim 2 in any of the following:

(1) The application in preparing anti-influenza virus products;

4. A use according to claim 3, characterized in that: the influenza virus comprises human influenza virus and avian influenza virus; the human influenza virus is H1N1 subtype influenza virus or H3N2 subtype influenza virus, and the avian influenza virus is H3N2/N8, H4N6, H5N1, H6N2/N8, H7N9, H8N4, H10N3 or H11N2/N6/N9 subtype influenza virus.

5. A product comprising the lncRNA of claim 1 as an active ingredient; the product has the effect of inhibiting influenza virus replication.

6. A lncRNA lipid nanoparticle, characterized in that the method of preparing the lncRNA lipid nanoparticle comprises:

7. The lncRNA lipid nanoparticle of claim 6, wherein the ultrasound is at 80W for 10min.

8. The lncRNA lipid nanoparticle of claim 6, wherein the dialysis purification is dialysis purification for 16 hours using a dialysis bag with a molecular weight cut-off of 1000.

9. Use of the lncRNA lipid nanoparticle of any of claims 6-8 in any of the following:

(1) The application in preparing anti-influenza virus products;

10. The use according to claim 9, characterized in that: the influenza virus comprises human influenza virus and avian influenza virus; the human influenza virus is H1N1 subtype influenza virus or H3N2 subtype influenza virus, and the avian influenza virus is H3N2/N8, H4N6, H5N1, H6N2/N8, H7N9, H8N4, H10N3 or H11N2/N6/N9 subtype influenza virus.