Use of an RNA-binding protein inhibitor in the preparation of a drug for inhibiting hepatitis B virus replication

Abstract

The present invention discloses the use of an RNA-binding protein inhibitor in the preparation of a drug for inhibiting the replication of hepatitis B virus, wherein the RNA-binding protein is insulin-like growth factor 2 mRNA-binding protein 1. The present invention for the first time confirms that the host factor insulin-like growth factor 2 mRNA-binding protein 1 can promote the replication of hepatitis B virus, and this promoting effect is achieved by insulin-like growth factor 2 mRNA-binding protein 1 recognizing and binding to the m6A modification on hepatitis B virus RNA and increasing its stability. Therefore, the present invention provides a new target for anti-hepatitis B virus. Anti-hepatitis B virus effects can be exerted by targeting and inhibiting the expression of insulin-like growth factor 2 mRNA-binding protein 1 or using cucurbitacin B to inhibit the binding of insulin-like growth factor 2 mRNA-binding protein 1 to hepatitis B virus RNA, thereby providing new ideas for the research and development of new anti-hepatitis B virus drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use of RNA binding protein inhibitors in the preparation of drugs for inhibiting the replication of hepatitis B virus. Background Art

[0002] Hepatitis B virus (HBV) infection is the main cause of chronic hepatitis B (CHB), liver cirrhosis and hepatocellular carcinoma. As a hepatotropic DNA virus, the infection and replication process of HBV needs to be completed through the interaction between viral and host factors. However, due to the insufficient understanding of the HBV life cycle, there is currently a lack of effective drugs for curing CHB.

[0003] Similar to host cell mRNA, HBV transcripts also need to undergo a series of processing before they can become mature mRNA, exit the nucleus and translate viral proteins. The post-transcriptional regulation of HBV RNA mainly depends on the unique post-transcriptional regulatory elements (PREs) in its structure, and is also affected by the methylation modification of HBV RNA, and a variety of host RNA binding proteins (RBPs) are involved. RBPs can form ribonucleoprotein complexes (RNPs) with HBV RNA and regulate the metabolism of HBV RNA in a highly dynamic interaction manner.

[0004] The source of HBV virus replication is the covalently closed circular DNA (cccDNA) in the form of minichromosomes present in the nuclei of infected liver cells. The cccDNA can transcribe five types of viral RNAs with lengths of 3.5, 2.4, 2.1 kb, and 0.7 kb. These HBV RNAs have different transcription start positions but share the same polyadenylation (polyA) signal, so their transcription terminates at the same position. Among them, the 3.5-kb preC mRNA can be translated in the cytoplasm to produce HBeAg, and the pgRNA, also approximately 3.5 kb, can serve as an mRNA to translate core protein and P protein, and can also be incorporated into the nucleocapsid as a template for viral reverse transcription. The 2.4-kb PreS1 mRNA is translated to form the large surface antigen (L-HBsAg), and the 2.1-kb PreS2 / S mRNA is translated to form the middle surface antigen (M-HBsAg) and the small surface antigen (S-HBsAg). L-, M-, and S-HBsAg can all be distributed on the envelope of mature viruses (Dane particles), while M- and S-HBsAg can also form tubular and spherical subviral particles without viral nucleocapsids. The 0.7-kb XmRNA can be translated to form the HBx protein, which plays an important role in both the transcriptional regulation of cccDNA and HBV carcinogenesis.

[0005] N6-methyladenosine (m6A) modification is a common base modification. More than 25% of mammalian transcripts carry m6A modification and affect multiple biological processes. In eukaryotic cells, m6A modification sites are mostly located in the 3’ and 5’ untranslated regions (UTRs) and the coding region of mRNA. m6A modification is co-regulated by methyltransferases and demethylases and is reversible. The mRNAs with simultaneous m6A modification can be bound by m6A recognition proteins, thereby regulating processes such as mRNA stability, translation, and nuclear export. Studies have found that HBV RNA contains multiple m6A modification sites, among which the m6A modification level at the A1907 site in the lower stem of the ε region is the highest. The 3’ ends of all five types of HBV RNAs have m6A modification at the A1907 site, but only the 5’ end of the 3.5-kb HBV RNA has the A1907 m6A modification site.

[0006] Therefore, in-depth analysis of all aspects of the HBV life cycle and determination of the molecular mechanism of the interaction between HBV and the host to provide new potential targets for the development of new anti-HBV drugs have important scientific significance and social value.

[0007] The information in the background art is only for explaining the general background of the present invention, and should not be regarded as an admission or an implication in any form that this information constitutes the prior art known to those of ordinary skill in the art. Summary of the Invention

[0008] To solve the technical problems in the prior art, the present invention discovers through research that RNA-binding proteins can specifically bind to HBV RNA by recognizing the m6A modification on HBV RNA, enhance its stability, and thereby promote the replication of HBV virus. By targeting and knocking down IGF2BP1, HBV replication can be inhibited. In addition, cucurbitacin B can inhibit HBV replication by inhibiting the binding of IGF2BP1 to HBV RNA. Specifically, the present invention includes the following content.

[0009] In a first aspect of the present invention, there is provided the use of an RNA-binding protein inhibitor in the preparation of a drug for inhibiting the replication of hepatitis B virus, wherein the RNA-binding protein is insulin-like growth factor 2 mRNA-binding protein 1.

[0010] In certain embodiments, according to the use of the present invention, the RNA-binding protein inhibitor includes at least one of the following:

[0011] (1) A gene expression inhibitor that can down-regulate the expression of insulin-like growth factor 2 mRNA-binding protein 1;

[0012] (2) A protein activity inhibitor that can reduce or inhibit the activity of insulin-like growth factor 2 mRNA-binding protein 1 or inactivate it;

[0013] (3) A protein blocker that can inhibit or block the binding of insulin-like growth factor 2 mRNA-binding protein 1 to hepatitis B virus RNA.

[0014] In certain embodiments, according to the use of the present invention, the RNA-binding protein inhibitor inhibits the replication of hepatitis B virus by inhibiting or blocking the binding of insulin-like growth factor 2 mRNA-binding protein 1 to m6A-modified hepatitis B virus RNA.

[0015] In certain embodiments, according to the use of the present invention, the gene expression inhibitor includes the CRISPR / Cas9 system, small interfering RNA, antisense oligonucleotides, and / or methylation inhibitors.

[0016] In certain embodiments, according to the use of the present invention, the small interfering RNA has the sequence shown below: GGCUCAGUAUGGUACAGUA.

[0017] In a second aspect of the present invention, there is provided a method for screening a compound useful for inhibiting the replication of hepatitis B virus, which comprises:

[0018] (1) constructing a cell or animal model using an insulin-like growth factor 2 mRNA-binding protein 1 overexpression reagent;

[0019] (2) measuring the amount of hepatitis B virus DNA in the cell or animal model;

[0020] (3) administering the test compound to the cell or animal model;

[0021] (4) measuring the amount of hepatitis B virus DNA after administering the compound, wherein if the amount of hepatitis B virus DNA after administering the compound decreases, the test compound is useful for inhibiting the replication of hepatitis B virus; if the amount of hepatitis B virus DNA after administering the compound remains unchanged or increases, the test compound is not useful for inhibiting the replication of hepatitis B virus.

[0022] In certain embodiments, in the method for screening a compound useful for inhibiting the replication of hepatitis B virus according to the present invention, the overexpression reagent comprises a genetic engineering reagent, preferably a recombinant plasmid that causes overexpression of insulin-like growth factor 2 mRNA-binding protein 1.

[0023] In certain embodiments, in the method for screening a compound useful for inhibiting the replication of hepatitis B virus according to the present invention, the cell is an in vitro cell.

[0024] In certain embodiments, in the method for screening a compound useful for inhibiting the replication of hepatitis B virus according to the present invention, the animal is a mammal.

[0025] In certain embodiments, in the method for screening a compound useful for inhibiting the replication of hepatitis B virus according to the present invention, the mammal includes a rat, a mouse, a rabbit or a monkey.

[0026] The present invention has first confirmed that the host factor IGF2BP1 promotes the replication of HBV, and this promotion is achieved by IGF2BP1 recognizing and binding to the m6A modification on HBV RNA and increasing its stability. The present invention provides a new anti-HBV target IGF2BP1, and anti-HBV effects can be exerted by targeting and inhibiting IGF2BP1 expression or inhibiting the binding of cucurbitacin B to HBV RNA, thereby providing new ideas for the research and development of new anti-HBV drugs. Description of the Drawings

[0027] Figure 1Two days after co-transfecting the prcccDNA / pCMV-Cre plasmid and the IGF2BP1 plasmid into HepG2 cells. A: Detect HBV DNA in the cell culture supernatant by qPCR. B & C: Detect HBeAg and HBsAg in the cell culture supernatant by chemiluminescent immunoassay analyzer. D: Detect core protein and preS1 protein in the cells by western blot. E: Detect intracellular HBV RNA by northern blot experiment. F: Detect intracellular HBV RNA by RT-qPCR experiment. Two days after overexpressing the IGF2BP1 plasmid in the stable HBV-producing HepAD38 cells. G: Detect HBV DNA in the cell culture supernatant by qPCR. H & I: Detect HBeAg and HBsAg in the cell culture supernatant by chemiluminescent immunoassay analyzer. J: Detect core protein and preS1 protein in the cells by western blot. K: Detect intracellular HBV RNA by northern blot experiment. L: Detect intracellular HBV RNA by RT-qPCR experiment. (Statistical analysis was performed after three repeated experiments: **, P < 0.01; *, P < 0.05; ns, no statistical significance).

[0028] Figure 2 Two days after transiently transfecting the prcccDNA / pCMV-Cre plasmid and knocking down IGF2BP1 in HepG2 cells. A: Detect HBV DNA in the cell culture supernatant by qPCR. B & C: Detect HBeAg and HBsAg in the cell culture supernatant by chemiluminescent immunoassay analyzer. D: Detect core protein and preS1 protein in the cells by western blot. E: Detect intracellular HBV RNA by northern blot experiment. F: Detect intracellular HBV RNA by RT-qPCR experiment. Two days after knocking down IGF2BP1 in the stable HBV-producing HepAD38 cells. G: Detect HBV DNA in the cell culture supernatant by qPCR. H & I: Detect HBeAg and HBsAg in the cell culture supernatant by chemiluminescent immunoassay analyzer. J: Detect core protein and preS1 protein in the cells by western blot. K: Detect intracellular HBV RNA by northern blot experiment. L: Detect intracellular HBV RNA by RT-qPCR experiment. (Statistical analysis was performed after three repeated experiments: ***, P < 0.001; **, P < 0.01; *, P < 0.05).

[0029] Figure 3To infect HepG2-NTCP cells with concentrated HBV virus and knockdown IGF2BP1 on the 3rd day after infection. A: Detection of HBV DNA in cell culture supernatant by qPCR. B & C: Detection of HBeAg and HBsAg in cell culture supernatant by chemiluminescence immunoassay analyzer. D: Detection of core protein in cells by western blot. E: Detection of intracellular HBV RNA by RT-qPCR experiment. (Statistical analysis was performed after three repeated experiments: ***, P < 0.001; **, P < 0.01; *, P < 0.05).

[0030] Figure 4A: Specific antibodies against IGF2BP1 and isologous IgG were used to co-incubate with the lysates of HepG2 cells transfected with the prcccDNA / pCMV-Cre recombinant plasmid system, and RT-qPCR detection and agarose gel electrophoresis experiments were performed on the precipitated RNA. B: Specific antibodies against IGF2BP1 and isologous IgG were used to co-incubate with the lysates of stably virus-producing HepAD38 cells, and RT-qPCR detection and agarose gel electrophoresis experiments were performed on the precipitated RNA. C: Biotin-labeled preCRNA, pgRNA, 2.4 kb, 2.1 kb, and 0.7 kb HBV RNA were respectively co-incubated with the lysates of HepG2 cells, and pulldown was performed using streptavidin magnetic beads to detect the proteins bound to different HBV RNAs. D: Actinomycin D was added to HepG2 cells with knocked-down IGF2BP1 and transiently transfected with the prcccDNA / pCMV-Cre plasmid to inhibit the production of nascent RNA. Cells were harvested at different time points after drug addition and normalized to 0 h to detect the relative expression levels of 3.5 kb HBV RNA at different time points. E: Actinomycin D was added to HepG2 cells with overexpressed IGF2BP1 and transiently transfected with the prcccDNA / pCMV-Cre plasmid to inhibit the production of nascent RNA. Cells were harvested at different time points after drug addition and normalized to 0 h to detect the relative expression levels of 3.5 kb HBV RNA at different time points. F: Actinomycin D was added to stably virus-producing HepAD38 cells with knocked-down IGF2BP1 to inhibit the production of nascent RNA. Cells were harvested at different time points after drug addition and normalized to 0 h to detect the relative expression levels of 3.5 kb HBV RNA at different time points. G: Actinomycin D was added to stably virus-producing HepAD38 cells with overexpressed IGF2BP1 to inhibit the production of nascent RNA. Cells were harvested at different time points after drug addition and normalized to 0 h to detect the relative expression levels of 3.5 kb HBV RNA at different time points. H: RNA immunoprecipitation was performed on the lysates of HepG2 cells with knocked-down METTL3 and METTL14 and transiently transfected with the prcccDNA / pCMV-Cre recombinant plasmid system using specific antibodies against IGF2BP1 and isologous IgG, and RT-qPCR detection was performed on the precipitated HBV RNA. I: RNA immunoprecipitation was performed on the lysates of stably virus-producing HepAD38 cells with knocked-down METTL3 and METTL14 using specific antibodies against IGF2BP1 and isologous IgG, and RT-qPCR detection was performed on the precipitated HBV RNA.J: The cell lysates of HepG2 cells transiently transfected with the mutated prccc-A1907C plasmid and the wild-type prccc-WT plasmid and the pCMV-cre plasmid were subjected to RNA immunoprecipitation using a specific antibody against IGF2BP1 and isologous IgG, respectively, and the precipitated HBV RNA was detected by RT-qPCR. K: A specific antibody against IGF2BP1 and isologous IgG were co-incubated with the cell lysates of HepG2 cells transfected with 1.2x HBV plasmid, 1.2x HBV-3’A1907 plasmid, 1.2x HBV-5’A1907C plasmid and 1.2x HBV-5’&3’A1907C plasmid, and the precipitated RNA was detected by RT-qPCR. (Statistical analysis was performed after three repeated experiments: ***, P<0.001; **, P<0.01; *, P<0.05).

[0031] Figure 5 A: After culturing HepAD38 with stable HBV production in the presence of 1 nM cucurbitacin B for 48 hours, the cell lysates were subjected to RNA immunoprecipitation using a specific antibody against IGF2BP1 and isologous IgG, respectively, and the precipitated HBV RNA was detected by RT-qPCR. B: 0.5 nM and 1 nM CuB were added to the HepAD38 cells with stable HBV production and cultured for 3 days and 5 days, respectively, and the HBV DNA levels in the cell culture supernatants were detected. C: 1.2x HBV plasmid was hydrodynamically injected into the tail veins of 6-8-week-old C57BL / 6J mice, and 1 ng / ml or 10 ng / ml CuB was administered to the mice by tail vein injection on the 2nd, 5th, 7th, 9th, and 12th days after hydrodynamic injection. Blood was collected from the medial canthus of the mice to detect the expression of HBsAg in the mouse serum, and part of the mice were sacrificed on the 9th day after hydrodynamic injection into the tail vein, and the liver was taken for HBcAg immunohistochemical staining and detection of HBV RNA and protein levels in the liver. D&E: Expression levels of HBsAg in the mouse serum at different time points after hydrodynamic injection of 1.2x HBV plasmid into the tail vein. F: HBcAg immunohistochemical staining of mouse liver tissue on the 9th day after hydrodynamic injection into the tail vein. G: The AOD values of the HBcAg immunohistochemical staining samples were analyzed using ImageJ (3 samples in each group). H: Western blot was used to detect the protein expression in the mouse liver tissue on the 9th day after hydrodynamic injection into the tail vein. I: RT-qPCR was used to detect the HBV RNA level in the mouse liver tissue on the 9th day after hydrodynamic injection into the tail vein. (Statistical analysis was performed after three repeated experiments: ***, P<0.001; **, P<0.01; *, P<0.05). Detailed implementation mode

[0032] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation manners of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value therebetween are specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Use

[0036] In one aspect of the present invention, there is provided the use of an RNA-binding protein inhibitor in the preparation of a drug for inhibiting the replication of hepatitis B virus (hereinafter referred to as HBV for short), wherein the RNA-binding protein is insulin-like growth factor 2 mRNA-binding protein 1 (hereinafter referred to as IGF2BP1 for short). The inventors of the present invention first discovered and verified that IGF2BP1 can recognize and bind to HBV RNA, participate in the transport of HBV mRNA, regulate the stability and translation ability of target HBV RNA, enhance the expression of target HBV RNA, and regulate HBV replication. The inventors of the present invention also found that IGF2BP1 can bind to m6A-modified HBV RNA, thereby inhibiting HBV replication.

[0037] In the present invention, the term "RNA-binding protein inhibitor" refers to any small molecule substance (such as a small molecule compound) and / or macromolecule substance (such as nucleic acid, polypeptide, antibody, protein, etc.) that can inhibit IGF2BP1. The inhibitory effect of the inhibitor should be understood in a broad sense, including but not limited to at least one of the following situations: inhibiting or blocking the binding of IGF2BP1 to target RNA (i.e., HBV RNA), inhibiting, reducing or downregulating the expression of IGF2BP1, inhibiting or reducing the amount of IGF2BP1, inhibiting or reducing the activity of IGF2BP1 or inactivating it.

[0038] In the present invention, inhibiting or reducing the activity of IGF2BP1 or inactivating it means that the activity of IGF2BP1 treated with an inhibitor is reduced by at least 50%, preferably at least 80%, more preferably at least 90%, further preferably at least 95%, such as at least 96%, 97%, 98%, 99%, 99.9%, or even 100% compared to untreated IGF2BP1. This activity includes its property of binding to HBV RNA, especially m6A-modified HBV RNA, and further inhibiting the replication of HBV.

[0039] In a preferred embodiment, the RNA-binding protein inhibitor is a gene expression inhibitor that can down-regulate the expression of IGF2BP1. Preferably, the gene expression inhibitor includes the CRISPR / Cas9 system, small interfering RNA, antisense oligonucleotides, and / or methylation inhibitors. More preferably, the gene expression inhibitor is siRNA or shRNA. Most preferably, the siRNA has the sequence shown below: GGCUCAGUAUGGUACAGUA.

[0040] In a preferred embodiment, the RNA-binding protein inhibitor is an anti-IGF2BP1 antibody that specifically binds to IGF2BP1, thereby changing its conformation and reducing or inactivating the activity of IGF2BP1. In certain embodiments, in the present invention, the antibody can be a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, murine antibody, or a fragment thereof, and the fragment can be Fab, Fab’, F(ab)2, F(ab’)2, scFv, scFv Fc fragment, or single-chain antibody ScAb.

[0041] In a preferred embodiment, the RNA-binding protein inhibitor is cucurbitacin B, which inhibits the replication of hepatitis B virus by inhibiting or blocking the binding of IGF2BP1 to HBV RNA.

[0042] In the present invention, the inhibition of hepatitis B virus replication can be determined by measuring the level (quantity) of HBV DNA. In addition, the level (quantity) of IGF2BP1 can be determined by any method known in the art, and there is no particular limitation. Those skilled in the art are familiar with how to determine the expression level of a relevant gene or the quantity of a relevant protein, such as using an antibody that specifically binds to the above protein, a probe for the relevant gene, or a primer designed to amplify the relevant gene.

[0043] Screening method

[0044] The present invention further provides a method for screening compounds useful for inhibiting chronic hepatitis B virus replication, which comprises: (1) constructing a cell or animal model using an insulin-like growth factor 2 mRNA-binding protein 1 overexpression reagent; (2) measuring the amount of chronic hepatitis B virus DNA in the cell or animal model; (3) administering a test compound to the cell or animal model; (4) measuring the amount of chronic hepatitis B virus DNA after administering the compound.

[0045] Herein, the IGF2BP1 overexpression reagent can be any reagent capable of increasing the amount of IGF2BP1 protein, such as a genetic engineering reagent. Such reagents include, but are not limited to, vectors capable of overexpressing IGF2BP1, such as plasmids. Overexpression can be carried out by methods known in the art. For example, a cytomegalovirus vector overexpressing IGF2BP1 is constructed, and after transfection of the vector into host cells, the virus is collected; the virus solution is added dropwise to infect cells; the virus is purified and concentrated; and IGF2BP1 is overexpressed by intravenous injection into mammals such as mice. In addition, an IGF2BP1 agonist can also be used in the present invention, and examples of such agonists can be small molecule compounds or nucleic acids known in the art.

[0046] When determining whether a compound or candidate drug is effective, the cell or animal model can be measured. For example, the amount of HBV DNA at different time points from the cell or animal model, such as before and after administering the test compound or candidate drug, is used to determine whether the test compound or candidate drug is effective in inhibiting HBV replication. In a preferred embodiment, the cell model of the present invention is hepatoma cells (HepG2). In another preferred embodiment, the animal model of the present invention is a rat or a mouse.

[0047] In the present invention, the test compound or candidate drug includes, but is not limited to, small molecule compounds, polypeptides, proteins, antibodies, etc.

[0048] Examples

[0049] 1. Experimental methods

[0050] 1.1 Fluorescent quantitative PCR

[0051] Take 20 μl of the cDNA product and dilute it to 100 μl with double-distilled water, and mix well. Take 2 μl as the template for real-time fluorescent quantitative PCR, and detect it using a Roche Light Cycle 480II PCR instrument. Reaction conditions: 95 °C for 5 minutes; 95 °C for 10 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, for 40 cycles. Analyze the specificity of the primers through the melting curve. According to the threshold cycle number Ct value of each reaction well, using the 2 -△△Ct method, with CtBP1 as the internal reference, calculate the relative expression level of the gene.

[0052] 1.2 Western blot assay

[0053] Cells were lysed in pre - cooled RIPA buffer (Invitrogen, California, USA) containing protease inhibitors (Roche, Basel, Switzerland). Then the cell lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected and the protein concentration was determined using a BCA protein assay reagent (Pierce, Illinois, USA). Protein lysates were separated on a NuPAGE Bis - Tris gel (Invitrogen, California, USA) and transferred to a PVDF membrane (Millipore, Massachusetts, USA). Hybridization was performed with antibodies. Protein - antibody complexes were visualized using an Odyssey Imager (LI - COR Biosciences, North Carolina, USA).

[0054] 1.3 Transfection of siRNA

[0055] Cells in the logarithmic growth phase were digested with trypsin, counted, and seeded into 6 - well culture plates at a density of 3×10 5 cells / mL. Two RNase - free EP tubes were taken and 600 μl of Opti - MEM solution was added to each. In one tube, 180 pmol of siRNA was added, and in the other, 15 μl of Lipofectamine RNAiMAX was added. After mixing, the tubes were left at room temperature for 5 minutes. The above two solutions were mixed, gently vortexed, and left at room temperature for 20 minutes. The mixture was added to the 6 - well culture plates, 200 μl per well. The cells were cultured in a 5% CO2, 37°C incubator.

[0056] 1.4 RNA half - life detection (RNA turnover)

[0057] After the cells were treated accordingly, actinomycin D was added at a final concentration of 5 μg / mL to inhibit the production of nascent RNA. Cells were harvested at different time points after adding the drug. RNA was extracted using TRIzol (Invitrogen, California, USA) and chloroform, and the relative expression levels of RNA at different time points were detected by RT - qPCR experiments. Using the 2 -ΔΔCt method, CtBP1 was used as an internal reference to calculate the relative gene expression levels. The expression values at other time points were normalized based on the 0 - h time point to calculate the relative expression levels.

[0058] 1.5 Northern blot hybridization

[0059] Harvest the cell pellets of the HBV in vitro replication model and the liver tissues of HBV-replicating mice in different treatment groups and the control group, and extract tissue or cell RNA using TRIzol reagent. Take 5 μg of total RNA for denaturation treatment, prepare an agarose gel, and perform electrophoresis for 5 - 7 h under a constant voltage of 70 V. After transferring to a nylon membrane, perform ultraviolet cross-linking fixation, pre-hybridization, probe hybridization, blocking, and antibody incubation in sequence, and conduct comparative analysis through ECL development.

[0060] 1.6 RNA Immunoprecipitation (RIP)

[0061] Lyse the cells in Polysome lysis buffer, and perform an RNA immunoprecipitation experiment on the cell lysate using Dynabeads Protein G (Invitrogen, California, USA) mixed with IGF2BP1 antibody (proteintech, Rosemont, USA). Then, wash the magnetic beads with NT2 buffer and extract the RNA pulled down by the magnetic beads using acid phenol-chloroform. Detect the expression level of RNA through RT-qPCR experiment.

[0062] 2. Experimental Results

[0063] 2.1 Overexpression of exogenous IGF2BP1 promotes HBV replication

[0064] To study the role of IGF2BP1 in HBV replication, the present invention overexpressed IGF2BP1 exogenously in HepG2 cells transiently transfected with the prcccDNA / pCMV-Cre plasmid. The results showed that overexpression of IGF2BP1 significantly increased the levels of HBV DNA, HBeAg, and HBsAg in the culture supernatant, as well as the levels of intracellular core protein and preS1 protein ( Figure 1 A - D). At the same time, overexpression of IGF2BP1 increased the intracellular HBV RNA level ( Figure 1 E&F). In addition, similar results were also observed in the HBV replication model HepAD38 cells ( Figure 1 G - L). In summary, the present invention confirmed in the HBV in vitro replication cell model that overexpression of IGF2BP1 can promote HBV replication.

[0065] 2.2 Knockdown of IGF2BP1 inhibits HBV replication

[0066] To further confirm the role of IGF2BP1 in HBV replication, the present invention knocked down endogenous IGF2BP1 using siRNA in HepG2 cells transiently transfected with the prcccDNA / pCMV-Cre plasmid. The results showed that knockdown of IGF2BP1 significantly reduced the levels of HBV DNA, HBeAg, and HBsAg in the cell culture supernatant, as well as the levels of intracellular core protein and preS1 protein ( Figure 2 A-D). Meanwhile, knockdown of IGF2BP1 decreased the intracellular HBV RNA level ( Figure 2 E&F). In addition, similar results were also observed in HepAD38 ( Figure 2 G-L) and the HBV in vitro infection cell model ( Figure 3 ). In summary, the present invention confirmed in the HBV in vitro replication and infection cell models that knockdown of endogenous IGF2BP1 can inhibit HBV replication.

[0067] 2.3 IGF2BP1 can bind to HBV RNA and enhance its stability

[0068] To confirm the interaction between IGF2BP1 and HBV RNA, the present invention co-incubated the specific antibody of IGF2BP1 and isotype IgG of the same species with the lysates of HepG2 cells transfected with the prcccDNA / pCMV-Cre recombinant plasmid system or the lysates of stable virus-producing HepAD38 cells, and then detected the precipitated RNA by RT-qPCR. The results showed that the levels of HBV RNA precipitated by the IGF2BP1 antibody were significantly higher than those of the control IgG ( Figure 4 A-B). Biotin-labeled preC RNA, pgRNA, 2.4 kb, 2.1 kb, and 0.7 kb HBV RNA were co-incubated with the lysates of HepG2 cells respectively, and pulldown was performed using streptavidin magnetic beads. The results showed that the five biotin-labeled HBV RNAs could bind to IGF2BP1 in the cell lysates in vitro ( Figure 4 C), further confirming the binding between IGF2BP1 and HBV RNA.

[0069] To explore the mechanism by which IGF2BP1 regulates HBV RNA expression after binding to HBV RNA, in this invention, actinomycin D was added to HepG2 cells transfected with the prcccDNA / pCMV-Cre recombinant plasmid system with IGF2BP1 knocked down and overexpressed respectively to inhibit the production of nascent RNA. Cells were harvested at different time points after adding the drug, and standardized with 0 h as the benchmark to detect the relative expression level of 3.5 kb HBV RNA at different time points, so as to explore the effect of IGF2BP1 on the degradation rate of HBV RNA. The results showed that the degradation rate of 3.5 kb HBV RNA became faster after knocking down IGF2BP1 ( Figure 4 D); overexpressing IGF2BP1 slowed down the degradation rate of 3.5 kb HBV RNA ( Figure 4 E). In addition, similar results were also observed in HepAD38 cells ( Figure 4 F-G). In summary, this invention confirmed that overexpressing IGF2BP1 can enhance the stability of HBV RNA, and knocking down IGF2BP1 reduces the stability of HBV RNA.

[0070] 2.4 IGF2BP1 binds to HBV RNA by recognizing the m6A modification at site A1907 on HBV RNA

[0071] To further explore the specific site where IGF2BP1 binds to HBV RNA, after knocking down the methyltransferases METTL3 and METTL14 to inhibit the m6A modification level of intracellular RNA in this invention, RNA immunoprecipitation was performed on the lysates of HepG2 cells transfected with the prcccDNA / pCMV-Cre recombinant plasmid system using the specific antibody of IGF2BP1 and isotype IgG respectively. The results showed that the amount of HBV RNA bound by IGF2BP1 decreased after the m6A modification level was reduced ( Figure 4 H). Similar results were also observed in HepAD38 cells ( Figure 4 I), indicating that the binding of IGF2BP1 to HBV RNA depends on the m6A modification on HBV RNA. The A1907 site on the prcccDNA plasmid was mutated so that it could not undergo m6A modification. The prccc-WT and the mutated prccc-A1907C plasmids were transiently transfected into HepG2 cells together with the pCMV-cre plasmid, and RNA immunoprecipitation was performed on the cell lysates using the specific antibody of IGF2BP1 and isotype IgG respectively. The results showed that the amount of HBV RNA bound by IGF2BP1 decreased after the m6A modification site A1907C was mutated ( Figure 4J). Since there is an A1907 site capable of m6A modification at each of the 5' and 3' ends of the 3.5 kb HBV pgRNA, the present invention constructed plasmids with single mutations and double mutations at the A1907 sites at the 5' and 3' ends respectively on the basis of the pBB4.5-1.2xHBV plasmid. After transfection into HepG2 cells, it was found that the 3.5 kb HBV RNA bound by IGF2BP1 decreased after single mutations at the 5' and 3' ends respectively, and further decreased after double mutations at the 5' and 3' ends ( Figure 4 K). This indicates that IGF2BP1 can bind to the A1907 sites at both ends of the 3.5 kb HBV RNA. After the A1907C mutation, although the HBV RNA bound by IGF2BP1 decreased, there was still some binding. Therefore, it is speculated that in addition to A1907, there are other IGF2BP1 binding sites on the HBV RNA.

[0072] 2.5 Cucurbitacin B inhibits HBV replication by inhibiting the binding of IGF2BP1 to HBV RNA

[0073] To explore whether cucurbitacin B (CuB) affects HBV replication, the present invention first added 1 nM cucurbitacin B to stably producing HepAD38 and cultured for 48 hours. Then, RNA immunoprecipitation was performed on the cell lysate using a specific antibody against IGF2BP1 and isotype IgG. The results showed that the HBV RNA bound by IGF2BP1 decreased after treatment with 1 nM CuB ( Figure 5 A). This indicates that CuB can inhibit the binding of IGF2BP1 to HBV RNA.

[0074] Furthermore, the present invention explored whether CuB can inhibit HBV replication. 0.5 nM and 1 nM CuB were added to stably producing HepAD38 cells and treated for 3 days and 5 days. The HBV DNA levels in the cell culture supernatant were detected respectively. It was found that CuB can reduce the HBV DNA level, and the inhibitory effect of low-concentration (5 nM) CuB on HBV DNA is better ( Figure 5 B).

[0075] In addition, a hydrodynamic injection of 1.2x HBV plasmid into the tail vein of 6-8-week-old C57BL / 6J mice was used to construct a HBV replication mouse model. PBS or 1 ng / ml or 10 ng / ml CuB was administered to the mice by tail vein injection on the 2nd, 5th, 7th, 9th, and 12th days after injection. Blood was taken from the medial canthus of the mice to detect the expression of HBsAg in the mouse serum. On the 9th day after hydrodynamic injection of the tail vein, some mice were sacrificed, and the liver was taken for immunohistochemical staining of HBcAg and detection of HBV RNA and protein levels in the liver (Figure 5 C). Intravenous injection of CuB can inhibit the level of HBsAg in the serum of mice ( Figure 5 D&E), and the results of immunohistochemistry and western blot show that CuB inhibits the expression of HBcAg in the liver tissue of mice ( Figure 5 F-H), and at the same time, the level of HBV RNA in the liver is also inhibited by CuB ( Figure 5 I). Consistent with the intracellular results, CuB can inhibit the replication of HBV in the HBV replication model of mice, and the inhibitory effect on HBV replication is stronger at a low concentration (1 ng / ml).

[0076] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes can be made to the exemplary embodiments of the specification of the present invention without departing from the scope or spirit of the present invention. The scope of the claims should be construed in the broadest manner to cover all modifications and equivalent structures and functions.

Claims

1. Use of an RNA binding protein inhibitor in the preparation of a drug for inhibiting the replication of chronic hepatitis B virus, characterized in that: The RNA binding protein is insulin-like growth factor 2 mRNA binding protein 1, and the RNA binding protein inhibitor is a gene expression inhibitor having a siRNA sequence shown as GGCUCAGUAUGGUACAGUA.

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