CN115466802B - Application of TP53BP2 in regulation and control of interferon signal path and antiviral - Google Patents

Abstract

The invention discloses the use of TP53BP2 in regulating interferon signaling pathways and anti-virus. The present invention confirms that TP53BP2 can relieve the negative regulatory effect of SOCS1/2/3 proteins on interferon response to a certain extent by inhibiting/downregulating the level of SOCS, and enhances the antiviral effect of interferon as an immunomodulator. The present invention can not only judge and predict an individual's therapeutic response to interferon based on the level of TP53BP2, but also regulate the activity of TP53BP2 through different measures to enhance the clinical therapeutic effect of interferon.

Description

The use of TP53BP2 in regulating interferon signaling pathways and anti-virus

Technical field

The present invention relates to the field of anti-virus, and specifically relates to the use of apoptosis-stimulating p53 protein 2 (TP53BP2) in predicting the response to interferon anti-viral treatment and enhancing the effect of interferon anti-viral treatment.

Background technique

Chronic hepatitis B (hereinafter referred to as chronic hepatitis B) is a chronic inflammatory disease of the liver caused by persistent infection with hepatitis B virus (HBV). Chronic hepatitis B is an important cause of severe hepatitis B, liver fibrosis, cirrhosis and primary hepatocellular carcinoma (HCC). More than 85% of hepatocellular carcinomas in my country have a background of chronic hepatitis B, so chronic hepatitis B is still a serious problem. One of the important infectious diseases endangering the health of our people.

Currently, the first-line clinical anti-HBV therapeutic drugs mainly include interferon (IFN) and nucleoside (acid) analogs (NAs). IFN mainly activates the JAK-STAT pathway and induces the expression and activation of antiviral host factors such as interferon-stimulated genes (ISGs), thereby exerting immune regulation and antiviral functions. The IFNs currently used clinically for the treatment of chronic hepatitis B are mainly interferon alpha (IFNα) and pegylated interferon alpha (PEG-IFNα). Among them, PEG-IFNα is also called long-acting interferon because of its long half-life. Studies have shown that standardized antiviral treatment can effectively inhibit the replication of HBV virus, alleviate the progression of liver disease, and effectively reduce the occurrence of cirrhosis and liver cancer.

The most ideal clinical treatment endpoint for chronic hepatitis B is to enable patients to achieve serum HBsAg negative conversion or seroconversion and achieve "clinical cure". However, the role of NAs and IFN treatment in achieving "clinical cure" is very limited. The cumulative HBsAg negative conversion rate of the former after long-term treatment is only about 1%. Compared with NAs, the clinical cure rate of chronic hepatitis B patients who receive limited courses of long-acting interferon treatment is around 9%-11%. However, due to the relatively obvious side effects of IFN, clinical guidelines recommend a limited course of treatment of 48 weeks, and recommend that patients with poor efficacy stop interferon treatment as early as possible and switch to relatively safer long-term treatment with NAs to reduce the occurrence of unnecessary side effects. . The main means clinically used to predict the antiviral response in patients with chronic hepatitis B is to monitor virological indicators such as HBV-related antigens and blood biochemical indicators such as serum alanine aminotransferase (ALT) levels. Each of these methods has its own advantages. There are also shortcomings in sensitivity, specificity and timeliness. The above indicators are all indicators for testing the therapeutic effect during the treatment process and predicting the subsequent treatment effect. There is currently a lack of indicators that can predict the IFN response effect before treatment, so some patients who are not suitable for interferon treatment may be included. treatment, thereby suffering unnecessary side effects, and also causing some patients to lose the chance of clinical cure.

The above current situation fully demonstrates that there is an urgent need to supplement indicators for predicting the response to interferon treatment of chronic hepatitis B and achieving clinical cure. Host anti-hepatitis B virus immunity plays an important role in controlling HBV replication and clearing the virus. Considering that interferon mainly exerts antiviral effects by regulating the host immune response, and host immunity is determined by its genetic background, it is necessary to explore the factors involved in affecting host immunity. Genetic markers to identify patients with good response and viral clearance to interferon therapy.

The information in the Background is merely illustrative of the general background of the invention and should not be construed as an admission or in any way implying that the information constitutes the prior art that is already known to those of ordinary skill in the art.

Contents of the invention

In order to solve at least part of the technical problems in the prior art, the present invention provides the use of TP53BP2 in regulating interferon signaling pathways and anti-virus. Specifically, the present invention includes the following contents.

A first aspect of the present invention provides a method for regulating interferon signaling-related pathways, which includes the step of regulating the amount or activity of TP53BP2.

In certain embodiments, the method for regulating interferon signaling related pathways according to the present invention, wherein the regulating is activating, upregulating and/or enhancing the interferon signaling related pathways; and the regulating is enhancing, Increase and/or up-regulate the amount or activity of TP53BP2, or increase the amount of TP53BP2 gene, or promote the expression of TP53BP2 gene.

In some embodiments, according to the method for regulating interferon signaling-related pathways of the present invention, the regulating includes at least one of the following situations:

(a) Reduce, down-regulate or block the amount or activity of SOCS protein through TP53BP2, or reduce, down-regulate or block the expression or activity of SOCS gene;

(b) Inducing the activation of interferon-stimulated genes in the presence of interferon through TP53BP2, or enhancing its expression;

(c) Enhance or improve the interferon response effect through TP53BP2;

(d) Promote STAT phosphorylation in hepatocytes stimulated by interferon through TP53BP2;

In certain embodiments, according to the method for regulating interferon signaling-related pathways of the present invention, the interferon response effect includes an anti-HBV effect.

A second aspect of the present invention provides the use of TP53BP2 or its gene in the preparation of drugs for interferon antiviral treatment.

In certain embodiments, the use according to the present invention is for enhancing or improving the antiviral therapeutic effect of interferon.

A third aspect of the present invention provides the use of a reagent for displaying the amount or activity of TP53BP2 or its gene in the preparation of a diagnostic agent for predicting or evaluating the antiviral therapeutic effect of interferon.

A fourth aspect of the present invention provides a method for screening compounds, which includes the following steps:

(1) The step of measuring the amount or activity of TP53BP2 or its gene from the model to obtain the first measured value T _t1 ;

(2) The step of applying the test compound to the model;

(3) measuring the amount or activity of TP53BP2 in the model after administration of the test compound to obtain a second measured value T _t2 ; and

(4) The step of comparing the first measured value T _t1 and the second measured value T _t2 ; when the second measured value T _t2 is greater than or equal to the first measured value T _t1 , the compound to be tested is Screening for compounds that can enhance or promote the antiviral therapeutic effect of interferon.

A fifth aspect of the present invention provides a method for screening compounds, which includes the following steps:

(1') The step of measuring the amount or activity of SOCS from the model to obtain the first measured value T _s1 ;

(2') the step of applying the test compound to the model;

(3') measuring the amount or activity of SOCS of the model after administration of the test compound to obtain a second measured value T _s2 ; and

(4') The step of comparing the first measured value T _s1 and the second measured value T _s2 ; when the second measured value T _s2 is less than the first measured value T _s1 , screen the compound to be tested It is a compound that can enhance or promote the antiviral therapeutic effect of interferon.

The present invention confirms that TP53BP2 can relieve the negative regulatory effect of SOCS1/2/3 proteins on interferon response to a certain extent by inhibiting/downregulating the level of SOCS, and enhances the antiviral effect of interferon as an immunomodulator. This discovery can not only predict an individual's therapeutic response to interferon based on the level of TP53BP2, but can also enhance the clinical therapeutic effect of interferon by regulating TP53BP2 activity through different measures.

Description of the drawings

Figure 1 shows the results of the effect of interferon on HBV replication in TP53BP2 knockdown HepAD38 cells.

Figure 2 shows that TP53BP2 enhances hepatocyte response to interferon.

Figure 3 shows that TP53BP2 promotes STAT phosphorylation in hepatocytes stimulated by interferon.

Figure 4 shows that TP53BP2 inhibits the expression level of SOCS2.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and every intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

In the present invention, the term "amount" means the content, level, absolute amount, relative amount, etc. of TP53BP2. The amount or level of the gene expressing TP53BP2 includes the content or level of the mRNA.

In the present invention, the terms "reagent" and "detection agent" are used interchangeably to include any reagent capable of displaying the amount or activity of TP53BP2 or its gene.

Methods for regulating interferon signaling related pathways

The present invention provides a method for regulating interferon signaling-related pathways, which is sometimes also called "a method for regulating interferon signaling-related pathways in vitro."

In certain embodiments, modulating includes promoting, activating, up-regulating and/or enhancing interferon signaling related pathways; and the modulating includes enhancing, increasing and/or up-regulating the amount or activity of TP53BP2, or increasing the amount of the TP53BP2 gene, or Promote the expression of TP53BP2 gene. Preferably, the interferon signaling related pathways include: JAK-STAT pathway.

In certain embodiments, regulating includes at least one of the following:

(a) Reduce, down-regulate or block the amount or activity of SOCS protein, or reduce, down-regulate or block the expression or activity of SOCS gene;

(b) Inducing the activation of interferon-stimulated genes in the presence of interferon, or enhancing its expression. Preferably, the interferon-stimulated genes include at least one of MX1, MX2, OAS1, OAS2, OAS3, ISG15 and ISG20;

(c) Enhance or improve the interferon response effect. Preferably, the interferon response effect includes the anti-HBV effect;

(d) Promote STAT phosphorylation in hepatocytes stimulated by interferon, preferably, increase the phosphorylation level of STAT protein, and preferably also increase the levels of p-STAT1 and p-STAT2;

(e) Reduce viral load and/or activity;

(f) Reduce the amount and/or activity of HBsAg and/or HBeAg.

In the present invention, "the amount and/or activity of TP53BP2 or its gene", "the amount and/or activity of SOCS protein", "the amount and/or activity of STAT protein", "the amount and/or activity of p-STAT1 and p-STAT2" /or activity", "the amount and/or activity of HBsAg and/or HBeAg" can be measured and obtained by methods known in the art. Determination methods include but are not limited to chemiluminescent immunoassay, enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR, gene chip technology or immunoblotting.

Use of TP53BP2 or its gene in preparing drugs for interferon antiviral treatment

The present invention further provides the use of TP53BP2 or its gene-related therapeutic agents in the preparation of drugs for interferon antiviral treatment. In certain embodiments, TP53BP2 or its gene-related therapeutic agents include any small molecule or macromolecule that can enhance, increase and/or upregulate the amount or activity of TP53BP2, or increase the amount of TP53BP2 gene, or promote the expression of TP53BP2 gene Examples of substances and therapeutic agents include, but are not limited to, TP53BP2 agonists, TP53BP2 promoters, TP53BP2 activators, and TP53BP2 enhancers.

In certain embodiments, uses of the present invention include use to enhance or enhance the antiviral therapeutic effect of interferon.

In certain embodiments, the present invention provides a method for improving the antiviral therapeutic effect of interferon in a subject, which includes the step of administering to the subject a therapeutically effective amount of the above therapeutic agent or drug. The present invention confirms that TP53BP2 can relieve the negative regulatory effect of SOCS1/2/3 proteins on interferon response to a certain extent by inhibiting/downregulating the level of SOCS, enhances the phosphorylation of STAT proteins, and further promotes interferon-stimulated genes. The expression of interferon enhances the antiviral effect of interferon as an immunomodulator.

Methods for predicting or evaluating the effect of interferon antiviral treatment

The present invention provides the use of a reagent for displaying the amount or activity of TP53BP2 or its gene in the preparation of a diagnostic agent for predicting or evaluating the effect of interferon antiviral therapy (sometimes referred to herein as "predicting or evaluating interferon antiviral therapy"). effect method").

In certain embodiments, the method for predicting or evaluating the antiviral therapeutic effect of interferon includes the step of using the amount or activity of TP53BP2 or its gene in a biological sample collected from the subject or model as a marker, specifically including the steps (1)-(3).

step 1)

Step (1) of the present invention is a step of using a reagent to measure the amount or activity of TP53BP2 or its gene in a biological sample collected from the subject or model as a first measurement value. The reagent refers to any reagent that can be used to display the amount or activity of TP53BP2 or its gene to be tested. Preferably, the agent includes, but is not limited to, an antibody or antigen-binding fragment thereof. Antibodies herein specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains in the antibody that exhibit the desired biological activity and other parts.

The term "monoclonal antibody (mAb)" refers to an antibody that is highly specific and directed against a single antigenic determinant (epitope). Therefore, the term "monoclonal" refers to an antibody directed against the same epitope and should not be understood as requiring the production of the antibody by any particular method. It is understood that monoclonal antibodies may be prepared by any technique or method known in the art; including, for example, the fusionoma method (Kohler et al., 1975, Nature 256:495), or recombinant DNA methods known in the art (see, e.g., U.S. Patent No. 4,816,567), or by using a phage antibody library and isolating recombinantly produced monoclonal antibodies using techniques described in: Clackson et al., 1991, Nature 352:624-628; and Marks et al., 1991, J. Mol. Biol. 222:581-597.

In the present invention, "antibody fragment" refers to a molecule that is different from an intact antibody, contains a portion of an intact antibody, and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments sexual antibodies.

The reagents of the present invention may include other components in addition to the above-mentioned antibodies. Examples of other ingredients include, but are not limited to, diluents, developing solutions, stopping solutions, washing solutions, and the like. In certain embodiments, any one of the above-mentioned substances may exist separately from the other substances and be stored in different containers (eg, vials), as long as they can be in contact with each other during use. In addition, preferably, any two or more of the above-mentioned substances may be mixed to exist as a mixture.

In certain embodiments, other ingredients may be provided in solution, such as in an aqueous solution. In the case of existing in an aqueous solution state, the concentration or amount of these substances can be easily determined by those skilled in the art according to different needs. For example, a substance may be present in a higher concentration for storage purposes and, when in operation or in use, the concentration may be reduced to a working concentration by, for example, diluting the higher concentration solution.

The reagents in the present invention can be further prepared as diagnostic agents for predicting or evaluating the antiviral therapeutic effect of interferon. The diagnostic agent may be in the form of a diagnostic composition, a diagnostic kit, or any other form in which multiple separate reagents are used in combination.

Herein, the type of sample is not limited, and examples thereof include but are not limited to tissue samples or fluid samples. Tissue samples include somatic cell samples, and fluid samples include blood or its components such as plasma, serum, etc. The biological sample may be a sample of any mammalian origin, preferably a sample of human origin. Examples of biological sample types that may be used in the present invention include, but are not limited to, one or more of the following: urine, feces, tears, whole blood, serum, plasma, blood components, bone marrow, cells, tissues, organs, body fluids, saliva, Cheek swabs, lymph fluid, cerebrospinal fluid, lesion exudates, and other fluids produced by the body. Biological samples may also be frozen, fixed, paraffin embedded, or fresh biopsy samples.

Step (2)

Step (2) of the present invention is a step of comparing the first measured value with a second measured value obtained after administering the therapeutic agent or drug of the present invention to a subject or model.

In certain embodiments, both the first measurement and the second measurement are obtained by the same method.

Step (3)

Step (3) of the present invention is a result determination step. Specifically, when the second measured value is greater than or equal to the first standard value, it is considered to enhance or promote the antiviral therapeutic effect of interferon. When the second measured value is less than the first standard value, then It is not thought to enhance or promote the antiviral therapeutic effect of interferon.

Methods used to screen compounds

The present invention further provides a method for screening compounds, sometimes referred to herein simply as a "screening method."

In certain embodiments, screening methods of the invention include:

(1) The step of measuring the amount or activity of TP53BP2 or its gene from the model to obtain the first measured value T _t1 ;

(2) The step of applying the test compound to the model;

(3) measuring the amount or activity of TP53BP2 in the model after administration of the test compound to obtain a second measured value T _t2 ; and

(4) The step of comparing the first measured value T _t1 and the second measured value T _t2 ; when the second measured value T _t2 is greater than or equal to the first measured value T _t1 , the compound to be tested is Screening for compounds that can enhance or promote the antiviral therapeutic effect of interferon.

In certain embodiments, the screening methods of the invention include the steps of:

(1') The step of measuring the amount or activity of SOCS from the model to obtain the first measured value T _s1 ;

(2') the step of applying the test compound to the model;

(3') measuring the amount or activity of SOCS of the model after administration of the test compound to obtain a second measured value T _s2 ; and

(4') The step of comparing the first measured value T _s1 and the second measured value T _s2 ; when the second measured value T _s2 is less than the first measured value T _s1 , screen the compound to be tested It is a compound that can enhance or promote the antiviral therapeutic effect of interferon.

In the present invention, subjects or models include HBV-infected animal models or in vitro cells, for example, rats, mice, dogs, pigs, monkeys, orangutans, etc. Such animals can be artificially induced to become infected with HBV. In vitro cells include, but are not limited to, human liver cancer tissue cells, examples of which include HepG2, Huh7, Hep3B, SMMC-7721, MHCC97 and their cultured progeny cell lines.

Reagent test kit

The present invention further provides a kit for predicting or evaluating the antiviral therapeutic effect of interferon, which includes measuring the amount or activity of TP53BP2 or its gene in a biological sample collected from the subject or model. value reagent. The reagents mentioned above can be used as reagents in the kit of the present invention. I won’t go into details here.

Example

1. Experimental methods

1. Fluorescent quantitative PCR

Dilute 20 μl of cDNA product to 100 μl with double distilled water and mix thoroughly. Take 2 μl as the template for real-time fluorescence quantitative PCR and use Roche Light Cycle 480II PCR instrument for detection. Reaction conditions: 95°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 40 cycles. Primer specificity was analyzed by melting curves. Based on the threshold cycle number Ct value of each reaction well, the 2- ^△△Ct method was used, with ACTB as the internal reference gene, to calculate the relative expression of the target gene.

2. Western blotting experiment

Cells were lysed in pre-chilled RIPA buffer (Invitrogen, California, USA) containing enzyme inhibitors (Roche, Basel, Switzerland). The cell lysate was then centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was collected and the protein concentration was determined by BCA protein assay reagent (Pierce, Illinois, USA). Protein lysates were separated on NuPAGE Bis-Tris gels (Invitrogen, California, USA) and transferred to PVDF membranes (Millipore, Massachusetts, USA). TP53BP2 (ABclonal, Wuhan, China), CtBP (Santa Cruz, California, USA) and p-STAT1 (CST, Massachusetts, USA), STAT1 (CST), p-STAT2 (Millipore, Massachusetts, USA), STAT2 ( Santa Cruz), p-STAT3 (CST), STAT3 (Proteintech, Illinois, USA), SOCS1 (CST), SOCS2 (CST), SOCS3 (CST), β-actin (ABclonal) and other antibodies were used for hybridization. Protein-antibody complexes were visualized using Odyssey Imager (LI-COR Biosciences, North Carolina, USA).

3. Transfection of siRNA

Cells in the logarithmic growth phase were digested with trypsin, counted and then seeded into a 6-well culture plate at a density of 3×10 ⁵ cells/ml. Take two RNase-free EP tubes and add 600 μl Opti-MEM solution respectively. Add 180 pmol siRNA to one tube and add 15 μl Lipofectamine RNAiMAX to the other tube. Mix well and let stand at room temperature for 5 minutes. Gently mix the above two tubes of solution and let stand at room temperature for 20 minutes. Add the mixture to a 6-well culture plate, adding 200 μl to each well. Cultivate in 5% CO ₂ and 37°C incubator.

4. In vitro cell interferon treatment experiment

The IFNα-2b storage solution has a concentration of 4.6 million IU/ml. Before use, take 1 μl, add ddH ₂ O, adjust the volume to 460 μl, and prepare a working solution of 100 IU/μl.

Cells growing in the logarithmic phase were counted and then inoculated into a 24-well culture plate at a density of 1×10 ⁵ cells/ml, 0.5 ml per well, and cultured overnight; remove the culture medium from the cell culture plate, and inoculate it into each well. Add 0.5 ml of new culture medium. Add 5 μl of IFNα-2b working solution to the cells in each well to make the final concentration of interferon 1000IU/ml. Culture in 5% CO ₂ , 37°C incubator.

5. Rat tail hydrodynamic injection experiment

The pBB4.5-HBV1.2 plasmid carrying 1.2 times the C genotype HBV was used for the mouse tail hydrodynamic injection experiment. Inject 2 mL of PBS containing 10 μg of plasmid into the tail veins of 6-week-old male TP53BP2 knockout mice and control BALB/c mice within 5 seconds. On the third day after the hydrodynamic injection of the rat tail, the mouse serum was taken and HBsAg and HBeAg were measured as the baseline. Each mouse was then injected with 20 mg of murine interferon-α every day for 4 consecutive days until the 7th day. Mouse sera were collected 7 days after injection for analysis of HBsAg and HBeAg levels.

6.RNA-seq sequencing and analysis

Control or TP53BP2 siRNA was transfected into HepG2 cells by Lipofectamine RNAiMAX. After 48 hours, HepG2 was treated with interferon-α-2b or PBS for one hour (1000IU/mL). Total RNA from cells was isolated using Trizol reagent, and RNA sequencing and analysis were completed by Majorbio (Shanghai, China).

2. Experimental results

1.TP53BP2 enhances the anti-HBV effect of interferon

In order to study whether TP53BP2 is involved in the antiviral effect of interferon, the effect of interferon on HBV replication in TP53BP2 knockdown HepAD38 cells was first detected. The results showed that compared with the siControl group, the inhibitory efficiency of interferon-α-2b on HBsAg and HBeAg in HepAD38 cells knocking down endogenous TP53BP2 expression was weakened (Figure 1A-D). These in vitro results indicate that TP53BP2 can enhance the antiviral effects of interferon in hepatocytes.

Next, pBB4.5-HBV1.2 HBV plasmid was injected into wild-type and Trp53bp2 ^+/- mice via hydrodynamic injection of the mouse tail. After 3 days, the mouse serum was collected, and HBsAg and HBeAg were quantitatively measured, proving that the plasmid expression was successful. Neither HBsAg nor HBeAg differed significantly between the WT and Trp53bp2 groups and were therefore considered the baseline levels for each mouse (Figures 1E and 1F). Mice were then treated with mouse interferon-α (20 mg/day per mouse), and sera were collected 4 days after treatment for analysis of HBsAg and HBeAg levels. It was found that in wild-type mice, HBsAg and HBeAg were significantly reduced after interferon-α treatment compared with baseline (Figures 1E and 1F). The inhibitory efficiency of interferon-α in the WT group against HBsAg and HBeAg was 72% and 84% respectively (Figure 1G-1H). In Trp53bp2 ^+/- mice, HBsAg was not significantly reduced after interferon-α treatment (Figure 1E), and the inhibitory efficiency of HBsAg by interferon-α was only 7% (Figure 1G). Although HBeAg was also significantly reduced after interferon-α treatment (Figure 1F), the inhibitory efficiency of HBsAg by interferon-α was also much lower than that in wild-type mice (71% vs 84%, P<0.01).

Therefore, TP53BP2 can enhance the anti-HBV effect of interferon at both in vivo and in vitro levels.

2.TP53BP2 enhances the response of hepatocytes to interferon

In order to explore the potential mechanism by which TP53BP2 increases the interferon-mediated anti-HBV effect, the effect of TP53BP2 knockdown on the HepG2 transcriptome before and after interferon stimulation was analyzed by analyzing RNA-seq. Enrichment analysis showed that the specific differentially expressed genes between the siControl-IFN and siTP53BP2-IFN groups were mainly enriched in virus-related and interferon-related pathways (Figure 2A). In addition, the effect of TP53BP2 on the expression of type I interferon signaling pathway genes was analyzed. The results showed that compared with the siControl-IFN group, all 22 type I interferon signaling pathway genes were reduced in the siTP53BP2-IFN group (Figure 2B). This result strongly suggests that activation of the interferon pathway in hepatocytes by interferon-α is TP53BP2-dependent.

The above results indicate that TP53BP2 is involved in the regulation of the IFN pathway. Since the antiviral activity of type I interferon is mainly mediated by interferon-stimulated genes (ISGs), the GEO database was next used to analyze the correlation between the expression levels of ISGs and TP53BP2 in the livers of CHB patients. The results showed that the expression level of TP53BP2 in the liver of CHB patients was significantly positively correlated with ISGs, including MX1, MX2, OAS1, OAS2, OAS3, ISG15 and ISG20 (Figure 2C). To confirm these observations, TP53BP2 was knocked down in HepG2 cells and the above genes were quantified. Although there was no obvious effect on ISG levels in the absence of interferon stimulation, TP53BP2 knockdown could significantly reduce the up-regulation effect of interferon on the expression levels of MX1, OAS1, OAS2, OAS3 and ISG15 in HepG2.

3.TP53BP2 promotes STAT phosphorylation in hepatocytes stimulated by interferon

The JAK-STAT pathway is the main regulator of ISG transcription. In order to study whether the TP53BP2-mediated upregulation of ISG expression is related to the activation of the JAK/STAT pathway, the effects of TP53BP2 knockdown on baseline and interferon-stimulated STATs phosphorylation and the effect of interferon-α stimulation were analyzed in HepG2 cells ( Figure 3A-3B). The results showed that the levels of p-STAT1 and p-STAT2 in HepG2 cells were significantly up-regulated after interferon-α stimulation, and reached a peak value 15-30 minutes after stimulation (Figure 3A-3B). For TP53BP2 knockdown cells, although interferon-α-2b can still activate p-STAT, the activation intensity and duration are significantly lower than those of control cells, and the total STAT1 and STAT2 levels do not change significantly. baseline or upon interferon-alpha stimulation (Figures 3A-3B). Next, the phosphorylation of STAT proteins in the livers of wild-type and Trp53bp2 ^+/- mice after interferon stimulation was detected. As expected, IFN-induced p-Stat1 and p-Stat2 levels in Trp53bp2 ^+/- mice were also significantly lower than in wild-type BalB/c mice (Fig. 3C-3D). In summary, the results of in vitro and in vivo experiments indicate that TP53BP2 enhances the phosphorylation of STAT proteins, and activation of STATs further promotes the expression of ISGs.

4.TP53BP2 inhibits the expression level of SOCS2

In order to explore the reason why TP53BP2 promotes the activation of the interferon pathway, the transcriptomes of multiple cell lines TP53BP2 knockdown cell lines were analyzed. The results found that the mRNA level of SOCS2 was significantly increased after TP53BP2 was knocked down. Further Western blot experiments found that TP53BP2 could enhance the protein levels of SOCS1, SOCS2, and SOCS3, whether at baseline or under interferon stimulation (Figure 4A). In vivo experiments also showed that the expression levels of SOCS1, SOCS2, and SOCS3 in the liver of Trp53bp2 ^+/- mice were also significantly higher than those in the wild group (Figure 4B).

Next, we analyzed whether the activation effect of TP53BP2 on the interferon pathway is dependent on SOCS family genes, and explored the role of SOCS2 in it. The results showed that in the SOCS2 knockdown cell line, the expression level of TP53BP2 no longer affected the effect of interferon on the phosphorylation of STAT1 and STAT2 (Figure 4C-4D). This suggests that the effects of TP53BP2 on the interferon pathway are at least partially SOCS2-dependent.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments described herein without departing from the scope or spirit of the invention. The scope of the claims should be given the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims (4)

1. A method for regulating interferon signaling related pathways in vitro, characterized in that it includes regulating the amount of cellular TP53BP2 gene in vitro and reducing, downregulating or blocking the amount or activity of SOCS protein, or reducing, downregulating or The step of blocking the expression or activity of the SOCS gene and then regulating the amount or activity of SOCS, wherein the regulation includes activating, up-regulating and/or enhancing the interferon signaling related pathways, and the regulation includes increasing the amount of the TP53BP2 gene, or Promote the expression of TP53BP2 gene. 2. The method for regulating interferon signaling-related pathways in vitro according to claim 1, characterized in that the regulation includes at least one of the following situations: (a) Inducing the activation of interferon-stimulated genes in the presence of interferon, or enhancing their expression; (b) Enhance or improve the effect of interferon response; (c) Promote STAT phosphorylation in hepatocytes stimulated by interferon; (d) reduce viral load and/or activity; (e) Reduce HBsAg and/or HBeAg. 3. The method for regulating interferon signaling-related pathways in vitro according to claim 2, wherein the interferon-stimulated genes include MX1, MX2, OAS1, OAS2, OAS3, ISG15 and ISG20. 4. The method for regulating interferon signaling-related pathways in vitro according to claim 2, wherein the interferon response effect includes an anti-HBV effect.