KR20170130875A - Pharmaceutical composition comprising interferon and DNA methylation inhibitor as an active ingredient for prevention or treatment of hepatitis virus B infection - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating hepatitis virus B (HBV) infection, comprising an interferon inducing the expression of TRIM22 protein and a DNA methylation inhibitor as effective components. Specifically, the interferon of the present invention is secreted when infected with HBV to increase the expression of an anti-viral protein, TRIM22; and the DNA methylation inhibitor of the present invention inhibits the methylation of a TRIM22 gene promotor and a CpG(+71) island of 5UTR, which HBV epigenetically regulates to avoid immune responses of a host cell, and thus allows the expression level of TRIM22 protein to be significantly restored by IFN. Accordingly, the pharmaceutical composition comprising the interferon and the DNA methylation inhibitor of the present invention can inhibit the progression into chronic hepatitis B infection when infected with HBV, by enhancing anti-HBV treatment effects of interferon drugs conventionally used against HBV infection.

Description

A pharmaceutical composition for the prevention and treatment of hepatitis B virus infection comprising interferon and DNA methylation inhibitor as an active ingredient, the pharmaceutical composition comprising interferon and DNA methylation inhibitor as an active ingredient for prevention and treatment of hepatitis virus infection,

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infectious disease, which comprises interferon and DNA methylation inhibitor which induce expression of TRIM22 protein as an active ingredient.

Fighting between the viral infection and the host's protective system can lead to the onset of the disease or can be determined by the treatment of the disease. During a viral infection, the host will recognize the virus and effectively remove the virus through its innate immunity or acquired immunity. However, viruses also have a variety of strategies to counteract them, so that the host's immune system can be avoided.

Interferon (IFN), which is expressed early in infection, is a major cytokine that can effectively work against various antigens in the innate immune response stage. There are various types of IFNs, such as type 1 (IFN ?, IFN? And IFN?), Type 2 (IFN?) And types 3 (IFN? 1, IFN? 2 and IFN? 3), which are antigens activated by an antigen recognition receptor - induces the expression of the relevant molecule. IFN is secreted when the virus is infected and induces the expression of various antiviral and interferon-stimulated genes (ISG) through the IFN signaling pathway. The induced interferon-inducible gene directly exhibits antiviral activity or immunoregulatory activity, and thus can effectively remove the virus. Therefore, IFNs and their signaling pathways can effectively remove viruses, so viruses infected with host cells also have a mechanism capable of avoiding the host's immune system by targeting such IFNs and their signaling .

Hepatitis B virus (HBV) is known to cause chronic hepatitis B, liver cirrhosis, or hepatocellular carcinoma. More than 350 million people in the world reportedly have liver disease caused by HBV infection. HBV, a non-cytopathic, liver-affinity DNA virus, is a strategy to replicate its own genome through reverse transcription using pre-genomic RNA (pregenomic RNA, pgRNA) Have. The synthesis of pgRNA and other viral RNAs is regulated by a viral enhancer and its associated promoter. The HBV X protein (HBx) is a viral protein with various functions related to dysregulation of cell function and antigenicity. Specifically, it has been reported through various studies that host proteins involved in HBV-mediated liver lesion can be genetically regulated by HBx.

Up to now, studies have been reported relating to IFN inhibition of HBV replication. IFNa inhibits HBV replication through subsequent regulation of degradation of pg RNA associated with cccDNA and MyD88. IFNγ is involved in the antiviral activity of cytotoxic T lymphocytes (CTLs) specific for HBV. Both type 1 and type 2 IFNs contain pgRNA and serve to inhibit HBV replication through inhibiting the formation of nucleic acid capsids capable of replication and accelerating their collapse. HBV replication can also be inhibited by the antiviral function of various ISGs such as MxA and APOBEC3G.

Therefore, PEGylated IFNα is widely used as a dietary therapy for antiviral treatment for HBV infection developed so far. However, after treatment with PEGylated IFNa, it has been reported that only about 30 to 44% of patients with chronic hepatitis B have satisfactory viral and serological responses. In addition, HBV exhibits relatively low susceptibility to IFN when compared to hepatitis C virus (HCV). Consequently, through these series of studies it is known that HBV has a resistance to treatment with IFN, because it has a mechanism to avoid or respond to IFN-associated antiviral activity for their survival. As evidence of this, mechanisms involving viral polymerase and HBx protein have been reported (CHEN, Jieliang, et al. Hepatology, 2013, 57.2: 470-482 .; KUMAR, Manish, et al. Journal of Virology, 2011, 85.2: 987-995; LUTGEHETMANN, Marc, et al., Gastroenterology, 2011, 140.7: 2074-2083. However, although these studies have been reported, no exact mechanism has been reported for HBV to avoid IFN-related immune processes.

The tripartite motif protein (TRIM) family is known as a protein involved in various biological processes including tumor formation, apoptosis and antiviral immunity. Specifically, a large number of TRIM proteins are induced in IFN and are involved in innate immune and antiviral protection responses. Of these TRIM proteins, TRIM22, which is one of the closest forms to TRIM5a paralogues, is known to exhibit anti-retroviral activity similar to that of TRIM5a. Previous studies have shown that transcription of human immunodeficiency virus-1 (HIV) through the inhibition of long terminal repeat (LTR) and disruption of intracellular trafficking of viral Gag proteins It is known to inhibit replication.

In addition, TRIM22 exhibits E3 ligase activity, which inhibits influenza virus by degrading the nuclear protein or degrading the viral 3C protein hydrolytic enzyme to inhibit encephalomyocarditis virus (EMCV) And TRIM22 has been known to exhibit antiviral activity against a wide range of viruses. Recently, TRIM22 has been reported to exhibit antiviral activity against HBV by inhibiting the HBV-associated promoter (Gao B, Duan Z, Xu W, et al. Hepatology 2009; 50: 424-433). In addition, there has been reported a full-length microarray analysis method that shows a virus cure associated with TRIM22 in chimpanzees infected with HBV (WIELAND, Stefan, et al., Proceedings of the National Academy of Sciences, 2004, 101.17: 6669-6674.). However, studies on the viral viability through TRIM22 have not yet been accurately reported, despite studies on the antiviral function of TRIM22.

Thus, the present inventors have studied the process of HBV to avoid the immune response of infected host cells, thereby improving the anti-HBV therapeutic effect of the conventional interferon therapeutic agent for HBV, As a result, the expression of TRIM22 protein, which shows antiviral activity among IFN-induced proteins secreted during HBV infection, is increased.

Thus, in order to avoid the immune response in hepatocytes, HBV protein HBx inhibits transcription of TRIM22 mRNA and expression of TRIM22 protein, Through the methylation of the CpG (+71) islet of the TRIM22 gene promoter and 5'UTR. Furthermore, by confirming that the expression level of TRIM22 protein by IFN was significantly restored when a DNA methyltransferase inhibitor capable of inhibiting TRIM22 gene methylation by HBx was treated, the interferon and DNA methylase inhibitor of the present invention The inventors have confirmed that the expression of TRIM22 protein can be restored in HBV-infected cells and used as an active ingredient of a pharmaceutical composition for the prevention and treatment of liver disease caused by HBV infection, thus completing the present invention.

 Sadler, Anthony J., and Bryan RG Williams. Nature reviews immunology 8.7 (2008): 559-568.   Gao, Bo, et al. Hepatology 50.2 (2009): 424-433.  Lim, Keo-Heun, et al. PLoS One 6.8 (2011): e22258.  Park, Eun-Sook, et al. Hepatology 58.2 (2013): 762-776.

Accordingly, the present inventors have developed a method of recovering the expression of TRIM22 anti-HBV active protein inhibited by the hepatitis virus B (HBV) in order to avoid the immune response of infected host cells Thereby completing the invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating HBV infection.

It is still another object of the present invention to provide a screening method for treating hepatitis B virus infection.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infection, which comprises interferon and a DNA methylation inhibitor as an active ingredient.

According to a preferred embodiment of the present invention, the interferon induces the expression of the TRIM22 protein.

According to a preferred embodiment of the present invention, the DNA methylation inhibitor is preferably a DNA methyltransferase inhibitor, and the DNA methyltransferase inhibitor is 5-aza-2'-deoxycytidine (5 ' aza-2'deoxycytidine, 5-AzaC).

According to a preferred embodiment of the present invention, the hepatitis B virus infection is preferably any one of chronic hepatitis B, liver cirrhosis, or hepatocellular carcinoma.

The present invention also relates to

(i) treating interferon and a test compound in a liver tissue sample derived from a hepatitis B patient;

ii) the liver tissue sample treated in step i) Measuring TRIM22 (tripartite motif 22) mRNA transcription level or TRIM22 protein expression level;

iii) TRIM22 measured in step ii) mRNA transcription level or TRIM22 protein expression level with an untreated control; And

iv) selecting a test compound which is increased in comparison with the untreated control group in the step iii), and screening the therapeutic agent for hepatitis B virus infection.

According to a preferred embodiment of the present invention, the interferon is preferably at least one selected from the group consisting of interferon alpha, interferon beta and interferon gamma.

According to a preferred embodiment of the present invention, the test compound is preferably a DNA methylation inhibitor.

According to a preferred embodiment of the present invention, the hepatitis B virus infection is preferably any one of chronic hepatitis B, liver cirrhosis or liver cancer.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infectious disease comprising interferon and DNA methylation inhibitor as an active ingredient.

The interferon of the present invention may be secreted during HBV infection to increase the expression of the antiviral protein TRIM22, and the DNA methylation inhibitor of the present invention may be used as an antimicrobial agent such as TRIM22, which HBV is epigenically regulated in order to avoid immune response of host cells Methylation of the CpG (+71) islet of the gene promoter and 5'UTR is inhibited, and the expression level of TRIM22 protein by IFN is significantly restored.

Accordingly, the pharmaceutical composition comprising the interferon and DNA methylation inhibitor of the present invention can be used to enhance the anti-HBV therapeutic effect of interferon therapeutics conventionally used for HBV, thereby inhibiting progression to chronic B-type infection when infected with HBV So it is effective.

Figure 1 shows screening at the protein level to screen for proteins that are desensitized by interferon (IFN) treatment in HBx expressing cells:
1A is a schematic diagram of a screening process;
FIG. 1B is a van diagram dividing the proteins whose expression levels change according to IFN treatment and HBx expression into groups; FIG.
1C is a diagram showing a heat map of the expression of a protein having an immune response and an antiviral function; And
FIG. 1D shows the expression level of TRIM22 protein according to HBx expression and IFN treatment.
Figure 2 shows the inhibition of the expression of IFN-induced TRIM22 through HBx of viruses:
FIGS. 2A and 2B are graphs showing inhibition of TRIM22 mRNA transcription by HBx expression level in HBx-expressing cells;
FIGS. 2c and 2d are diagrams showing TRIM22 mRNA transcription with time after IFN addition in HBx expressing cells;
FIG. 2E shows the induction of various ISG expression according to the type of IFN treated;
FIG. 2f shows the level of expression of TRIM22 protein according to the type of IFN treated;
Figure 2g shows the level of IFN-induced TRIM22 protein expression in HBx expressing cells;
FIG. 2h shows the level of expression of TRIM22 protein according to IFN treatment concentration in HBx-expressing cells; And
FIG. 2I shows the effect of HBx on TRIM22 expression over time after IFNγ treatment. FIG.
Figure 3 shows the expression position of TRIM22 expressed in cells.
Figure 4 shows the identification of the major components acting on TRIM22 expression inhibition by HBx:
4A is a schematic diagram showing the construction of a plasmid capable of searching for a part related to inhibition of TRIM22 expression by HBx;
4B is a graph showing the results of luciferase analysis of TRIM22 promoter activity at baseline level;
FIG. 4C is a view showing IFN-induced TRIM22 promoter activity; And
FIG. 4d shows IFN-induced TRIM22 promoter activity in HBx-expressing cells. FIG.
5 shows the confirmation of the relationship of IRF1 in inhibition of TRIM22 transcription by HBx:
FIG. 5A shows the level of IFN-induced IRF1 expression in HBx expressing cells;
FIG. 5b shows the level of IFN-induced IRF1 expression in HBV infected cells; And
FIG. 5c shows the level of IFN-induced IRF1 protein expression in HBV-infected or HBx-expressing cells.
Figure 6 shows confirmation of the postchemical regulatory process involved in the inhibition of TRIM22 expression by HBx:
FIG. 6A is a diagram showing the location of DNA methylation for inhibiting TRIM22 transcription by HBx; FIG.
FIG. 6B is a graph showing the transcription level of TRIM22 mRNA in HBx-expressing cells according to the treatment with 5-AzaC, a DNA methyltransferase inhibitor; And
FIG. 6C is a graph showing the expression level of TRIM22 protein in HBx-expressing cells according to the treatment with 5-AzaC, a DNA methyltransferase inhibitor.
7 shows the change in the expression pattern of DNA methyltransferase in the inhibition of TRIM22 transcription by HBx:
7A is a graph showing the correlation between DNA methyltransferase and TRIM22 protein expression levels; And
FIG. 7B is a graph showing the mRNA expression level of DNA methyltransferase according to IFN addition in HBx-expressing cells.
Figure 8 shows the identification of DNA methylation and control related elements for inhibition of IFN-induced TRIM22 expression by HBx:
8A is a schematic view of a mutant sequence prepared to confirm the specific position of TRIM22 promoter methylation by HBx;
FIG. 8B shows the activity of IFN-induced promoter in the TRIM22 promoter mutant;
Figure 8C shows the expression of IFN-induced mutant TRIM22 in HBx expressing cells Lt; / RTI > activity of the promoter;
Figure 8d shows the sequence of the [32P] labeled probe for IRF1 used for EMSA analysis;
FIG. 8E shows the interaction between IRF1 and probe through an antibody super shift assay; FIG. And
FIG. 8f shows the results of immunohistochemistry using HBx expressing cell genomic DNA and IRF1 antibody TRIM22 Lt; / RTI > shows the ChIP analysis of the promoter region.
Figure 9 shows the confirmation of anti-HBV viral effect of IFN induction via TRIM22:
9A is a graph showing HBV protein and TRIM22 protein expression levels according to IFN gamma treatment in HBV infected cells; And
FIG. 9B shows the level of expression of HBV genome according to IFN gamma treatment in HBV infected cells.
10 shows the change in viral gene cloning activity according to TRIM22 expression knockdown:
10A is a graph showing the expression level of TRIM22 protein after knocking down TRIM22 with siRNA in TRIM22 overexpressing cells;
FIG. 10B is a graph showing the expression level of TRIM22 protein after knockdown of TRIM22 in cells treated with IFNγ; And
FIG. 10c shows HBx expression level after TRIM22 knockdown in HBx-expressing cells.
Figure 11 shows the anti-HBV activity of TRIM22 through inhibition of HBV promoter activity in HBx expressing cells:
FIG. 11A shows changes in the activity of HBV promoter amplifiers in TRIM22 overexpressed or knockdown cells;
FIG. 11B is a graph showing changes in HBV genome DNA and protein expression levels according to TRIM22 expression level in HBx-expressing cells; And
FIG. 11C is a graph showing changes in HBV genome DNA and protein expression levels according to IFN treatment in HBx-expressing cells. FIG.
Fig. 12 is a schematic diagram showing a manufacturing process of a mouse model of HBV infection.
Figure 13 shows confirmation of IFN-induced TRIM22 expression and HBV immune evasion responses in vivo in HBV infection.
13A is a graph showing the level of HBV replication in liver tissue in an HBV-infected mouse model;
FIG. 13B shows the expression of HBV surface antigen in liver tissue in an HBV-infected mouse model; And
13C is a view showing the expression level of HBx in an HBV-infected mouse model.
Figure 14 shows IFN-induced TRIM22 promoter activity in HBV infected mouse models:
14A shows expression of mouse IFNa, IFN [beta] and IFN [gamma] mRNA according to the type of plasmid injected into an HBV-infected mouse model;
14B is a diagram showing TRIM22 promoter activity in a mouse model expressing HBx; And
14c is a graph showing TP6 activity according to HBx expression in an HBV-infected mouse model.
Figure 15 shows IFN-induced TRIM22 promoter activity following treatment with DNA methyltransferase inhibitor in an HBV infected mouse model:
15A is a schematic diagram showing steps of 5-AzaC administration experiment in an HBV-infected mouse model; And
15B is a graph showing recovery inhibition of TRIM22 promoter activity by HBx according to administration of 5-AzaC.
Figure 16 shows HBV characteristics that inhibit IFN-induced TRIM22 expression in primary human hepatocytes (PHH): < RTI ID = 0.0 >
16A is a schematic diagram showing an experimental procedure using PHH;
Figure 16b is a graph showing TRIM22 mRNA transcript levels following 5-AzaC treatment in HBV infected PHH; And
16C is a graph showing the level of TRIM22 protein expression by 5-AzaC treatment in HBV-infected PHH.
FIG. 17 is a schematic diagram showing an immunity-evoking mechanism of HBV induced by IFN-induced antiviral activity, which has been confirmed in the present invention, through proliferative regulation of TRIM22 to avoid an immune response.

Hereinafter, the present invention will be described in more detail.

As described above, interferon (IFN) therapeutic agents have been widely used as therapeutic agents against hepatitis virus B (HBV), but they exhibit low sensitivity in practical use. On the other hand, it is known that HBV has a resistance against the treatment using IFN because it has a mechanism capable of avoiding or coping with the antiviral activity induced by IFN, but the specific action mechanism and the target have been studied none.

The interferon of the present invention may be secreted during HBV infection to increase the expression of the antiviral protein TRIM22, and the DNA methylation inhibitor of the present invention may be used as an antimicrobial agent such as TRIM22, which HBV is epigenically regulated in order to avoid immune response of host cells Methylation of the CpG (+71) islet of the gene promoter and 5'UTR is inhibited, and the expression level of TRIM22 protein by IFN is significantly restored.

Accordingly, the pharmaceutical composition comprising the interferon and DNA methylation inhibitor of the present invention can be used to enhance the anti-HBV therapeutic effect of interferon therapeutics conventionally used for HBV, thereby inhibiting progression to chronic B-type infection when infected with HBV Therefore, it is effective in the prevention and treatment of liver disease caused by HBV infection.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infectious disease comprising interferon and DNA methylation inhibitor as an active ingredient.

The " interferon " of the present invention preferably induces the expression of TRIM22 protein in a cell. The term " interferon " is preferably one or more selected from the group consisting of interferon alpha, interferon beta and interferon gamma. More preferably, the interferon alpha or interferon gamma or both is more preferably interferon gamma , But it is not limited thereto. Any interferon known in the art may be used as a factor inducing the TRIM22 protein in the cell.

The "DNA methylation inhibitor" of the present invention is preferably a DNA methyltransferase inhibitor. More specifically, the DNA methyltransferase inhibitor may be 5'aza-2'-deoxycytidine having a structure represented by the following formula (1) -2'deoxycytidine, 5-AzaC), but is not limited thereto:

[Chemical Formula 1]

.

.

The hepatitis B virus infection according to the present invention is preferably any one of hepatitis, liver cirrhosis or hepatocellular carcinoma. More preferably, the hepatitis is chronic hepatitis B, but is not limited thereto. Do not.

In a specific example of the present invention, the inventors of the present invention conducted screening (FIG. 1A) of a gene targeted by HBV in order to avoid interferon-related immune response upon hepatitis B virus infection, IFN-induced TRIM22 was selected as a protein showing immunological and antiviral activities among proteins (ISP) (Fig. 1B, Fig. 1C and [Table 1]), and the expression of TRIM22 was actually inhibited by HBx when IFNγ was actually treated (Fig. 1d).

Further, the present inventors have further confirmed the process of inhibiting the expression of TIM22 in order to avoid the immune response of the host after infection with HBV. As a result, in the cells transfected with the HBV genome or HBx, TRIM22 mRNA and / It was confirmed that expression of TRIM22 protein was significantly inhibited (Fig. 2A to Fig. I and Fig. 3).

The present inventors constructed a plasmid to express a promoter region and a luciferase reporter essential for transcription of TRIM22 (Fig. 4A), and thereby, by HBx in the TRIM22 promoter region As a result of confirming the positions associated with the inhibition of the expression, it was confirmed that the 5 'UTR (+ 1 to +211 ) region of the TRIM22 gene plays an important role by changing the activity of the TRIM22 promoter by IFN treatment or HBx expression (FIGS. 4d). However, despite the fact that the 5'UTR of the TRIM22 gene is an IFN regulatory factor-1 (IRF1) -binding region, it was confirmed that HBx does not affect IFR1 expression (FIGS. 5A to 5C).

Further, the inventors of the present invention found that the 5'UTR (+ 1 to +211) of TRIM22 was found in the 5'UTR (+ 1 to +211 ) in order to confirm whether the HBV regulation that inhibits transcriptional activity was epigenetic regulation (Fig. 6A), confirming that the suppression of TRIM22 expression by HBx was due to post-sexual control through methylation of the CpG (+71) region of the TRIM22 promoter region (Figs. 6B and 6C ) 6c). In addition, TRIM22 transcriptional repression by HBx confirmed that DNA methylation was through regulation of activity rather than expression level of DNA methyltransferase (FIGS. 7 and 7b).

In addition, the present inventors confirmed that inhibition of TRIM22 expression by HBx was confirmed by confirming whether IRF1 binding was involved in suppression of TRIM22 expression by HBx in order to more specifically confirm posterior control through methylation of the CpG (+71) region of TRIM22 promoter region (Fig. 8A). One CpG island (+71) plays an important role in TRIM22 desensitization by HBx (Fig. 8B and Fig. 8C), and the IFNγ-induced TRIM22 expression (Fig. 8D to Fig. 8F).

The present inventors further confirmed the anti-HBV activity induced by IFN-induced TRIM22 and the posttreatment control of HBV to avoid the anti-HBV activity induced by IFN-γ. As a result, the expression levels of RNA and protein of HBV (FIG. 9A and FIG. 9B). When TRIM22 was overexpressed or knocked down with siRNA (FIG. 10), the promoter activity of HBV was decreased as the expression level of TRIM22 increased It was confirmed that the anti-HBV activity induced by IFN gamma is due to TRIM22 (Figs. 11A to 11C). In addition, when 5-AzaC, an inhibitor of DNA methyltransferase, was treated, expression level of TRIM22 induced by IFNγ was increased and HBV transcription level was decreased (FIG. 11d).

In addition, the present inventors produced a HBV-infected mouse model to confirm the expression of IFN-inducible TRIM22 and the immune avoidance of HBV against HBV infection in vivo ( in vivo ) (FIGS. 12 and 13A to 13C) . IFN-induced TRIM22 promoter activity was then significantly inhibited by HBx in the HBV-infected mouse model (FIGS. 14a-14c), and when 5-AzaC was co-administered, the HBx- , Indicating that TRIM22 promoter activity was restored (Figs. 15A and 15B).

In addition, the present inventors have also found that HBV-infected primary human hepatocyte model (FIG. 16A), which is prepared for confirming the expression of IFN-induced TRIM22 and HBV immunoreactive response in human tissue cells when HBV is infected Similarly, TRIM22 mRNA transcription and protein expression levels induced by IFN gamma were reduced upon HBV infection and were restored when 5-AzaC treatment was performed (FIGS. 16B and 16C).

Therefore, the interferon of the present invention can be secreted during HBV infection to increase the expression of the antiviral protein TRIM22, and the DNA methylation inhibitor of the present invention can be used to prevent HBV from epigenetically regulating the immune response of host cells And the 5'UTR CpG (+71) isomerization of the TRIM22 gene promoter and the expression level of the TRIM22 protein by IFN can be significantly restored. Therefore, the interferon and the DNA methylation inhibitor can prevent the HBV infection And as an active ingredient of a pharmaceutical composition for therapeutic use.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing interferon and DNA methylation inhibitor as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration, It is not.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing the interferon and DNA methylation inhibitor of the present invention as an active ingredient may be administered parenterally, and the parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathecal injection. In this case, in order to formulate the composition for parenteral administration, a pharmaceutical composition containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, Or vial unit dosage forms. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

In addition,

(i) treating interferon and a test compound in a liver tissue sample derived from a hepatitis B patient;

ii) the liver tissue sample treated in step i) Measuring TRIM22 mRNA transcription level or TRIM22 protein expression level;

iii) TRIM22 measured in step ii) mRNA transcription level or TRIM22 protein expression level with an untreated control; And

iv) selecting a test compound which is increased in comparison with the untreated control group in the step iii), and screening the therapeutic agent for hepatitis B virus infection.

The " interferon " of the present invention preferably induces the expression of TRIM22 protein in a cell. The term " interferon " is preferably one or more selected from the group consisting of interferon alpha, interferon beta and interferon gamma. More preferably, the interferon alpha or interferon gamma or both is more preferably interferon gamma , But it is not limited thereto. Any interferon known in the art may be used as a factor inducing the TRIM22 protein in the cell.

The "test compound" of the present invention is preferably a DNA methylation inhibitor, and more preferably a DNA methyltransferase inhibitor, but is not limited thereto.

The hepatitis B virus infection according to the present invention is preferably any one of hepatitis, liver cirrhosis or hepatocellular carcinoma. More preferably, the hepatitis is chronic hepatitis B, but is not limited thereto. Do not.

The TRIM22 protein of the present invention is an anti-viral active protein induced by interferon during viral infection. In order to avoid the immune response of the infected host cell, the TRIM22 protein is desensitized by methylation of the TRIM22 gene promoter, When the transferase inhibitor is treated with interferon, the expression of TRIM22 mRNA transcript or TRIM22 protein is restored and may exhibit anti-HBV activity. Therefore, the test compound is treated with interferon to confirm TRIM22 mRNA transcription or TRIM22 protein expression level May be useful for screening methods for treating hepatitis B virus infection.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Hepatitis B virus (Hepatitis Virus B, HBV ) Infection, interferon ( Inteferon , IFN) to avoid associated immune responses HBV Targeted  Screening of genes

<1-1> Preparation of target cell line

Various viruses have been reported to be able to avoid IFN-related immune responses in host cells, and antiviral activity associated with IFN and ISG (interferon-stimulating gene) can be suppressed through this avoidance reaction (Garcia-Sastre , Adolfo, and Christine A. Biron, Science 312.5775 (2006): 879-882 .; Sen, Ganes C. Annual Reviews in Microbiology 55.1 (2001): 255-281.). In connection with the present invention, it is known that HBx protein, which is a viral protein expressed by HBV, plays an important role in protein expression of host cells through widespread genetic control (Non-Patent Document 4) Cell lines expressing HBx were prepared and cultured.

Specifically, a human liver cancer cell line, HepG2 cell line (ATCC) or Huh7 cell line was cultured in DMEM medium supplemented with 10% heat-inactivated FBS (Gibco BRL, USA) and 1% penicillin / streptomycin (Gibco BRL) Respectively. Cells capable of expressing HBx were obtained from YI Lee (KRIBB, Korea) using HBx expression plasmid-transfected cells, and cells transfected with pcDNA plasmid were used as control cells. For transfection, Lipofectamine 2000 (Invitrogen, Canada) was used according to the protocol provided by the manufacturer.

<1-2> On HBx  Suppressed by IFN  Inducible protein ( IFN -stimulating protein, ISP)

In order to search for a target protein that may be involved in immune avoidance of HBV from IFN, the expression levels of HBx and control protein (pcDNA) Respectively.

Specifically, the pcDNA or HBx transfected HepG2 cell line prepared in Example <1-1> was cultured and treated with 0 (no treatment) or 500 units / ml IFNγ and cultured for 16 hours. After incubation, cells were harvested and cells were lysed using RIPA buffer. Proteins in the dissolved cells were quantitatively analyzed by BCA analysis (Pierce) and then trypsinized using the modified FASP method (5). The peptide digested with trypsin was eluted by centrifugation at 14,000 x g and the eluate was vacuum dried in a vacuum pump. It was then pre-cleared using the 18c column (Thermo) and following the manufacturer's protocol using a 3100 OFFGEL fractionator and IPG strip (Agilent, pH 3 to 10) The trypsin-digested peptides were separated into 12 samples. A total of 10 specimens were prepared from the 12 samples, in which the peptide concentrations in the samples were lower than those of the other samples, and the 11th and 12th samples were combined. Ten samples were desalted using a C18 column and then dried using a vacuum pump.

Each prepared peptide sample was analyzed using an LTQ-Orbitrap hybrid mass spectrometer (Thermo Finnigan, CA) equipped with a Nano-ACQUITY UPLC system (Waters Corporation, USA). The assay conditions were solvent conditions starting with 90% solvent I (0.1% formic acid aqueous solution) / solvent II (acetonitrile containing 0.1% formic acid) to a linear gradient varying to 50% solvent I / II, 300 nl / min for a total of 65 minutes. For separation, reverse phase chromatography using a linear trap (C18 sample trap) with elution system (C18 analytical column) was performed. For the mass spectrometry, the conditions were appropriately set according to the results (top 8 and 30s exclusion duration).

The measured MS / MS results were retrieved using the IPI.HUMAN.v.3.87 database (91,491 entries) using the SEQUEST algorithm (Thermo Finnigan). The variables of the SEQUEST algorithm are as follows: The ion mass error of the precursor was ± 2da, the ion mass error of the slice was ± 1da, and the number of positions at which trypsin was not cleaved was set to 2, which was due to oxidation of methionine (+ 15.995Da) Carbamidomethylation (+ 57.034). Then, among the peptides identified using Scaffold3 (Proteome Software, USA), a set of peptides having a PeptideProphet probability of 0.95 or more was selected, and again a list of proteins having a Peptide Probability probability of 0.99 or more was created.

The list of prepared proteins includes HBx non-expressing and IFN gamma untreated cells (IFN gamma- / pcDNA); HBx expression and IFNγ untreated cells (IFNγ- / HBx); HBx naïve and IFNγ treated cells (IFNγ + / pcDNA); And HBx expression and IFNγ-treated cells (IFNγ + / HBx). In the van diagram, only the proteins contained in only one group were selected and named clusters A, B, C, and D, respectively.

As a result, as shown in FIG. 1B, it was confirmed that the total number of proteins related to the change of expression level according to HBx expression and IFNγ treatment was 1406, and when they were divided into 4 groups, 885 IFNγ- / pcDNA , 946 IFNγ- / HBx, 832 IFNγ + / pcDNA, and 822 IFNγ + / HBx (FIG. 1b). Also, in clusters divided into proteins alone in one group, 116 proteins that are hyperactivated by HBx under IFNγ stimulation belonging to cluster D, and 116 proteins belonging to cluster C are IFN-inducible Protein (ISP) (Fig. 1B).

<1-3> Of HBV  Immune avoidance Target  Screening of ISP Proteins

In order to search for proteins targeted by HBV to avoid IFN-induced immunity, the target proteins of HBV were searched for as a function of selected proteins as IFN-inducible proteins (ISP) inhibited by HBx.

Specifically, all of the proteins identified in Example <1-2> were classified according to a gene ontology using the DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov). Among these proteins classified according to their biological functions, proteins having a function related to "immune response" and "antiviral" are selected, and the target of HBV immunity avoidance is finally determined through comparison of the expression amount of the selected protein (heat map) Were selected.

As a result, eight proteins having functions related to the &quot; immune response &quot; were selected (Table 1) while being included in the cluster C, which means an ISP suppressed by HBx as shown in Fig. 1C and Table 1 below Two proteins, TRIM22 and TWF2, were selected as proteins having a function related to &quot; antiviral &quot; (Fig. 1C).

<1-4> Identification of TRIM22 as a target ISP protein for the immunity-evoking mechanism of HBV

Since the selected TRIM22 protein has been reported to have antiviral activity against various viruses such as HBV (Hattlmann, Clayton J., Jenna N. Kelly, and Stephen D. Barr. Molecular biology international 2012 (2012) TRIM22 was identified at the protein expression level to be a target of HBV.

Specifically, the cells transfected with the HBx expression plasmid prepared in Example <1-1> or the cells transfected with the pcDNA plasmid were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin Lt; / RTI &gt; After inoculation, 500 units / ml of IFN gamma was added to the medium and cultured for 16 hours to obtain cells, which were treated with 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1 (50 mM Tris-HCl buffer, pH 7.4) containing 0.1% SDS, 1 mM PMSF, 1 μg / ml aprotinin, 1 μg / ml leupeptin and 0.5% proteinase inhibitor cocktail ) In RIPA lysis buffer (Sigma, USA). The dissolved cells were centrifuged to obtain supernatant, followed by Western blotting according to known methods to confirm the expression level of TRIM22 protein (Non-Patent Document 3).

As a result, as shown in FIG. 1D, TRIM22 was induced by IFNγ in HBV-non-infected cells, whereas TRIM22 expression was significantly suppressed by HBx even when treated with IFNγ (FIG. 1d ).

Virus HBx  through IFN - inductive Of TRIM22  Confirmation of expression inhibition

&Lt; 2-1 > TRIM22  Confirmation of transcription level inhibition

In order to more specifically confirm the inhibition of TIM22 expression in order to avoid host immune response after HBV infection, the expression level of TRIM22 was confirmed in HBV infected cells according to HBx expression.

Specifically, HBV genome (Wt HBV1.2mer) capable of replication, HBV genome lacking HBx (HBV1.2 (X-)) or HBV genome (HBV genome) lacking HBx was added to HepG2 or Huh7 cells cultured in Example <1-1> HBx expression gene (pcDNA-HBx-HA; subtype: ayw ) was transfected with 0, 0.5 or 1 μg of each gene. Cells cultured for 48 hours after transfection were harvested and the cells were lysed with a trizol solution (Invitrogen, USA) and RNA was isolated. 2 μg of isolated total cellular RNA and oligo dT were mixed and primary cDNA was synthesized using M-MLV reverse transcription polymerase (Intron Biotechnology, Korea). Then, using the synthesized cDNA as a template, quantitative RT-PCR was carried out using the TRIM22, HBx or GAPDH primers of the sequence shown in [Table 2] below. Quantitative RT-PCR results were analyzed by electrophoresis on agarose gel to compare the level of TRIM22 gene expression with HBV genome or HBx gene expression.

A specific primer sequence for performing RT-PCR Gene name Primer sequence TRIM22
Forward 5'-AGCTCTGGGTTTGCTTTTGA-3 ' Reverse 5'-CTCCGTGGTTTGTGACATTG-3 ' HBx
Forward 5'-AAAAAGTTGCATGGTGCTGGTGAAC-3 ' Reverse 5'-GCGCGGGACGTACTTTGT-3 ' mouse IFNα1
Forward 5'-ATGGCTAGGCTCTGTGCTTTC-3 ' Reverse 5'-TTGGCTCAGGACTCATTTCTC-3 ' mouse IFN ??
Forward 5'-GCCTGGCTTCCATCATGAAC-3 ' Reverse 5'-GAGGCATCAACTGACAGGTC-3 ' mouse IFN ??
Forward 5'-GCCATCGGCTGACCTAGAG-3 ' Reverse 5'-CGAATCAGCAGCGACTCCTT-3 ' STAT1
Forward 5'-CCGTTTTCATGACCTCCTGT-3 ' Reverse 5'-TGAATATTCCCCGACTGAGC-3 ' STAT2
Forward 5'-GAGGCCTCAACTCAGACCAG-3 ' Reverse 5'-GCGTCCATCATTCCAGAGAT-3 ' PKR
Forward 5'-AATGATGGAAAGCGAACAAGGAGTA-3 ' Reverse 5'-CTTCCACACAGTCAAGGTCCTTAGT-3 ' Mx1
Forward 5'-GCTTGCTTTCACAGATGTTTCG-3 ' Reverse 5'-AAGGGATGTGGCTGGAGATG-3 ' RNaseL
Forward 5'-TCATTCATCGTCTCTTCCATCCT-3 ' Reverse 5'-ACATTCCGAAGCGTCCTATAGC-3 ' Myd88
Forward 5'-ATGGTGGTGGTTGTCTCTGA-3 ' Reverse 5'-GACAGTGATGAACCTCAGGA-3 ' OAS1
Forward 5'-TCCACCTGCTTCACAGAACTACA-3 ' Reverse 5'-TGGGCTGTGTTGAAATGTGTTT-3 ' OAS2
Forward 5'-TACCTGAAGCCCTACGAAGAATG-3 ' Reverse 5'-TCAGCTTATCCCCAGTTTTATCG-3 ' OAS3
Forward 5'-CCCTGGTCTGAGACTCACGTTT-3 ' Reverse 5'-GACTTGTGGCTTGGGTTTGAC-3 ' OAS L
Forward 5'-CGTGAAACATCGGCCAACTAAG-3 ' Reverse 5'-GTACCCATTTCCCAGGCATAGA-3 ' IRF1
Forward 5'-CAAATCCCGGGGCTCATCTGG-3 ' Reverse 5'-CTGGCTCCTTTTCCCCTGCTTTGT-3 ' DNMT1
Forward 5'-GACTACATCAAAGGCAGCAACC-3 ' Reverse 5'-GAGTGGACTTGTGGGTGTTCTC-3 ' DNMT3A1
Forward 5'-CAGCGTCACACAGAAGCATATC-3 ' Reverse 5'-ACCACATTCTCAAAGAGCCAGA-3 ' DNMT3A2
Forward 5'-GGATGAAGATCAGAGCCGAGAA-3 ' Reverse 5'-CAGGCACTCCACACAGAAACAC-3 ' GAPDH
Forward 5'-CGTCTTCACCACCATGGAGA-3 ' Reverse 5'-CGGCCATCACGCCACAGTTT-3 ' ChIP (IRF1)
Forward 5'-TCAGGCAAGCTGTATAACTGT-3 ' Reverse 5'-CCAGATTCACTCACCAAGCC-3 ' ChIP
(Negative control) Forward 5'-GAGGCAGCTTGTGAACTGTC-3 ' Reverse 5'-AAGGGGTAGGATTTGCGTCA-3 '

As a result, as shown in Fig. 2, cells transfected with a HBV genome capable of replication (Huh7-Wt HBV1.2) and cells transfected with a pcDNA plasmid containing an HBx expression gene in the HBV genome (Huh7-HBx-HA ) Revealed that the expression level of TRIM22 gene was decreased according to the expression level of HBx. However, the level of mRNA expression of TRIM22 was significantly increased in the cells transfected with HBx-deficient HBV genome (Huh7-HBV1.2 (X-)) (Fig. 2A and Fig. 2B).

<2-2> Confirmation of inhibition of TRIM22 expression level by HBx under IFN treatment

It was confirmed that the HBx protein inhibited TRIM22 gene transcription of the host cell when HBV was infected. Thus , it was confirmed whether or not the expression of TRIM22 , which is an ISG, was inhibited by HBx when IFN was treated.

Specifically, the cells (HepG2-HBx) transfected with the HBx expression plasmid prepared in Example <1-1> or hepG2-pcDNA transfected with the pcDNA plasmid were cultured in DMEM medium, IFNγ (type 2 IFN; LG Biotechnology, Korea) was treated with the culture medium for 6 hours. Cells were obtained at 0, 2, and 6 hours after IFN treatment, and cDNA was synthesized by performing the same method as in Example <2-1>, and PCR amplification was performed to perform RT-PCR and real-time PCR Transcript levels of relative TRIM22 mRNA were determined by analysis. Real-time PCR was performed on cells obtained 2 hours after IFN treatment. Relative TRIM22 gene expression level was confirmed based on the expression level of TRIM22 gene in the experimental group not treated with IFN in HepG2- pcDNA .

As a result, it was confirmed that the expression of TRIM22 gene was induced by treatment with IFNγ as shown in FIG. 2C and FIG. 2D, but the expression of TRIM22 was suppressed by HBx (FIGS. 2C and 2D).

<2-3> IFN  Depending on the type On HBx  by TRIM22  Identification of expression level inhibition

It was confirmed that the expression of TRIM22 , which is an ISG, was suppressed by HBx even when IFN was treated. Therefore, it was confirmed whether or not the level of TRIM22 expression was changed by HBx depending on the type of IFN treated.

Specifically, HepG2-pcDNA cells, a HepG2 cell line; Or Huh7-Wt HBV1.2, Huh7-HBx-HA or Huh7-HBV1.2 (X-) cells were inoculated in DMEM medium. IFN?, IFN? (Type 1 IFN; PBL InterferonSource, USA) or IFN? Was treated at a concentration of 500 units / ml in each culture medium and cultured for 24 hours.

Next, HepG2- pcDNA cells were first obtained, and the expression level of the TRIM22 gene and the gene selected as ISG in the above Example <1-2> was analyzed by real-time PCR in the same manner as in Example <2-2> And Western blot was performed in the same manner as in Example <1-4> to compare TRIM22 protein expression levels. In addition, Huh7-Wt HBV1.2, Huh7-HBx-HA and Huh7-HBV1.2 (X-) cells were obtained and Western blot was performed to compare the expression levels of HBx and TRIM22 protein.

As a result, as shown in FIG. 2E, FIG. 2F and FIG. 2G, transcription levels of TRIM22 gene in IFNα and IFNγ in HepG2- pcDNA cells (FIG. 2e) increased and expression levels of TRIM22 protein increased (FIG. 2f) , It was confirmed that IFN [beta] did not induce the expression of TRIM22 significantly. However, it was confirmed that the HBx-expressing cells Huh7-Wt HBV1.2 and Huh7-HBx-HA cells showed inhibition of TRIM22 protein expression by HBx even when treated with IFNa or IFNγ (FIG. 2g).

<2-4> Confirmation of inhibition of TRIM22 expression by HBx expression level

In order to confirm the suppression of TRIM22 expression by HBx, it was confirmed that the expression of TRIM22 was inhibited by the expression level of HBx, since it was confirmed that HBx inhibited the expression of TRIM22 in HBV-infected cells.

Specifically, in preparing Huh7-Wt HBV1.2, Huh7-HBx-HA and Huh7-HBV1.2 (X-) cells in the same manner as in Example <2-1>, the corresponding plasmid Were transfected with 0, 1, 2, or 4 μg each. Then, each cell was inoculated with DMEM medium and treated with IFNγ at a concentration of 500 units / ml for 24 hours. Then, each cell was obtained and the level of TRIM22 protein expression was compared according to the amount of expression of HBx protein via Western blot. The amount of total DNA (4 μg) was determined using pcDNA3.1. For TRIM22 expression, a TRIM22-flag plasmid was inserted into pcDNA3.1 (+) using a Jurket cell cDNA library to overexpress the TRIM22 protein for use as a positive control.

As a result, it was confirmed that the expression of TRIM22 protein was significantly inhibited as the expression level of HBx protein expressed in the cells was increased as shown in FIG. 2h (FIG. 2h).

<2-5> Confirmation of inhibition of TRIM22 expression by HBx according to time after IFN treatment

More specifically, in order to confirm the suppression of TRIM22 expression by HBx, the expression level of TRIM22 was confirmed in HBx-expressing cells according to time after treatment with IFN.

Specifically, IFNγ was cultured in DMEM medium inoculated with HepG2-HBx or HepG2-pcDNA cells at a concentration of 500 units / ml for 24 hours, and cultured at 0, 8, 12, 16, and 24 hours HepG2-HBx and HepG2-pcDNA cells were obtained and RT-PCR was performed to confirm the transcription level of TRIM22 gene. Western blot analysis was performed to confirm the expression level of TRIM22 protein. In order to quantitatively analyze protein expression levels, expression levels of TRIM22 protein were quantitatively compared using GelQuant.NET analysis software (Biochem Lab Solution) after Western blotting.

As a result, as shown in FIG. 2 (i), expression levels of TRIM22 gene and protein were increased with time after treatment with IFNγ, but expression of TRIM22 gene and protein was inhibited in HBx-expressing cells 2C and 2I).

<2-6> Determination of antiviral activity level by TRIM22 in HBV-infected cells

It is known that TRIM22 protein, which HBV targets as a target of immune defense, exhibits anti-HBV activity, which appears through core promoter inhibition of HBV in the nucleus (Gao B, Duan Z, and Xu W, et al. Hepatology 2009; 50: 424-433), and the expression level of TRIM22 in the nucleus was confirmed.

Specifically, IFNγ was cultured in DMEM medium inoculated with HepG2-HBx or HepG2-pcDNA cells at a concentration of 500 units / ml and cultured for 16 hours. Then, cells were harvested, and the cells were treated with Nuclear and Cytoplasmic Extraction Reagents; Pierce, USA) to separate and obtain the cytoplasmic and nuclear fractions. And the level of expression of TRIM22 protein was confirmed by Western blot analysis using the nuclear fraction and the cytoplasmic fraction obtained as a sample. For use as markers in nuclear and cytoplasmic fractions, the expression of alpha -tubulin and lamin was also confirmed.

As a result, as shown in Fig. 3, it was confirmed that the TRIM22 protein induced by IFN gamma migrated into the nucleus and the expression level of TRIM22 protein was inhibited in the HBx-expressing cell, so that the expression level was not increased ).

On HBx  by TRIM22  Identification of the key factors that affect expression inhibition

<3-1> TRIM22  Plasmid construction for confirmation of promoter region activity essential for transcription

In order to determine which is engaged element in the process is by HBx inhibiting the expression of TRIM22 in detail, the upper portion (upstream) from the parts related to TRIM22 expression inhibition by HBx the transcription initiation part of TRIM22 gene As shown in the schematic diagram Fig. 4a Was constructed.

Specifically, a DNA sequence of about 2.5 kb was obtained from the transcription start site of the TRIM22 gene from the genomic DNA of HepG2 cells by PCR. The obtained DNA sequence includes 5'UTR and promoter of TRIM22 gene, and the DNA sequence region (TP1 ~ TP5) at the upper part of TRIM22 gene was selected in a total of 5 ranges by deleting the DNA length in various ranges. The DNA sequences of the TP1 to TP5 regions were then inserted into pGL3-Basic vector (Promega, USA) so that they could be expressed together with the luciferase reporter gene. In addition, the sequence containing the TP5 sequence and the 5 'UTR of TRIM22 was selected as TP6. In addition, the pGL3-Basic vector was constructed TP5 sequences, by selecting a sequence containing the intron 1 sequence, some of the 5 'UTR and TRIM22 of TRIM22 to TP7, TP6 or TP7 sequence also to express with a luciferase reporter. The TP7 sequence included the p53 response element (p53-RE), which is essential for the promoter activity of TRIM22 . The pGL3-Basic vectors constructed by this method were named pTP1-Luc to pTP7-Luc vectors, respectively, and they were constructed so as to include the promoter portion of the TRIM22 gene.

<3-2> TRIM22  Identification of regions essential for transcription of the promoter

In the promoter region involved in transcription of TRIM22 , luciferase activity was measured using the constructed plasmid in order to identify the essential promoter region affecting the transcription level.

Specifically, hepatoma cell line HepG2 cells or Huh7 cells were inoculated into 12-well plates and cultured at a density of about 4 × 10 ⁴ cells or 1 × 10 ⁵ cells. Then, a DNA mixture prepared by mixing the respective luciferase reporter plasmids pTP1-Luc to pTP7-Luc constructed in the above Example <3-1> and the TRIM22-flag containing the β-gal reporter clone, The cells were transfected. To control the total amount of DNA for transfection, pcDNA3.1 (+) was used. After transfection, the cells were cultured for 48 hours. Cells were harvested, lysed in lysis buffer (Promega), and luciferase activity was measured using Luciferase Assay System (Promega). The measured luciferase activity levels were normalized on the basis of? -Galactosidase activity.

As a result, the luciferase activity in the baseline level not treated with IFN as indicated at 4b was confirmed to have a significant increase in strong promoter activity in pTP6-Luc and pTP7-Luc, 5 of TRIM22 gene through which 'UTR (+ 1 to +211 ) region plays an important role in TRIM22 transcription (FIG. 4B).

<3-3> IFN  Processing and HBx  According to expression TRIM22  Promoter's transcription Increase of level  Confirm

The effect of IFN treatment or HBx expression on the transcription induction of the TRIM22 promoter was confirmed in order to confirm the position of the TRIM22 promoter essential for the increase of IFN-induced TRIM22 transcription and to confirm the change of the promoter activity depending on the expression of HBx .

Specifically, cells transfected with pTP1-Luc to pTP7-Luc vectors were prepared in HepG2-pcDNA cells, Huh7 cells, or HepG2-HBx cells in the same manner as in Example <3-2> With IFN [alpha] or IFN [gamma] at a concentration of 500 units / ml and then cultured for 24 hours. After culturing, each cell was obtained and luciferase activity was measured in the same manner as in Example <3-2> above.

As a result, it was confirmed that the 5'UTR region also plays an important role in TRIM22 transcription induced by IFNa or IFNγ as shown in FIGS. 4C and 4D (FIG. 4C). In addition, the level of luciferase activity of the pTP6-Luc and pTP7-Luc plasmids was rapidly increased by induction of IFNγ in HepG2-pcDNA cells, and the expression of pTP6-Luc and pTP7-Luc It was confirmed that the 5'UTR region was also involved in the inhibition of TRIM22 expression by HBx through the fact that the luciferase activity level of the plasmid was not increased (FIG. 4D).

<3-4> On HBx  by TRIM22  Warrior In suppression Of IRF1  Confirm association

The 5'UTR region, which is found to be an important factor in the inhibition of TRIM22 expression by HBx, is an area that is induced by IFNγ and is known to bind to IFN regulatory factor-1 (IRF1). Thus, it was confirmed whether or not the transfer of the TRIM22 inhibition induced by HBx whether by adjustment of IRF1.

Specifically, HepG2-pcDNA, HepG2-HA, HepG2-Wt HBV1.2 or HepG2-HBV1.2 (X-) cells were prepared by transfection and treated with IFNa or IFNγ for 24 hours. Cultured cells were then obtained and the level of expression of IRF1 was compared by performing real-time PCR.

As a result, as shown in FIG. 5, the relative expression level of IRF1 was significantly increased by IFNγ treatment, and the expression level of IRF1 was not significantly changed depending on the expression of HBx, so that HBV or HBx affected IRF1 expression (Fig. 5A to Fig. 5C).

On HBx  by TRIM22  Involved in suppression of expression Post-sexual  Identification of the conditioning process

<4-1> On HBx  by TRIM22  Warrior In suppression  DNA methylation and location

It has previously been reported that HBV and HBx expressed therefrom can regulate the expression of host proteins that can exhibit antiviral activity through epigenetic regulation such as DNA methylation. Therefore, it has been confirmed that the 5'UTR (+ 1 to +211) of TRIM22 plays an important role in transcriptional activity and also plays an important role in suppression of IFN-induced and HBx-induced TRIM22 expression. + 1 ~ + 211) was confirmed by DNA methylation by HBx. Methods for identifying DNA methylated regions include the treatment of bisulfite with 5'aza-2'deoxycytidine (5-AzaC), an inhibitor of DNA methyltransferase, ) Sequence analysis.

Specifically, Huh7-pcDNA or Huh7-HBx-HA cells were treated with 10 uM 5-AzaC (Sigma, USA) and cultured for 3 days. The cells were cultured in DMEM medium supplemented with 5-AzaC every 24 hours. Three days after the start of the culture, the genomic DNA of the cells was obtained, and 2 μg of the genomic DNA was subjected to a CpGenome fast DNA modification kit (Millipore) according to the manufacturer's protocol and reacted at 50 ° C. for 18 hours, Was treated with sodium bisulfite. Then, the amplified DNA was subjected to PCR amplification using a sodium hydrogen sulfite-treated genomic DNA as a template and inserted into a pGEM-T easy vector. Finally, the nucleotide sequence of the inserted DNA was analyzed to confirm the methylated region in the DNA sequence of the TP6 region (-500 to +211) of TRIM22.

As a result, as shown in FIG. 6A, it was confirmed that there were four CpG islands in the TP6 sequence, and the positions were -265, -182, +71, and +150, respectively. Furthermore, in cells that do not express HBx, the CpG islands are demethylated in the TRIM promoter regardless of whether the 5-AzaC inhibitor is a DNA methyltransferase inhibitor. However, in the HBx-expressing cells, the methylation level of each of the CpG islands Respectively. In addition, when 5-AzaC was treated with HBx-expressing cells, the degree of methylation was stable in the CpG region at positions -265 and +150, whereas the degree of demethylation was significantly increased in the third CpG region (+71) (Fig. 6A). It was confirmed that methylation at CpG island at position +71 of TRIM22 gene plays an important role in the transcriptional repression of TRIM22 by HBx.

&Lt; 4-2 > HBx  Induction TRIM22  Confirming transcriptional repression modulation effect

It was confirmed that the TRIM22 transcriptional repression by HBx was caused by DNA methylation in the +71 locus CpG region of 5'UTR. Therefore, whether or not the suppression of TRIM22 transcription by HBx can be changed by controlling the DNA methylation level of the CpG region Respectively.

Specifically, the medium was inoculated with Huh7-pcDNA, Huh7-Wt HBV1.2, Huh7-HBx-HA or Huh7-HBV1.2 (X-) cells and treated with 10 μM 5-AzaC and 500 unit / And cultured for 24 hours. After culturing, cells were obtained, and the level of TRIM22 mRNA expression was determined by qRT-PCR according to 5-AzaC treatment. Western blot analysis was performed to confirm the expression level of TRIM22 protein according to treatment with 5-AzaC and IFNγ.

As a result, as shown in FIG. 6B and FIG. 6C, the expression of TRIM22 was suppressed even though IFNγ was treated in cells expressing HBx (FIG. 2g) MRNA and protein expression levels of TRIM22 were significantly increased and expression was restored (Figs. 6B and 6C). This suggests that inhibition of TRIM22 expression by HBx is a result of post-sexual control through methylation of the TRIM22 promoter region.

<4-3> On HBx  by TRIM22  Warrior In suppression  Identification of changes in the expression pattern of DNA methyltransferase

We confirmed that transcriptional inhibition of TRIM22 by HBx occurs through DNA methylation. Therefore, the expression levels of DNMT1, DNMT3A1 and DNMT3A2, which are intracellular DNA methyltransferases, were determined by HBx expression.

Specifically, HepG2 cells transfected with pcDNA, HBx-HA, DNMT1, DNMT3A1 or DNMT3A2 expression plasmids were inoculated into the medium and cultured for 24 hours with 500 units / ml IFNγ. After culturing, cells were obtained, Western blot analysis confirmed the expression of TRIM22 protein by expression of HBx or DNA methyltransferase, and the level of DNA methyltransferase mRNA expression was confirmed by the increase of HBx expression.

As a result, it was confirmed that overexpression of DNA methylation enzymes such as DNMT1, DNMT3A1 and DNMT3A2 as shown in Fig. 7 did not affect the protein expression level of TRIM22 (Fig. 7A). In addition, it was confirmed that even when HBx expression level was increased, gene expression level for DNA methyltransferase did not change significantly (FIG. 7b).

On HBx  by IFN  Inductive TRIM22  Identification of DNA methylation and control factors related to inhibition of expression

&Lt; 5-1 > TRIM22  Specific location of promoter methylation

(+71) in the expression of TRIM22, which is an IFN-inducible TRIM22, can be restored by inhibiting hypermethylation of the third CpG island (+71) of the TP6 sequence by treatment with 5azaC, Can play an important role. Therefore, in order to confirm this more specifically, a total of five TP6 variant reporter clones containing CpG mutation sequence in each of four CpG islands in the TP6 sequence were prepared , and the induction activity of the promoter of TRIM22 was determined depending on IFN treatment Respectively.

Specifically, using the pTP7-Luc vector constructed in the above Example <3-1> as a template, five pTP6 mutant sequences were produced through the site-directed mutagenesis. The pTP6 mutant sequence thus prepared was designated as pTP6-M1-Luc to pTP6-M5-Luc vector as shown in FIG. 8A. Subsequently, the pTP6-M1-Luc to pTP6-M5-Luc vectors were transfected into HepG2 or HepG2-HBx cells, IFNα or IFNγ was treated at a concentration of 500 units / Lt; / RTI &gt; After culturing, each cell was obtained and luciferase activity was measured in the same manner as in Example <3-2> above.

As a result, mutants transfected with pTP6-M1-Luc to pTP6-M4-Luc vectors lost TRIM22 transcription induction activity by IFNa or IFNγ as shown in FIG. 8B and FIG. 8C, It was confirmed that TRIM22 transcription-inducing activity by IFN [alpha] or IFN [gamma] was increased in the pTP6-M6 mutant in which the nucleotide sequence of the CpG island was substituted (Fig. In addition, when HBx was expressed, it was confirmed that the promoter activity of pTP6-M5 was strongly inhibited regardless of treatment with IFNγ (FIG. 8C), and one CpG island (+71) was found to be important in TRIM22 desensitization by HBx And it was confirmed that it is a role position.

<5-2> On HBx  by TRIM22  Promoter methylation and IRF1  Identify the relationship between bonds

As shown in FIG. 8A, since the CpG island at position +71 binds to IRF1, it was confirmed whether or not IRF1 binding essential for the expression of IFNγ-induced TRIM22 was blocked when the CpG island was methylated. To confirm this, electrophoretic mobility shift assay (EMSA) analysis was performed using four DNA probes having radioactivity (FIG. 8D).

Specifically, as a double-stranded DNA oligomer to be subjected to EMSA analysis, the DNA oligomer was prepared in the same manner as in [Table 2] so as to confirm IRF1 binding, and each DNA oligomer was labeled [32P]. In addition, poly (dI-dC) and salmon sperm DNA (1 μg) were mixed with nuclear extract (2 μg) of HepG2 cells and pre-cultured, and then each labeled [32 P] -labeled DNA oligomer was added. Prior to addition of the [32P] -labeled DNA oligomer, IRF1 binding was confirmed by treating the unlabeled oligomer as a cold competitor, IRF1 antibody for supershift, and IFNγ, respectively. Relative IRF1 binding affinities were quantified using a BAS-phosphorimager.

As a result, it was confirmed that IRF1 induced by IFN gamma in the nucleus, as shown in FIG. 8E, binds to a wild-type probe containing a CpG (+71) region, which is the same in cold competition and antibody super shift results 8e). In addition, it was confirmed that expression of IRF1 in the nucleus occurs only in the case of treatment with IFNγ, and it was confirmed that the binding affinity to IRF1 is reduced in both the AG probe and the TG probe (Fig. 8 (e), right). These results are in agreement with the disappearance of the TRIM22 transcription induction activity of pTP6-M2 and pTP6-M3 (Fig. 8 (b)), as compared with the IRF1 strong binding band observed in wild-type probes. EMSA In the analysis, it was confirmed that the binding affinity of IRF1 was significantly decreased. This confirmed that the binding of IRF1 and TRIM22 promoter sites was blocked by methylation of CpG (+71) position, confirming that CpG (+71) island plays a very important role in IRF1 binding and TRIM22 transcription induction .

<5-2> HBx  According to expression TRIM22  Promoter and IRF1  Identify the relationship between bonds

Since the CpG (+71) island plays a very important role in IRF1 binding and TRIM22 transcription induction, chromatin immunoprecipitation (CHIP) analysis was performed to confirm the interaction between the IRF1 and TRIM22 promoter regions in HBx expressing cells Respectively.

Specifically, 500 units / ml IFN gamma and 10 uM 5-AzaC were treated while culturing Huh7-HBx-HA cells and control Huh7-pcDNA cells, respectively. After culturing, cells were obtained, the cells were lysed by ultrasonication, and the lysed cell lysates were incubated with IRF1 antibody according to the manufacturer's protocol using a ChIP assay kit (Milipore, USA). Then, the immunoprecipitated DNA fragment was detected by PCR amplification using the primers of the nucleotide sequences shown in Table 2 above.

As a result, as shown in FIG. 8F, HBx does not inhibit the expression of IRF1, and IFNγ-induced IRF1 can function at the binding site in cells that do not express HBx, whereas in cells expressing HBx, (+71) position was significantly decreased (Fig. 8 (f)). In addition, when 5-AzaC was treated, the binding of IRF1 was completely restored in the HBx-expressing cells, and the binding of IRF1 was blocked by CpG methylation by HBx, thereby down-regulating IFN-induced TRIM22 expression Respectively.

By IFN  Induced On TRIM22  The anti- HBV  Active and to avoid it Of HBV Post-sexual  Confirm adjustment

<6-1> IFN  Induction of anti- HBV  Check for viruses

It has been reported that TRIM22 has anti-HBV activity through inhibition of transcriptional activity of HBV core promoter during intracellular HBV infection. Therefore, in order to avoid such anti-HBV virus activity, HBx HBx expresses TRIM22 . To confirm this more specifically, it was once again confirmed that IFN inhibits replication and transcription of infected HBV through TRIM22 induction.

Specifically, HepG2-Wt HBV1.2 cells were treated with IFNγ at a concentration of 0, 500 or 1000 unit / ml and cultured for 48 hours. After incubation, cells were obtained and subjected to Southern blot to confirm viral genome replication level in HBV infected cells and Western blot performed to confirm TRIM 22 protein and viral surface / core protein expression levels. Northern blot analysis was also performed to determine the level of pgRNA / subgeneric RNA transcription of the virus, and the level of relative HBV RNA transcription was analyzed using a phosphorimager.

As a result, it was confirmed that the expression level of the HBV genomic DNA and virus proteins (HBs and core protein) decreased as the amount of IFN gamma treatment increased as shown in Fig. 9 (Fig. 9A). Transcription of HBV genomic RNA and subgenomic RNA was also significantly reduced by IFNy (Fig. 9b). In other words, the expression level of TRIM22 was inversely proportional to HBV replication level (Fig. 9a), confirming that IFNγ plays an important role in anti-HBV activity.

<6-2> TRIM22  Expression In knockdown  Identification of viral gene cloning activity changes

In order to confirm whether the anti-HBV activity of IFN identified in Example <6-1> was related to TRIM22 , viral RNA, DNA and protein enantiomer activity were analyzed in cells knocked down by TRIM22 expression with siRNA.

Specifically, the nucleotide sequence of the siRNA for knockdown of TRIM22 was as shown in Table 3 below, and the one synthesized in ST Pharm (Korea) was used. Each siRNA was transfected into cells using lipofectamine 2000 at a concentration of 20 nM and Western blot analysis confirmed knockdown of TRIM22 protein expression. GL3 siRNA and Universal C siRNA were used as negative control for siRNA.

SiRNA nucleotide sequence for TRIM22 knockdown Gene name siRNA base sequence TRIM22
sense 5'-CAAUAUGGCUACUGGGUUATT-3 ' Antisense 5'-UAACCCAGUAGCCAUAUUGTT-3 ' GL3
sense 5'-CUUACGCUGAGUACUUCGATT-3 ' Antisense 5'-UCGAAGUACUCAGCGUAAGTT-3 ' Universal C
sense 5'-AUGAACGUGAAUUGCUCAATT-3 ' Antisense 5'-UUGAGCAAU UCACGUUCAUTT-3 '

As a result, as shown in FIG. 10, the expression level of TRIM22 was also decreased by siTRIM22 in TRIM22-flag transfected cells to overexpress TRIM22, and the level of TRIM22 expression was also decreased in cells expressing TRIM22 by treatment with IFNγ (Fig. 10).

<6-3> HBV  Through inhibition of promoter activity Of TRIM22  term- HBV  Verify Active

Since TRIM22 is known to inhibit the activity of the amplicon / core promoter of HBV, the effect of TRIM22 on the promoter activity of the virus was confirmed.

Specifically, the DNA sequence of the EnhI-EnhII region (+957 to +1798), which is the enantiomer of HBV, was obtained by PCR amplification using the nucleotide sequence of Wt HBV1.2, and a vector containing luciferase pGL3-Basic and inserted to construct a pEnHII-Luc vector. Then, HepG2 cells were transfected with pEnHII-Luc vector (0.4 μg), pcDNA-TRIM22-flag vector and pβ-gal vector (0.25 μg), and 20 nM siRNA was transfected to knock down TRIM22. Transfected HepG2 cells were treated with 500 unit / ml IFNγ for 24 hours, and the activity of the amplified product was determined by measuring the expression of TRIM22 and the expression level of luciferase.

As a result, as shown in FIG. 11A, when TRIM22 was overexpressed, the activity of the amplicon I / II decreased as the amount of TRIM22 expression increased. In contrast, when TRIM22 knocked down, the activity of the amplicon I / II (Fig. 11A).

<6-4> HBx  In expression cells HBV  Through inhibition of promoter activity Of TRIM22  term- HBV  Verify Active

The anti-HBV activity through TRIM22 overexpression or IFN gamma treatment was significantly reduced by siTRIM22, thus confirming how the anti-HBV activity of TRIM22 changes in HBx expressing cells.

Specifically, siRNA, pcDNA-TRIM22-flag vector or pβ-gal vector (0.25 μg) was transfected into HepG2-Wt HBV1.2 cells and treated with 0 or 500 unit / ml IFNγ for 24 hours. From the cells obtained after culturing, Northern blots were performed to confirm the RNA transcription level of the virus. Then, relative HBV RNA transcription levels were analyzed using a phosphorimager, Western blot was performed, and TRIM 22 protein and virus surface / core protein expression levels Respectively. In addition, the Enzygnost HBsAg monoclonal kit (Dade Behring, Germany) was used according to the manufacturer's protocol to determine the level of HBV surface antigen (HBsAg) secreted in the culture medium.

As a result, as shown in FIG. 11B and FIG. 11C, the RNA transcription of the virus and the expression of the viral core / surface protein were reduced by TRIM22 at the early stage of HBV infection, and it was also confirmed that the antigen secreted to the medium environment was also inhibited ). However, when TRIM22 knocked down, the level of RNA and protein expression of HBV was increased, and the expression of TRIM22 was significantly restored when IFNγ was added in siTRIM22 knockdown cells (Fig. 11C, left). When TRIM22 was knocked down, transcript levels of HBV genomic RNA (3.5 kb), which is a template for genomic DNA replication and mRNA for core protein, increased, and the total virus and subgeneric RNA (2.4 / 2.1 kb, mRNA ) (Fig. 11c, right). As a result, it was confirmed that anti-HBV activity induced by IFNγ was induced by TRIM22.

<6-4> HBx  DNA methyltransferase inhibitor treatment in expressing cells Of TRIM22  Identification of anti-HBV activity

Since the antiviral effect against HBV is effected by IFN-induced TRIM22 and HBV also has a mechanism of inhibiting the expression of TRIM22 by post-translational processing through HBx in order to avoid the antiviral effect, 5-AzaC And the DNA methyltransferase was inhibited to confirm whether the anti-HBV activity induced by IFN was changed.

Specifically, HepG2-HBV1.2 transfected cells were treated with 10 [mu] M 5-AzaC and 500 unit / ml IFN [gamma] and cultured. Northern blot and Western blot were performed to obtain RNA transcription and protein expression level Respectively.

As a result, as shown in FIG. 11D, when TRIM22 expression was induced by treatment with IFNγ, the level of HBV transcription was decreased, and when IFNγ and 5-AzaC were treated together, the expression of TRIM22 was further increased and HBV transcription level was further reduced (Fig. 11D). This confirms that anti-HBV activity induced by IFN gamma is related to TRIM22 and that HBV epigenetically inhibits TRIM22 through HBx in order to avoid such anti-HBV activity induced by IFN gamma.

In vivo ( in vivo ) Expression of IFN-induced TRIM22 in HBV infection and confirmation of immune avoidance of HBV

<7-1> Preparation of mouse model of HBV infection

To confirm the expression of IFN-induced TRIM22 and the immune avoidance of HBV against HBV in vivo, an HBV-infected mouse model was prepared (Fig. 12). Since there is no human TRIM22 gene in the mouse, a TRIM22 promoter clone was introduced by hydrodynamic injection in order to confirm the role of IFN-induced TRIM22 expression and HBx.

Specifically, the luciferase reporter plasmid containing the promoter sequence of TRIM22 20μg (pTP5-Luc or pTP6-Luc) and pβ-gal vector to 20 μg of pcDNA, HBx-HA, Wt HBV1.2 or HBV1.2 (X- ) Cells in the tail vein of mice. After 48 hours of administration, fluid-injected mice were dissected to obtain liver tissue.

The pieces of liver tissue obtained were deparaffinized by the addition of xylene to stain immunohistochemistry and citrate buffer (pH 6.0) was added to recover the antigen. After the addition, the piece of liver tissue was treated with a methanol solution containing 3% hydrogen peroxide to inactivate the extracellular peroxidase, and then treated with anti-HBs or anti-beta-gal antigen and incubated overnight. After incubation, the antibodies were blocked by treatment with 10% standard goat serum. After blocking, the tissue pieces were washed with PBS and reacted by treating the secondary antibody. Then, the reacted tissue fragment was developed with a 3, 3 'diaminobenzidine tetrahydrochloride chromogen solution (DAKO).

In order to use the obtained liver tissue as a sample for biochemical analysis, the mouse was homogenized in a RIPA dissolution buffer and centrifuged to obtain a supernatant as a liver tissue sample. Southern blot analysis was performed on hepatocyte samples to confirm the difference in HBV replication level according to HBx expression in the liver of HBV-infected mouse model, and Western blot was performed to confirm the expression of HBx-HA protein.

As a result, as shown in FIG. 13, it was confirmed that HBx-HA protein was significantly expressed in hepatic tissues compared with control mice (pcDNA injection) in the HBV mouse model injected with HBx-HA expressing cells (FIG. 13c) , It was confirmed that the replication of the virus was not significantly observed in the HBV mouse model in which HBV1.2 (X-) injected with no HBx was compared with the liver tissue of the HBV mouse model injected with Wt HBV1.2 13A and 13B).

<7-2> HBV  In the infected mouse model IFN  Inductive TRIM22  Identification of Promoter Activity

The activity of the TRIM22 promoter (pTP6-Luc) containing 5'UTR (+ 1 to + 211) was confirmed in order to confirm whether the characteristics of IFN-induced TRIM22 expression could be confirmed in an HBV mouse model prepared by introducing external DNA Respectively.

Specifically, the level of mRNA expression of mouse IFN in the sample was confirmed by performing qRT-PCR on liver tissue samples of the HBV mouse model prepared in Example <7-1> above. In addition, the activity of the TRIM22 promoter according to HBx expression in mouse liver tissue samples was measured through the activity of luciferase.

As a result, as shown in Fig. 14, it was confirmed that the expression of the first type IFN (IFN? And IFN?) And the second type IFN (IFN?) Was strongly induced in the HBV mouse model prepared by introducing the external DNA ). In addition, IFN-induced TRIM22 promoter activity was significantly increased in the liver tissues of the HBV mouse model injected with the TRIM22 promoter (pTP6-Luc) containing 5'UTR (+ 1 to + 211) In HBV mouse model injected with HBx expressing cells, the TRIM22 promoter activity was significantly decreased by HBx (Fig. 14B). In addition, HBx can be expressed from viral genomic DNA, and the activity of TRIM22 promoter can be strongly inhibited in Wt HBV1, which can express HBx compared to HBV1.2 (X-), which does not contain HBx gene (Fig. 14C).

<7-3> HBV  DNA methyltransferase inhibitor treatment in infected mouse models IFN  Inductive TRIM22  Identification of Promoter Activity

It was confirmed that IFN-induced TRIM22 promoter activity could be inhibited by HBx expression in the HBV mouse model. Thus , it was confirmed that the activity of the IFN-inducible TRIM22 promoter could be restored when DNA methyltransferase activity was inhibited.

Specifically, 5-AzaC was administered intravenously to mice at a dose of 5 mg / kg on day 1, and 5 mg / kg of 5-AzaC was administered again on day 2, , The promoter sequence of TRIM22 and the HBV-related cells were fluid-injected, and 5 mg / kg of 5-AzaC was again administered on day 3 (Fig. 15A). Then, the mice injected with fluids on the 4th day were incised to obtain liver tissue, and homogenization was performed to measure the activity of the TRIM22 promoter by measuring the luciferase activity in the obtained liver tissue sample.

As a result, as shown in FIG. 15B, in the HBx-HA-expressing mouse model without 5-AzaC treatment, the TRIM22 promoter activity was significantly inhibited compared with the control pcDNA- injected mouse whereas the 5-AzaC- It was confirmed that the TRIM22 promoter activity was significantly restored in the HA-expressing mouse model (Fig. 15B). Thus, it was confirmed that the 5 'UTR region of TRIM22 is an essential element for suppression of TRIM22 expression in vivo by HBx.

Primary human hepatocytes hepatocytes , PHHs )in HBV  When infected IFN  Inductive TRIM22  Expression and Of HBV  Identification of immune avoidance response

An HBV - infected cell model was constructed to confirm the expression of IFN - induced TRIM22 and HBV immunoreactive response in human tissue cells when HBV was infected.

Specifically, human hepatic tissue specimens not infected with the virus were obtained by partial hepatectomy for therapeutic purposes. Living primary hepatocyte (PHH) was isolated from the tissue samples obtained using a two-step collagenase perfusion technique as known (Seeger, Christoph, and William S. Mason. Microbiology and Molecular Biology Reviews 64.1 ): 51-68.). The isolated primary hepatocytes infected with HBV-derived HepAD38 at a concentration of 1000 HBV per cell (Bouchard, Michael J., and Robert J. Schneider. Journal of virology 78.23 (2004): 12725-12734) An infected cell model was prepared. Then, the produced HBV-infected cell model was subjected to real-time PCR and Western blot analysis of TRIM22 mRNA transcription and TRIM22 protein expression by IFNγ or 5-AzaC treatment for 7 days after HBV infection according to the procedure shown in Fig. 16 Respectively.

As a result, as shown in Fig. 16B and Fig. 16C, the expression level of TRIM22 expressing basal level in PHH was less than that of HepG2. In addition, it was confirmed that HBV core protein was expressed in the HBV-infected cell model (FIG. 16c), and TRIM22 mRNA transcription and protein expression levels, which were significantly increased by IFNγ treatment, were significantly reduced upon HBV infection 16b and 16c). Thus, when 5-AzaC was treated to examine changes in DNA methylation, both the mRNA transcription of TRIM22 and the expression level of TRIM22 protein were significantly increased in the HBV-infected cell model (FIGS. 16B and 16C).

Claims (10)

A pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infection, comprising interferon and a DNA methylation inhibitor as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the interferon is any one or more selected from the group consisting of interferon alpha, interferon beta, and interferon gamma.
The pharmaceutical composition for preventing and treating hepatitis B virus infection according to claim 1, wherein the interferon induces the expression of the TRIM22 protein.
The pharmaceutical composition for preventing and treating hepatitis B virus infection according to claim 1, wherein the DNA methylation inhibitor is a DNA methyltransferase inhibitor.
The DNA methyltransferase inhibitor according to claim 4, wherein the DNA methyltransferase inhibitor is 5'aza-2'deoxycytidine (5-AzaC) having the structure of the following formula (1) A pharmaceutical composition for preventing and treating hepatitis B virus infection, comprising:
[Chemical Formula 1]

.
The pharmaceutical composition according to claim 1, wherein the hepatitis B virus infection is any one of hepatitis B, liver cirrhosis, and hepatocellular carcinoma.
(i) treating interferon and a test compound in a liver tissue sample derived from a hepatitis B patient;
ii) the liver tissue sample treated in step i) Measuring TRIM22 (tripartite motif 22) mRNA transcription level or TRIM22 protein expression level;
iii) TRIM22 measured in step ii) mRNA transcription level or TRIM22 protein expression level with an untreated control; And
iv) selecting a test compound that is increased in comparison with the untreated control group in the step iii), and screening the therapeutic compound for hepatitis B virus infection.
8. The method of screening a therapeutic agent for hepatitis B virus infection according to claim 7, wherein the interferon is any one selected from the group consisting of interferon alpha, interferon beta and interferon gamma.
The method for screening a therapeutic agent for hepatitis B virus infection according to claim 7, wherein the test compound is a DNA methylation inhibitor.
The method for screening a therapeutic agent for hepatitis B virus infection according to claim 7, wherein the hepatitis B virus infection is any one of chronic hepatitis B, liver cirrhosis, or liver cancer.

Images