KR101777514B1 - Pharmaceutical composition comprising TRAIL and autophagy 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 a TNF-related apoptosis-inducing ligand (TRAIL) and an autophagy inhibitor as effective components. The TRAIL of the present invention can recognize HBV-infected cells and induce apoptosis thereof, and the autophagy inhibitor of the present invention can enhance apoptosis of HBV cells induced by the TRAIL by blocking the breakdown of DR5 protein induced by HBx of HBV, and therefore the TRAIL and the autophagy inhibitor may be beneficially used as effective components of a pharmaceutical composition for preventing and treating HBV infection. Additionally, the DR5 protein is an immunity-related ligand protein capable of inducing apoptosis by binding to the TRAIL when infected with virus, and although TRAIL-induced apoptosis is inhibited as HBV breaks down the DR5 protein by autophagy to avoid immune responses of the infected host cells, when the autophagy inhibitor is treated with TRAIL, the autophagy-mediated DR5 breakdown can be inhibited, and therefore these processes may be beneficially exploited in a method for screening a therapeutic agent for HBV infection.

Description

A pharmaceutical composition for prevention and treatment of hepatitis B virus infection comprising TRAIL and an autophage inhibitor as an active ingredient, the pharmaceutical composition comprising a TRAIL and an autophagy inhibitor as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infectious disease, which comprises as an active ingredient an inhibitor of TRAIL (TNF-related apoptosis-inducing ligand) and autophagy.

Autophagy is an intracellular major catalytic function to break down or reuse degraded cellular organs and end-of-life proteins. An autophagosome surrounds the cell organs or proteins to form an autolysosome ). ≪ / RTI > Autophage is not only essential for maintaining intracellular homeostasis, but plays an important role in various physiological and pathological activities such as inhibiting tumor growth or blocking the replication of antigens. It is known that, under stressed conditions such as malnutrition, chemotherapy, and viral infection, autophage is rapidly activated and affects viral infection or survival of transformed cells.

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. On this line, it has been reported that some viruses have a strategy to respond to autophagy while other viruses can induce or utilize the autophagy mechanism as a viral factor favoring viral replication (JACKSON, William T. Virology , 2015 , 479: 450-456).

Hepatitis B virus (HBV) is a non-cytolytic and liver-friendly DNA virus and is well known as the major antigen causing liver disease worldwide. More than 350 million people in the world reportedly have liver disease caused by HBV infection, and HBV infection can cause chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma.

As HBV vaccine was developed, new HBV infection was greatly reduced, but HBV vaccine was not helpful for patients with chronic infection who were already infected with HBV, and patients with chronic HBV infection were classified as high risk group that could progress to liver cirrhosis or liver cancer do.

The HBV X protein (HBx) is an essential element in virus replication and maintenance of viral infection, and has been reported in various studies in HBV-infected mouse models and various cultured cell models. HBx-mediated host protein can be regulated genetically by HBx, and upregulation of beclin1 (BECN1) expression can induce autophagy in liver cells (TANG, Hong , et al., Hepatology, 2009, 49.1: 60-71.). HBx stimulates autophagy by directly reacting with a group III phosphoinositide 3-kinase (PI3K) to favor viral replication.

Despite the fact that HBx induces autophagy in this way, the role of autophagy in hepatic pathology in relation to HBV has not yet been studied. Therefore, there has not been developed a method for treating HBV infection that targets the induction of an autoimmune reaction by autophagy during HBV infection, and it is possible to prevent the development of viable HBV-infected cells into chronic hepatitis B infection There is a demand for a treatment method.

TNF-related apoptosis-inducing ligand (TRAIL) is expressed on the surface of cells that perform immune surveillance such as natural killer cells and cytotoxic T lymphocytes, and binds to DR5, a deadly ligand Is known to be a typical killing ligand that induces apoptosis of virus-infected or transformed cells. In the binding of the ligand, the DR5 protein induces caspase cascade as a major receptor molecule for the cell membrane and kills the virus-infected cells or tumor cells as well as immune cells that terminate the immune response , Which is known to reduce damage to tissues and maintain homeostasis even after the end of control for infection. In addition, the amount of DR5 on the cell surface can measure the intensity of apoptosis through the serological level of TRAIL.

In addition, death receptor signals have been treated as important targets for viruses to avoid cell death by TRAIL. It has been reported that the death receptor is a direct target for adenovirus and human cytomegalovirus and can downregulate expression on the cell surface (SMITH, Wendell, et al. Cell host & microbe , 2013, 13.3: 324- 335). In addition, other viruses may inhibit the signaling of death receptors by upregulating anti-apoptotic proteins (RAHMAN, Masmudur M .; MCFADDEN, Grant. PLoS Pathog , 2006, 2.2: e4.).

TRAIL is abundant in the serum of liver tissue and chronic hepatitis B patients (CHB), suggesting that TRAIL signaling may be associated with antiviral response and liver damage in CHB patients. However, despite the reports of this antiviral response, the characteristics of HBV to induce chronic infection throughout life have not been studied yet, and only immune evasion responses have been reported.

Therefore, the present inventors have studied the process of HBV to avoid the immune response of infected host cells, and have attempted to develop a therapeutic agent for TRAIL-mediated HBV infection associated with liver damage in chronic hepatitis B patients. As a result, , It was confirmed that DR5 protein was degraded by the HBx protein in the intracellular and cell surface by the autophage action and thus the cell death process involving TRAIL was blocked and HBV infected cells could survive. In order to block the host immune response avoidance process of HBV, when TRAIL and autophagy inhibitor were treated together, apoptosis of HBV infected cells was induced and cell viability was decreased. It has been confirmed that the autophagy inhibitor can be used as an active ingredient of a pharmaceutical composition for prevention and treatment of liver disease caused by HBV infection by blocking DR5 protein degradation in HBV infected cells and completed the present invention.

Accordingly, the present inventors have developed a method of inducing apoptosis by blocking hepatitis B virus (HBV) in order to avoid an immune response of an infected host cell and thereby blocking autophagy degradation of DR5 protein Thus completing the present 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 method for preventing and treating infectious disease of hepatitis B virus (HBV), which comprises an effective component of TRAIL (TNF-related apoptosis-inducing ligand) and autophagy inhibitor A pharmaceutical composition is provided.

According to a preferred embodiment of the present invention, the autopage inhibitor may be any one selected from the group consisting of chloroquine, bafilomycin A1 and 3-methyladenine (3-MA) Or more.

According to a preferred embodiment of the present invention, the autophagy inhibitor is preferably an siRNA composed of the nucleotide sequence of SEQ ID NO: 1, which is complementary to the mRNA of the LC3B (microtubule-associated protein 1 light chain 3B) gene .

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 TRAIL and a test compound with a liver tissue sample derived from a hepatitis B patient or a cell line infected with HBV;

ii) analyzing the characteristics of the liver tissue sample or the liver cell line treated in the step i); And

iii) a step of treating the result of the step ii) with a non-treatment step.

According to a preferred embodiment of the present invention, the test compound of step i) may be an autophage inhibitor.

According to a preferred embodiment of the present invention, the characteristics of the liver tissue sample or liver cell line of step ii) may be any one of the following a) to e):

a) reduced cell viability;

b) increased levels of apoptosis;

c) increased levels of intracellular DR5 protein expression;

d) increased levels of DR5 protein expression on the cell surface; And

e) Decreased levels of intracellular BECN1 protein expression.

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.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatitis B virus (HBV) infectious disease comprising TRAIL (TNF-related apoptosis-inducing ligand) and autophagy inhibitor as an active ingredient do.

The TRAIL of the present invention recognizes HBV-infected cells and induces apoptosis. The autophagy inhibitor of the present invention blocks HBV-induced HBV-induced autophage-mediated degradation of DR5 protein, Lt; RTI ID = 0.0 > HBV < / RTI >

Accordingly, the pharmaceutical composition comprising the TRAIL and the autophagy inhibitor of the present invention can effectively treat the HBV-infected cells by enhancing the anti-HBV therapeutic effect of TRAIL, which can be used as a therapeutic agent for HBV infected cells, It is effective because it can inhibit progression to hepatitis.

Figure 1 shows the confirmation of intracellular and surface DR5 protein expression upon HBV infection or HBx expression:
FIG. 1A shows the level of expression of DR5 mRNA and protein in HepG-X cells and HepG-M cells;
FIG. 1B is a graph showing the expression level of DR5 protein according to HBx expression time in HBx-HA cells;
Figure 1C shows the level of expression of DR5 mRNA and protein in HepG2.2.15 cells transfected with whole HBV genome;
FIG. 1D shows the level of expression of DR5 protein in HBV1.2 cells transfected with HBV1.2mer, a replication-element of the HBV construct;
FIG. 1E shows the expression of DR5 and HBx proteins in cells expressing fluorescently labeled HBx; And
FIG. 1F is a graph showing the level of DR5 protein expression on the cell surface according to HBx expression.
Figure 2 shows confirmation of a method of downregulating DR5 protein by HBx:
Figure 2A shows the level of DR5 expression in HepG2 cells and HepG2.2.15 cells following treatment with the lysozyme inhibitor chloroquine or the proteasome inhibitor MG132; And
FIG. 2B shows the level of DR5 expression by treatment with chloroquine or MG132 in HepG-M cells and HepG-X cells.
Figure 3 shows confirmation of autophage induction in HBV infected cells:
Figure 3A shows the formation of autophagosomes and autolysosomes in HBx-HA cells;
FIG. 3B shows the formation of autogranular and autolytic bodies in HepG-M and HepG-X cells;
Fig. 3C shows the formation of autogranular and autolysed bodies in HepG2 cells and HepG2.2.15 cells; Fig.
Figure 3D shows the expression of an autopagitic marker protein in HBx-HA cells;
FIG. 3E shows the expression of an autopagitic marker protein in HepG-M and HepG-X cells;
Figure 3F shows the expression of autopagitic marker protein in HepG2 cells and HepG2.2.15 cells; And
FIG. 3G shows the expression of a protein associated with apoptosis in HepG-M and HepG-X cells.
Figure 4 shows confirmation of DR5 degradation by autophagy induced by HBx:
FIG. 4A is a graph showing the level of DR5 degradation due to inhibition of autolytic activity in HepG-M and HepG-X cells;
FIG. 4B is a graph showing the level of DR5 degradation according to BFA treatment, which is an inhibitor of autolysin activity, in HepG2 cells and HepG2.2.15 cells;
FIG. 4C shows the level of DR5 expression according to LC3 knockdown using siRNA in HBV-infected cells;
FIG. 4D shows the level of DR5 expression in HepG-X cells according to 3-MA treatment as an autophagy inhibitor;
FIG. 4E shows the level of DR5 expression in HepG-X cells cultured in starvation; And
FIG. 4F is a graph showing the expression level of DR5 according to LC3 knockdown using siRNA or 3-MA treatment as an autophagy inhibitor on the surface of HBV infected cells.
Figure 5 shows confirmation of the correlation of HBx, DR5 and autophage-related proteins in HBx expressing cells:
FIG. 5A is a graph showing the expression of an autophage-related protein in a sample immunoprecipitated with LC3B; FIG.
FIG. 5B is a graph showing the expression of an autophage-associated protein in a sample in which DR5 has been immunosuppressed;
FIG. 5C is a graph showing the expression of an autophage-related protein in a sample in which HA has been immunosuppressed;
FIG. 5D is a graph showing the expression of an autophage-related protein according to LC3B knockdown in a sample immunohistochemically reacted with HA; And
5E is a schematic diagram showing an auto-phage process for DR5 degradation by HBx.
FIG. 6 is a diagram showing the measurement of the staining intensity of DR5 protein analyzed by immunohistochemical staining. FIG.
Figure 7 shows the identification of autophagic activity and DR5 expression by HBV in chronic hepatitis B tissue:
FIG. 7A shows the level of DR5 protein expression in chronic hepatitis B tissues; FIG.
FIG. 7B shows the level of expression of BECN1 in chronic hepatitis B tissue; And
FIG. 7C shows the expression of DR5 protein in chronic hepatitis B tissue of BECN1-positive and negative.
FIG. 8 shows the degree of degradation of DR5 according to TRAIL treatment time in HepG-M cells and HepG-X cells.
Figure 9 shows the cytoprotective activity of HBx against TRAIL-induced apoptosis in HBV infected cells:
FIG. 9A is a graph showing cell viability according to TRAIL treatment in HepG-M cells and HepG-X cells;
FIG. 9B is a view showing cell death levels according to TRAIL treatment in HepG-M cells and HepG-X cells;
FIG. 9C is a graph showing the activity of caspase according to TRAIL treatment in HepG-M cells and HepG-X cells;
FIG. 9D shows cell viability and cell death levels in TRAIL-treated HepG-M cells subjected to knockdown or 3-MA treatment of LC3B;
FIG. 9E is a graph showing the caspase activity of TRAIL-treated HepG-M cells knocked down by LC3B; And
FIG. 9F shows the cell survival rate and cell death level according to TRAIL treatment in Hep3.2.15 cells treated with LC3B knockdown or 3-MA treatment.

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

Although TRAIL signaling has been reported to be associated with liver damage in patients infected with hepatitis virus B (HBV), HBV has been shown to inhibit TRAIL signaling through immunosuppression The specific mechanism of action and the therapeutic agent using the agent have not been studied.

The TRAIL of the present invention recognizes HBV-infected cells and induces apoptosis. The autophagy inhibitor of the present invention blocks HBV-induced HBV-induced autophage-mediated degradation of DR5 protein, Lt; RTI ID = 0.0 > HBV < / RTI >

Therefore, the pharmaceutical composition comprising the TRAIL and the autophage inhibitor of the present invention can be used as a therapeutic agent for HBV-infected cells by effectively killing the HBV-infected cells by enhancing the anti-HBV therapeutic effect of TRAIL, It is effective for 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 TRAIL (TNF-related apoptosis-inducing ligand) and autophagy inhibitor as an active ingredient do.

The "autophage inhibitor" of the present invention is preferably one or more selected from the group consisting of chloroquine, bafilomycin A1 and 3-methyladenine (3-MA) 3-MA. However, the present invention is not limited thereto, and any one known in the art may be used as an auto-phage inhibitor capable of blocking at least one step of the auto-phage process.

It is preferable that the " autophagy inhibitor " of the present invention is an siRNA that complementarily binds to the mRNA of the LC3B (microtubule-associated protein 1 light chain 3B) gene, and more specifically, it is an siRNA composed of the nucleotide sequence of SEQ ID NO: 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.

In a specific example of the present invention, the present inventors confirmed the expression levels of DR5 protein in cells and on the surface by HBV infection or HBx expression. As a result, DR5 mRNA expression level was significantly While no difference was observed, the expression level of the DR5 protein was significantly decreased (Figs. 1A to 1F).

In addition, the present inventors determined that HBx of HBV induces degradation of DR5, and confirmed that the DR5 protein was degraded by HBx-induced lysosomes when HBV was infected (FIGS. 2A and 2B) 2B), indicating that HBx induces the progress of autophagy as a whole in host cells, leading to the degradation of host protein DR5 (FIGS. 3A to 3G).

In order to further confirm DR5 degradation by autophagy, the inventors of the present invention found that an autoxidation inhibitor, an siRNA for LC3B knockdown, or a 3-methyladenine (3-methyladenine, 3- MA) showed that DRx5 protein expression level was dependent on class III PI3K, and that this signal transduction pathway plays an important role in the down regulation of HBx-induced DR5 protein (Figures 4A-4F).

In addition, the present inventors confirmed the correlation between HBx, DR5 and autophage-related proteins in HBx-expressing cells. As a result, HBx induced autophagy and bound between LC3-I and DR5 proteins to decompose DR5 (Fig. 5A to Fig. 5E).

In addition, the present inventors confirmed the autophagic activity and DR5 expression by HBV in chronic hepatitis B tissues. As a result, DR5 expression level in hepatic tissues of chronic hepatitis B patients was significantly lower than that of the normal control group (FIGS. 6 and 7A), the expression level of BECN1 was found to increase (Fig. 7B and Fig. 7C).

In addition, the inventors of the present invention found that the HBx cell protection activity against TRAIL-induced apoptosis in HBV-infected cells was inhibited by treatment with TRAIL in HBx-expressing cells, and the survival rate of infected cells (Fig. 8 and Figs. 9A to 9C). When the autophagy inhibitor was treated with TRAIL in HBV-infected cells, it was confirmed that HBx inhibited TRAIL-mediated cell death (Fig. 9D to Fig. 9F).

Therefore, TRAIL of the present invention recognizes HBV-infected cells and induces apoptosis. The autophagy inhibitor of the present invention blocks HBV-induced HBV-induced autophage-mediated degradation of DR5 protein, The TRAIL and autophagy inhibitor can be effectively used as an active ingredient of a pharmaceutical composition for the prevention and treatment of HBV infection because it can enhance the HBV cell killing effect by TRAIL.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing TRAIL and an autophage 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 TRAIL and autophage inhibitor of the present invention as an active ingredient can be administered parenterally, and the parenteral administration is by injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic 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 TRAIL and a test compound with a liver tissue sample derived from a hepatitis B patient or a cell line infected with HBV;

ii) analyzing the characteristics of the liver tissue sample or the liver cell line treated in the step i); And

and iii) comparing the result of the analysis of step ii) with that of the untreated control group, thereby screening the therapeutic agent for hepatitis B virus infection.

The "cell line" of the step i) of the present invention may be any cell line derived from human liver as long as it is known in the art. Specifically, it is preferable to use a HepG2 cell line, but not limited thereto.

The " test compound " of step i) of the present invention is preferably, but not limited to, an autophage inhibitor.

The "liver tissue sample or liver cell line property" of step ii) of the present invention is preferably, but not limited to, any one of the following a) to e)

a) reduced cell viability;

b) increased levels of apoptosis;

c) increased levels of intracellular DR5 protein expression;

d) increased levels of DR5 protein expression on the cell surface; And

e) Decreased levels of intracellular BECN1 protein expression.

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 DR5 protein of the present invention is an immunoglobulin protein capable of binding to TRAIL and inducing apoptosis upon viral infection. In order to avoid the immune response of the infected host cell, HBV decomposes DR5 protein into autophagy, However, when the autophagy inhibitor is treated together with TRAIL, the degradation of DR5 by autophagy can be suppressed. Therefore, the test compound is treated with TRAIL to inhibit the expression of DR5 protein, the survival rate of HBV-infected host cells or the cell death 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.

HBV  Infection or HBx  Identification of intracellular and surface DR5 protein expression by expression

<1-1> Preparation of target cell line

In connection with the present invention, HBx protein, which is a viral protein expressed by HBV, is known to play an important role in protein expression of host cells. Therefore, in order to carry out the embodiment of the present invention, a cell line expressing HBx is prepared, Respectively.

Specifically, a HepG2 cell line (ATCC) as a human liver cancer cell line was cultured in a DMEM medium (culture medium) supplemented with 10% heat-inactivated FBS (Gibco BRL, USA) and 100 U / ml penicillin / streptomycin (Gibco BRL) ) And cultured in a 5% carbon dioxide incubator at 37 ° C.

For the production of the HBV-infected cell model, a plasmid was constructed to continuously express HBx by inserting the HBV amplicon and the HBx gene in place of the CMV promoter of the pcDNA3.1 vector (Invitrogen). HBV1.2mer (strain ayw ) plasmid was used as an HBV construct having elements for replication of HBV (CHA, Man-Young, et al. Journal of General Virology, 2009, 90.4: 978-986.). For use as a plasmid for transient expression of HBx, genes coding for HBx-HA or HBx-flag were inserted into the pcDNA3.1 vector to construct each plasmid. To transfect the plasmid into the HepG2 cell line, Lipofectamine 2000 (Invitrogen, Canada) was used according to the protocol provided by the manufacturer.

Various types of HBV-infected cell models were prepared as shown in the following Table 1 by transfection of the constructed plasmid (Table 1).

The type of HBV-infected cell model prepared in the present invention Cell name Transgenic
Plasmid Contents HepG-X cells Includes HBV Amplifier and HBx
pcDNA3.1 vector HepG2 cells continuously expressing HBx HepG-M cells pcDNA3.1 Normal control group 2.2.15 cells HBx-flag included
pcDNA3.1 vector HepG2 cells containing HBV whole genome
Fluorescent marker can be labeled for HBx HBx-HA cells Includes HBx-HA
pcDNA3.1 vector HepG2 cells transiently overexpressing HBx HBV1.2 cells HBV 1.2mer plasmid Contains elements for HBV replication

<1-2> HBx  Comparison of Expression Levels of DR5 Gene and Protein in Expression According to Expression

Since it is well known that HBx protein is an essential factor in maintaining a persistent infection state through non-lesional viral replication in host cells, HBx downregulates TRAIL receptor signaling, In order to confirm whether or not it could be avoided from the reaction, the level of DR5 expression was first confirmed in HBx expressing cells.

Specifically, an appropriate plasmid was transfected into HepG2 cells by the method of Example <1-1> to prepare an HBV-infected cell model. Cells were obtained after transfection 24, 48 or 72 hours, respectively, 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 performed using the DR5 or HBx primer of the sequence shown in [Table 2] below. Quantitative RT-PCR results were confirmed by electrophoresis on agarose gel.

In addition, immunoblotting was performed to confirm protein expression. The HBV-infected cell model obtained by the transfection and culturing was obtained and dissolved in RIPA lysis buffer (Sigma, USA). The lysed cells were centrifuged to obtain a supernatant and Western blot was performed according to known methods to confirm the level of expression of the DR5 and HBx proteins. Anti-DR5 antibody (Cell signaling Technology, USA), anti-HBx antibody (BioVendor, Germany) and anti-GAPDH antibody (Santa Cruz, USA) were used as primary antibodies for immunoblotting. Anti-mouse antibody and anti-rabbit antibody (Sigma, USA) with horseradish peroxidase conjugated thereto was used as the secondary antibody. Analysis of signal intensity for quantitative analysis of protein expression was quantitatively analyzed using Multi Gauge software (Fujifilm, Japan). The DR5 signal intensity was corrected based on the GAPDH expression intensity and the level of DR5 expression in HepG-X cells was relatively expressed based on the expression level in normal control cells as 100%.

A specific primer sequence for performing RT-PCR Gene name Primer sequence DR5
Forward 5'-CAG AGG GAT TGT GTC CAC CT-3 ' Reverse 5'-TAC GGC TGC AAC TGT GAC TC-3 ' HBx
Forward 5'-AAA AAG TTG CAT GGT GCT GGT GAA C-3 ' Reverse 5'-GCG CGG GAC GTA CTT TGT-3 ' GAPDH
Forward 5'-CGT CTT CAC CAT GGA GA-3 ' Reverse 5'-CGG CCA TCA CGC CAC AGT TT-3 '

As a result, as shown in FIG. 1A to FIG. 1D, the expression level of DR5 mRNA was significantly increased in HepG-X cells, which continuously express HBx under the control of HBV amplifiers / promoters, as compared with the normal control group HepG-M cells While no difference was observed, the expression level of DR5 protein was reduced to about 40% level (Fig. 1A). The expression level of DR5 protein was also decreased with the time of inducing transient HBx expression by transfection of the plasmid into HBx-HA cells, and the expression level of DR5 protein was reduced to about 40% level after 72 hours of transfection (Fig. 1B).

In addition, HepG2.2.15 cells, which were transfected with whole HBV genome, showed no significant difference in DR5 mRNA expression level compared to the normal control group, while DR5 protein expression level was 30 ± 10% (Fig. 1C). In addition, down-regulation of DR5 protein was also observed in HBV1.2 cells transfected with HBV1.2-mer, a replication-element of the HBV construct (Fig. 1D).

<1-3> HBx  Comparison of DR5 Protein Expression Levels by Intracellular Expression

Immunofluorescence analysis was performed on HepG2.2.15 cells expressing fluorescently labeled HBx in order to more specifically confirm downward regulation of DR5 protein in HBx expressing cells.

Specifically, HepG2 cells were inoculated on coverslips and HBx-flag plasmid was transfected to prepare HepG2.2.15 cells. After 24 hours of transfection, HepG2.2.15 cells were fixed with 3.7% paraformaldehyde (Sigma, USA) for 15 minutes at room temperature. After fixation, the cells were washed with PBS, and the cells were labeled with anti-DR5 antibody (Novus Biologicals, USA) and anti-Flagag2 antibody (Sigma) at 4 ° C overnight. Then, the cells were washed three times with PBS, and the secondary antibody, Alexa Fluor 488 anti-rabbit IgG antibody, Alexa Fluor 546 anti-mouse IgG antibody (Invitrogen, USA) was added and incubated at room temperature for 40 minutes. Antibody-bound HBx protein (red) and DR5 protein (green) were visualized by fluorescence imaging with a Carl Zeiss Axiovert 200 microscope.

As a result, as shown in FIG. 1E, it was confirmed that HepG2.2.15 cells expressing HBx remarkably decreased DR5 protein expression level to 37 +/- 5% level when compared with normal control HepG-M cells 1E).

<1-4> HBx  Comparison of DR5 Protein Expression Levels on Cell Surface by Expression

The expression level of DR5 expressed on the cell surface has been reported to be an important factor determining the degree of apoptosis according to the physiological TRAIL concentration. Thus, it is possible to determine whether HBx can decrease the expression level of DR5 expressed on the cell surface Respectively.

Specifically, HepG2.2.15 cells or HepG-M cells were each cultured in a culture medium, separated from the culture plate with trypsin, washed with PBS and suspended in PBS containing 2% FBS. Each suspended cell was stained with ice-added DR5 antibody (R & D system, USA) for 30 minutes with phycoerythrin-conjugated DR5 antibody. The stained cells were analyzed using a FACScalibur flow cytometer (Becton Dickinson, USA) and flow cytometry software (University of Turku, Finland). DR5 expressed on the cell surface was expressed as percentage compared to the fluorescence intensity in the control cells.

As a result, it was confirmed that the level of cell surface expression of DR5 was markedly decreased in cells infected with HBV as well as cells transiently or continuously expressing HBx, as shown in Fig. 1F (Fig. 1F) . It was confirmed that HBx protein down-regulates intracellular and surface DR5 protein expression when HBV infected hepatocytes.

On HBx  Identification of down-regulation of DR5 protein by

HBx-induced downregulation of DR5 did not show a significant difference in DR5 mRNA expression level, and therefore it was expected that HBx would induce DR5 degradation expressed. To confirm this possibility, DR5 protein expression levels were determined after administration of proteosomes or lysosomal inhibitors.

Specifically, HepG2 cells, HepG2.2.15 cells, HepG-M cells or HepG-X cells prepared in Example <1-1> were inoculated on each culture plate and cultured. In culture, 20 μM chloroquine (CQ) (Invitrogen) or MG132 (MG) (protease inhibitor) (Calbiochem, USA), which is a lysosome inhibitor, was added to the culture medium in the presence of 25 μM Respectively, and cultured for 16 hours. Then, each cultured cell was obtained, and the degree of expression of DR5 protein was confirmed by Western blotting.

As a result, as shown in FIG. 2, HBx-induced DR5 degradation was completely blocked by the lysozyme inhibitor chloroquine, but when treated with the proteasome inhibitor MG132, HBx or HBV-expressing cell lines (Fig. 2A and Fig. 2B). These results confirm that DR5 protein is degraded by HBx - induced lysosomes when HBV is infected.

HBV  Within infected cells Auto Phage  Confirmation of induction

<3-1> HBV  In an infected cell model Autogenous body  And Autolytic  Confirmation of occurrence

HBx protein degrades DR5 protein through lysosomes. Proteolysis by lysosomes is the last step of autophagy. Therefore, LC3 protein, an autophagy marker, was identified and HBV induced complete autophage I can confirm that I can do it.

Specifically, HepG-X, HepG-M, Hep2.2.15 and HBx-HA cells were cultured on cover slip, respectively, and mRFP-GFP tandem fluorescence-labeled LC3 plasmids (mRFP-GFP tandem fluorescent- tagged LC3, tfLC3 plasmid ; Dr. HJ Lee, Konkuk University, Korea) were transfected into each cell. After the transfection, the cells were cultured for 16 hours and observed with a Carl Zeiss Axiovert 200 fluorescence microscope. The intracellular LC3 protein was identified, and the degree of fluorescence was quantitatively analyzed using the software provided by the manufacturer. LC3 spots that produce both red and green fluorescence were identified as autophagosomes, and LC3 spots identified only as red fluorescence were identified as autolysosomes, - positive cells was expressed as a percentage based on the total number of mRFP-positive cells. Cell counts were repeated three times or more based on a single experiment that contained more than 100 mRFP-positive cells per sample.

As a result, as shown in FIG. 3A to FIG. 3C, autoporosis bodies can be displayed as red and green fluorescence points due to GFP and RFP, but GFP is unstable in acid in the lysosome, Therefore, the autolysosome is displayed only with the red RFP-LC3 point. Therefore, in the image obtained by combining the GFP color development and the RFP color development, the autopagococcus is displayed as yellow, and the autolysosome is red (FIG. 3A to FIG. 3B).

Fluorescence microscopy revealed that the numbers of autophagous and autolysed cells were significantly increased in HBx-HA cells compared to control cells, and the proportion of autophagic cells containing autophagic cells was remarkably increased (Fig. 3A). In addition, in both HepG-X and HepG2.2.15 cell lines expressing HBx, it was confirmed that the proportion of cells expressing autogranular and autolysin was markedly increased as compared with the normal control (FIGS. 3A and 3C). These results suggest that HBx induces the entire process of autophagy, inducing the formation of autogranular bodies and inducing maturation by autolysin.

<3-2> HBV  In infectious cell model Self-predation Marker  Confirmation of expression

HBx-infected cells induced autophagy in host cells, HBx expression induced intracellular p62 / SQSTM1 (autoregulatory flow marker) and LC3-II (autoradiographic marker) And so on.

Specifically, the HepG2 cells, HBx-HA cells, HBV1.2 cells, HepG2.2.15 cells, HepG-M cells or HepG-X cells prepared in Example <1-1> Respectively. Each cell was obtained at 24, 48 and 72 hours after transfection and Western blot was performed. (Anti-p62 / SQSTM1 antibody (Santa Cruz), anti-BECN1 antibody (Atlas Antibodies, Sweden), anti-capase 8 antibody-anti -FADD antibody, anti-cIAP-2 antibody, anti-c-FLIP antibody and anti-Mcl-1 antibody were used. Quantitative analysis of Western blotted protein bands was performed using Multi Gauge software and the expression levels were corrected for each sample based on the level of expression of GAPDH and compared to the expression levels of LC3II and p62 / SQSTM1 in normal control cells Relative expression levels.

As a result, in the HBx-HA cells transiently expressing HBx, the level of p62 / SQSTM1 was decreased for 48 hours after the HBx-HA gene transfection as compared with the normal control cells, as shown in Figures 3D to 3G, And the level of LC3-II was increased (FIG. 3D). In addition, the level of p62 / SQSTM1 was decreased 72 hours after transfection in HBV1.2 cells transfected with HBV genome (Fig. 3E), compared with control cells (Fig. 3E) and HepG-X and HepG2.2.15 In both cell lines, p62 / SQSTMl and intracellular LC3-II levels were all reduced (Fig. 3F). However, it was confirmed that expression levels of the proteins associated with apoptosis such as FADD, cIAP-2, c-FLIP and Mcl-1 were not affected by HBx expression (Fig. 3G). The decrease in the level of LC3-II in normal controls without HBx expression was due to the degradation of LC3-II at the end of autophagy (NI, Hong-Min, et al. Autophagy, 2011, 7.2: 204.), confirming that HBx induces the progress of autophagy in host cells and induces degradation of the host protein.

HBx  Inducing Otto Phage  Confirmation of DR5 degradation by

<4-1> HBx  In expression cells Autolysis body  Confirm DR5 degradation level by inhibition of activity

In order to confirm whether the expression level of DR5 is reduced by HBx-induced autophage, the level of DR5 protein expression was confirmed after treatment with bafilomycin A1 (BFA), a self-decomposing inhibitor.

Specifically, HepG-X, HepG-M, HepG2.2.15 and HepG2 cells prepared in Example <1-1> were cultured. The culture medium was treated with 100 nM BFA (Invitrogen, USA), and each cell was obtained at 0, 6 and 16 hours after treatment and Western blot was performed to determine intracellular DR5, LC3-I, LC3- .

As a result, the expression levels of p62 / SQSTM1 and LC3-II were increased in cells treated with BFA compared with cells not treated with BFA (0 hour) as shown in FIGS. 4A and 4B, and increased p62 / SQSTM1 and The expression level of LC3-II was restored to a level similar to that of the normal control (Figs. 4A and 4B). The results showed that BFA significantly inhibited autogenous plumage and DR5 was degraded by HBx - induced autopagic flow.

<4-2> HBx  In the expression cells, LC3 In knockdown  Of DR5 expression levels

To further confirm the autophagy induced by HBx to digest DR5, we examined whether the level of autophagy activity and DR5 expression was altered in cells knocked down with LC3 by siRNA.

Specifically, siRNA (SEQ ID NO: 1: 5'-GAU AAU UAG AAG GCG CUU A-3 ') for knockdown of LC3 was synthesized from ST Pharm (Korea). siRNA was transfected into HepG2, HBx-HA, HepG-M, HepG-X or HepG2.2.15 cells using lipofectamine 2000 at a concentration of 100 nM, and cells were obtained after 48 to 72 hours of transfection, The expression levels of LC3-I, LC3-II, p62 and DR5 proteins were analyzed by analysis. As the solvent control (con), LC3 expressing cells without siRNA transfection were used.

As a result, LC3B expression was decreased and p62 / SQSTM1 level was increased in all experimental cells transfected with LC3B siRNA as shown in Fig. 4C (Fig. 4C). Also, it was confirmed that the expression level of DR5 in HBx expressing cells was restored to the control level under the condition that the expression of LC3B was decreased.

<4-3> HBx  In expression cells Auto Phage  Identification of DR5 expression level by inhibitor treatment

To further confirm the autophagy induced by HBx to digest DR5, the autophagy activity and DR5 expression level in the culture environment treated with 3-methyladenine (3-MA), an autophagy inhibitor, .

Specifically, HepG-X and HepG-M cells transfected with the tfLC3 plasmid in Example <3-1> were treated with 5 mM 3-MA and cultured for 16 hours. Then, cultured cells were obtained, and the level of expression of LC3, p62 / SQSTM1 and DR5 in cells by 3-MA treatment was confirmed by fluorescence microscopy analysis and immunoblot analysis. As a solvent control, cells treated with DMSO instead of 3-MA were cultured and used.

As a result, as shown in FIG. 4D, the spot formation of RFP-LC3 was increased in HBx-expressing cells in which autophagy was blocked by treatment with 3-MA, and p62 / SQSTM1 expression level and DR5 expression level were increased, Lt; RTI ID = 0.0 &gt; (Figure 4D). &Lt; / RTI &gt; Thus, autophagy stimulated by HBx is class III PI3K-dependent, confirming that this signaling pathway plays an important role in the down-regulation of HBx-induced DR5 protein (CHA, Man-Young, et al. Journal of General Virology, 2009, 90.4: 978-986.).

<4-4> HBx  Identification of DR5 expression level in cultured cells according to culture medium nutritional status

To confirm more specifically the autophagy induced by HBx to degrade DR5, we confirmed whether DR5 degradation could occur under starvation conditions, since the cells increase the autophagy in the nutritional starvation state.

Specifically, the HepG-X and HepG-M cells transfected with the tfLC3 plasmid in Example <3-1> were cultured in EBSS (Earle's balanced salt solution; Welgene Co., Korea) for 16 hours. Then, cultured cells were obtained, and fluorescence microscopic analysis and immunoblot analysis were performed to confirm the levels of intracellular LC3, p62 / SQSTM1 and DR5 expression according to the nutrient conditions of the culture medium. As a non-treated control group, cells were cultured in DMEM medium.

As a result, when the cells expressing HBx were cultured in a nutrient-poor medium as shown in FIG. 4E, the DR5 expression level was remarkably decreased compared with the nutrient-treated control (FIG. 4E) , And that autophagy induced by environmental conditions could promote HBx - induced DR5 degradation.

<4-5> HBx  In expression cells Auto Phage  Inhibitor treatment or LC3 In knockdown  The level of DR5 expression on the cell surface

It was confirmed that degradation of intracellular DR5 was increased through autophagy induced by HBx. Thus, it was confirmed whether the expression level of DR5 expressed on the cell surface was changed.

Specifically, HepG-M and HepG2.2.15 cells were knocked down with LC3B siRNA in the same manner as in Example <4-2> or in 5 mM 3-MA treatment medium in the same manner as in Example <4-3> And cultured. Then, each cell was obtained, and the level of DR5 expression was confirmed on the cell surface by flow cytometry. As untreated controls (con), cells that did not knock down LC3B or cells that did not receive 3-MA treatment were used.

As a result, as shown in FIG. 4F, the cell surface expression of DR5, which was reduced by HBx in all experimental groups, was significantly recovered as compared with the untreated control group. Thus, The expression of DR5 on the cell surface was also down-regulated from the DR5 protein degradation (Fig. 4F).

HBx  In expression cells HBx DR5, and Auto Phage  Related protein correlation  Confirm

It was confirmed that DR5 degradation was increased by HBx-induced autophagy in HBV-infected cells. Immunoprecipitation confirmed whether HBx, DR5 and autophage system had a specific relationship with each other Respectively.

Specifically, the HBx-HA cells prepared in Example <1-1> were cultured for 48 hours, and then immersed in a solution buffer (10 mM NaCl, 1% NP-40 and a protease inhibitor cocktail for immunological infiltration In 50 mM Tris-HCl buffer, pH 7.2). The dissolved cells were centrifuged to obtain a supernatant as a lysate. The lysate was pretreated with protein A agarose (Sigma) for 1 hour at 4 ° C, and the anti-LC3B antibody, anti-DR5 antibody or anti- The anti-HA antibody (Sigma) was treated and incubated overnight at 4 ° C. Then, the cells were incubated with protein A agarose for 4 hours at 4 ° C, washed three times with a lysis buffer, heated, and the heated sample was immunoblotted to determine the relationship between LC3B, DR5 and HBx Respectively. Immunomodulatory reactions were also performed in the same manner in cells knocked down with LC3B by siRNA.

As a result, as shown in Fig. 5, anti-LC3B antibody bound by immunoinfusion,

Both anti-DR5 and anti-HA antibodies have been implicated in HB5 as well as DR5 and LC3B proteins in immunoblotting (Figures 5A-5C). It was also confirmed that the binding between DR5 and LC3B increased with the expression of HBx (Fig. 5A and Fig. 5B). In addition, it was confirmed that HBx still binds to DR5 in cells that do not express by knockdown of LC3B, confirming that LC3B is not involved in binding between HBx and DR5 proteins (FIG. 5D). Through this, it was confirmed that HBx can be selected as a target of DR5 for the autophagy system through binding between two proteins (Fig. 5E).

In chronic hepatitis B tissue On HBV  by Auto Phage  Identification of active and DR5 expression

<6-1> Determination of DR5 protein expression level in chronic hepatitis B tissues

In order to determine whether the autophagy induction and DR5 degradation process by HBV infection in the HBV-infected cell model were identical in vivo, immunohistochemical staining of hepatic tissues of chronic hepatitis B patients (CHB) Expression levels were confirmed.

Specifically, a liver slice for analysis (LV208112; US Biomax, Inc., USA) was prepared and used to detect DR5 and BECN1 inducing autophagy. Normal control liver tissue samples and CHB liver tissue samples were prepared to include 16 samples / 32 pairs of samples, respectively. Information on the histopathology of normal control and CHB patients was searched on the manufacturer's website (http://www.biomax.us/tissue-arrays/). The sample slurry prepared above was baked at 60 DEG C for 30 minutes, de-paraffinized with xylene, and dehydrated with ethanol. The cells were then incubated with blocking buffer to inhibit nonspecific antigen binding, then incubated with anti-DR5 antibody or anti-BECN1 antibody overnight at 4 ° C for antigen retrieval. After incubation, the cells were stained contrastively with hematoxylin. Immunohistochemistry (IHC) imaging was standardized on a Pannoramic MIDI automated slide scanner (3DHistech Ltd., Hungary) to check the leveled signal strength and minimize experimental errors. To determine the expression level of DR5, signal intensity was analyzed using NIH-ImageJ software with the IHC Profiler plugin (http://rsb.info.nih.gov/ij/). The signal intensity of DR5 is represented by inverted median pixel value (IMPV). When IMPV was over 150 (> 150 IMPV), it was judged to be strongly positive for DR5. 150-100 IMPV was moderately positive, 100 to 50 IMPV was weakly positive, and less than 50 IMPV were negative.

As a result, as shown in FIG. 6 and FIG. 7A, IMPV was higher in a positive tissue with a high DR5 expression level (FIG. 6) and DR5 expression level was significantly positive (mean expression level = 1421 IMPV) in a liver of a normal control group On the contrary, DR5 expression level was weakly positive (124 IMPV) in liver tissue of CHB patient, and DR5 expression was significantly down-regulated in liver tissue of CHB patients (Fig. 7A).

<6-2> Chronic Hepatitis B Tissue BECN1  Identify expression levels

In order to confirm whether the autophagic induction and DR5 degradation process by the HBV-infected cell model are identical in vivo, immunohistochemical staining of hepatic tissues of chronic hepatitis B patients (CHB) Lt; RTI ID = 0.0 &gt; BECN1 &lt; / RTI &gt;

Specifically, the same method as in Example <6-1> was performed to immunohistochemically stain the liver of CHB and then the expression of BECN1 was confirmed. The expression level of BECN1 was confirmed by the level of microencapsulated BECN1, and the BECN1 expression site was selected from the scanned IHC images and the nuclei were stained contrastively. Within the selected area, the percentage of cells containing BECNl micro-aggregates is calculated so that the score of the area of less than 20% is 1+, the score of the area of 20 to 40% is 1+, the score of the area of 40-70% , And a score of 70 to 100% in the region was defined as 3+.

As a result, as shown in FIG. 7B, BECN1 was not detected in the liver samples of the normal control group, whereas dark brown spots were formed in the liver tissue samples of CHB, indicating that BECN1 was positive (FIG. 7B). Among the 32 CHB liver tissue samples, 47 of the 15 samples showed strong positive for BECN1, whereas in the normal control liver samples, only 4 of 32 samples showed a weak positive (12%) , And BECN1 positive, the autophagy activity was increased in CHB patients hepatocytes (FIG. 7B).

<6-3> In chronic hepatitis B tissues, DR5 and BECN1  Confirm relationship

Expression levels of DR5 in BECN1 positive and negative tissues were compared in order to determine whether the autophagy induction and DR5 degradation process by HBV infection in the HBV infected cell model were identical in vivo.

Specifically, DR5 expression levels in tissues were examined and compared in tissue samples identified as BECN1 positive (n = 19) or negative (n = 45) in Example <6-2> above.

As a result, it was confirmed that DR5 expression is strongly positive (average expression level = 132 IMPV) in BECN1 negative tissue as shown in FIG. 7C, while BECN1 positive tissue is weakly positive (average expression level = 121 IMPV) 7C). In a related statistical analysis, BECN1 positive was found to be associated with a decrease in DR5 expression level in the liver of CHB patients, with an inverse relationship (P = 0.0339) to DR5 expression in clinical liver tissue .

HBV  Of TRAIL-induced apoptosis in infected cells Of HBx  Check cell protection activity

&Lt; 7-1 > HBV  Cells of infected cells Survival rate  Confirm

To confirm the functional consequences of downregulation of DR5 involved in autophagy, cell viability and cell death levels were determined in TRAIL treatment.

Specifically, the HepG-M or HepG-X cells prepared in Example <1-1> were treated with TRAIL at a concentration of 50 ng / ml or 100 ng / ml and cultured for 16 hours. First, each cell was harvested after 1, 3 and 5 hours of TRAIL treatment and the level of DR5 protein following TRAIL treatment was confirmed by immunoblotting. Then, after completion of the culture for 16 hours, each cell was treated with 50 μg / ml MTT and incubated for 2 hours, and the absorbance was measured at 560 nm using a SpectraMAX microplate reader (Molecular Devices, USA). Relative cell viability (%) was expressed as a percentage of the number of viable cells in the untreated control cell of TRAIL.

The number of apoptotic cells was determined by measuring the amount of DNA in hypodiploid cells. Cells treated with TRAIL and cultured were harvested, the cells were fixed with cold 70% ethanol, and the cells were fixed with propidium iodide / RNase staining solution (Cell Signaling Technology, Inc.) The amount of DNA was measured according to the protocol provided by the manufacturer. Cells were analyzed using a FACScalibur flow cytometer and the amount of intracellular DNA was analyzed using WinMDI software (Scripps Research Institute, USA). In addition, immunoblotting was performed using anti-caspase-8 antibody, anti-caspase-3 antibody and anti-PARP-1 antibody as primary antibodies.

As a result, as shown in FIG. 8 and FIGS. 9A to 9C, it was confirmed that the DR5 protein was rapidly degraded in the HBx-expressing cells and the DR5 protein was degraded rapidly (FIG. 8) (Fig. 9A and Fig. 9B), caspase-8, caspase-3 and PARP-1 activity were remarkably inhibited compared to control cells without HBx (Fig. 9C). This confirms that HBx limits the apoptosis of TRAIL-involved.

<7-2> HBV  In infected cells Auto Phage  Depending on the inhibitor treatment HBV  Identification of infecting cell death effect

It was confirmed that DR5 protein was degraded by autophagy caused by HBx in HBV-infected cells, and the effect of TRAIL-induced apoptosis was blocked. Therefore, it is possible to obtain the killing effect of HBV-infected cells when the autophagy inhibitor is treated with TRAIL Respectively.

Specifically, HepG-X or HepG2.2.15 cells prepared in Example <1-1> were treated with 5 mM 3-MA and 100 ng / ml TRAIL and cultured for 16 hours. In Example 4-2, TRAIL was treated with HepG-X or HepG2.2.15 cells inhibiting LC3B expression with siRNA (siLC3B) at a concentration of 100 ng / ml and cultured for 16 hours. After incubation, cells treated with 3-MA and cells knocked down with LC3B were obtained, and cell viability was confirmed by MTT assay. The DNA amount of hypodiploid cells was measured to determine the number of apoptotic cells Respectively. In addition, immunoblot analysis was performed to confirm the expression levels of LC3-I, LC3-II and PARP-1 proteins.

As a result, in the experimental group in which autophagy was inhibited by LC3B knockdown or 3-MA treatment as shown in FIGS. 9D to 9F, the cell viability of HBV-infected cells was decreased even after the TRAIL treatment, despite the expression of HBx (Fig. 9D and Fig. 9F). In addition, it was confirmed that the expression of PARP-1 was restored in HBx-expressing cells through LC3B knockdown (Fig. 9E).

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Pharmaceutical composition comprising TRAIL and autophagy          inhibitor as an active ingredient for prevention or treatment          hepatitis virus B infection <130> 1060602 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> LC3B siRNA <400> 1 gauaauuaga aggcgcuua 19

Claims (8)

As a pharmaceutical composition for the prophylaxis and treatment of chronic hepatitis B infection caused by infection with Hepatitis Virus B (HBV) comprising, as an active ingredient, TRAIL (TNF-related apoptosis-inducing ligand) and autophagy inhibitor ,
Wherein the autopage inhibitor is an siRNA consisting of the nucleotide sequence of SEQ ID NO: 1.
delete The pharmaceutical composition according to claim 1, wherein the siRNA is complementarily bound to the mRNA of the LC3B (microtubule-associated protein 1 light chain 3B) gene, for the prophylaxis and treatment of chronic hepatitis B infection by hepatitis B virus infection Gt;
delete i) treating TRAIL and the test compound with a liver tissue sample or HBV-infected cell line derived from a chronic hepatitis B patient;
ii) analyzing the characteristics of the liver tissue sample or the liver cell line treated in the step i); And
iii) screening the result of the analysis of step ii) with a non-treated control group.
6. The method according to claim 5, wherein the test compound of step i) is an autophage inhibitor of siRNA complementarily binding to the mRNA of LC3B (microtubule-associated protein 1 light chain 3B) gene. A method for screening a therapeutic agent for chronic hepatitis B by
6. The method for screening a therapeutic agent for chronic hepatitis B virus infection according to claim 5, wherein the characteristics of the liver tissue sample or liver cell line in step ii) are any one of the following a) to e)
a) reduced cell viability;
b) increased levels of apoptosis;
c) increased levels of intracellular DR5 protein expression;
d) increased levels of DR5 protein expression on the cell surface; And
e) Decreased levels of intracellular BECN1 protein expression.
delete

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