KR100930282B1 - SiRNA for the NiV gene and a liver disease treatment comprising the same - Google Patents

Abstract

The present invention relates to siRNA for NF-κB induced kinase (NIK) gene and gene therapy of liver disease by HBV infection using the same, wherein siRNA that specifically binds to mRNA of NIK gene is NF, which is a lower level of NIK. By regulating the signaling pathway of -κB, it can be used to prevent or treat liver disease caused by HBV infection.

Description

SiRNA of NIV gene and liver disease therapeutic agent comprising same {siRNA of NIK gene and therapeutic agent for liver diseases comprising}

The present invention relates to the gene therapy of siRNA for NF-κB induced kinase (NIK) gene and liver disease by HBV infection using the same, which is a substep of NIK using siRNA that specifically binds to mRNA of NIK gene. It relates to a method for regulating the signaling pathway of NF-κB.

Chronic hepatitis B virus (HBV) is the 10th leading cause of death worldwide, with an estimated 350 million virus carriers alone. In Asia, 1 in 10 people have the virus. 75% of the world's owners live in Asia, and hepatitis B virus is estimated to account for 5-8% of the population. Most infection routes in areas with high prevalence, including Korea, are vertical infections. In adults, hepatitis B infection cannot be cured and chronically progresses to less than 5%. However, maternal infection is more than 90% chronic. The problem is even worse. Chronic hepatitis B can develop into liver cancer after 20 to 40 years, and 30-40% of chronic patients can develop cirrhosis or liver cancer.

The nuclear factor-kappaB (NF-κB) signaling pathway is an important regulatory pathway in connection with liver disease. The transcription factor NF-κB regulates the balance between apoptosis and cell proliferation, and all cells, including cancer cells, can survive and proliferate by the NF-κB signaling system. In particular, HBV is known to induce NIK (NF-κB inducing kinase) -dependent NF-κB activity, and it has also been reported that inhibition of NF-κB activity is associated with liver cancer caused by HBV (Mozer- Lisewska et al . , Biochem. 52 (1): 56-61 2006). Inhibition of NF-κB and its higher levels of NIK in the NF-κB signaling system known to control the progression of liver cancer due to chronic hepatitis B will have an effective effect in the treatment of HBV-related liver disease.

RNA interference (RNAi) is a sequence specific mechanism that is stimulated by siRNAs that cause degradation of complementary target single stranded mRNAs and "silencing" of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet. 3: 737 2002). RNAi functions by enzymatic digesting long RNA strands into "short-interfering RNA" (hereinafter siRNA) sequences having a biological activity of about 18 to 23 nucleotides in length (Elbashir, et. al ., Genes Dev. 15: 188 2001). siRNAs can be used to specifically lower or silence the expression of target genes having sequence homology to siRNAs. Preferably, the lowering of genes associated with various diseases and disorders is exemplified.

In the RNAi process, siRNA is first recognized and cleaved into 18-23 bp fragments called siRNA by an enzyme called Dicer with RNase III activity (Cullen, Nat. Immunol. 3: 597-599 2001; Hannon, Nature 418: 244-251 2002; Tuschl, Chembiochem 2: 239-245 2001; Sharp, Genes Dev. 15: 485-490 2004; Moss, Curr. Biol. 11: R772-R775, 2001); SiRNA fragments then specifically bind to multicomponent nucleases to form a complex called an RNA-induced silencing complex (RISC); Finally, RISC hybridizes to target mRNAs with complementary bands and the target mRNA is cleaved in the middle of the 18-23 bp band. Thus, mRNA is not functioning.

Methods of suppressing the expression and replication of RNAi-based HBV viruses are Guang-Li Ren (Ren et al., Biochem Biophys Res Commun 335: 1051-1059 2005) and Xiangrong Ren (Ren et al., J Hepatol 44: 663- 670 2006), a method of inhibiting RNAi based HBV target gene expression is described in International Patent Publication WO0478181. The target genes include pre-Surface 1, pre-Surface 2, surface genes (coding HBsAg), pre-Core, and Core genes (coding HBeAg); Core gene (encoding HBcAg), P gene (encoding polymerase); And the X gene. In addition, a method comprising the step of inhibiting the expression of the Nox4 gene by introducing into the cell siRNA capable of complementary hybridization with the nucleotide of the Nox4 gene to inhibit the activity of NF-κB. Reported on 0020139.

However, in order to suppress NF-κB activity by regulating the NF-κB signaling pathway, a method of inhibiting the expression of the NIK (NF-κB inducing kinase) gene, which is a higher level of NF-κB in a host cell, is RNAi-based. It has not been reported yet. Therefore, the inventors of the present invention revealed that the isolated siRNA of the present invention inhibits the activity of NIK and NF-κB by directly injecting an isolated siRNA capable of complementarily hybridizing to the nucleotides of the NIK gene into a host cell. Completed.

An object of the present invention is to introduce a small interfering RNA (siRNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene into a host cell, thereby inhibiting NF-κB activity, thereby inhibiting HBV (hepatitis B virus). To treat liver disease).

In order to achieve the above object,

The present invention provides a small interfering RNA (siRNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene.

The present invention also provides a therapeutic agent for liver disease comprising the siRNA as an active ingredient.

The present invention also provides a method of inhibiting the activity of NF-κB by introducing the siRNA into the host cell at an effective concentration that inhibits the expression of the NIK gene.

In addition, the present invention provides a method for treating liver disease caused by HBV infection by administering the siRNA at an effective concentration that inhibits the expression of the NIK gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a small interfering RNA (siRNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene.

The siRNA is composed of 17 to 25 mer sense sequences selected from the mRNA base sequence (SEQ ID NO: 1) of the NIK gene and antisense sequences complementarily bound thereto, and the sense sequences described in SEQ ID NOs: 2, 4, and 6; It is preferred that the antisense sequence is complementary thereto.

The present invention also provides a therapeutic agent for liver disease comprising the siRNA as an active ingredient.

The present inventors have found that siRNA 1180, SEQ ID NO: 4 and SEQ ID NO: 2 and 3, which will inhibit the expression of NIK, a higher level of NF-κB, to block the NF-κB signaling system that progresses to liver cancer in chronic B-type infections. SiRNA 1269, described as 5 and siRNA 1397, as depicted in SEQ ID NOs: 6 and 7. First, MTT detection was performed to determine the efficacy of siRNA in cancer cell lines. Survival was reduced for siRNA in both A549 (human lung cancer cell line), HepG2 (human HepG2 liver cancer cell line), and HepG2.2.15 (human hepG2 liver cancer cell line stably transformed with HBV genome). 1). Subsequently, to examine whether NF-κB activity by HBV is inhibited by NIK-specific siRNA, a luciferase assay was attempted using a reporter plasmid. As a result, the activity of NF-κB in the cell line introduced into the siRNA of the present invention was reduced than the cell line transduced with pNIK DN (dominant negative), it can be seen that the activity of NF-κB is inhibited by 30% in all cell lines (See Figure 2). Although NF-κB has been shown to be a gene that affects the transition from chronic hepatitis B to liver cancer, NF-κB is also found in cell proliferation, B cell maturation, and inflammation. Plays an important role. Therefore, it was expected that the excavated siRNA only inhibited the activity of HBV-associated NF-κB, but the other cells could be inhibited.

Furthermore, RT-PCR was performed on cell lines transduced with siRNA. Decreased NIK expression was found in hepatocellular carcinoma cells, but not in lung cancer cells. In particular, mRNA levels of NIK genes were reduced by more than 50% by siRNA 1180 and siRNA 1397 in HepG2.2.15 cell lines infected with HBV. (See Figure 3).

In addition, siRNA 1397, which significantly reduced the mRNA expression level of the NIK gene in HepG2.2.15, was treated at different concentrations to investigate the inhibition of NIK expression in each cell line. As a result, in the lung cancer cell lines A549 and HepG2 cell line, no significant change in the expression level of NIK was observed by siRNA 1397, but in HepG2.2.15, the expression of NIK was decreased. It was confirmed that expression was suppressed (see FIG. 4). Therefore, the siRNA of the present invention can be used as an effective treatment of liver cancer derived from type B infection.

The siRNA is usually administered as a pharmaceutical composition. The administration can be performed by known methods, wherein the nucleic acid is introduced into the target cell in vitro or in vivo. In this case, the introduction of siRNA into the cell is not particularly limited, but siRNA may be directly incorporated into a host cell or transformed into a cell with a recombinant vector expressing siRNA, and the expression vector is not particularly limited thereto. Do not. Recombinant vectors expressing siRNAs will be viral vectors selected from the group consisting of plasmids or adeno-associated viruses, retroviruses, vaccinia viruses, and cancer cell soluble viruses.

Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods (Graham, FL and van der Eb, AJ (1973) Virol. 52, 456; McCutchan, JH and Pagano, JS (1968), J. Natl. Cancer Inst. 41, 35 1; Chu, G. et al. (1987), Nucl. Acids Res. 15, 1311; Fraley, R. et al. (1980), J. Biol. Chem. 255, 10431; Capecchi, MR (1980), Cell 22, 479). A recent addition to these techniques for introducing nucleotides into cells is the use of cationic liposomes (Felgner, P.L. et al. (1987), Proc. Nati. Acad. Sci. USA 84, 7413). Commercially available cationic lipid preparations are, for example, Tfx 50 (Promega) or Lipofectamin 20O (Life Technologies). The method of directly incorporating siRNA capable of hybridizing with the NIK gene directly into the cell is not particularly limited thereto, but may be lipofection for 15 to 40 minutes by mixing 2 to 6 µg of cationic liposomes per µg of siRNA. Is preferably performed.

The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances which facilitate the administration of pharmaceutically active substances. For example, an excipient can impart a shape or viscosity or can also act as a diluent. Suitable excipients include, but are not limited to, stabilizers, moisturizers, and emulsifiers, salts capable of varying osmolality, encapsulating agents, buffers, skin penetration promoters. Excipients and formulations for oral and parenteral drug delivery are presented in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

The composition may be used for diagnostic or therapeutic applications in human medicine or veterinary medicine. For diagnostic or therapeutic applications, the compositions may be in the form of solutions, such as injectable solutions, creams, ointments, tablets, suspensions and the like.

The composition may be administered in any suitable manner, such as by injection, oral, topical, nasal, rectal application, and the like. The carrier can be any suitable pharmaceutical carrier. Preferably, a carrier is used that can increase the efficiency with which the RNA molecule is introduced into the target-cell. Suitable examples of such carriers are liposomes, especially cationic liposomes. More preferred method of administration is injection.

The present invention also provides a method of inhibiting the activity of NF-κB by introducing the siRNA into the host cell at an effective concentration that inhibits the expression of the NIK gene.

The host cell can be an isolated cell that expresses the NIK gene or a cultured cell. Host cells can be included in a mammal without being isolated. Examples of mammals include non-human mammals (eg, dogs, cats, horses, pigs, sheep, cattle, goats, rodents; hamsters, mice, rats, and primates) and humans. Host cells expressing the target gene can be germ cells or somatic cells, totipotent or pluripotent cells, isolated or non-isolated cells, parenchyma cells or epithelial cells, immortal cells or transformed cells and the like. The host cell may be a stem cell or a differentiated cell. The host cell can be a cell derived from the liver of a mammal. In the present invention, human lung cancer cell line (A549), liver cancer cell line (HepG2), and liver cancer cell line (HepG2.2.15) in which the HBV genome was stably transformed were used in the hepatoma cell line.

The phrase “inhibition of gene expression” refers to the ability of gene silencing for the NIK gene of the siRNA of the invention. Inhibition of expression of the NIK gene is achieved when the test value based on the control is about 90%, preferably 50%, more preferably 25-0%. Suitable assay, for example, known to those skilled in the art techniques, for example, dot blot, Northern blot, in situ (in situ ) hybridization, ELISA, immunoprecipitation, enzyme function, as well as investigation of protein or mRNA levels using phenotypic assays known to those skilled in the art.

The "effective concentration" of an siRNA is an amount sufficient to provide a reduction in the expression of the NIK gene compared to the normal expression level detected in the absence of the desired effect, for example siRNA. siRNA may be introduced in an amount that allows delivery of at least one copy per cell. The higher the amount of double stranded material introduced (e.g., at least 5, at least 10, at least 100, at least 500, or at least 1000 copies per cell), the higher the inhibitory efficiency, and the lower the amount of introduction for specific applications. It may be advantageous.

The phrase "introduced into host cell" means that the siRNA of the present invention is introduced into a cell by "systemic delivery" or "local delivery" to a cell expressing the NIK gene. As used herein, "systemic delivery" refers to delivery that causes widespread biodistribution of a compound in an organism. Some dosing techniques cause systemic delivery to certain compounds, others may not. Systemic delivery means that a useful, preferably therapeutically effective amount of a compound is exposed to most of the body. In general, in order to obtain a broad biodistribution, fast nonspecific cell binding or by fast disassembly or removal of the compound (e.g., the first passage organ (liver, lung, etc.) before reaching the disease site far from the site of administration. Life expectancy in the blood is required. Systemic delivery of nucleic acid-lipid particles may be any means known in the art, including intravenous, subcutaneous, intraperitoneal, and in preferred embodiments, systemic delivery of nucleic acid particles is directed to intravenous delivery. Is made by As used herein, "topical delivery" refers to the delivery of a compound directly to a target site in an organism. For example, the compound can be delivered locally by injection directly into a disease site, such as a tumor or other target site, such as an inflammatory site or target organ, such as the liver, heart, pancreas, kidney, or the like.

In addition, the present invention provides a method for treating liver disease caused by HBV infection by administering the siRNA at an effective concentration that inhibits the expression of the NIK gene.

The siRNA of the present invention is preferred for the treatment of liver cancer caused by chronic hepatitis B.

As described above, siRNA capable of complementarily hybridizing with the mRNA of the NIK gene of the present invention inhibits the expression of the NIK gene and inhibits the activity of NF-κB, which is a lower signaling step, resulting in HBV infection. It can be used to prevent or treat a disease.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example 1> Preparation of siRNA hybridization to the NIK gene

SiRNAs that hybridize to the nucleotides of the NF-κB induced kinase (NIK) gene represented by SEQ ID NO: 1 are the most suitable oligomers among the candidate sequences obtained through the siRNA prediction program (http://rnaidesigner.invitrogen.com/rnaiexpress/). Based on the base sequence of

A specific nucleotide sequence of 19 nucleotides from NIK cDNA (SEQ ID NO: 1) was selected for siRNA preparation. Table 1 shows the sequences of siRNAs represented by SEQ ID NOS: 2 to 7, which complementarily hybridize to the nucleotides of the NIK gene of the present invention. SiRNAs having the sequence of Table 1 were prepared through oligonucleotide synthesis (Curebio, Korea).

siRNA sequence SEQ ID NO: name order SEQ ID NO: 2 sense of siRNA 1180 UCC AGU GGA CCU CUU CUC GAU ACU C SEQ ID NO: 3 antisense of siRNA 1180 GAG UAU CGA GAA GAG GUC CAC UGG A SEQ ID NO: 4 sense of siRNA 1269 UUU GAC AGC ACA CUG GAA GCC UGU C SEQ ID NO: 5 antisense of siRNA 1269 GAC AGG CUU CCA GUG UGC UGU CAA A SEQ ID NO: 6 sense of siRNA 1397 AGC AGU UCC AUG AAG AUG UUC ACC C SEQ ID NO: 7 antisense of siRNA 1397 GGG UGA ACA UCU UCA UGG AAC UGC U

Example 2 Host Cell Culture

Example 2-1 Host Cell Line

Host cells expressing the NIK gene of the present invention include A549 (human lung cancer cell line; KCLB-Korea Cell Line Bank, no.10185), HepG2 (human HepG2 liver cancer cell line; KCLB-Korea Cell Line Bank, no.58065) and HepG2.2.15 ( Liver cancer cell line stably transformed with HBV genome in human HepG2 liver cancer cell line; ATCC, USA).

A549 (human lung cancer cell line) is a RPMI1640 medium containing 10% fetal bovine serum (hereinafter, FBS; WelGENE Inc., Korea), 150 μg / ml of penicillin-streptomycin (Gibco-BRL, USA). Were grown in WelGENE Inc., Korea) and stored in a 37 ° C., 5% CO ₂ incubator (MCO-17A, SANYO, Japan).

HepG2 (human HepG2 liver cancer cell line) and HepG2.2.15 (liver cancer cell line stably transformed with HBV genome in human HepG2 liver cancer cell line) are DMEM (Dulbecco's Modified Eagle's) containing 10% FBS, 150 μg / ml penicillin-streptomycin. Medium) was grown in medium (WelGENE Inc., Korea) and stored in 37 ° C., 5% CO ₂ incubator.

Example 2-2 Host Cell Culture

The medium was aspirated out and trypsin-EDTA: trypsin-phenol (WelGENE Inc., South Korea) was mixed at a ratio of 1: 1, and an amount corresponding to 1/10 of the total volume of the cell culture flask was added. . After incubation for about 3 minutes in a 37 ° C., 5% CO ₂ incubator, it was centrifuged at 20 rpm for 5 minutes at room temperature (EBA3S, Hetlich, Germany). Aspirate out the medium again and fill with fresh medium.

Example 2-3 Freezing of Host Cells

The medium was aspirated off and the trypsin-EDTA: trypsin-phenol (WelGENE Inc., South Korea) was mixed at a ratio of 1: 1, and an amount corresponding to 1/10 of the total volume of the cell culture flask was added. After incubation for about 3 minutes at 37 ℃, 5% CO ₂ incubator, centrifuged at room temperature for 5 minutes at 20 rpm. Aspirate out the medium again and fill with fresh medium. Subsequently, freezing media (10% FBS + 10% DMSO; Sigma, USA) filtered using a 0.2 μm membrane filter (NALGENE, USA) was added and suspended. 1 ml aliquots were placed in cryo vials (NUNC, Denmark), and stored in cryo container (NALGENE, USA) for 1 to 2 days in a deep freezer (Sanyo, Japan) at -70 ° C. , Stored in a liquid nitrogen tank (MVExc 33/22, Chart Ind., USA).

Example 3 Survival Rate by siRNA Introduction

When the siRNA of the present invention was introduced into a human cancer host cell, an MTT test was performed to measure the relative survival rate of the host cell by the siRNA.

The host cells were prepared by inoculating the host cells cultured by the method of Examples 2-1 to 2-2 with 3 × 10 ⁵ cells in 6-well plates and exchanging them with serum-free medium after 24 hours. Next, 100 pM of siRNA was mixed with 250 μl of serum-free medium containing the host cell and cultured for 5 minutes to introduce into the host cell. In addition, 10 μl of the transfection solution (Lipofectamine ^™ 2000 transfection reagent, invitrogen, USA) was also incubated together for 5 minutes, mixed with the host cells, gently mixed, and then incubated for another 15 minutes. After another 5 hours, the serum-free medium was aspirated and drained, and serum-containing medium was added thereto and cultured for 24 hours. Through the above process, siRNA was stably transfected into cells. Host cells were incubated in 24 well plates (NUNC, Denmark) until 1 × 10 ⁴ , followed by an additional 24 hours. After 24 hours, 10 μl of stock MTT (Sigma, USA) solution (10 mg / ml in PBS) was added. After 3-4 hours, the medium and the MTT solution were removed and 100 μl of DMSO (Sigma, USA) was added to elute the MTT-formazan crystal. Finally, the survival rate was measured by measuring the absorbance at 570 nm (ELISA reader- Bio TEK, USA).

As a result, it was found that the survival rate in all cells regardless of the cell line was approximately 30% inhibited by the HBV-specific siRNAs of the present invention (FIG. 1).

Example 4 Inhibition of NF- κB Activity by Introducing siRNA

Example 4-1 Direct Incorporation of siRNA and Reporter Plasmids Complementary to and Complementary to the NIK Gene into Host Cells

The host cells were prepared by inoculating the host cells cultured by the method of Examples 2-1 to 2-2 with 3 × 10 ⁵ cells in 6-well plates and exchanging them with serum-free medium after 24 hours. Next, 4 μg of the NF-κB sensitive promoter luciferase reporter plasmid (Viromed, Korea) and 100 pM of siRNA were mixed with 250 μl of serum-free medium containing host cells and incubated for 5 minutes. Introduced into cells. In addition, 10 μl of the transfection solution (Lipofectamine ^™ 2000 transfection reagent, invitrogen, USA) was also incubated together for 5 minutes, mixed with the host cells, gently mixed, and then incubated for another 15 minutes. After another 5 hours, the serum-free medium was aspirated and drained, and serum-containing medium was added thereto and cultured for 24 hours. Through this procedure, the NF-κB-sensitive promoter luciferase reporter plasmid and siRNA were stably transfected into cells.

< Example  4-2> Luciferase  Essay

HBV-related NF-κB activity is associated with the upregulated NIK gene. To demonstrate that HBV-associated NF-κB activity is inhibited by NIK-specific siRNAs prepared by Example 1 of the present invention, a luciferase assay was performed using a pNF-κB-luciferase vector as a reporter plasmid.

Cells incubated for 24 hours were lysed with lysis buffer (25 mM Tris-Phosphate, 2 mM DTT, 2 mM EDTA, 1% Triton-X 100, 10% Glycerol, Protease Inhibitor, Distilled Water; Sigma, USA) and luciferase Add 100 μl of detection substrate (luciferase assay kit, Promega Corp., USA) to the luciferase detection tube (luciferase assay kit, Promega Corp., USA), and fluorescence using a luminometer (TUNER DESIGNS, USA). The degree was measured.

As a result, NF-κB activity in host cells transfected with siRNAs was reduced compared to that in host cells transfected with pNIK DN (dominant negative; plasmid that dominates the activity of NIK gene; Viromed, Korea). 2). In addition, in the A549 and HepG2.2.15 cell lines, the expression of NF-κB was inhibited by more than 30% by siRNAs, and more than 20% was observed in HepG2. In other words, NIK-specific siRNAs also showed a decrease in NF-κB activity, indicating that NIK and NF-κB are related, and that the siRNAs of the present invention inhibit both expression levels of NIK and NF-κB. Seemed to

Example 5 NIK Expression by siRNA Introduction

< Example  5-1> RNA Separation of

Cells transfected with the method of Example 3 were further incubated for one day and then centrifuged at 1500 rpm for 5 minutes (Scientific Industries, USA). To inhalate the medium, break the cells and extract RNA, 1 ml of Trizol (Invitrogen, USA) was added and then vortexed (Scientific Industries) for 10 seconds. Then, 200 μl of chloroform (Sigma, USA) was added and vortexed for 10 seconds. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 10 minutes at 4 ° C. The supernatant was transferred to a new tube (e-tube, Sorenson, USA) and 400 μl of isopropanol (Sigma, USA) was added. After inverting 2-3 times, the cells were incubated at room temperature for 10 minutes, and then centrifuged at 13,000 rpm for 10 minutes at 4 ° C. Subsequently, the supernatant was removed, 70% ethanol (Sigma USA) was added, followed by three times of purification after centrifugation at 4 ° C. for 10 minutes at 13,000 rpm after inverting 2-3 times. The supernatant was removed and dried for less than 5 minutes and 20-50 μl of DEPC (Sigma, USA) solution was added to give total RNA.

< Example  5-2> Reverse transcription Polymerase Chain Reaction  ( Reverse Transcription Polymerase  Chain Reaction ; RT - PCR )

2.25 μl of 2 × reaction buffer, RNA template, 1 μl forward primer, 1 μl reverse primer, 0.5 μl RT taq polymerase, 1.25 μl DEPC solution were added to the total 12 μl in the e-tube (RT-PCR kit, Invitrogen , USA).

Each primer is as follows.

GAPDH specific omni-directional (5'- CTCAGACACCATGGGGAAGG-3 '), SEQ ID NO: 8

GAPDH specific reverse (5′- GTGGCAGTGATGGCATGGA-3 ′), SEQ ID NO: 9

NIK specific omni-directional (5'- TACGCCACTCACGAAGGAT-3 '), SEQ ID NO: 10

NIK specific reverse (5'- CAAGGGAGGAGACTTGTTTG-3 '), SEQ ID NO: 11

After keeping the e-tube in ice to maintain a low temperature, PCR was performed in the PCR actuator (MJ mini, BioRad, USA) under the following conditions.

CDNA synthesis of GAPDH was performed at 55 ° C for 30 minutes, initial denaturation at 94 ° C for 2 minutes, then denaturation at 94 ° C for 15 seconds, annealing at 60 ° C for 30 seconds, and elongation at 68 ° C for 1 minute ( The procedure of extention) was repeated 40 times and PCR was performed by final elongation at 68 ° C. for 5 minutes.

NIK performed cDNA synthesis for 30 minutes at 55 ° C, initial denaturation at 95 ° C for 5 minutes, followed by denaturation at 95 ° C for 50 seconds, annealing at 60 ° C for 50 seconds, and stretching at 72 ° C for 50 seconds. PCR was performed by final elongation at 72 ° C. for 7 minutes.

PCR results stained with loading dye were electrophoresed on 1% agarose gel (ROTH, USA) (BioRad, POWER PACK 300, USA). Based on the amount of GAPDH based on the amount of the PCR product constant, the degree of change in the amount of NIK expression was observed.

As a result, the decrease in NIK activity was found in liver cancer cells, but not in lung cancer cells. In other words, mRNA expression level of NIK gene could not be observed in A549 human lung cancer cell regardless of siRNA type, whereas mRNA expression level of NIK gene was decreased by siRNA in HepG2 and HepG2.2.15 human liver cancer cell lines. I could see it. In HepG2 cells, the mRNA expression of the NIK gene was dim but slightly decreased by siRNA 1180. In contrast, the mRNA expression of the NIK gene was reduced by more than 50% in HepG2.2.15 cells by siRNA 1180 and siRNA 1397. That is, the mRNA expression of the NIK gene was reduced by 100 pM of NIK-specific siRNA 1180 and siRNA 1397 (FIG. 3).

< Example  5-3> siRNA  Liver Cancer Cell Specificity of 1397 NIK  Inhibit gene expression

In addition, mRNA expression of the NIK gene was expressed only in HepG2.2.15, a cell line that transformed the HBV genome to develop liver cancer to confirm whether the siRNA of the present invention specifically inhibited NF-κB activity by NIK activated by HBV. Only siRNA 1397 which significantly reduced the amount was selected and treated with different concentrations at 250 pM, 12.5 pM, 0.625 pM and 0.0625 pM, and further PCR was carried out in the same manner as in Examples 5-1 and 5-2.

As expected, no significant change in the mRNA expression level of the NIK gene was detected by siRNA 1397 in the A549 and HepG2 cell lines. In addition, in the HepG2.2.15 cell line, the mRNA expression level of the NIK gene was most markedly reduced when the siRNA 1397 was treated with 250 pM (FIG. 4).

As a result of the analysis by the above method, it was found that siRNA 1397 of the present invention reduced the expression level of the mRNA of the NIK gene, thereby inhibiting the activity of NIK and NF-κB.

Figure 1 shows the survival rate of cancer cells by introduction of siRNA of the present invention in A549, HepG2 and HepG2.2.15 MTT (3- [4,5-dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide) It is a result diagram measured by the detection method,

FIG. 2 shows the introduction of NF-κB by the introduction of siRNA of the present invention in human lung cancer cell line (A549), liver cancer cell line (HepG2) and liver cancer cell line (HepG2.2.15) stably transformed with HBV genome in the hepatoma cell line. It is the result of measuring the extent to which activity was suppressed by the luciferase detection method,

Figure 3 is a result of measuring the expression of the NIK gene by the introduction of the siRNA of the present invention in A549, HepG2 and HepG2.2.15 by RT-PCR method,

Figure 4 is a result of measuring the expression level of the NIK gene by the introduction of the siRNA 1397 concentration of the present invention in A549, HepG2 and HepG2.2.15 by RT-PCR method.

<110> KIM, JIN-SEOK <120> siRNA of NIK gene and therapeutic agent for liver diseases          configuring <130> 7P-05-43 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 2829 <212> DNA <213> Homo sapiens <400> 1 atggccgtga tggaagtggc ctgcccgggc actcctgggt cagcagtcgg gcagcagaag 60 gagcttgcca aagccaagga gaagacacag tcactgggga agaagcagag ctgcatcttc 120 aagcttgagg ccgtggagaa gagccccgtg ttctgtggga agtgggagat cctaaacgac 180 gtgatcacca aaggcacagc caaggacggc tctgagggag gaccaccggc catctccatc 240 atcgcccagg ctgaatgtga gaatagccaa gagttcagcc ccaccttctc agagcgcatt 300 ttcatcgcgg ggtcacagca gtacagccag tctgagagtc tcgatcaaat ccccaacaat 360 gtggcccatg caactgaagg caaaatggcc cgtgtgtgcc ggaggggaaa acgtcacggc 420 aaagcccgaa aaaaacgtag gaagaagagg tcgaagtcac tggcccaggc aggagtggcc 480 ttagccaagc ccctgcccag aacccctgag caagagagct gtaccatccc agtacaggaa 540 gatgagtctc cactaggcaa cctctatgcc agaaatgtct cccagttcac caagcctctg 600 gggggaccag gccttggcca cctgtgcttt aagaaacaag atgaaggcct gcgaccggta 660 ctgcctcgac cagaactcca caaactgatc agccccttgc aatgtctaaa ccacgtgtgg 720 aaactccacc acccccaggc cacaggcccc cggccccacc cgactcaccc cttcccctac 780 agcggaatgc cccatccttt cccattctac cccctggagc cctggaaacc ctatatgctg 840 gactctgccg tcctggacaa actagccggt gtcagcggcc agcggcctct gcctggccca 900 ccgcatctaa gccaactggc ccatggagac agtcagaagc cgctgcctgg cccacacctg 960 gagtccagct gcccgtctcg gggtgcccta gaaaaggttc ccgtggagga atacctggtg 1020 catgcgctcc aaggaagtgt gagctcaggc caggcccaca gcctggccag cctggctaag 1080 acatggtcct cgggaagcgc caagctgcag aggctcggcc ccgaaactga ggacaacgag 1140 ggggtcctgc ttactgagaa actcaagcca gtggattatg agtatcgaga agaggtccac 1200 tggatgacac accagcctcg ggtgggcaga ggctccttcg gcgaggtcca cagaatgaag 1260 gacaagcaga caggcttcca gtgtgctgtc aaaaaggtac gactcgaggt gtttcgggta 1320 gaggaactag tggcctgtgc tggtctgagc tcgcccagaa tcgtccctct ctatggagct 1380 gtgagagaag gcccgtgggt gaacatcttc atggaactgc tagaaggtgg ctcgctgggt 1440 cagctcataa agcaaatggg ctgtctgcca gaagaccgag ccctttacta cctgggccag 1500 gccctggagg ggctggagta cctccacaca cgcaggattc tgcatggcga tgtcaaagct 1560 gacaacgtgc tcctgtccag tgatggaagc cgagcggccc tctgcgactt tggccacgcc 1620 ttgtgcctgc aacctgacgg cctagggaaa tccttgctca caggggacta cattcctggc 1680 acggagaccc acatggcacc agaagtggtg atgggaaagc cctgcgatgc caaggtggac 1740 atctggagca gctgctgcat gatgctccac atgctcaacg gctgccaccc ctggactcag 1800 tacttccgag gcccgctttg tctcaagatt gccagcgagc ctccaccgat cagggagatc 1860 ccaccttcct gcgcacccct cacagcccag gccatccaag aggggctgag gaaagagccc 1920 gtccaccgag catctgccat ggagcttcgg aggaaagtgg gcaaggcact acaggaagtg 1980 ggaggtctga aaagcccttg gaaaggagaa tataaagaac caagacctcc accccaagac 2040 caagccacct gccaccagac cctacctact ccgccgagag agaacccacc agccaaggcc 2100 aacacagacg gggctcctga gcctcagcct cctctaccgc cagaaccacc agaaccgagc 2160 aaagcgccag ccctgaacct gagcaaggag gagtctggca catgggaacc cctgcctctg 2220 tcctccctgg acccagccac tgccaaaggc cccagcttcc cagaccggag ggcaaccttg 2280 ccagagctgg agctacagca actggagata gaactgtttc tcaacagcct gtcccagccg 2340 ttctctctgg aggaacagga acaaatcctc tcctgcctca gcatcgacag cctctcgctg 2400 tcagatgaca gtgaaaagaa tccatcgaag gcctctcaga gctcacggga caccctgagt 2460 tctggcgtgc actcttggaa cagccaagct gaggcaagaa cctgcagctg cagcacggcg 2520 ctggcccggg ggcggcctac tgacatcccg agctacttca acggggtcaa ggtccagatc 2580 cagtctctca atggcgaaca cctgcatatc cgggaattcc accgcgtcaa ggtgggagac 2640 attgccaccg gcatcagcag ccagatccca gccacagctt tcagcctggt gaccaaagat 2700 ggacagcctg tttgctatga catggaggtg ccagactcgg gcatcgacct gcagtgcacc 2760 ctggcccctg atggcagctt tgcttggacc tggagggtca agcatggtca gctggagaac 2820 cgaccctag 2829 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Sense primer of siRNA 1180 <400> 2 uccaguggac cucuucucga uacuc 25 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Antisense primer of siRNA 1180 <400> 3 gaguaucgag aagaggucca cugga 25 <210> 4 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Sense primer of siRNA 1269 <400> 4 uuugacagca cacuggaagc cuguc 25 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Antisense primer of siRNA 1269 <400> 5 gacaggcuuc cagugugcug ucaaa 25 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Sense primer of siRNA 1397 <400> 6 agcaguucca ugaagauguu caccc 25 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Antisense primer of siRNA 1397 <400> 7 gggugaacau cuucauggaa cugcu 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH specific forward primer <400> 8 ctcagacacc atggggaagg 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH specific reverse primer <400> 9 gtggcagtga tggcatgga 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NIK specific forward primer <400> 10 tacgccactc acgaaggat 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NIK specific reverse primer <400> 11 caagggagga gacttgtttg 20  

Claims (13)

By specifically complementing the mRNA of the NIK (NF-κB induced kinase) gene to inhibit the expression of NIK mRNA, it inhibits the activity of NIK in liver cancer cells infected with hepatitis B virus (HBV) , SiRNA (small interfering RNA) consisting of the sense RNA set forth in SEQ ID NO: 6 and the corresponding antisense RNA. delete delete delete delete delete delete delete delete delete delete delete delete

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