KR20150103874A - Use of miR-101 for NLK expression inhibitor - Google Patents

Abstract

The present invention relates to a use of miR-101 as a NLK expression inhibitor and, more specifically, to a novel use of miR-101 which can treat liver cancer by inhibiting NLK expression and cell proliferation of a liver cancer cell line through miR-101 binding to NLK 3′UTR. The present invention first investigates miR-101 is a regulator which inhibits NLK expression, wherein the miR-101 which inhibits NLK expression can be helpfully used in a method for preventing or treating liver cancer induced by NLK overexpression and can be effectively useful in treating other various diseases induced by NLK overexpression.

Description

Use of miR-101 as an inhibitor of expression of NLK {Use of miR-101 for NLK expression inhibitor}

The present invention relates to the use of miR-101 as an inhibitor of the expression of NLK, and more particularly to the use of miR-101 as an inhibitor of NLK, and more particularly, to a method for inhibiting the proliferation of miR- 101 < / RTI >

Liver cancer is the fifth most common cancer in men and the seventh most common cancer in women. It is the world's second most serious cancer worldwide. Of these, hepatocellular carcinoma is the most common hepatocellular carcinoma, and more than 500,000 new cases are reported. Although hepatitis B and hepatitis C viruses, aflatoxin B1 and alcohol are known to be the main causes of hepatocellular carcinoma, the survival rate of patients with hepatocellular carcinoma is very low despite the research of 20 years. Many previous studies have shown that abnormal regulation of cell growth signaling pathways such as ERK / MAPK pathway and Wnt / β-catenin pathway is closely correlated with hepatocellular carcinoma tumor formation, The molecular mechanism of onset is still poorly understood.

NLK (Nemo-like kinase) is an evolutionarily conserved MAP-kinase-like kinase that regulates a variety of signaling processes through the phosphorylation of several transcription factors. NLK is known to regulate the Wnt / β-catenin signaling pathway and the Notch signaling pathway in mammalian cell lines and regulate many transcription factors. Recently, NLK expression and activity have been reported to correlate with cancer progression, but there is no consensus on the role of NLK in tumorigenesis. The present inventors have previously reported that NLK is overexpressed in hepatocellular carcinoma and that NLK positively regulates the expression of cyclin D1, a key component of cell cycle regulation. Activated Wnt / β-catenin signal is associated with malignant mutation and proliferation of hepatocytes. NLK is known to promote cyclin D1 expression by positively regulating Wnt / β-catenin signaling in neuro-2a cell lines. Thus, NLK could enhance Wnt / β-catenin signaling activity and contribute to the growth of hepatocellular carcinoma. Conversely, NLK functions as a tumor suppressor in prostate cancer and glioma. Thus, the function of NLK is tumor specific and its function may vary depending on the stage of tumor progression.

MicroRNAs (miRNAs) are non-coding RNAs composed of 21 to 25 nucleotides that bind to the 3'-untranslated region of the target gene mRNA to induce mRNA degradation and inhibit mRNA translation do. Much research has shown that miRNAs play a crucial regulatory role in the progression of many human cancers, and the loss and gain of miRNA function are upregulated on oncogenes or tumor suppressor genes It is known to induce silencing and to affect cancer development. Abnormal expression of miRNAs such as miR-125, miR-29 and miR-221 in hepatocellular carcinoma is known to regulate tumor cell growth, apoptosis, cell migration and invasion process. However, miRNAs that regulate NLK in liver cancer have not yet been identified.

In order to identify miRNAs targeting NLK in hepatocarcinogenesis, the present inventors applied miRNA profiles in human hepatocellular carcinoma tissues to miRWARK miRNA target prediction program and National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database Whereby the present invention can be completed.

Korean Patent No. 10-131557

Accordingly, an object of the present invention is to provide a novel method for preventing or treating liver cancer by inhibiting the expression of NLK (nemo-like kinase).

Another object of the present invention is to provide a novel method for inhibiting the expression of NLK (nemo-like kinase) which is a pathogenic factor of liver cancer.

It is still another object of the present invention to provide a composition for inhibiting NLK expression.

In order to achieve the above object, the present invention provides a method for preventing or treating liver cancer by inhibiting expression of NLK (nemo-like kinase), comprising the step of treating miR-101 comprising the nucleotide sequence of SEQ ID NO: 1 A method for preventing or treating liver cancer comprising the steps of:

In one embodiment of the present invention, miR-101 may directly bind to the 3'UTR of NLK to inhibit the expression of NLK.

In one embodiment of the present invention, the miR-101 may inhibit cell growth of a liver cancer cell line.

In addition, the present invention provides a method for inhibiting the expression of NLK (nemo-like kinase), which is a pathogenic factor of liver cancer, by treating miR-101 consisting of the nucleotide sequence of SEQ ID NO: 1 in vitro.

In one embodiment of the present invention, miR-101 may directly bind to the 3'UTR of NLK to inhibit the expression of NLK.

Further, the present invention provides a composition for inhibiting NLK expression comprising miR-101 consisting of the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, miR-101 may directly bind to the 3'UTR of NLK to inhibit the expression of NLK.

The present invention is the first to demonstrate that miR-101 is a regulator that inhibits the expression of NLK. MiR-101 inhibits the expression of NLK and is useful as a therapeutic method for preventing or treating liver cancer induced by overexpression of NLK And may be effectively utilized for the treatment of various diseases induced by overexpression of other NLKs.

Figure 1 shows abnormal regulation of NLK in human hepatocellular carcinoma. (A) The expression of NLK mRNA in hepatocellular carcinoma was confirmed by RT-PCR. GAPDH means loading control, N means non-tumor tissue, and T means tumor tissue. (B) Expression of NLK protein in human liver cell line was confirmed by Western blotting, and GAPDH means loading control. (C) Results of analysis of microarray data from GEO (Gene Expression Omnibus) database. GSE data sets with accession numbers of GSE 14520 and GSE 36376 show that NLK mRNA expression is significantly different in hepatocellular carcinoma and non-tumor groups (unpaired Student t- test, mean ± SD, * P <0.05; * P < 0.01; *** P < 0.001). (D) GAPDH was used as loading control as a result of Western blotting of Hep3B with negative control siRNA (NC) and two different Dicer-siRNAs (si-Dicer # 1, # 2)
Figure 2 identifies miRNAs targeting NLK in human hepatocellular carcinoma, and miR-101 was down-regulated in hepatocellular carcinoma. (A) miRNA expression based on microarray in primary hepatocellular carcinoma (n = 16) and normal liver tissue (n = 8). The log ₂ intensity was assigned to each category and expressed as a scatter plot, These differences were determined by unpaired Student t test (* P <0.05, vs non-tumor). Primary miRNA microarray data is available in the NCBI, GEO database (Accession No. GSE 39678). (B) Positive miRNA expression in large cohort hepatocellular carcinoma patients. MiRNA microarray data were obtained from NCBI, GEO database (Accession No. GSE22058). Relative expression of miRNAs was visualized as a scatter plot in patients with non-tumor and hepatocellular carcinoma tumors. The mean expression level of each group was expressed horizontally. miRNA expression levels are indicated in the markers (log2 intensity). Differential expression of miRNA in hepatocellular carcinoma was determined by unpaired Student t test (ns; no significant and ** P <0.01 and *** P <0.001 versus Non-tumor). (C) The Kaplan-Meier survival curve of the GSE31384 data set showed that the 5-year survival rate of miR-101 highly expressed patients in tumor tissues was significantly higher than that of non-tumor tissues (*** P <0.001). The expression of miR-101 in tumor tissues was normalized to U6 snRNA (mean ± standard deviation, n = 3, *** P &Lt; 0.001). The results of qRT-PCR analysis of miR-101 in (E) normal hepatocyte (MIHA, HLK-3) and subgroup of hepatocellular carcinoma cell lines (Hep3B, Huh7, PLC / PRF / 5, SK- HepI, SNU- Expression of miR-101 was normalized to U6 snRNA (mean ± standard deviation, n = 3, *** P <0.001).
Figure 3 shows the results of confirming that miR-101 functions as an internal regulator of NLK in hepatocellular carcinoma. (A) shows the target site of NLK 3'UTR by miR-101 and (B) shows the wild type 3'UTR After insertion into the psi-CHECK-2 plasmid, co-transfection was performed with Hep3B and Huh7 cell lines with miR-101 and luciferase assay was performed. All analyzes were performed in triplicate (mean ± standard deviation, n = 3, *** P <0.001). (C) Hep3B and Huh7 cell lines were transfected with miRNA mimics of 100 nM miR-101, and the amount of miR-101 expression was analyzed by qRT-PCR. In this case, small nuclear RNA U6 was used for normalization (mean ± standard deviation, n = 3, * P <0.05; ** P <0.01; *** P <0.001). (D) Hep3B cell line was transfected with 100 nM si-Dicer transfection and 100 nM miRNA mimic. Western blot analysis was performed to analyze the amount of Dicer and NLK protein expression, and GAPDH was used as a loading control.
Figure 4 shows the results of (A) and (B) BrDU incorporation assay that MiR-101 directly inhibited the growth of hepatocellular carcinoma cells by targeting NLK directly. Ectopic expression of miR-101 showed cell proliferation of Hep3B and Huh7 Respectively. The upper panel shows NLK inhibition by ectopic expression of miR-101 by Western blotting. The lower panel shows the cell proliferation after 24 and 28 hours after miR-101 transfection by BrdU assay (mean ± SD, n = 3, ** P &lt;0.01; *** P < 0.001). (C) and (D) were the results of MTT assay for the inhibition of Hep3B and Huh7 cell growth by the ectopic expression of miR-101 (mean ± standard deviation, n = 3, ** P <0.01; P < 0.001).

Hereinafter, the present invention will be described in detail.

NLK (Nemo-like Kinase) is a nemo-like kinase that is involved in cell motility (omatidia rotation) in the development of fruit flies, while wild-type flies form a hexagon- The mutation forms a square eye. The vertebrate homolog of the nemo gene was discovered by the Brott team in 1998, and it was named "Nemo-like kinase" and NLK was found to be evolutionarily conserved from larva to human. NLK phosphorylates LEF1 in human cell lines and positively regulates the Wnt / β-catenin signaling pathway. In addition, NLK regulates many transcription factors such as c-Myb and Foxo. In human cancer, NLK is known to function as a tumor suppressor in the progression of various cancers. For example, it is known that the ectopic expression of NLK induces apoptosis and inhibits androgen receptor-mediated prostate cancer. NLK, Inhibit cell proliferation and cell migration activity. In addition, overexpression of NLK has also been reported to inhibit the growth of cancer cells in human colon cancer and induce apoptosis.

Meanwhile, the present inventors have previously found that NLK is abnormally regulated in hepatocellular carcinoma and NLK cleavage inhibits cell division of tumor cells by inhibiting transcription of cell cycle regulators such as CDK2 and cyclin D1 in liver cancer There is a bar. Thus, the possibility of NLK as a carcinogenesis in cancer progression is closely related to the fact that NLK inhibits lung cancer progression by negative mutation of Wnt signaling and functions as a major regulator of malignant growth of gallbladder cancer cells.

Therefore, in order to investigate the mechanism of NLK regulation, the miRNA expression data set of hepatocellular carcinoma can be used to study the mechanism of regulation of NLK, which plays an important role in the development and progress of cancer, , And that miR-101 functions as a major negative regulator of hepatocellular carcinoma. That is, ectopic expression of miR-101 was found to inhibit the expression of NLK in hepatocellular carcinoma cell lines.

miRNAs regulate gene expression in small noncoding RNAs that are conserved evolutionarily. Much research has shown that miRNA expression is down-regulated in cancer, miRNA overexpression or silencing is closely related to cancer development have.

The present inventors confirmed that NLK protein expression was increased by inducing knockdown of Dicer, an miRNA-producing enzyme, and it was found that up-regulation of NLK in the late transcription stage inhibited or lost miRNAs selectively targeting NLK in hepatocarcinoma And the like.

Thus, we have identified four miRNAs targeting HDAC2 that are downregulated significantly in hepatocellular carcinomas through extensive miRNA expression analysis of human hepatocarcinoma cells (see FIG. 2). Among them, miR-101 was found to function as a tumor suppressor and inhibit the expression of NLK in cells by inhibiting NLK, a carcinogenesis gene in hepatocellular carcinogenesis (see FIGS. 3 and 4).

In the present invention, it was confirmed that miR-101 acts as a tumor suppressor in hepatocarcinoma cells and inhibits proliferation and growth of hepatocellular carcinoma cells. In particular, miR-101 functions as a tumor suppressor by inhibiting NLK overexpression in hepatocarcinogenesis. Expression of miR-101 is down-regulated in hepatocellular carcinoma cell line. Ectopic expression of miR-101 is expressed in NLK mRNA And suppress tumor growth by directly inhibiting translation of the tumor.

Accordingly, the present invention provides a method for preventing or treating liver cancer by inhibiting the expression of NLK (nemo-like kinase), comprising the step of treating miR-101 comprising the nucleotide sequence of SEQ ID NO: 1 to prevent or treat liver cancer . &Lt; / RTI &gt;

In one embodiment of the present invention, the miR-101 binds directly to the 3'UTR of NLK to inhibit the expression of NLK, thereby inhibiting the cell growth of the liver cancer cell line.

In addition, the present invention provides a method for inhibiting the expression of NLK (nemo-like kinase), which is a pathogenic factor of liver cancer, by treating miR-101 consisting of the nucleotide sequence of SEQ ID NO: 1 in vitro.

The present invention also provides a pharmaceutical composition for inhibiting NLK expression comprising miR-101 comprising the nucleotide sequence of SEQ ID NO: 1.

Accordingly, the NLK expression inhibiting method and the composition for inhibiting NLK expression as described above can be utilized as a therapeutic method and a pharmaceutical composition for treating various diseases known to be caused by overexpression of NLK.

Diseases known to be caused by overexpression of NLK include, but are not limited to, liver cancer.

The pharmaceutical composition of the present invention may be administered together with a pharmaceutically acceptable carrier. In the oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, A buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed and used for injections, and a base, an excipient, a lubricant and a preservative may be used for topical administration. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms.

The effective dosage range of the pharmaceutical composition of the present invention may vary depending on various factors such as sex, severity, age, administration method, target cell, expression level, and the like, and can be easily determined by those skilled in the art .

The pharmaceutical composition according to the present invention is orally or parenterally administered to humans and animals such as intravenously, subcutaneously, intranasally or intraperitoneally. Parenteral administration includes injection and drip, such as subcutaneous, intramuscular, and intravenous. Other pharmaceutical compositions of the invention may be prepared according to techniques commonly used in the form of various formulations.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Material preparation

A total of 10 hepatocellular carcinoma tissues and corresponding normal tissues were obtained from patients at Yonsei University College of Medicine. Informed consent was obtained in accordance with the Helsinki Declaration on the obtaining of samples. A written informed consent was obtained from all patients and the present invention was conducted under the approval of the Institutional Review of Board of the College of Medicine, Catholic University of Medicine (IRB approval number: MC12EISI0106). MiRNA101 used in the present invention is miR-101-3p (Accession No .: MIMAT0000099) and has 5'-UACAGUACUGUGAUAACUGAA-3 '.

< Example  2>

Cell culture

Human hepatocellular carcinoma cell lines Hep3B, Huh7, PLC / PRF / 5, SK-Hep-I and SNU-182 were purchased from KCLB (Korean Cell Line Bank, Seoul, South Korea). Liver cell lines MIHA and HLK-3 were purchased from ATCC (Manassas, VA, USA). Each cell line was cultured in RPMI 1640 or DMEM medium supplemented with 10% FBS (Sigma, St. Louis, MO) and 100 units / ml penicillin-streptomycin (Invitrogen, Carlsbad, Calif. And cultured in a 5% CO ₂ , 37 ° C wet incubator.

< Example  3>

Statistical analysis

All experiments were performed at least three times, and all samples were tested three times or more and analyzed statistically. Results were expressed as mean ± standard deviation, and statistical differences between the experimental groups were evaluated by unpaired two-tailed student t-test using Graphpad ^™ 5.0 software. Only P <0.05 was considered as statistically significant data.

< Example  4>

In liver cancer tissue and surrounding normal tissue NLK Expression level  compare

In order to confirm the overexpression of NLK in hepatocarcinoma cell line, expression pattern of NLK mRNA was confirmed by RT-PCR in 10 hepatocellular carcinoma cell lines and liver normal tissue cells adjacent to hepatocarcinoma.

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, Calif.) And 1 μg of total RNA was reverse transcribed with cDNA using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, Calif.) According to the manufacturer's protocol. PCR was performed with nTaq-tenuto DNA Polymerase (Enzynomics, KP) and GAPDH gene was used as a control.

As a result, as shown in FIG. 1A, the NLK expression of the hepatocellular carcinoma cell line was significantly increased in all 10 samples compared with the NLK expression in the surrounding normal tissues.

In addition to the above experiments, we further examined the expression of NLK protein in normal liver and hepatocellular carcinoma cell lines.

For this, the cells were suspended in protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na ₂ P ₂ O ₇ , 1 mM Na ₃ VO ₄ , (Bio-Rad, Hercules, Calif., USA). The lysates containing the same amount of protein were analyzed by SDS-PAGE. ). The blots were then blaked with 5% skim milk solution and reacted with anti-NLK and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) antibody. Immobilon ^™ Western blotting detection system (Millipore, MA) was used to identify the bound antibody. The intensity of the band was quantified by LAS 3000 (Fuji Photo Film Co., Japan).

As a result, as shown in FIG. 1B, relatively high NLK expression patterns were observed in human hepatoma cell lines Hep3B, Huh7, PLC / PRF / 5, SK-Hep1 and SNU-182 as compared with normal liver cell lines MIHA and HLK- Respectively.

NLK gene expression was confirmed in a large population of patients with hepatocellular carcinoma identified through National Center for Biotechnology Information (NCBI) and Gene Expression Omnibus (GEO) databases (accession numbers: GSE14520, GSE36376, GSE22058 and GSE22097) 1C, the amount of NLK mRNA was significantly increased compared with the normal control.

< Example  5>

NLK  As an expression regulator miRNA The presence of

A series of processes known to be associated with cancer, such as apoptosis, cell proliferation, cell survival and metastasis, are known to be regulated by miRNAs, small regulated non-coding RNAs composed of approximately 19-25 nucleotides. Thus, the fact that NLK is expressed in hepatocellular carcinomas may be normalized by intracellular miRNAs that selectively regulate NLK mRNA translation in liver cells.

To determine whether NLK expression was regulated by miRNAs in hepatocellular carcinoma cell lines, we transfected Dicer specific siRNAs (si-Dicer # 1, # 2) to inhibit control siRNA (NC) and miRNA production in Hep3B cell line .

siRNA was synthesized in Genolution (seoul, Korea) and control RNA duplex was purchased from Ambion (Austin, TX). Transfection was carried out using lipofectamine 2000 and RNAiMAX (Invitrogen, Carlsbad, Calif.) According to the manufacturer's protocol.

As a result, as shown in FIG. 1D, knockdown of Dicer in Hep3B hepatocellular carcinoma cell line was found to increase the amount of NLK protein expression. Therefore, miRNAs that regulate NLK were present inside these cells.

< Example  6>

Bioinformatics  Used NLK  As an expression regulator miR -101 Identification

Gene expression and miRNA expression profiling data sets were obtained from National Center for Biotechnology Information (NCBI) and Gene Expression Omnibus (GEO) to identify miRNAs that regulate NLK in hepatocellular carcinoma cell lines (Accession No. GSE 14520, GSE 25097, GSE31376, GSE22058, GSE10694, GSE21362 and GSE31384).

MiRWALK (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was applied to identify miRNAs that regulate NLK in hepatocellular carcinoma cell lines. First, eight prediction programs (RNA22, miRanda , miRDB, TargetScan, RNAhybrid, PITA, PICTAR, and Diana-microT), 33 miRNAs targeting 3'UTR of NLK were identified in at least seven programs. Of these, the four miRNAs miR-101, miR-140-5p, miR-152 and miR-181a, which are significantly down-regulated in the hepatocellular carcinoma control compared to the normal control, were confirmed in the HCC miRNA signature data set (GSE 39678 2A).

On the other hand, confirmation of the expression patterns of these miRNAs in two different GEO data sets (GSE 22058 and GSE 21362) confirmed that only miRNA-101 was down regulated in two different HCC populations (see FIG. 2B).

In addition, the Kaplan-Meier survival curves of patients with hepatocellular carcinoma showed that the 5-year overall survival of patients exhibiting low miR-101 expression was much lower than that of patients exhibiting high miR-101 expression (see FIG. 2C). On the other hand, other miRNAs in hepatocellular carcinoma patients did not show a significant correlation with survival rate.

We next examined the expression of miR-101 in 10 randomly selected hepatocellular carcinoma tissues and adjacent normal tissues in order to confirm miR-101 inhibition in patients with hepatocellular carcinoma. As a result, miR-101 expression of nine hepatocellular carcinoma tissues among 10 hepatocellular carcinoma tissues was found to be decreased as compared with normal liver tissues (see Fig. 2D).

In addition, the expression of miR-101 was inhibited in liver cancer cell lines as compared to normal liver cell lines (see FIG. 2E).

In view of these results, the present inventors confirmed that miR-101 is an miRNA that plays an important role as a regulator of NLK expression in liver tumorigenesis.

< Example  7>

miR -101 NLK  Tumor Inhibition by Expression Control

To confirm whether miR-101 specifically regulates NLK expression through direct binding to the 3'UTR of the NLK gene, the 3'UTR of NLK was compared with the expression level of psiCHECK2-NLK_3'UTR wild type (NLK 3'UTR) And cloned into a reporter vector. This vector was transfected into liver cancer cell lines Hep3B and Huh7 cell lines. As a result, miR-101 was found to inhibit the activity of the reporter gene in both Hep3B and Hu7 cell lines (see Figs. 3A and 3B). From these results, it was confirmed that miR-101 can regulate NLK expression through direct binding with 3'UTR of NLK in liver cancer cell line.

On the other hand, it was confirmed that ectopic expression of miR-101 could affect NLK inactivation in hepatocarcinoma cell line. As a result, it was found that overexpression of miR-101 showed an inhibitory effect of NLK (FIGS. 3C and 3C) 3D).

< Example  8>

miR -101 on Hepatocellular Carcinoma Cell Line Growth

These results indicate that miR-101 is a NLK-specific modulator in the liver. Therefore, the present inventors tried to confirm whether ectopic expression of miR-101 affects cell growth of liver cancer cell line.

For this purpose, Hep3B and Huh7 cells were plated in 24 well plates and siRNA or miRNA were transfected for 72 hours. Bromodeoxyuridine (BrdU) incorporation assay was performed with BrdU cell proliferation assay kit (Millipore, MA) according to the manufacturer's method.

As a result, miR-101 expression suppressed NLK expression and effectively suppressed the proliferation of Hep3B and Huh7 cell lines (see FIGS. 4A and 4B).

In addition, to further confirm the effect of miR-101 on cell proliferation of liver cancer cell lines, Hep3B and Huh7 cells were cultured for 16 hours in a 6-well plate at about 30%, and siRNA and miRNA were transfected , Treated with 5 mg / ml of MTT, and reacted at 37 ° C. After this, the cormazan crystal was dissolved in DMSO and the absorbance was measured with a VICTOR3 ^TM multilabel plate reader (PerkinElmer, Boston, Mass.). As a result, it was confirmed that miR-101 effectively inhibited cell growth of Hep3B and Huh7 cell lines (see FIGS. 4C and 4D).

These results indicate that miR-101 can act as a tumor suppressor that regulates the abnormal activity of NLK in hepatoma production as a direct regulator of NLK in the cells.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Use of miR-101 for NLK expression inhibitor <130> PN1401-009 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> miR-101-3p RNA sequence <400> 1 uacaguacug ugauaacuga a 21

Claims (7)

A method for preventing or treating liver cancer by inhibiting the expression of NLK (nemo-like kinase), comprising the step of treating miR-101 comprising the nucleotide sequence of SEQ ID NO: 1 . The method according to claim 1,
Wherein said miR-101 binds directly to the 3'UTR of NLK to inhibit the expression of NLK. 3. The method of claim 2,
Wherein said miR-101 inhibits cell growth of a liver cancer cell line. A method for inhibiting the expression of NLK (nemo-like kinase), a factor of liver cancer, by treating miR-101 consisting of the nucleotide sequence of SEQ ID NO: 1 in vitro. 5. The method of claim 4,
Wherein said miR-101 binds directly to the 3'UTR of NLK to inhibit the expression of NLK. 1. A pharmaceutical composition for inhibiting NLK expression comprising miR-101 consisting of the nucleotide sequence of SEQ ID NO: 1. The method according to claim 6,
Wherein said miR-101 binds directly to the 3'UTR of NLK to inhibit the expression of NLK.

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