KR20130051520A - Composition for inhibiting expression of dlk-1 gene - Google Patents

Abstract

PURPOSE: A composition for suppressing Dlk-1 gene expression is provided to suppress cancer cell progression induced by pref-1 expression, and to develop a cell therapeutic agent or an anticancer therapeutic agent using stem cells. CONSTITUTION: A composition for preventing or treating cancer contains an miR-143 nucleic acid molecule as an active ingredient. The molecule contains a base sequence in sequence number 1. The molecule suppresses Dlk-1 gene expression. The composition is used for treating cancer in which pref-1 is overexpressed. The cancer is selected from the group consisting of small cell lung cancer, neuroendocrine tumor, neuroblastoma, glioma, type-1 neurofibromatosis, liver cancer, kidney cancer, ovarian cancer, and acute leukemia. The molecule contains a vector for intracellular expression.

Description

Composition for inhibiting Dlk-1 gene expression {Composition for inhibiting expression of Dlk-1 gene}

The present invention relates to a composition for inhibiting Dlk-1 gene expression, and more particularly, to a composition for inhibiting Dlk-1 gene expression comprising miR-143, a microRNA that inhibits the expression of Dlk-1, 143, a composition for preventing or treating cancer that inhibits the expression of Dlk-1 overexpressed in cancer cells, and miR-143, a microRNA that inhibits the expression of Dlk-1 that is overexpressed in undifferentiated cells and inhibits differentiation of undifferentiated cells, The present invention relates to a composition for promoting differentiation.

Lung cancer is a lung carcinoma originating from epithelial cells, mainly divided into small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer (NSCLC) is sometimes treated with surgery, while small cell lung cancer (SCLC) is usually more responsive to chemotherapy and radiation.

In order to treat small-cell lung cancer that can be treated by non-surgical methods, studies have been actively conducted to investigate the cause of the disease. Recently, attention has been focused on Dlk-1, which is known to be involved in the pathogenesis of lung cancer .

Human Dlk-1 (delta-like 1 homolog, hDlk-1) has a total of 383 amino acid residues, has six EGF-like motifs in the extracellular domain, and is a membrane- It is known that the extracellular domain is known to be homologous to the Notch / Delta / Serrate family and may be referred to as Pref-1, pG2, SCP-1 and ZOG. The gene coding for hDlk-1 was cloned as a factor inhibiting the differentiation of a molecule expressed in a gastroprokinetic cell-derived cell line of GRP (gastrin releasing peptide) response or a pre-adipocyte cell. The hDlk-1 expressed from the gene is transferred to the cell membrane, the extracellular domain is cleaved by the protease, and the cleaved protein fragment is secreted into the blood. The protein fragments secreted into the blood have about 225 to 262 It is known as FA-1 (Fetal antigen-1), a glycoprotein composed of amino acids.

The hDlk-1 gene and its gene products are expressed at high levels in undifferentiated and highly proliferating fetal cells, and are expressed at high levels in fetal liver, fetal kidney, fetal skeletal muscle, fetal brain, (WO 2005/052156) that expression is not confirmed in most tissues, and that expression in normal adipose tissues is restricted in adrenals, placenta, hypothalamus, undifferentiated stem cells and progenitor cells (WO 2005/052156). In particular, it is known that hDlk-1 is expressed in mesenchymal stem cells, which are undifferentiated and multipotent liver stromal cells, bone / cartilage / adipocyte hepatocytes in adult liver, and hDlk- It is expected to play an important role in maintaining the undifferentiated function in the rat.

The hDlk-1 gene and its gene products are known to be expressed at high levels in various cancers and tumors. In addition to the aforementioned small cell lung cancer, neuroendocrine tumors, neuroblastoma, glioma, type 1 neurofibromatosis, It is known that hDlk-1 is expressed at a high level in tissues of patients suffering from kidney cancer, ovarian cancer, acute myelogenous leukemia, and the like (WO 02/081625; Japanese Patent Application Laid-Open No. 2001-269174). In addition, it has been reported that hDlk-1 gene is introduced into K562 cell, which is an erythroleukemia cell line (erythroleukemia), and cell proliferation is enhanced by introducing hDlk-1 gene into gliadinoma cell. Is expected to be involved in the oncogenesis mechanism, and therefore, researches have been actively conducted to use it as a target of cancer treatment. For example, Korean Patent Laid-Open Publication No. 2009-88893 discloses an anti-hDlk-1 antibody capable of specifically binding to hDlk-1 and a pharmaceutical composition for treating cancer comprising the antibody. However, since the antibody acts after hDlk-1 is expressed and is located on the cell membrane, cancer can be prevented from progressing, but it is disadvantageous in that cancer is fundamentally inadequate.

On the other hand, small RNA (small RNA, sRNA) means a ribonucleic acid having a length of about 17 to 25 nucleotides, which plays a role in regulating gene expression in vivo. sRNAs are classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs) depending on how they are generated. miRNAs are derived from partially double-stranded hairpin RNAs, and siRNAs are derived from long double stranded RNA (dsRNA). In general, sRNAs that play an important role in various regulatory processes in vivo are miRNAs, and sRNAs used to control the expression of specific genes are classified as siRNAs.

The miRNA is produced by an endogenous hairpin-shaped transcript as a single strand RNA (ssRNA) molecule of 19-25 nt length. miRNAs complementarily bind to the 3 'untranslated regions (UTRs) of the target mRNA and act as post-transcriptional gene suppressors and inhibit translation by inhibiting translation and mRNA destabilization. miRNA plays an important role in various processes such as development, differentiation, proliferation, apoptosis and metabolism. These processes are sometimes disturbed during tumor formation, and many miRNAs appear to play a role in tumorigenesis as they are kept low in human cancers.

The strong association between miRNA and cancer has recently been demonstrated and is emerging as a new area of research in cancer biology, where expression of miRNA dramatically changes during development and cell differentiation, and through previous analysis of the regulatory network for miRNA function, I was able to understand how molecules are generated and controlled.

Accordingly, the inventors of the present invention have conducted intensive research to develop a method for preventing the development of various cancers including the small-cell lung cancer. As a result, miR-143, a kind of miRNA, can be used to inhibit the expression of hDlk-1 gene And the present invention was completed.

It is an object of the present invention to provide a composition for preventing or treating cancer comprising miR-143 as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting Dlk-1 gene expression comprising miR-143 as an active ingredient.

It is still another object of the present invention to provide a composition for promoting differentiation comprising miR-143 as an active ingredient.

In one embodiment of the present invention, the present invention provides a composition for preventing or treating cancer comprising miR-143 nucleic acid molecule as an active ingredient.

The term "miR-143 nucleic acid molecule" (miR-143 ")" of the present invention refers to a single or double strand of miR-143 which is a type of microRNA that is well preserved in vertebrates, It is a direct transcription target of serum reactive genes myocardin and nkx2-5, which is located close to miR-145 at position 33 of chromosome 5 in humans. . For the purpose of the present invention, miR-143 is used as an inhibitor to suppress the expression of the Dlk-1 gene overexpressed in cancer tissues and undifferentiated cells. The miR-143 nucleic acid molecule may be, but is not limited to, preferably an animal including a human, such as monkeys, pigs, horses, cows, sheeps, dogs dogs, cats, mice, rabbits, and the like.

MiR-143 of the present invention may have a nucleotide length of 18 to 100 nt, preferably 19 to 25 nt in length as mature miR-143, more preferably 21, 22 or 23 nt in length, May be an RNA having the nucleotide sequence of SEQ ID NO: 1 having a length of 21 nt. In addition, miR-143 of the present invention may be provided as a precursor miR-143 having a length of 50 to 100 nt, preferably 65 to 95 nt.

miR-143: 5'-UGAGAUGAAGCACUGUAGCUC-3 '(SEQ ID NO: 1)

MiR-143 used in the present invention is modified by deletion, substitution or insertion of a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the base sequence of miR-143, It is a concept that includes variants that can function in the same way as miR-143.

MiR-143 of the present invention may exist in single-stranded or double-stranded form. The mature miR-143 is predominantly single-stranded, but the precursor miR-143 is at least partially self-complementary (e.g., stem-and-loop-structure), which can primarily form double-stranded portions. In addition, the nucleic acid molecule of the present invention can be configured in the form of RNA, DNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA).

The nucleic acid molecules of the present invention may be isolated or prepared using standard molecular biology techniques, such as chemical synthesis or recombinant methods, or may be commercially available.

In addition, the composition may include not only miR-143 itself, but also other substances capable of increasing the expression rate of miR-143 in cells such as a compound, a natural product, a novel protein and the like.

The term "prevention" of the present invention means all actions that inhibit cancer formation or delay the onset of cancer by administration of the composition, and "treatment" means that the administration of the composition causes all the actions it means.

According to one embodiment of the present invention, the Dlk-1 gene pref-1 is overexpressed in undifferentiated cell follicle cells, but the expression level of pref-1 is decreased while the cell follicle cells are differentiated, whereas miR-143 Level, and the expression level of pref-1 was further lowered when an analog of miR-143 was introduced from the outside (Fig. 1). Further, it was confirmed that miR-143 directly reacted with pref-1 and miR-143, and it was confirmed that miR-143 directly acts on 3 'UTR of pref-1 (FIG. 2). In addition, when miR-143 inhibitor is treated, the expression level of pref-1 is slightly increased in the differentiated cells, and the degree of phosphorylation of erk1 / 2 downstream of pref-1 is decreased when miR-143 is overexpressed (Fig. 3).

Thus, miR-143 inhibits the expression of the Dlk-1 gene pref-1. The pref-1 inhibits the expression of small cell lung cancer, neuroendocrine tumors, neuroblastoma, glioma, type 1 neurofibromatosis, It is known that cancer cells such as kidney cancer, ovarian cancer, and acute myelogenous leukemia are overexpressed. When the activity of Dlk-1 is inhibited at the protein level by using an anti-Dlk-1 antibody in the cancer cells, proliferation of cancer cells is suppressed Inhibiting the expression of pref-1 in cancer cells using miR-143 as described above, it is possible to inhibit the development of cancer and to exhibit a preventive effect or inhibit the proliferation of cancer It was predicted to have the effect of treating cancer. It was also expected that the composition containing miR-143 would have the same effect.

As described above, the composition comprising miR-143 as an active ingredient provided in the present invention can inhibit the expression of Dlk-1, which is known to be overexpressed in various cancers, Prevention or treatment. At this time, in order to exhibit more effective anticancer activity, it may be used in combination with a conventionally known anticancer agent.

The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition may be any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories It can have a formulation.

In another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising administering a composition comprising the miR-143 nucleic acid molecule.

The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and is effective The dose level will depend on the species and severity, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent drugs and other well known factors in the medical field Can be determined. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

In another aspect of the present invention, there is provided a composition for inhibiting Dlk-1 gene expression comprising miR-143 nucleic acid molecule as an active ingredient.

The term "Dlk-1 gene" of the present invention means a gene encoding a membrane-type 1-type membrane protein having a total length of 383 amino acid residues and having six EGF-like motifs in the extracellular region, It may also be referred to as pref-1, pG2, SCP-1 and ZOG in some cases. As is well known, the Dlk-1 gene is expressed in undifferentiated cells and not only inhibits the differentiation of undifferentiated cells, but also expresses in various cancer cells to promote cancer proliferation.

According to another aspect of the present invention, there is provided a composition for promoting differentiation comprising miR-143 nucleic acid molecule as an active ingredient.

The term " composition for promoting differentiation " of the present invention means a composition for inducing or promoting the differentiation of undifferentiated cells. For the purpose of the present invention, since undifferentiated cells overexpressing the Dlk-1 gene are inhibited from differentiation, the expression of the Dlk-1 gene is inhibited by using the miR-143-containing composition to inhibit the expression of the Dlk- May be used to induce or promote differentiation of cells.

The composition comprising miR-143 of the present invention not only promotes the differentiation of pre-differentiated cells maintained by the expression of pref-1 by decreasing the expression level of Dlk-1 gene (pref-1) The present invention can be widely applied to the development of a cell therapy agent or an anti-cancer therapeutic agent using stem cells.

1 is a graph and a photograph showing changes in expression levels of Dlk-1 gene (pref-1) by expression of pref-1 and miR-143 and overexpression of miR-143. Total RNA was obtained from 3T3-L1 cells at each reaction time after induction of differentiation into 3T3-L1 cells, and quantitative real-time RT-PCR was performed to measure pref-1 and miR-143. Correction for Pref-1 mRNA was performed with GAPDH and miR-143 was corrected with U6 snRNA. The table is marked with means ± standard error. Protein expression of pref-1 by differentiation was measured by westen blot. A 50 uM miR-143 analog or negative control was introduced into 3T3-L1 cell follicular cells. Total RNA was extracted 48 hours after the introduction and real-time PCR was performed. The mRNA expression level of Pref-1 was corrected by GAPDH. Expression of Dlk-1 by overexpression in miR-143 was measured by Western blot analysis.
Figure 2 (A) is a schematic diagram showing the target site of miR-143 located in the 3 'UTR of pref-1. The single binding site of miR-143 located in the 3 'UTR of mouse pref-1 is indicated in bold. (B) is a cleavage map schematically showing the reporter plasmid. pGL3-pref-1 wt cloned pref-13 'UTR at the back of the luciferase gene in the pGL3-control vector. pGL3-pref-1 mu introduced a site-specific mutation in which the binding site of pref-13 'UTR miR-143 in the pGL3-pref-1 wt vector was italicized. (C) is a graph showing the specific binding of miR-143 to pref-13 'UTR. (B) was introduced into COS7 cells alone or in combination with miR-143. Forty - eight hours after the induction, they were analyzed using the dual luciferase assay kit. The activity of Renilla luciferase was normalized to firefly luciferase. The graph is denoted as means ± standard error.
3 is a photograph showing the regulation of Dlk-1 function by miR-143. (A) was prepared by completely culturing 3T3-L1 cells, introducing miR-NC (negative control: non-targeting miRNA) and miR-143 inhibitor, and incubating for 48 hours with growth medium Total RNA was extracted 48 h after cells treated with differentiation medium (DM), and the expression level of pref-1 was analyzed by RT-PCR. (B) shows that when 3T3-L1 cells are completely cultured, the miR-NC and miR-143 analogs are introduced. After 2 days, the DM is treated for 2 days and the protein is extracted from the cells to measure the activation of Erk1 / 2 Phosphor-erk1 / 2 was measured by Western blot analysis and normalized to total erk1 / 2.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: method

Example  1-1: Plasmid composition

Firefly luciferase reporter pGL3- The control vector (Promega) was constructed to contain the 3 'UTR of the mouse Dlk-1 gene (pref-1) (GenBank Accession No. NM_003836). The fragment was generated by PCR. The PCR product was digested with restriction enzyme XbaI to generate appropriate extruded ends. Site specific mutations of the luciferase promoter (pGL3-pref-1 wt) were performed according to the user's manual using the QuikChange site-directed mutagenesis kit (Stratagene). The original sequence TCATCTC was mutated to TCAGACC in the miR-143 target region of the Dlk-1 3 'UTR. The nucleotide sequence was analyzed to confirm whether the mutation was properly performed.

Example  1-2: cell culture

3T3-L1 cells were cultured in DMEM medium containing 10% bovine serum and cultured to fill the culture dish. After 2 days had elapsed (Day 0), cell differentiation of cells was carried out using DMEM medium containing 10 μg / ml insulin, 1 μM DEX, 0.5 mM MIX (3-Isobutyl-1-methylxanthine) and 10% FBS . After 48 hours (Day 2), the culture medium was replaced with DMEM medium containing 10 μg / ml insulin and 10% FBS, and the medium was changed every other day. At 2 days after another day, intracellular triglycerides were observed, and the cells were completely differentiated at 8 days from the start of differentiation.

Example  1-3: Real Time PCR  analysis

Total RNA was isolated from 3T3-L1 cells using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA). CDNA was synthesized from the total RNA using MMLV-reverse transcriptase (Takara, Shinga, Japan) and oligo dT primer (Invitrogen, Carlsbad, CA, USA). Real-time quantification was performed according to the user's manual using BIO-RAD iCycler iQ system (BioRad, Hercules, CA, USA). Fluorescence threshold values were calculated using iCycle iQ system software. The reverse transcription reaction mixture was reacted with iQ SYBR Green supermix (BioRad, Hercules, CA, USA). The data were processed by the comparative cycle threshold method and expressed as a relative increase rate relative to the baseline transcription level. The amount of target mRNA was normalized according to the level of GAPDH mRNA.

The oligonucleotides used are as follows:

Dlk-1 F: 5'-TGGCTTCTCAGGCAACTTCT-3 '(SEQ ID NO: 2)

Dlk-1 R: 5'-CTTGCACAGACACTCGAAGC-3 '(SEQ ID NO: 3)

GAPDH F: 5'-GACTTCAACAGCAACTCCCAC-3 '(SEQ ID NO: 4)

GAPDH R: 5'-TCCACCACCCTGTTGCTGTA-3 '(SEQ ID NO: 5)

Total RNA was extracted using TRIzol (Invitrogen) to perform miR qRT-PCR; Primers and probes specific for human miR-143 and internal control U6 snRNA were obtained from Applied Biosystems (Foster City, CA, USA); Amplification and detection were performed using a BIO-RAD iCycler iQ system (40 cycles, denaturation 95 ° C. 15 sec, ligation / extension 60 ° C. 60 sec).

This was carried out by reverse transcription at 42 DEG C for 30 minutes and denaturation at 85 DEG C for 5 minutes.

Example  1-4: Temporary introduction and Luciferase  Reporter Analysis

To introduce miRNAs transiently, miR-143 mimic or miR-143 inhibitor (GenePharma, Shanghai, China), or control oligonucleotides were added to 3T3-L1 cells according to their user manual using Oligofectamine (Invitrogen) Lt; / RTI > To analyze luciferase activity, reporter plasmids were introduced into COS7 cells according to their user manual using lipofectamine 2000 reagent (Invitrogen). The luciferase activity was measured 48 hours after the introduction using the dual-luciferase assay kit (Promega). COS7 cells were co-transfected with pcDNA3.1 or pcDNA3.1-miR-143 in a reporter plasmid. Luciferase activity was measured using a GloMax 20/20 luminometer (Turner BioSystem, USA).

Example  1-5: Western Blot  analysis

The cells were washed with chilled PBS and resuspended in chilled cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor cocktail (Roche, IN, USA) Na ^ 3VO ^ 4). Protein extracts were separated by SDS-PAGE and western blotted on nitrocellulose transfer membrane. At this time, the blocking was performed for 1 hour at room temperature with TBS-T containing 5% skimmed milk powder, and then an anti-Erk1 / 2 anti-phospho-Erk1 / 2 antibody (cell signaling) 1; Santa Cruz) or anti-actin (Sigma) antibody. Washed with PBS, and reacted by adding HRP-conjugated secondary antibody to the membrane. Protein signal was visualized with a chemiluminescence kit (Santa Cruz Inc., CA, USA).

Example  2: Result Analysis

Example 2-1: Change of miR- 143 by adipocyte differentiation

In order to examine the potential regulatory activity of miR-143, miR-143 expression pattern was examined at the differentiation of adipocytes in 3T3-L1 cells. miR-143 was more expressed in cells differentiated into adipocytes than in undifferentiated adipocytes (Fig. 1A). The expression level of pref-1, the target of miR-143, decreased as adipocyte differentiation progressed (Fig. 1 A). It was confirmed that the decrease of pref-1 was decreased not only at the gene level but also at the protein level according to differentiation (Fig. 1B). To determine whether the decrease in expression of pref-1 was due to the increase of miR-143, overexpressing miR-143 analogues and confirming the expression level of pref-1 resulted in transient introduction of miR-143 and overexpression. It was confirmed that the protein level as well as the mRNA of -1 was reduced (C and D of FIG. 1).

As shown in the above results, miR-143 expression level was increased during fat differentiation, and overexpression of miR-143 decreased expression of pref-1. Thus, when adipocytes were differentiated, miR- And the expression of the gene was regulated.

Example  2-2: pref -1 of the 3 ' UTR Present in miR Identification of target site of -143

To determine whether miR-143 directly down-regulated pref-1, the 3 'UTR region of pref-1 was examined with targetscan and miRANDA.

pref-1 was predicted to have a target portion of miR-143 at the 246 to 252 nucleotide position of the 3 ' UTR (Fig. 2A). In order to confirm whether the predicted portion was recognized by miR-143, a reporter plasmid containing a portion predicted to react with miR-143 and a plasmid in which a site-specific mutation was introduced at the portion thereof were respectively prepared 2B). Expression of the reporter vector containing the 3 'UTR of pref-1 was inhibited by more than 60% when miR-143 was overexpressed (FIG. 2C). However, in the case of the vector into which the mutation was introduced at the target site of miR-143, it was confirmed that the reporter activity was not changed even when miR-143 was overexpressed (FIG. 2C).

From the above results, it was found that inhibition of pref-1 expression by overexpression of miR-143 was caused by direct reaction with 3 'UTR.

Example  2-3: pref -1 affects activity miR Effect of -143

The expression of pref-1 was decreased at the differentiation of cell follicle cells by external factors, so that the decrease was due to the increase of miR-143 by differentiation.

The inhibitor of miR-143 was treated when 3T3-L1 was cultured, treated with growth medium and differentiation medium 2 days later, and again the level of pref-1 expression was measured 2 days later. As a result, when the cells were treated with the differentiation medium (control group), the expression level of pref-1 was greatly decreased, but when the inhibitor of miR-143 was treated and the differentiation medium was treated, the expression of pref-1 was increased (Fig. 3A).

Since pref-1 inhibition of lipid differentiation is known to occur through activation of erk1 / 2, the effect of erk1 / 2 on the downstream of pref-1 by miR-143 expression Respectively. After overexpression of miR-143 and addition of differentiation medium, the degree of phosphorylation of erk1 / 2 was measured. As a result, it was confirmed that erk1 / 2 phosphorylation was reduced when miR-143 was overexpressed (FIG.

From the above results, it can be seen that miR-143 is a regulator that reduces the expression of pref-1 upon differentiation of adipocytes.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition for inhibiting expression of Dlk-1 gene <130> PA110842 / KR <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-143 <400> 1 ugagaugaag cacuguagcu c 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dlk-1 F primer <400> 2 tggcttctca ggcaacttct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dlk-1 R primer <400> 3 cttgcacaga cactcgaagc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F primer <400> 4 gacttcaaca gcaactccca c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R primer <400> 5 tccaccaccc tgttgctgta 20

Claims (9)

Cancer prevention or treatment composition comprising miR-143 nucleic acid molecule as an active ingredient.
The method of claim 1,
The miR-143 nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 1.
The method of claim 1,
Wherein said miR-143 nucleic acid molecule inhibits Dlk-1 gene expression.
The method of claim 1,
A composition in which pref-1 is used for the treatment of cancer diseases overexpressed.
5. The method of claim 4,
The cancer disease is selected from the group consisting of small cell lung cancer, neuroendocrine tumor, neuroblastoma, glioma, type 1 neurofibromatosis, liver cancer, kidney cancer, ovarian cancer and acute myeloid leukemia.
The method of claim 1,
Wherein said miR-143 nucleic acid molecule is included in a vector for intracellular expression.
The method of claim 1,
Wherein the composition further comprises a pharmaceutically acceptable diluent, excipient or carrier.
Composition for inhibiting Dlk-1 gene expression comprising the miR-143 nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.
A composition for promoting differentiation comprising the miR-143 nucleic acid molecule of SEQ ID NO: 1 as an active ingredient.

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