KR20130093378A - Use of hdac2 as a target for liver cancer suppressor - Google Patents

Abstract

PURPOSE: HDAC2 is provided to control transcriptional activity of modulator, and to suppress breeding of cell by suppressing G1/S stage among the cell cycle through the transcriptional activity modulation. CONSTITUTION: A pharmaceutical composition for preventing or curing liver cancer comprises HDAC2 gene expression inhibitor or HDAC2 protein activity inhibitor as an active ingredient. The HDAC2 is overexpressed in liver cancer cell. The inhibitor is HDAC2-specific antisense oligonucleotide, siRNA, aptamer, or antibody. A method of providing information for predicting or diagnosing liver cancer comprises the steps of: measuring a level of HDAC2 gene expression or a level of protein that the gene codes from the biological sample of a patient; and comparing the measured levels with the normal levels.

Description

Use of HDAC2 as a target for liver cancer suppressor}

The present invention relates to the use of HDAC2 as a target for inhibiting liver cancer, and more particularly, a pharmaceutical composition for preventing or treating liver cancer comprising HDAC2 gene expression inhibitor or HDAC2 protein activity inhibitor as an active ingredient, liver cancer using HDAC2. A method for predicting or diagnosing onset and a method for screening a liver cancer drug using HDAC2.

Histone deacetylases (HDACs), gene expression inhibitors, interact with transcription inhibitors such as RB, mSin3a, and N-CoR to form large protein complexes, and the complexes form a chromatin structure. It is known to be involved in inhibiting gene expression by deacetylating histone proteins (Laszlo Nagy, Hung-Ying Kao, Debabrat Chakravarti, Richard J. Lin, Christian A. Hassig, Donald E. Ayer, Stuart L. Schreiber and Ronald M. Evans, Cell, 89: 373-380 (1997); Robin X. Lwo, Antonio A. Postigo, and Douglas C. Dean, Cell, 92: 463-473 (1998)). It is commonly found in eukaryotic cells such as fruit flies, yeast, and corn. In yeast, five types (Rpd3, Hda1 and HOS1, 2 and 3), and in humans, 17 similar forms have been found (Steven G. , Gray and Tomas J. Ekstrom, Experimental Cell Research, 262: 75-83 (2001).

Human histone deacetylases are broadly divided into two groups: HDAC 1, 2, 3 and 8 of group I, similar to Rpd3 of yeast, and HDAC 4, 5, 6, and 7 of group II, similar to Hda1 (Steven). G., Gray, Tomas J. Ekstrom, Experimental Cell Research, 262: 75-83 (2001). The molecular weight of group I is about 45-55 kDa, and in the case of group II, its size is about 80-131 kDa. Unlike group I, the non-catalyst is included at the N-terminus. In the case of group I, deacetylase enzyme functions by combining with mSin3a or NuRD (nucleosome remodeling and deacetylating) complex, while group II is known to perform its function without interaction with these mediators (Eric Y. Huang, Jinsong Zhang, Eric A. Miska, Matthew G. Guenther, Tony Kouarides and Mitchell A. Lazar, Gene Development 14: 45-54 (2000).

It is known that such human histone deacetylase is expressed in tissues and environmental factor stimulus and is expressed in a large amount in various cancer cells and undifferentiated leukemia cells (Wen-Ming Yang, Ya-Li Yao, Jian-). Min Sun, James R. Davie and Edward Seto, Journal of Biological Chemistry, 272: 28001-28007 (1997). Therefore, a number of inhibitors capable of inhibiting histone deacetylase have been developed and studied as anticancer agents in various solid and hematological cancers.

Meanwhile, liver cancer is the most common cancer in Korea, the Far East Asia and Southern Africa. In particular, liver cancer is known to have the highest mortality rate from gastric cancer and liver cancer. Recent advances in molecular biology research have revealed that cancers have been shown to be a significant cause of cancer suppressor genes and mutations in cancer genes. In addition, most of these cancer-related genes are known to be intercellular regulators of their expression and function as cell cycle regulators. However, the research on molecular biological mechanisms in the various forms of liver cancer are insignificant.

The present inventors have identified molecular biological mechanisms based on the organic correlation of cell cycle regulators according to the expression level of HDAC2, a type of histone deacetylase, in liver cancer development. The present invention has been completed by experimentally clearly demonstrating that it acts as an important factor for development.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer through inhibiting overexpression of HDAC2.

Another object of the present invention is to provide a method for providing information for predicting or diagnosing the onset of liver cancer using HDAC2.

It is still another object of the present invention to provide a method for screening a liver cancer therapeutic agent using HDAC2.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising an inhibitor of HDAC2 gene expression or an inhibitor of activity of HDAC2 protein as an active ingredient.

In one embodiment of the invention, the HDAC2 may be overexpressed in liver cancer cells.

In one embodiment of the invention, the inhibitor may be selected from the group consisting of antisense oligonucleotides, siRNAs, aptamers and antibodies specific for HDAC2.

In one embodiment of the present invention, the inhibitor may exhibit the effect of preventing or treating liver cancer by inhibiting transcriptional activity inhibition through p21 ^{WAF1 / Cip1} Sp1-binding site of ^HDAC2 .

The present invention also includes the steps of (a) measuring the expression level of the HDAC2 gene or the protein encoded by the gene from a biological sample of a patient suspected of liver cancer; And (b) comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene of the normal control sample. Provide a way to.

In one embodiment of the invention, the measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, ELISA (enzyme linked) immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay.

The present invention also comprises the steps of: (i) contacting a sample to be analyzed to liver cancer cells or tissues comprising the HDAC2 gene or its expression protein; (Ii) measuring the amount of expression of said selected gene or the amount or activity of said expressed protein; And (iii) determining that the sample is a material for preventing or treating liver cancer when the amount of expression of the selected gene or the amount or activity of the expressed protein is reduced as a result of the measurement in step (ii). It provides a method for screening a material for the prevention or treatment of.

If HDAC2 gene of the present invention is abnormal over-expression, a is the activity of CDK2, CDK4 and cyclin D1 increases with cell cycle regulators of p16 ^INK4A, and inhibition of expression of p21 ^{WAF1 / Cip1.} In particular, since HDAC2 can regulate the transcriptional activity of p21 ^{WAF1 / Cip1} through the Sp1-binding site (proximal to the TATA box) of p21 ^{WAF1 / Cip1} , a regulator capable of inhibiting the cell cycle, when using HDAC2 inhibitors, It is possible to inhibit the proliferation of cells by inhibiting the progression of the G1 / S phase of the cell cycle through. Therefore, the composition of the present invention comprising an inhibitor of expression of HDAC2 gene or an inhibitor of activity of HDAC2 protein as an active ingredient can be usefully used for the prevention or treatment of liver cancer.

1 is measured by immunoblotting the expression of HDAC2, β-catenin, cyclin D1 and c-Myc in 10 tissues of human hepatocellular carcinoma compared to non-tumor tissue.
Figure 2 is measured by immunoblotting the expression of HDAC2, β-catenin, cyclin D1 and c-Myc in human liver cancer cell line.
Figure 3 immunoblotting the expression of HDAC2 protein according to the β-catenin, cyclin D1 or c-Myc reduction through knock-down experiments using siRNA of β-catenin, cyclin D1 and c-Myc genes in Hep3B cells It is measured through.
FIG. 4 shows the expression levels of various HDACs (HDAC1 to HDAC8) in Hep3B cells through qRT-PCR.
5 is an immunoblotting measurement of accumulation of acetylated histones H3 and H4 following HDAC2 reduction in Hep3B cells.
Figure 6 is a graph comparing the growth rate of cells transformed with HDAC2 siRNA and untreated or negative control (scrambled siRNA transfectant (Scr)) in order to measure the cell growth rate according to the inhibition of HDAC2.
Figure 7 shows the apoptotic cells through flow cytometry after 48 hours after transforming with HDAC2 siRNA.
8 is a 48- and 72-hour period after transforming Hep3B cells with HDAC2 siRNA in order to determine whether it causes an anti-mitogenic effect of HDAC2 reduction in Hep3B cells, and then measured cell proliferation activity through thymidine incorporation assay. It is.
FIG. 9 shows DNA contents of PI-stained cells after 48 and 72 hours after transforming Hep3B cells with HDAC2 siRNA using flow cytometry.
10 is a DNA content of PI-stained cells in an experimental group treated with HDAC exogenous Valproic acid for 72 hours to Hep3B cells by flow cytometry.
FIG. 11 shows that p21 ^{WAF1 / Cip1} expression was measured by immunoblotting after transforming Hep3B cells with HDAC2 siRNA (Scr: negative control group, VPA (Valproic acid): positive control group).
12 is 72 hours after transforming Hep3B cells with HDAC2 siRNA (# 1, # 2), and the expression levels of cyclins, CDKs and CDKIs (G / 1 transition inhibitors) were measured by immunoblotting. None: no siRNA, Reag: oligofectamin only, Scr: 100 nmol / L scarmbled siRNA.
FIG. 13 is 72 hours after transforming Hep3B cells with HDAC2 siRNAs (# 1, # 2), and the expression level of pRb and E2F / DP1 target genes was measured by immunoblotting (None: no siRNA). , Reag: oligofectamin only, Scr: 100 nmol / L scarmbled siRNA).
FIG. 14 shows p21 ^{WAF1 / Cip1} , CDK2 and PCNA protein expression in immunohistoblotting in 10 tissues of human hepatocellular carcinoma compared to non-tumor tissues.
15 is a by introducing HDAC2 shRNA in Hep3B cells by measuring the immune blotting for p16 ^INK4A and p21 ^{WAF1 / Cip1} selective induction of the CDK2, the inhibition of cyclin D1 and PCNA effects of the functional inactivation of HDAC2.
Figure 16 shows the growth rate of cells over time of Hep3B_HDAC2KD (introducing HDAC2 shRNA into Hep3B cells), Hep3B_Mock (control 1) and Hep3B mother cell line (control 2).
FIG. 17 shows that Hep3B_HDAC2KD (introducing HDAC2 shRNA into Hep3B cells), Hep3B_Mock (Control 1), and Hep3B mother cell line (Control 2) were synchronized in the G2 / M phase using nocodazole, followed by a cell cycle process. DNA content of PI-stained cells over time (0, 2, 6, 12, 24, 48h) was analyzed using a flow cytometer (left). The percentage of G1 phase in the cell is calculated and graphed (right).
18 shows that sustained inhibition of HDAC2 reduces clonal cell growth via in vitro colony forming assays.
19 shows tumor growth according to Hep3B cell xenografts (experimental group: Hep3B_HDAC2KD injection group, control group: Hep3B_Mock injection group) in order to evaluate the effect of abnormal HDAC2 regulation on the induction of tumor cells in vivo.
FIG. 20 shows tumor size and mice after 52 days after Hep3B cell xenografts.
FIG. 21 shows the induction of p21 ^{WAF1 / Cip1} transcriptional activity in ^{Hep3B_HDAC2KD} after transformation of Hep3B_HDAC2KD with reporter constructs containing wild type p21 ^{WAF1 / Cip1} promoter (-2323 to +1) linked to the luciferase reporter gene (pWWP-Luc). Luciferrace activity was measured to see (the experimental group treated with 1 μM Apicidin for 24 hours as a positive control).
Figure 22 is transformed Hep3B_HDAC2KD with the expression vector (F-HDAC2) containing FLAG-epitope tagged HDAC2 to express the HDAC2 gene in Hep3B_HDAC2KD p21 ^{WAF1 / Cip1} Luciferrace activity was measured to determine transcriptional activity induction.
Figure 23 shows the correlation with the p21 ^{WAF1 / Cip1} proximal promoter region of the HDAC2 protein by ChIP assay.
Figure 24 shows by investigating whether HDAC2 is possible to suppress the p21 ^{WAF1 / Cip1} expression through Sp1- binding site on the p21 ^{WAF1 / Cip1.}

The present inventors have identified molecular biological mechanisms based on the organic correlation of cell cycle regulators according to the expression level of HDAC2, a type of histone deacetylase, in liver cancer development. Experimentally clearly proved to be an important factor.

Histone deacetylases 2 (HDAC2) is one of histone deacetylases and is known to be overexpressed in some cancer tissues.

However, the molecular biological mechanism based on the organic correlation of cell cycle regulators according to the expression level of HDAC2 in hepatocellular carcinoma (HCC) of HDAC2 is not clearly known.

In the present invention, were investigated the molecular mechanism based on organic Correlation of cell cycle regulators in accordance with the level of expression of HDAC2, especially and HDAC2 an abnormal over-expression in liver cancer cell line, so that the cell cycle regulators of p16 ^INK4A and p21 ^{WAF1 / Cip1} It was first identified that hepatocellular carcinoma progressed through a cooperative mechanism of suppressing the expression of and increasing the activity of CDK2, CDK4 and cyclin D1. Also HDAC2 is p21 ^{WAF1 /} of ^Cip1 Sp1-binding site and to control the p21 ^{WAF1 / Cip1} enterprise activity through the (TATA box proximal), the progress G1 / S phase inhibition of the cell cycle through such a wide active control by being a cell It was first identified that it could inhibit proliferation.

Therefore, the present invention can prevent or treat liver cancer by inhibiting the expression of HDAC2 gene or inhibiting HDAC2 protein activity, the present invention is a drug for preventing or treating liver cancer comprising an inhibitor of the expression of HDAC2 gene or an activity inhibitor of HDAC2 protein as an active ingredient. Pharmaceutical compositions may be provided.

In general, the cell cycle is performed in a certain order by a mechanism established within the cell. However, if abnormalities occur in this order, the maintenance of the cell cycle will be difficult, and the regulators responsible for repairing cell cycle abnormalities are cyclin and cyclin-dependent kinase: (Hereinafter abbreviated as 'CDK') and cyclin-dependent kinase inhibitors (hereinafter abbreviated as 'CDKI'). In the early stage of the G1 phase, CDK4, 6, and 8 act on CDK4, 6, and 8 depending on the cell type, CDK2 acts on the G1 and S phases, and G2 to M CDK1 is known to play an important role.

In addition, binding of cyclins is essential for the activity of CDK. CDK4, 6, and 8 are activated by binding to cyclin D, and CDK2 binds to cyclins A and E. CDK1 binds to cyclin B and A, and other cyclins G and F are known. Since the specific cyclin-CDK complex is activated at each stage of the cell cycle, and the proteins that are specifically phosphorylated to CDK are responsible for cell cycle progression, the cell cycle may be named CDK cycle.

In addition, CDK acts as an essential factor for the activity of cyclin. Activated CDK-cyclin is divided into cyclin control unit and CDK activity unit. There are two ways to control CDK of cyclin. Cyclin and CDK bind to each other to induce structural changes of the protein, and the arrangement of the ATP phosphate group changes easily to transfer to the substrate protein. In addition, the location of the T loop, which blocks the protein substrate from accessing the CDK, is changed, making it easier to access the substrate. The activation of CDKs only at specific times in the cell cycle is due to the cyclin synthesis that occurs specifically in the cell cycle.

Cyclin D is mainly synthesized in mid-G1, and is mainly induced by mitogens such as cell growth factors. Cyclin D has three subtypes (D1, 2, and 3), which vary in degree depending on the cell type. For example, inhibition of the synthesis of cyclin D causes cell cycle G1 to stop, and overexpression of cyclin D shortens the G1 phase and initiates the cell cycle without mitogen.

The ^{p15 INK4B, p16 INK4A, p18 INK4C} , p19 INK4D, p27 Kip1 and p21 ^{WAF1 / Cip1} and cell cycle regulation such as characters are known as core control to inhibit cyclin D1 / CDK4,6 or Cyclin E / CDK2 complexes. Therefore, such cell cycle regulators are referred to as negative cell cycle regulators, and cell cycle progression can be suppressed when they are expressed.

The present inventors in the Examples, it was confirmed that the HDAC2 an abnormal over-expression within the liver cancer cell line (Fig. 1 reference to Fig. 4), if to inhibit the expression of HDAC2, cell cycle regulators of p16 ^INK4A and p21 ^{WAF1 / Cip1} It was confirmed that the inhibition of the expression and the activity of CDK2, CDK4 and cyclin D1 is increased (see Figs. 12 to 15).

In addition, in one embodiment of the present invention, when inhibiting the expression of HDAC2 using siRNA for HDAC2, it was confirmed that the G1 phase is increased by 12.8% compared to the negative control, similarly the HDAC exogenous foot In the positive control treated with prolactic acid (Valproic acid), it was confirmed that the G1 phase interruption was strongly observed in the cell cycle (see FIGS. 9 and 10).

In addition, in the following examples of the present invention, HDAC2 shRNA (Hep3B_HDAC2KD) is introduced into Hep3B cells, which is a hepatic cancer cell line, to suppress the proliferation of Hep3B cells when inducing continuous inhibition of HDAC2 using a transformant expressing minimal HDAC2. And cell growth obstruction (delay) proved to be due in particular to G1 arrest during the cell cycle (see FIG. 17).

In addition, in one embodiment of the present invention, it was investigated whether sustained inhibition of HDAC2 could attenuate the potential of tumorigenicity in hepatocellular carcinoma cells in vitro and in vivo. It was confirmed that the tumor formation can be significantly reduced (see FIGS. 18 to 20).

In addition, in one embodiment of the present invention, it was investigated whether HDAC2 is possible to suppress the p21 ^{WAF1 / Cip1} expression through the Sp1-binding site on the p21 ^{WAF1 / Cip1,} the result TATA box care of p21 ^{WAF1 / Cip1} It was found that the proximal site at was an important site for the regulation of p21 ^{WAF1 / Cip1} transcription by ^HDAC2 (see FIG. 24).

Therefore, through the above results, the present inventors expressed p21 ^{WAF1 / Cip1} , a cell cycle regulator that can stop cell cycle progression when inhibiting ^HDAC2 expression in liver cancer cells, ultimately inhibiting the proliferation of liver cancer cells. It was found that it has anticancer activity.

Therefore, the present invention can provide a composition for preventing or treating liver cancer as an active ingredient of an inhibitor of expression of HDAC2 gene or an inhibitor of activity of HDAC2 protein.

HDAC2 inhibitors that can be used as active ingredients in the compositions of the present invention include, but are not limited to, antisense oligonucleotides, RNAi, siRNA, shRNA, aptamers, antibodies, single chain variable region fragments, low molecular weight compounds or natural extracts. no.

In the present invention, the term "antisense oligonucleotide means a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to the complementary sequence in the mRNA to translate the mRNA into a protein. The antisense sequence of the present invention refers to a DNA or RNA sequence that is complementary to the mRNA of the gene and capable of binding to the mRNA, and translates the mRNA, translocation into the cytoplasm, and maturation. ) Or any other essential biological function.

In addition, the antisense nucleic acid can be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995). The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

The antisense oligonucleotides may be synthesized in vitro in a conventional manner and administered in vivo or may allow the synthesis of antisense oligonucleotides in vivo. An example of synthesizing an antisense oligonucleotide in a test tube is to use RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

In the present invention, "RNAi" refers to the RNA Interference, in Korean, it means the meaning of RNA interference. RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. It is thought to be a type of gene monitoring mechanism used by cells to defend against viral infections, to suppress transposons, or to remove abnormal mRNAs. In particular, gene suppression by small RNA is called RNA interference in a broad sense, and RNA interference in a narrow sense means mRNA degradation by siRNA. In addition, RNA interference may refer to a technique for inhibiting genes using siRNA.

In the present invention, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409, and 00 SiRNA is provided as an efficient gene knock-down method or gene therapy method because it can inhibit the expression of a target gene.

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to the mRNA sequence of the marker gene) and an antisense strand (a sequence complementary to the mRNA sequence) are positioned opposite to each other to form a double chain. The siRNA molecules of the present invention may have a single chain structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to the complete pairing of double-stranded RNA moieties in RNA pairs, but may be mated by mismatch (corresponding bases are not complementary), bulge (no bases corresponding to one strand) And a portion that is not achieved may be included. In addition, the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the marker gene can be suppressed by the RNAi effect, and the adhesive terminal structure has a 3'-terminal protrusion structure and a 5'- end. Both terminal protruding structures are possible.

In addition, the siRNA molecules of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is subjected to intramolecular hybridization. As a result, a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The method for preparing siRNA is to directly synthesize siRNA in vitro and then introduce it into the cell through a transformation process, and to siRNA expression vector or PCR-derived siRNA expression cassette prepared to express siRNA in the cell. There is a method of conversion or infection.

In addition, the composition of the present invention comprising a gene-specific siRNA may include an agent that promotes intracellular infiltration of siRNA. Agents that promote intracellular inflow of siRNA can generally include agents that promote nucleic acid inflow. Examples of such agents include liposomes or one lipophilic one of a number of sterols, including cholesterol, cholate, and deoxycholic acid Can be combined with the carrier. It is also possible to use poly-L-lysine, spermine, polysilazane, polyethylenimine (PEI), polydihydroimidazolenium, polyallylamine Cationic polymers such as chitosan, chitosan and the like may be used, and succinylated PLL, succinylated PEI, polyglutamic acid, polyaspartic acid, anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, hyaluronic acid, and the like, Can be used.

As used herein, the term "aptamer" refers to an oligonucleotide molecule having binding activity to a given target molecule. Aptamers can inhibit the activity of a given target molecule by binding to the given target molecule.

The aptamers of the invention can be RNA, DNA, modified oligonucleotides or mixtures thereof. The aptamers of the present invention may also be in the form of straight chain or cyclic. The length of the aptamer of the present invention is not particularly limited, and may usually be about 15 to about 200 nucleotides, but for example, about 100 nucleotides or less, preferably about 80 nucleotides or less, more preferably about 60 nucleotides or less, Most preferably about 45 nucleotides or less. The length of the aptamers of the invention can also be, for example, at least about 18, 20 or 25 nucleotides. Smaller total numbers of nucleotides are advantageous for easier chemical synthesis, chemical formula and mass production, economical, high in vivo stability and low toxicity.

The aptamers of the present invention are the SELEX method and its modifications [see, eg, Ellington et al., Nature, 1990 346, 818-822; Tuerk et al., Science, 1990 249, 505-510. The SELEX method is a method of selecting oligonucleotides that specifically bind to a target substance from a pool of oligonucleotides having about 10-14 different nucleotide sequences. The oligonucleotide used has a structure in which a random sequence of about 40 residues is inserted as a primer sequence. This oligonucleotide pool is associated with a target substance to recover only the oligonucleotides bound to the target substance using a filter or the like. The recovered oligonucleotide is amplified by RT-PCR and used as a template for the next round. By repeating this operation about 10 times, it is possible to obtain an aptamer that specifically binds to the target substance. In the SELEX method, by increasing the number of rounds or by using a competitive substance, it is possible to concentrate and select aptamers having stronger binding to the target substance. Thus, by adjusting the number of rounds of SELEX and / or changing the race state, it is possible to obtain aptamers with different binding forces, aptamers with different binding forms, and aptamers with the same binding force or binding form but different base sequences. In addition, although the SELEX method includes the amplification process by PCR, by providing a mutation by using manganese ions in the process, it becomes possible to perform a SELEX richer in diversity.

In addition, aptamers can be obtained using the Cell-SELEX technique for complex targets, ie living cells and tissues, in addition to the conventional SELEX technique (Guo et al., Int. J. Mol. Sci., 9 (4): 668). , 2008), the Cell-SELEX technique has the advantage of enabling the development of aptamers for diseased cells even when surface marker targets are unknown. In addition, the Cell-SELEX technique has advantages over the conventional SELEX procedure because the isolated protein may not exhibit its original properties, since the target protein in the physiological state allows for a more functional approach in the selection process. have.

On the other hand, aptamers bind to target substances by various binding modes such as ionic bonds using negative charges of phosphate groups, hydrophobic bonds and hydrogen bonds using ribose, and hydrogen bonds or stacking bonds using oligonucleotide bases. In particular, ionic bonds using negative charges of phosphate groups present in the number of constituent nucleotides strongly bind positive charges of lysine or arginine on the surface of the protein. For this reason, oligonucleotide bases not involved in direct binding to the target material can be substituted. In particular, since part of the stem structure is already made of base pairs and is directed inward of the double helix structure, the oligonucleotide base is difficult to bind directly with the target material. Therefore, even if the base pair is replaced with another base pair, the activity of the aptamer is often not reduced. Even in a structure in which a base pair is not formed, such as a loop structure, the base can be substituted when the oligonucleotide base is not involved in the direct bond with the target molecule. For example, at the 2 'position of the ribose, the hydroxy group may be a nucleotide in which it is substituted with any atom or group. As such arbitrary atoms or groups, for example, hydrogen atom, fluorine atom or -O-alkyl group (e.g. -O-CH3), -O-acyl group (e.g. -O-CHO), amino group (e.g. -NH2) And nucleotides substituted with. As such, aptamers retain their activity unless they substitute or eliminate the functional groups involved in direct binding to the target molecule.

In addition, the inhibitor of the HDAC2 of the present invention includes an HDAC2 protein activity inhibitor, and as such a protein activity inhibitor, an antibody, a single chain variable region fragment, a peptide, a low molecular weight compound or a natural extract that specifically binds to HDAC2 It can be illustrated.

Antibodies that specifically bind to and inhibit activity by the HDAC2 protein that can be used in the present invention are polyclonal or monoclonal antibodies. Antibodies to the HDAC2 protein can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler et al., European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. 4,816,56) or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). Can be prepared by General procedures for antibody preparation are described in Harlow, E. et al., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells that produce monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. . Polyclonal antibodies can be obtained by injecting an HDAC2 protein antigen into a suitable animal, collecting antisera from the animal, and then separating the antibody from the antisera using known affinity techniques.

In the present invention, the antibody may include a single chain variable region fragment (scFv). The single chain variable region fragment may be composed of "variable region (VL) -linker-heavy chain variable region (VH) of light chain". The linker means an amino acid sequence of a certain length that serves to artificially link the variable regions of the heavy and light chains.

In addition, the composition for preventing or treating liver cancer according to the present invention may further include a pharmaceutically acceptable carrier as a pharmaceutical composition for treating liver cancer. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention, together with a pharmaceutically acceptable carrier as described above, may be formulated into a suitable form according to a method known in the art. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods, and isotonic aqueous solutions or suspensions are preferred for injection formulations typical of parenteral administration formulations. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the administration may include introducing a predetermined substance into the patient by any suitable method And the route of administration of the agent may be administered through any conventional route so long as it can reach the target tissue.

In addition, the effective amount in the above means an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the disease type and severity of the patient, age, sex, weight, sensitivity to the drug, type of current treatment, administration method, target cell, and the like. It can be easily determined by experts. In addition, the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be single or multiple administrations. Preferably all of the above factors can be administered in an amount that can achieve the maximum effect in a minimum amount without side effects, more preferably 1 to 10000 ㎍ / weight kg / day, even more preferably 10 to 1000 mg It may be administered repeatedly several times a day at an effective dose of / kg body weight / day.

The present invention may also provide a composition for diagnosing liver cancer comprising a substance for measuring mRNA or protein level of HDAC2 gene.

In the present invention, the expression level of the gene, preferably means the mRNA level, that is, the amount of mRNA expressed in the gene, and the material capable of measuring the level may include a primer or probe specific for the gene. Can be. In the present invention, the primer or probe specific for the HDAC2 gene may be a primer or probe capable of specifically amplifying the entire gene or a specific region of the gene of the HDAC2, and the primer or probe may be obtained by a method known in the art. You can design.

The term primer in the present invention refers to a single-strand that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers. Oligonucleotides. The appropriate length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer of the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence of the gene that is the template, and it is sufficient that the primer has sufficient complementarity within the range capable of hybridizing with the gene sequence to perform the primer action. In addition, it is preferable that the primer according to the present invention can be used for gene amplification reaction.

The amplification reaction refers to a reaction for amplifying a nucleic acid molecule. The amplification reactions of these genes are well known in the art, and examples thereof include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR) (LCR), electron mediated amplification (TMA), nucleic acid sequencing substrate amplification (NASBA), and the like.

In the present invention, the term probra refers to a linear oligomer of natural or modified monomers or linkages, and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, and is present naturally. Or artificially synthesized. The probe according to the invention may be single chain, preferably oligodioxyribonucleotides. Probes of the invention can include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogs or derivatives. In addition, the probe of the present invention may also include a ribonucleotide. For example, the probes of the present invention can be used in combination with a framework-modified nucleotide such as a peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoamidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'- 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexy Tolyl DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, 7-deazapurines having C-7 substituents (the substituents being fluoro, bromo, chloro, bromo, chloro, , Iodo-, me -, ethyl-, vinyl-, formyl-, alkynyl -, alkenyl-, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-), inosine, and may include a diamino purine.

In addition, as a substance capable of measuring the level of the protein in the present invention, antibodies such as polyclonal antibodies, monoclonal antibodies, and recombinant antibodies that can specifically bind to the protein expressed from the HDAC2 marker gene of the present invention. It may include.

The "antibody" may be prepared by a person skilled in the art using a known technique, for the production of the antibody, for example, in the case of polyclonal antibodies, the antigen of the protein is injected into the animal and blood collected from the animal Can be produced by methods well known in the art for obtaining serum comprising antibodies, such polyclonal antibodies from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc. It can be manufactured. In the case of monoclonal antibodies, the hybridoma method (Kohler et al., European Jounral of Immunology, 6, 511-519, 1976) to be prepared, or phage antibody libraries using (Clackson et al, Nature, 352 , 624-628, 1991, Marks et al, J. Mol Biol, 222:.. 58 , ≪ / RTI > 1-597, 1991). In addition, the antibodies according to the invention may comprise functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In addition, the present invention provides a liver cancer diagnostic kit comprising the liver cancer diagnostic marker or the liver cancer diagnostic composition according to the present invention.

The kit for diagnosing liver cancer of the present invention may include a primer, a probe, or an antibody capable of measuring the expression level of the marker gene HDAC2 gene or the amount of the protein expressed from the gene, and the definition thereof is as described above. .

If the simplified diagnostic kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth) ), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, wherein the kit for diagnosing liver cancer of the present invention In the case, the kit of the present invention may optionally comprise a substrate of a secondary antibody and a label. Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

In addition, the present invention provides a liver cancer diagnostic microarray comprising the liver cancer diagnostic marker.

In the microarray of the present invention, a primer, probe or antibody capable of measuring the expression level of the marker protein or gene encoding the same is used as a hybridizable array element and immobilized on a substrate. . Preferred substrates may include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports have. The hybridization array elements are arranged and immobilized on the substrate, and such immobilization can be performed by chemical bonding methods or covalent bonding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid, it may be labeled and hybridized with an array element on a microarray. Hybridization conditions may vary, and detection and analysis of hybridization degree may be variously performed depending on the labeled substance.

In addition, the present invention may provide a method for predicting and diagnosing liver cancer through a method of measuring the expression level of the HDAC2 marker gene or the expressed protein level according to the present invention, preferably, the method comprises: (a) liver cancer Measuring the expression level of the HDAC2 gene or the protein encoded by the gene from a biological sample of the suspected patient; And (b) comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene in the normal control sample. Can be.

The above method of measuring the expression level of the gene or the amount of the protein can be carried out using a known technique including a known process of separating mRNA or protein from a biological sample.

In the present invention, the "biological sample" refers to a sample collected from a living body, which is different from a normal control group, in which the expression level or protein level of the gene according to the occurrence or progression of liver cancer is used. But not limited to, tissue, cells, blood, serum, plasma, saliva, urine, and the like.

The expression level of the gene is preferably to measure the level of mRNA, the method for measuring the level of mRNA reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern Blots and DNA chips, etc., but is not limited thereto.

The protein level can be measured using an antibody, in which case the marker protein in the biological sample and the antibody specific thereto form a conjugate, i.e., an antigen-antibody complex, wherein the amount of antigen-antibody complex formed is a detection label. It can be measured quantitatively through the magnitude of the signal of the (detection label). Such detection labels can be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules and radioisotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assays, FACS, protein chips, and the like.

Therefore, the present invention can determine the amount of mRNA expression or protein of the marker gene of the control group and the amount of mRNA expression or protein of the marker gene in liver cancer patients or suspected liver cancer patients through the above detection methods, the expression amount By comparing the degree of with the control group, it is possible to predict and diagnose the incidence, progression or prognosis of liver cancer.

More specifically, the method for predicting or diagnosing the onset of liver cancer may be determined that liver cancer is induced when the expression level of the liver cancer marker gene HDAC2 gene or the amount of the expressed protein thereof is increased compared to a normal control sample. Can be.

The present invention further comprises the steps of: (i) contacting a sample to be analyzed with a liver cancer cell or tissue comprising a marker gene for diagnosis of liver cancer according to the present invention or an expression protein thereof; (Ii) measuring the amount of expression of said selected gene or the amount or activity of said expressed protein; And (iii) determining that the sample is a material for preventing or treating liver cancer when the amount of expression of the selected gene or the amount or activity of the expressed protein is reduced as a result of the measurement in step (ii). A method for screening a material for the prophylaxis or treatment of can be provided.

According to the screening method of the present invention, first, a sample to be analyzed may be contacted with liver cancer cells containing the gene or protein. Herein, the sample means an unknown substance used in screening to test whether the gene expression level, protein amount or protein activity is affected. The sample may include, but is not limited to, chemicals, oligonucleotides, antisense-RNAs, small interference RNAs (shRNAs), shRNAs or natural product extracts. Thereafter, the amount of expression of the gene, the amount of protein or the activity of the protein can be measured in the cells treated with the sample. If so, the sample may be determined as a substance capable of treating or preventing liver cancer.

The method of measuring the expression amount of the gene, the amount of the protein or the activity of the protein in the above may be carried out through a variety of methods known in the art, for example, but not limited to reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion (radioimmunodiffusion) And immunoprecipitation assays.

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

< Example >

1. Materials and Methods

Ethics Statement

A total of 30 hepatocellular carcinomas with their normal status were obtained from Yonsei University College of Medicine. Prior consent was provided according to helsinki. Written informed consent was obtained from all subjects and the study was approved by the Catholic University Medical School Ethics Committee (IRB approval number; CUMC09U119). For animal studies, all animal samples were conducted according to the guidelines of the Committee on Laboratory Animal Operations of the Department of Laboratory Animals, Catholic University Medical School (approval number; CUMC-2009-0050-03).

Plasmid Preparation

The plasmids used in this study were previously described: pME18S-FLAG-HDAC2, FLAG epitope-tagged HDAC2 expressing [Weichert W, Roske A, Niesporek S, Noske A, Buckendahl AC, et al. (2008) Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo. Clin Cancer Res 14: 1669-1677.]; Minimal luciferase reporter plasmid, Sp1-luc [Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ, et al. (1998) Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391: 597-601 .; pWWP, three deletion-formed plasmids (pWPdel-BstX1, pWPdel-Smal) and specific Sp1-binding sites, produced by subcloning with the 2.4 kbp human wild-type p21WAF1 / Cip1 promoter inserted into the pBL3-Basic reporter vector With two variant plasmids (pWPmt-Sp1-3, pWPmt-Sp1-5,6) [Luo J, Su F, Chen D, Shiloh A, Gu W (2000) Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 408: 377-381.]. pSilencer ^™ 3.1-H1 neo plasmid (Ambion, Austin, TX, USA) was used for the preparation of shRNA-encoding. Two pairs of oligonucleotides were each annealed and subclone into the vector (see Table 1 below).

Oligonucleotide Sequences Used for HDAC2 shRNA Plasmid Constructs Primer sequence si- # 1_top 5'-GAT CCG GAT TAC ATC ATG CTA AGA TTC AAG AGA TCT TAG CAT GAT GTA ATC CTT TTT TGG AAA -3 ' si- # 1_bottom 5'-AGC TTT TCC AAA AAA GGA TTA CAT CAT GCT AAG ATC TCT TGA ATC TTA GCA TGA TGT AAT CCG-3 ' si- # 2_top 5'-GAT CCG GAA GAA GAT AAA TCC AAG TTC AAG AGA CTT GGA TTT ATC TTC TTC CTT TTT TGG AAA-3 ' si- # 2_bottom 5'-AGC TTT TCC AAA AAA GGA AGA AGA TAA ATC CAA GTC TCT TGA ACT TGG ATT TAT CTT CTT CCG-3 '

Cell culture and siRNA

Human hepatocellular carcinoma cell line Hep3B was supplied from ATCC (Manassas, VA, USA) and RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / mL) and streptomycin (100 μg / mL) , Carlsbad, CA, USA). Cells were maintained in a humidified incubator containing 37 ° C., 5% CO ₂ . siRNA transformation was performed with Oligofectamin (Invitrogen) according to the manufacturer's instructions. Hep3B cells were transformed with human β-cateninn, c-Myc, cyclin D1 or two pre-designed HDAC2 siRNAs (Ambion) demonstrated at a final concentration of 100 nM. Crambled siRNA was used as a control.

Two sense strand sequences of HDAC2 siRNA sequence HDAC2 si- # 1 5'-GGAUUACAUCAUGCUAAGATT-3 ' HDAC2 si- # 2 5'- GGAAGAAGAUAAAUCCAAGTT-3 '

HDAC2 shRNA Stable expression of

Cells were transformed with 1 μg of HDAC2-shRNA expression vector pSilencer-HDAC2- # 1 or pSilencer-HDAC2- # 2. At 48 hours after transformation, 0.5 mg / mL Geneticin (G418 from Introgen) was added to the medium and G418-resistant colonies were selected. Medium was changed every two days. After about 3 weeks, G418 resistant cells were isolated as single colonies and each of them was cultured.

qRT-PCR

First-strand cDNA was synthesized from total RNA from each sample using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA). qRT-PCR was performed using the iQTM5 Real-Time PCR Detection System (Bio-Rad Laboratorises, Philadephia, PA, US) according to the manufacturer's instructions.

Western Blasting

Cells were harvested using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.2% SDS, 100 μg / mL PMSF, 1% μg / mL aprotinin and 0.5% sodium orthovanadate). After lysis, SDS-PAGE and immunoblotting were performed. Antibodies were used in Table 3 below. Chemiluminescence was detected using ECL reagent (GE Healthcare).

Antibodies Used for Immunoblotting Antibody company Catalog number anti-HDAC1 Upstate Biotechnology # 05-614 anti-HDAC2 # 07-222 anti-Ac-histone H3 # 06-599 anti-Ac-histoneH4 # 06-598 anti-beta-catenin Santa cruz biotechnology sc-7963 anti-cyclin D1 sc-717 anti-c-Myc sc-40 anti-CDK2 sc-6248 anti-DP-1 sc-610 anti-E2F1 sc-193 anti-E2F5 sc-999 anti-HDAC2 sc-7899 anti-p130 sc-317 anti-p-p130 sc-16300 anti-GAPDH sc-32233 anti-cyclin E2 Cell signaling technology # 4132 anti-CDK4 # 2906 anti-CDK6 # 3136 anti-cdc2 # 9112 anti-cleaved caspase3 # 9661 anti-E2F1 # 3742 anti-p21 # 2946 anti-p16 # 4824 anti-PARP # 9542 anti-p-pRb # 9300 anti-PCNA # 2586 anti-cyclinA # 4656 anti-MCM2 BD Pharmingen 559542 anti-pRb # 14001A anti-a-tubulin Sigma T-5168 anti-beta-actin A-5441

Cell growth assay cell growth assay )

A trypan blue dye exclusion assay was used to determine the number of live cells present in the cell suspension. The cell suspension and 0.4% trypan blue solution were mixed and incubated at room temperature for 3 minutes. Trypan blue / cell mixture on hemacytometer was applied to hemacytometer, and unstained (live) and stained (dead) cells were calculated using binocular microscope respectively.

Cell proliferation assay

Thymidine incorporation was measured using [methyl- ³ H] -thymidine (New England Nuclear Life Science). Briefly, after transformation as described above, 1 μCi / mL methyl- ³ H] -thymidine was added to the culture medium 10 hours before each assay. 48 and 72 hours after transformation, cells were washed with PBS and 5% TCA and lysed using lysis buffer (0.5% M NaOH, 2% SDS). Aliquots were used to measure the thymidine mixture on the scintillation counter LS6500 (Beckman Coulter, Fullerton, CA, USA).

Detection of Apoptotic Cells

Apoptotic cells were quantified using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions (BD Biosciences, San Jose, Calif., USA). Cells were analyzed using FACSCalibur flow cytometry and the percentage of apoptosis was measured with CellQuest ™ software.

Cell cycle analysis

The cells were trypsinized and then washed with cold PBS and fixed overnight using 70% ethanol, pre-cooled at 20 ° C. To determine the DNA content, cells were stained using Propidium Iodide (PI) solution (50 μg / mL PI, 100 μg / mL RNase A, 0.05% Triton X-100 in PBS). And incubated for 30 minutes in the dark 37 ℃. DNA content was examined by flow cytometry using FACSCalibur (BD Biosciences) and FlowJo software.

Cell synchronization

Stable cell lines were treated with 100ng / mL nocodazole for 18 hours and synchronized in prometaphase, followed by the cell cycle. After washing three times with PBS, cells were incubated in fresh medium for another time as indicated experiment.

Tumor xenograft experiments

For xenograft tumorigenesis assay, 5 × 10 ⁶ cells of the indicated cell lines were suspended in 0.2% mL PBS pH 7.4 and mixed with 20% (v / v) Matrigel. Cell suspensions were injected subcutaneously in 5 week old SCID mice. Tumor size [V (mm ³ ) = 0.52 × (width) ² × (length)] was calculated weekly using formula.

Chromatin immunoprecipitation (CHiP) assays

Cells were incubated overnight in 10 mm dishes to 70-90% saturation, then cross reacted with formaldehyde. After harvesting the cells, chromatin Immunoprecipitation was performed. Most of the courses followed the standard protocol for ChIP as described with some variations on the University of California at Davis Genome web site (genomecenter.ucdavis.edu/farnham/). DNA was isolated using a PCR purification kit, and was analyzed by PCR using a primer on a portion of the p21 ^{WAF1 / Cip1} promoter region (supporting information, Table S5).

Luciferase  Essay( luciferase assay )

Cells (1.5 × 10 ⁵ cells / well) were transformed with the promoter reporter plasmid 500ng / well of p21 ^{WAF1 / Cip1} using Lipofectamine PLUS (Invitroenn). Twenty four hours after transformation, the medium was replaced with or without 1 μM apicidin. At 24 hours after incubation, luciferase activity was measured using the Dual-Luciferse Reporter Assay System (Promega, Madison, Wis., USA).

Statistical analysis

All experiments were performed three times and samples were analyzed three times. The results are shown as averages. Statistical differences between groups were evaluated by applying Student's t-test using Microsoft Office Excel (Microsoft Albuquerque, NM, USA) for unparied data.

2. Results

Whether the Wnt pathway or c-Myc activity affects abnormal regulation of HDCA2

In previous studies, increased HDAC2 expression was found in colon cancer, and the introduction of HDAC2 proved to be dependent on the Wnt pathway and c-Myc. Previous reports also showed overexpression of HDAC2 in human hepatocellular carcinoma [Weichert W, Roske A, Gekeler V, Beckers T, Ebert MP, et al. (2008) Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis. Lancet Oncol 9: 139-148., Miyake K, Yoshizumi T, Imura S, Sugimoto K, Batmunkh E, et al. (2008) Expression of hypoxia-inducible factor-1alpha, histone deacetylase 1, and metastasis-associated protein 1 in pancreatic carcinoma: correlation with poor prognosis with possible regulation. Pancreas 36: e1-9.]. It has been reported that the activity of the Wnt pathway in hepatocarcinogenesis may be caused by stabilizing mutations in the β-catenin gene (15-25% of cases) or inactivated mutations in the Axin1 gene (5% of cases). This fact suggests that increased HDAC2 expression can be regulated by the Wnt pathway in hepatocellular carcinoma progression.

Therefore, in order to confirm abnormal expression of HDAC2 and show its association with its regulation with the Wnt pathway, we performed immunoblotting of HDAC2, β-catenin, cyclin D1 and c-Myc in 10 pairs of human hepatocellular carcinoma. Was performed. As a result, as shown in Figure 1, it was confirmed that the high overexpression of HDAC2 in all 10 pairs of hepatocellular carcinoma compared to the non-tumor tissue. However, upregulation of β-catenin was observed in three of 10 hepatocellular carcinomas (patient # 1, 5 and 7). The frequency of β-catenin overexpression (30%) is consistent with previously reported reports of Wnt signaling activity by stabilizing β-catenin in hepatocellular carcinoma [Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, et al. (2001) Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 120: 1763-1773.]. In contrast, cyclin D1 and c-Myc have been shown to overexpress in almost all hepatocellular carcinomas compared to non-tumor controls.

We also examined protein expression associated with Wnt signaling in human liver cancer cell lines including THLE-3, an immortal liver cell line. As a result, as shown in FIG. 2, high levels of HDAC2 expression were detected in all cell lines tested. On the other hand, β-catenin was not detected in hepatoblastoma cell line HepG2, and c-Myc showed various expression patterns. This means that Wnt signaling and c-Myc activity do not affect abnormal regulation of HDCA2 in liver cancer.

Finally, the present inventors conducted a gene knock-down experiment of β-catenin, cyclin D1 and c-Myc in Hep3B cells to prove the above. As a result, as shown in Figure 3, it was confirmed that HDAC2 expression does not affect due to the decrease of β-catenin, cyclin D1 or c-Myc in Hep3B cells. These results supported that abnormal HDAC2 expression was not dependent on the Wnt pathway or c-Myc activity in human hepatocarcinogenesis.

Effect of Reduction of HDAC2 on Anti-mitogenic Effects in Hepatocellular Carcinoma

In this experiment, we investigated HDAC2, which represents major histone deacetylase activity among HDACs in hepatocellular carcinoma. To assess the intracellular expression levels of various HDACs in the Hep3B cell line, qRT-PCR was performed using primer sets specific to each of the HDACs shown in Table 4 below. As a result, as shown in Figure 4, HDAC2 showed the highest expression level among the HDACs. As also shown in FIG. 5, HDAC2 reduction in Hep3B cells resulted in the accumulation of H3 and H4 of acetylated histones indicating major deacetylase activity.

Primer Sequences for the HDACs Gene gene Position Sequence (5 '- &gt; 3') Length (bp) Product Length
(bp) HDAC1 Forward CCA GTA TTC GAT GGC CTG TT 20 428 Reverse GGC TTG AAA ATG GCC TCA TA-3 20 HDAC2 Forward GAC AGG GTC ATC CCA TGA AG 20 415 Reverse AAT TCA AGG ATG GCA AGC AC 20 HDAC3 Forward GCA AGG CTT CAC CAA GAG TC-3 20 490 Reverse GTT GAT AAC CGG CTG GAA AA-3 20 HDAC4 Forward GGA GCT GAA GAA TGG CTT TG-3 20 429 Reverse GAT GAC ACC AGC ACC ACA TC-3 20 HDAC5 Forward CAG CAG GCG TTC TAC AAT GA-3 20 495 Reverse GTT GAT GTT GGG CTT TTG CT-3 20 HDAC6 Forward AAC CAG GCA GCG AAG AAG TA-3 20 405 Reverse TGA ACC AAC ATC AGC TCT TCC-3 20 HDAC7 Forward ATG GGC TTC TGC TTC TTC AA-3 20 566 Reverse TCC ACC CTG TTA CCC AGA AG-3 20 HDAC8 Forward GGC CAG TAT GGT GCA TTC TT-3 20 429 Reverse TGC AGA TCC AAA TCC ACG TA-3 20

In addition, to investigate the biological significance of abnormal expression of HDAC2 in liver cancer, experiments were performed to inhibit HDAC2 expression by RNA interference. As a result, as shown in Figure 6, the growth rate of the cells transformed with HDAC2 siRNA was found to be significantly reduced compared to the scrambled siRNA transfectant (Scr) untreated group or negative control.

In the hepatocellular carcinoma, the effect of inhibiting cell growth due to HDAC2 reduction may be partially explained by interference (mediation) of cell growth regulation such as cellular apoptosis, cell cycle arrest or cellular aging. Therefore, the present inventors have examined the effects of HDAC2 inhibition on cellular apoptosis and cell cycle regulation. As a result, as shown in Figure 7, in the flow cytometry analysis of the measurement of the annexin V stained cells (flow cytomeric analysis) compared to the control (Scr siRNA) did not show a significant induction of apoptotic cells. In addition, HDAC2 reduction did not affect the expression of pro-apoptotic constituents such as ALF, Bax and Apaf-1, and did not appear to cause caspase-3 or PARP cleavage. These results confirmed that overexpression of HDAC2 did not affect apoptotic signals in hepatocellular carcinoma.

We also investigated thymidine incorporation assay to determine if HDAC2 reduction caused anti-mitogenic effects in hepatocellular carcinoma cells. At this time, the Scr siRNA treatment group was used as a negative control, and the Valacic acid treatment group, an HDAC exogenous agent, was used as a positive control. As a result, as shown in Figure 8, compared to the negative control group in the experimental group transformed with HDAC2 siRNA when 48 hours and 72 hours elapsed, it was confirmed that significantly reduced cell proliferation and new DNA synthesis by HDAC2 reduction. .

We further performed cell cycle analysis of PI-stained cells in HDAC2 siRNA transformants using a flow cytometer. As a result, as shown in FIG. 9 and FIG. 10, when 48 hours elapsed in the HDAC2 siRNA-transformed experimental group, the G1 phase increased by 5.3% in the S phase and 7.8% in the G2 / M phase and increased by 12.8%. Similarly, the positive control group treated with Valproic acid, an HDAC exogenous agent, showed a strong G1 phase interruption during the cell cycle. In addition, HDAC2 reduction was shown to strongly induce p21 ^{WAF1 / Cip1} expression in Hep3B cells, but interestingly, unlike the positive control, it was observed that cyclin-dependent kinase 2 (CDK2) expression was inhibited in HDAC2 siRNA transformed experimental group. (See FIG. 11).

Effect of Selective Regulation of HDAC2 on G1 / S Components of Cell Cycle Circuits in Hep3B Cells

The fact that the inhibition of HDAC2 induces G1 phase arrest in the cell cycle implies that HDAC2 can modulate the activity of cell cycle regulatory elements. In this experiment, the effects of HDAC2 on the regulatory elements of the G1 / S transition were investigated. In the G1 / S transition, a ^{p15 INK4B, p16 INK4A, p18 INK4C} , p19 INK4D, p27 Kip1 and p21 ^{WAF1 / Cip1} and cell cycle regulators, such that the key factor to inhibit the cyclin D1 / CDK4,6 or cyclin E / CDK2 complexes Grana X, Reddy EP (1995) Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11: 211-219.

As a result, as shown in Figure 12, it was able to confirm that the p16 ^INK4A and p21 ^{WAF1 / Cip1} selectively induced in accordance with the decrease HDAC2 in Hep3B cells, were also able to confirm that inhibit cyckin D1, CDK4 and CDK2 at the same time. These results means that it is possible to induce the p16 ^INK4A and p21 ^{WAF1 / Cip1} inhibition of HDAC2 is overexpressed CDKI (cyclin-dependent kinase inhibitor) . And at the same time, it means to induce the expression of their specific regulators such as cyclin D1, CDK4 and CDK2 in the G1 / S transiton of hepatocellular carcinoma cells.

In general, activated cyclin / CDK complexes may lead to hyperphosphorylation of pRb, which results in the loss of Rb's activity as tumor suppressor and E2F / DP1 transcriptional activity. The present inventors investigated whether abnormal regulation of cyclins and CDKs by HDAC2 influences E2F / DP1 transcriptional activity. As a result, as shown in Figure 13, HDAC2 inhibition in Hep3B cells resulted in dephosphorylation of p130, pRb isoform (Rb2). These results indicate that abnormal HDAC2 regulation affects the phosphorylation of pRb protein through inactivation of transcriptional activity and / or inhibitory regulators of cyclin / CDKs. This hypothesis supports the fact that E2F / DP1 target genes such as E2F1, PCMA, CDC2 and E2F5 are selectively downregulated by HDAC2 reduction.

In order to prove the systematic regulation of cell cycle components according to the expression level of HDAC2 in vivo, 10 pairs of hepatocellular carcinoma tissues were tested. As a result, as shown in FIG. 14, p21 ^{WAF1 / Cip1} expression was detected at a relatively high level in all normal (non-tumor) tissues, while it was confirmed that the hepatocellular carcinoma tissues were significantly reduced. In contrast, CDK2 and PCNA have been shown to be upregulated in all cancer cell cancer tissues compared to normal tissues. These results HDAC2 the p16 ^INK4A and p21 ^{WAF1 / Cip1} inhibition and CDK2, CDK4 and very strong cleavage promote stimulation of the cooperative mechanism of the activity of cyclin D1 leading to cell growth that is not controlled while the HCC proceeds according to the abnormal expression Could be inferred.

Effect of Sustained Inhibition of HDAC2 on Tumor Potential of Hep3B HCC Cells

Since the knock-down of transient HDAC2 showed strong anticancer activity in this experiment, the present study examined whether sustained inhibition of HDAC2 could attenuate the potential of tumorigenesis in hepatocellular carcinoma cells in vitro and in vivo. . For the analysis of empirical weighing, HDAC2 shRNA (Hep3B_HDAC2KD) was introduced into Hep3B cells to prepare transformants expressing minimal HDAC2. As a result, as shown in Figure 15, it was confirmed that according to the functional inactivation of HDAC2 showing the inhibitory effect of the selective induction of p16 ^INK4A and p21 ^{WAF1 / Cip1} and CDK2, cyclin D1 and PCNA. In an experiment evaluating the growth rate of Hep3B_HDAC2KD cells, Hep3B_HDAC2KD cells showed reduced growth rate compared to Hep3B_Mock (Control 1) or Hep3B mother cell line (Control 2) (see FIG. 16).

We also analyzed the cell cycle using established cell lines. For accurate analysis of cell cycle progression, the established cell lines were synchronized in the G2 / M phase using nocodazole, and then the G1 phase was analyzed by allowing the cell cycle process to proceed. As a result, as shown in FIG. 17, it was confirmed that the ratio of cells in the G1 phase in Hep3B_HDAC2KD was increased at all times compared to the control group (Hep3B_Mock). This suggests that inhibition of proliferation and cell growth inhibition (Delay) of Hep3B cells due to the decrease of HDCA2 is due to G1 arrest in the cell cycle.

On the other hand, in vitro colony forming assay, it was confirmed that the continuous inhibition of HDAC2 reduced clonal cell growth (see Figure 18). Finally, SCID mice were injected subcutaneously with Hep3B_HDAC2KD cell line to demonstrate that abnormal regulation of HDAC2 caused cell tumors in vivo. As a result, as shown in FIG. 19 and FIG. 20, the total tumor growth was significantly decreased in the Hep3B_HDAC2KD injection group (P ≦ 0.05), and the average mass volume was confirmed to be very small in the Hep3B_HDAC2KD injection group compared to the control Hep3B_Mock. (P ≦ 0.05). In addition, 5 tumors were present in all animals injected with Hep3B_Mock cell line, and 3 tumors were present in animals injected with Hep3B_HDAC2KD cell line.

HDAC2 is released via p1-binding site ^{WAF1 / Cip1} Regulation of transcriptional activity

Previous studies have shown that HDAC ^exerts strongly activate the expression of p21 ^{WAF1 / Cip1} through enhanced histone acetylation of the p21 ^{WAF1 / Cip1} promoter containing the Sp1-binding site. In this experiment, a reporter assay was performed to investigate whether HDAC2 can selectively regulate the expression of p21 ^{WAF1 / Cip1} through the Sp1-binding site.

First, wild-type p21 ^{WAF1 / Cip1} reporter constructs were transformed into Hep3B_HDAC2KD cells and compared with Hep3B_Mock, a control group, and the effects of ppic ^{WAF1 / Cip1} treatment on Hep3B_Mock cells were investigated. . As a result, as shown in Figure 21, compared to the control Hep3B_Mock Hep3B_HDAC2KD introduced HDAC2 shRNA was confirmed that significantly increased luciferase activity. In contrast, when HDAC2 was re-expressed in Hep3B_HDAC2KD, it was confirmed that p21 ^{WAF1 / Cip1} transcriptional activity was inhibited compared to the control group (see FIG. 22). These results indicate that the intracellular expression of p21 ^{WAF1 / Cip1} is regulated at the transcriptional level by HDAC2.

ChIP assay was performed to confirm the results as described above. As a result, as shown in FIG. 23, it was confirmed that the HDAC2 protein was associated with the p21 ^{WAF1 / Cip1} proximal promoter region (upper panel), and it was found that the same portion was responsible for the deacetylation of histone H4. (Lower panel).

We also investigated whether ^HDAC2 can inhibit the expression of p21 ^{WAF1 / Cip1} via the Sp1-binding site on the p21 ^{WAF1 / Cip1} phase. As a result, as shown in FIG. 24, Hep3B_HDAC2KD cells showed increased luciferase activity in the presence of Sp1-binding site containing Sp1-6 at least in Sp1-3 (pWWP, pWPdel-BstXI and pWP101). However, the transcriptional activity of p21 ^{WAF1 / Cip1} was not induced by the mutant form of Sp1-5,6 (pWPmt-Sp1-5,6) in Hep3B_HDAC2KD cells. Interestingly, transcription of p21 ^{WAF1 / Cip1} is still induced by two variant plasmids (pWPdel-Smal and Sp1-luc) with two or three tandem repeats of the Sp1-binding site near the start of the TATA box or transcription. Appeared. These results suggest that the proximal site in the TATA box of p21 ^{WAF1 / Cip1} in Hep3B cells is an important site for the regulation of p21 ^{WAF1 / Cip1} transcription by ^HDAC2 .

So far I looked at the center of the preferred embodiment for the present invention. 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.

Claims (7)

A pharmaceutical composition for preventing or treating liver cancer comprising an inhibitor of HDAC2 gene expression or an inhibitor of HDAC2 protein activity. The method of claim 1,
The HDAC2 is a pharmaceutical composition for preventing or treating liver cancer, characterized in that it is overexpressed in liver cancer cells. The method of claim 1,
The inhibitor is a pharmaceutical composition for preventing or treating liver cancer, characterized in that the antisense oligonucleotide, siRNA, aptamer or antibody specific for HDAC2. The method of claim 1,
The inhibitor is a pharmaceutical composition for preventing or treating liver cancer, characterized in that the inhibition of the transcriptional activity of the Sp1-binding site to the p21 ^{WAF1 / Cip1} of HDAC2. (a) measuring the expression level of the HDAC2 gene or the protein encoded by the gene from a biological sample of a patient suspected of liver cancer; And
(b) providing information for predicting or diagnosing the onset of liver cancer comprising comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene in a normal control sample. Way. The method according to claim 6,
The measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA). : A method for providing information for predicting or diagnosing the onset of liver cancer, characterized in that it is selected from the group consisting of radioimmunoassay, radioimmunodiffusion and immunoprecipitation assay. (Iii) contacting a sample to be analyzed with a liver cancer cell or tissue comprising the HDAC2 gene or an expression protein thereof;
(Ii) measuring the amount of expression of said selected gene or the amount or activity of said expressed protein; And
(Iii) when the amount of expression of the selected gene or the amount or activity of the expressed protein is reduced as a result of the measurement of step (ii), the sample includes the step of determining liver cancer as a substance for preventing or treating liver cancer. Method of screening for prophylactic or therapeutic substances.

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