KR100966333B1 - Agent for Inhibiting Large Intestine Cancer Cell Proliferation Comprising Gene Delivery Vehicle Containing a GABRA1 Gene - Google Patents

Abstract

The present invention relates to a proliferation inhibitor of colorectal cancer cells and a therapeutic agent for colorectal cancer, including a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ).

According to the present invention, the gene carrier or gamma-aminobutyric acid A receptor alpha 1 protein containing a gene encoding the gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) is colorectal cancer. Since it has an effect of inhibiting proliferation of cells, it is useful for the treatment of colon cancer.

Gamma-aminobutyric acid A receptor alpha 1, colorectal cancer, methylation, promoter

Description

Agent for Inhibiting Large Intestine Cancer Cell Proliferation Comprising Gene Delivery Vehicle Containing a GABRA1 Gene}

The present invention relates to a proliferation inhibitor of colorectal cancer cells and a therapeutic agent for colorectal cancer, including a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ).

In normal cells, oncogenes, tumor suppressor genes, and apoptosis regulating genes are complementarily regulated to grow and maintain in harmony, but the cells proliferate abnormally due to various causes. And it is known that cancer occurs by repeated growth, the root cause of which is caused by abnormality of genes in cells. These genetic abnormalities are largely genetic and epigenetic.

One of the epigenetic mutations, DNA methylation, occurs mainly in the cytosine of CpG islands at the promoter site of a particular gene, and methylation of CpG islands in promoters that regulate this gene expression inhibits expression of the gene of interest.

In particular, cancer suppressor genes ( p16INK4a , p15INK4b , p14ARF , p73 ), DNA damage repair genes ( hMLH1 ) and cancer metastasis and invasion related genes ( CDH1 , TIMP3 , DAPK ) are known to be inhibited by promoter methylation in many cancers. Esteller and Herman, J. Pathol ., 196: 1, 2002; Esteller et. al ., Cancer Res ., 61: 3225, 2001).

Extensive research is being conducted to treat cancer, which accounts for the highest proportion of human deaths. To date, known cancer treatment methods include surgery, radiation therapy, chemotherapy, immunotherapy, and gene therapy. Recently, researches have been conducted to discover new target genes using gene therapy and to efficiently introduce them into target cells to induce long-term expression and to improve gene delivery efficiency when used in clinical treatment.

Recently, a gene or a protein thereof is often used to treat cancer, and research on this continues. In order to find the most suitable therapeutic genes, research is being conducted in parallel with the human genome project to find genes that are deficient in cancer patients, genes that cause cancer, and genes having anti-cancer effects. There is a great need for the discovery of new cancer suppressor genes.

On the other hand, the inventors have applied for a patent application revealing that the cause of gene expression inhibition in colorectal cancer tissue cells is due to DNA methylation in the CpG island of the gene promoter region (Korean Patent Publication No. 10-2007-0098034). However, the therapeutic use of colorectal cancer using the same is not known.

Therefore, the present inventors have made efforts to develop a therapeutic agent for colorectal cancer using a novel cancer suppressor gene. As a result, the present inventors introduced a recombinant vector containing a gene encoding gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) into colon cancer cells. The proliferation inhibitory ability of the colon cancer cells was confirmed, and the present invention was completed.

A main object of the present invention is to provide a therapeutic agent using a gene encoding the gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) or a protein that inhibits the proliferation of colon cancer cells.

In order to achieve the above object, the present invention provides a proliferation of colorectal cancer cells comprising a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) Provide inhibitors.

The present invention also provides a composition for inhibiting proliferation of colorectal cancer cells containing gamma-aminobutyric acid A receptor alpha 1 protein as an active ingredient.

The present invention also provides a therapeutic agent for colorectal cancer comprising a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ).

The present invention also provides a pharmaceutical composition for treating colorectal cancer containing gamma-aminobutyric acid A receptor alpha 1 protein as an active ingredient.

In the present invention, the gene encoding the gamma-aminobutyric acid A receptor alpha 1 may be represented by SEQ ID NO: 1, the gamma-aminobutyric acid A receptor alpha 1 protein is represented by SEQ ID NO: 2 It may be characterized by.

The present invention is a composition for inhibiting the growth of colorectal cancer cells containing a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 and a colon cancer proliferation inhibitor containing gamma-aminobutyric acid A receptor alpha 1 protein Has the effect of providing.

A gene carrier or gamma-aminobutyric acid A receptor alpha 1 protein containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ), a cancer suppressor gene according to the present invention, Since it has an effect of inhibiting the proliferation of colon cancer cells, it is useful for the treatment of colon cancer.

In the present invention, a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) or a protein thereof, among genes in which a promoter for regulating gene expression in colorectal cancer cells is specifically methylated, may be used to treat colorectal cancer. It was.

Gamma-aminobutyric acid A receptor alpha 1 protein binds to gamma-aminobutyric acid, which plays a role in neurotransmission inhibition in the brain of mammals, and serves to initiate a salt channel.

In one aspect, the present invention relates to a proliferation inhibitor of colorectal cancer cells comprising a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1.

In the present invention, first, the expression of the GABRA1 gene in colorectal cancer was confirmed in relation to the methylation of the promoter.

That is, in one embodiment of the present invention, in order to confirm the expression of the GABRA1 gene in colorectal cancer, microarray hybridization was performed, and GABRA1 in total RNA isolated from normal tissue and tumor tissue near the tumor of colon cancer patients, respectively. The expression level of the gene was measured by an indirect comparison method.

In the present invention, "indirect comparison" evaluates the level of nucleic acid isolated from the first source and the same allele level in the nucleic acid isolated from the second source using a reference probe hybridized to the nucleic acid isolated from the first source and the second source, respectively. And comparing the results to determine the relative amount of nucleic acid present in the sample without competing direct binding to the reference probe.

As a result of the indirect comparison, it was confirmed that the expression of the gene was down-regulated in the tumor tissue and the GABRA1 gene was also down-regulated.

In addition, as a result of treatment with the dimethylating agent DAC to confirm whether the expression of the gene is regulated by the methylation of the promoter, the expression of the gene was increased when the DAC was treated, indicating that the gene expression is due to the methylation of the promoter. there was.

In another embodiment of the present invention, the promoter of the gene was detected GABRA1 the methylation status of the gene promoter GABRA1 the sequencing method, the pie in order to check whether the methylated in colon cancer cell lines and colon cancer tissue. At this time, the DNA is treated with bisulfite, while unmethylated cytosine is transformed into uracil, while methylated cytosine remains unchanged. After amplifying the bisulfite-treated DNA by PCR and analyzing the sequencing, it was found that the Caco-2 and HCT-116 colorectal cancer cell lines had a methylation index of 80% or more, and the colorectal cancer tissue was also normal. It was confirmed that methylation was higher in 85.7% of cancer tissue samples than in tissues. In other words, it was found that the decrease in expression in the colorectal cancer cell line and tissue of the GABRA1 gene is due to the methylation of the promoter.

In the present invention, the GABRA1 gene, which was confirmed that the decrease in gene expression was caused by the methylation of the promoter, was introduced into the colorectal cancer cell line to confirm the proliferation inhibitory ability of the colorectal cancer cells.

Introduction of the gene can be made by a conventional method, the technique of transferring the target gene is largely non-viral vector-based transfer method, using a synthetic phospholipid or synthetic cationic polymer, etc. There are physical methods such as non-viral delivery method and electroporation method that introduces genes by applying temporary electrical stimulation to cell membrane. Among the delivery technologies, viral transporter is used as a therapeutic gene. It is a preferred method for gene therapy because it is a vector that lacks the copying ability of some or all of the genes that have been replaced, and can efficiently transfer genes.

Preferably, the gene is included in a recombinant expression vector and introduced, and a virus used as a virus transporter or viral vector includes RNA viral vectors (retroviral vectors, lentivirus vectors, etc.) and DNA viral vectors (adenovirus vectors). , Adeno-associated viral vectors, etc.), but also include, but are not limited to, herpes simplex viral vector, alpha viral vector, and the like.

For the embodiment of the present invention to deliver GABRA1 gene, by inserting the gene into the pcDNA4 GABRA1 ^TM vector GABRA1 A GABRA1 gene expression vector capable of expressing a gene was constructed.

GABRA1 above The gene can be isolated from human tissue or synthesized according to known DNA synthesis methods. In the present invention, 1,371bp Homo sapiens ( Homo) GABRA1 gene (GenBank NM_000806, SEQ ID NO: 1) and gamma-aminobutyric acid A receptor alpha 1 protein ((GenBank NP — 000797.2, SEQ ID NO: 2)) were used in the cDNA sequence derived from sapiens .

Encoding the gamma-aminobutyric acid A receptor alpha 1 As a result of introducing the recombinant vector containing the gene into the colorectal cancer cells, it was confirmed that the proliferation of the colorectal cancer cells was suppressed. Thus, the gamma-aminobutyric acid A receptor alpha 1 encoding When recombinant vectors containing genes were introduced into colon cancer cells, they were found to be effective in treating colon cancer.

In the present invention, the pcDNA4 ^TM vector is used, but various expression vectors may be used depending on the purpose of expression, and sizes of genes, base sequences, etc., inserted into the foreign gene insertion site of the expression vector may be variously known. Can change. In particular, in the present invention, the adenovirus as an expression vector-by delivering a gene associated with a viral (adeno-associated virus, AAV), such as a virus specific example using the vectors, but, a known chain gene delivery viral vectors in vivo It will be apparent to those skilled in the art that proliferation of colorectal cancer cells in the stomach can be used as an inhibitor and a therapeutic agent for colorectal cancer.

In addition, in the present invention, there is no specific example in which gamma-aminobutyric acid A receptor alpha 1 protein inhibits the proliferation of colorectal cancer cells, but GABRA1 Overexpression of the gene inhibits the proliferation of colorectal cancer cells, thus encoding the gamma-aminobutyric acid A receptor alpha 1 When gamma-aminobutyric acid A receptor alpha 1 protein obtained by culturing a transformant into which a recombinant vector containing a gene was introduced is treated to colon cancer cells, the proliferation of colon cancer cells is suppressed. It will be apparent to those skilled in the art that it will be effective.

Therefore, in another aspect, the present invention relates to a composition for inhibiting colorectal cancer cells containing gamma-aminobutyric acid A receptor alpha 1 protein as an active ingredient.

The composition according to the present invention can be formulated or used in combination with drugs such as antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics that are already used as pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

Compounds containing the compositions of the present invention or pharmaceutically acceptable salts thereof can be used as the main ingredient or additives and auxiliaries of foods in the manufacture of various functional foods and dietary supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

In particular, the following examples illustrate only the pcDNA4 ^TM vector as a gene carrier, but it will be apparent to those skilled in the art that the same result can be obtained using other various gene carriers.

Example  1: Gamma in Colon Cancer Tissue and Colon Cancer Cell Lines- Aminobutyric acid  A receptor alpha 1 ( GABRA1 Expression measurement of the gene encoding

Microarray hybridization was performed to confirm the expression of the gene encoding gamma-aminobutyric acid A receptor alpha 1 in colorectal cancer. Microarray hybridization was performed using standard protocols (Schena et al ., Science , 270: 467, 1995).

Normal tissue (5 samples) and tumor tissue (5 samples) near the tumor were isolated from the colon cancer patient (Yonsei University Hospital Surgery) and total RNA was separated therefrom. In order to indirectly compare the relative differences in gene expression levels of normal tissues and tumor tissues near the paired tumors, standard comparative RNAs were prepared.

That is, to prepare a standard comparative RNA, lung cancer cell line A549 (Korean cell line Eunhaeng (KCLB) 10185), gastric cancer cell line AGS (KCLB 21739), kidney cancer cell line Caki-2 (KCLB 30047), colon cancer cell line HCT116 (KCLB 10247), Cervical cancer cell line Hela (KCLB 10002), blood cancer cell line HK-60 (KCLB 10240), HT1080 (KCLB 10121), breast cancer cell line MDA-MB231 (KCLB 30026), liver cancer cell line SK-hep1 (KCLB 30052), T cell-derived cell line Total RNA was isolated from 11 human cancer cell lines of Molt-4 (KCLB 21582) and brain cancer cell line U-87MG (KCLB 30014) using Tri Reagent (Sigma, USA) and total RNA isolated from the 11 cell lines. Mix in equal amounts and use this as internal control.

In order to compare relative gene expression levels of the paired near tumor and tumor tissue, RNA isolated from non-tumor and tumor tissue was compared indirectly with standard RNA. To this end, 50 μg of total RNA was labeled with Cy3-dUTP or Cy5-dUTP, that is, the standard comparative RNA was labeled with Cy3, and RNA isolated from the tissue was labeled with Cy5 and performed using a DNA microarray. The two cDNAs labeled Cy3- and Cy5- were purified using a PCR purification kit (Qiagen, Germany). The purified cDNA was mixed and concentrated to a final volume of 27 μl using Microcon YM-30 (Millipore Co., USA).

100 μl of the hybridization reaction solution (27 μl of the labeled cDNA target, 20 μl of 20X SSC, 8 μl of 1% SDS, 24 μl of formamide (Sigma, USA) and 20 μg of human Cot1 DNA (Invitrogen, USA)) Heated at 2 ° C. for 2 min and immediately hybridized to a human 35K oligonucleotide (GenomicTree, Inc., Korea) microarray at 42 ° C., 12-16 hours in HybChamber X (GenomicTree, Inc., Korea), humidity-controlled. The hybridized microarray slides were read using Axon 4000B (Axon Instrument Inc., USA). Signal and background fluorescence intensities were measured for each probe by averaging the intensity of all pixels inside the target site using GenePix Pro 4.0 software (Axon Instrument Inc., USA). Spots showing obvious abnormalities were excluded from the analysis. All data were subjected to normalization, statistical analysis and cluster analysis using GeneSpring 7.2 (Agilent, USA).

In addition, statistical analysis (ANOVA, p <0.05) for indirect comparisons was performed to determine relative differences in gene expression levels of non-tumor and tumor tissues. As a result, it was confirmed that all 301 genes are down regulated in tumor tissues compared to normal tissues near paired tumors, and GABRA1 It was confirmed that the gene is also downregulated by about 2.33 fold (Table 1).

In addition, in order to confirm whether expression of the gene is regulated by methylation of the gene promoter, 200 aM 5-aza-2-deoxycytidine (DAC, Sigma, USA) which is a dimethylating agent in the intestinal cancer cell line Caco-2 (KCLB 30037) Was treated for 3 days. The total RNA was isolated by treating the DAC-treated and untreated cell lines with Tri reagent.

In order to confirm gene expression changes by DAC treatment, the level of transcription between the untreated and treated cell lines was compared directly. The colorectal cancer cell lines were cultured at 37 ° C. and 5% CO ₂ concentration in RPMI culture medium (GIBCO / BRL, Grand Island, NY) containing 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin, respectively. Afterwards, the gene expression of the DAC treated group was measured as compared to the control not treated with DAC (Table 1). As a result, it was confirmed that the level of re-expression of GABRA1 gene by dimethylation increased 2.2 times.

TABLE 1 Expression levels of GABRA1 gene in colorectal cancer tissues and colorectal cancer cell lines

Downward expression in colorectal cancer tissue Reexpression Levels in Colorectal Cancer Cell Lines Average fold change p-value CaCo-2 GABRA1 0.43 <0.01 2.2

Example  2: In a colon cancer cell line  gamma- Aminobutyric acid  Determination of methylation of genes encoding A receptor alpha 1

In order to determine whether the re-expression of the GABRA1 gene confirmed in Example 1 in the colorectal cancer cell line by dimethylation, the degree of methylation was measured in the colorectal cancer cell lines Caco-2 and HCT116 (KCLB 10247).

For this purpose, GABRA1 is used as a pyrosequencing method. The methylation status of the gene promoter (GenBank NT_023133, SEQ ID NO: 3) was detected (FIG. 1). The whole genomic DNA was isolated, and bisulfite was treated with 200 ng of genomic DNA using the EZ DNA methylation-Gold kit (Zymo Research, USA). Treatment of DNA with bisulfite leaves unmethylated cytosine modified with uracil and methylated cytosine remains unchanged. The bisulfite treated DNA was eluted with 20 μl of sterile distilled water, and pyrosequencing was performed according to the manufacturer's instructions (Biotage, USA). 20 ng of bisulfite treated genomic DNA was amplified using PCR under the following conditions; A total of 40 treatments of 94 ° C. (1 min), 66 ° C. (1 min) and 72 ° C. (1 min) were performed in one cycle, and finally extended at 72 ° C. for 10 minutes. Template DNA labeled with biotin was purified and sequenced using PyroMark ID (Biotage, USA).

GABRA1 above PCR and sequencing primers for performing pyro sequencing on genes were designed using the PSQ assay design program (Biotage, USA). PCR and sequencing primers for the methylation measurement of the GABRA1 gene are shown in Table 2.

Table 2 PCR and Sequencing Primers

primer Sequence (5 '-> 3') SEQ ID NO: CpG position ^a Amplicon size Forward GGG AAG TAA ATT TGG GTG TGA A 4
+ 893, + 899, +905, +907


210 bp Reverse (5'-biotin) CCC CCT CTA AAC ACA AAA TCT CTT 5 Sequencing primer ATT AAG TGA GTG AGA GGT AG 6

^a nucleotide from the transcription initiation point (+1): the position on the genomic DNA of the CpG site used to measure methylation

GABRA1  Gene Pyro Sequencing  Site (SEQ ID NO: 7)

gg gaagtaaatt tgggtgtgaa atttttagta aaggagtayg tagagtttat gatggtttag attaagtgag tgagaggtag agygaggayg tttttttgtt ttggygygtt yggattygga ttygtagatt ygygttggtt ttagtttttt taygattttt ttttttagat tttttttygg ttttaagaga ttttgtgttt agaggggg; +819-+1,018 nt, underlined sequence indicates pyrosequencing site, y: CpG site

As a result, as shown in Figure 1, the promoter region of the GABRA1 gene was found to be methylated in the Caco-2 cell line and HCT-116 cell line showing an methylation index of 80% or more. This means that after demethylating agent treatment in colorectal cancer cell lines, re-expression of the GABRA1 gene is due to demethylation.

Example  3: gamma in colorectal cancer tissue Aminobutyric acid  Determination of methylation of genes encoding A receptor alpha 1

To determine whether the decrease in the expression of the gene encoding gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) in colorectal cancer tissues is due to methylation, GABRA1 in normal and cancerous tissues (35 pairs) of surgical tissue of colorectal cancer patients The methylation of was measured.

GABRA1 In order to determine the methylation of the gene promoter region, the pyro sequencing method was used. Pyro sequencing method was carried out in the same manner as the cell line.

As a result, as shown in FIG. 2, the promoter of the GABRA1 gene showed a low methylation index of 20% or less in normal tissue, while a high level of methylation index (p value = 0.0001) was found in cancer tissue (FIG. 2A). ROC (Receiver Operating Characteristic) curve analysis showed that when the methylation index was set at the methylation index cut-off, the sensitivity for diagnosis of colorectal cancer was 85.7% and the specificity was 100. It was found to be very good in% (FIG. 2B). Therefore, it can be seen that the degradation of the GABRA1 gene in colorectal cancer tissue is due to the methylation of the promoter.

Example  4: Gamma Aminobutyric acid  Construction of Expression Vectors of Genes Encoding A Receptor Alpha 1

To prepare gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ) gene expression vector, GABRA1 from 21C Human Gene Bank (Biotechnology Research Institute) A cDNA clone (hMU002273) of 3,440 bp (GenBank BC030696, SEQ ID NO: 8) containing the gene was received.

In order to clone the GABRA1 cDNA, GABRA1 primer containing the recognition of the plasmid clones hMU002273 and (Korea Biotechnology 21C Frontier Human GenBank) as the template, the restriction enzyme Eco RI, which contains the cDNA sequence (SEQ ID NO: 9) and the restriction enzyme Xho PCR was performed using a primer containing the recognition sequence of I (SEQ ID NO: 10).

SEQ ID NO: 5'-ATT AGA ATT CGC GAT GAG GAA AAG T-3 '

SEQ ID NO: 10'-TAA TCT CGA GGA TCT ATT GAT GTG GTG-3 '

The amplified PCR product was digested with Eco RI and Xho I restriction enzymes, and cloned back into the Eco RI and Xho I sites of the pcDNA4 ^™ / HisMax expression vector (Invitrogen) to produce a GABRA1 gene expression vector (FIG. 3).

Example  5: Gamma Aminobutyric acid  Introduction of gene encoding A receptor alpha 1 into colon cancer cells and measurement of cancer cell proliferation inhibition

Cell proliferation experiments were performed to determine the effect of inhibiting the proliferation of cancer cells by overexpression of gamma-aminobutyric acid A receptor alpha 1 in colorectal cancer cell lines.

Colorectal cancer cell line HCT116 in RPMI 1640 medium (Sigma, USA) supplemented with 10% FBS (Sigma, USA), penicillin (100 units / ml, WelGENE, Korea) and streptomycin (100 units / ml, WelGENE, Korea) , 37 ° C., 5% CO ₂ incubator (Forma Scientific Inc.). To carry out cell proliferation experiments, cells were inoculated at a concentration of 3 X 10 ³ in 96 well plates and incubated for 24 hours.

These cells were transfected with the gamma-aminobutyric acid A receptor alpha 1 gene expression vector prepared in Example 4 using Fugene HD transfection reagent (Roche Applied Science, Cat. No. 4709705001) according to the manufacturer's instructions. transfection). As a control, pcDNA4 ^TM / HisMax (control vector) containing no GABRA1 gene was transfected. After incubating the transformants for 48 hours, 10 μl of WST-1 reagent (Roche Applied Science) was added per well, and further cultured for 1 hour in a 37 ° C., 5% CO ₂ incubator. The culture was observed under a microscope, and the cell proliferation state was observed under a microscope by measuring the absorbance at 440 nm using an ELISA reader (BIO-RAD, Benchmark Plus Microplate Spectrophotometer).

As a result, as shown in FIG. 4, after the introduction of the control vector and the GABRA1 expression vector, the gamma-aminobutyric acid A receptor alpha-1 gene expression vector, the shape of colorectal cancer cells was taken under a microscope. In the cells into which the receptor alpha 1 gene was introduced, it was confirmed that cell proliferation was significantly suppressed (FIG. 4A). That is, the gamma-aminobutyric acid A receptor alpha 1 gene or protein can effectively inhibit the proliferation of lung cancer cells, which means that it is highly useful as a therapeutic agent for colorectal cancer.

In addition, as a result of measuring the amount of dead cells using ELISA, it was confirmed that the proliferation in colorectal cancer cells in which pGABRA1 expression vector was introduced to 0.75 ug / ml was significantly inhibited by 46% compared to cells in which control vectors were introduced. (FIG. 4B). This means that the GABRA1 gene or a protein thereof can effectively inhibit the proliferation of colorectal cancer cells and can be used as a therapeutic agent for colorectal cancer.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 is a measure of the methylation of the GABRA1 gene promoter in the colon cancer cell lines Caco-2 and HCT116 cell line.

Figure 2 is a measure of the methylation of the GABRA1 gene promoter in normal and colorectal cancer tissue (A: electrophoresis of methylation in normal and colorectal cancer tissue; B: methylation in normal and colorectal cancer tissue Plotting measurement results).

Figure 3 shows a schematic of the GABRA1 gene expression vector.

Figure 4 shows the results of performing a cell proliferation inhibition experiment after introducing the GABRA1 gene expression vector in colorectal cancer cell line.

<110> Genomictree <120> AGENT FOR INHIBITING LARGE INTESTINE CANCER CELL PROLIFERATION          COMPRISING GENE DELIVERY VEHICLE CONTAINING A GENE ENCODING          GABRA1 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 1311 <212> DNA <213> GABRA1 <400> 1 atgaggaaaa gtccaggtct gtctgactgt ctttgggcct ggatcctcct tctgagcaca 60 ctgactggaa gaagctatgg acagccgtca ttacaagatg aacttaaaga caataccact 120 gtcttcacca ggattttgga cagactccta gatggttatg acaatcgcct gagaccagga 180 ttgggagagc gtgtaaccga agtgaagact gatatcttcg tcaccagttt cggacccgtt 240 tcagaccatg atatggaata tacaatagat gtatttttcc gtcaaagctg gaaggatgaa 300 aggttaaaat ttaaaggacc tatgacagtc ctccggttaa ataacctaat ggcaagtaaa 360 atctggactc cggacacatt tttccacaat ggaaagaagt cagtggccca caacatgacc 420 atgcccaaca aactcctgcg gatcacagag gatggcacct tgctgtacac catgaggctg 480 acagtgagag ctgaatgtcc gatgcatttg gaggacttcc ctatggatgc ccatgcttgc 540 ccactaaaat ttggaagtta tgcttataca agagcagaag ttgtttatga atggaccaga 600 gagccagcac gctcagtggt tgtagcagaa gatggatcac gtctaaacca gtatgacctt 660 cttggacaaa cagtagactc tggaattgtc cagtcaagta caggagaata tgttgttatg 720 accactcatt tccacttgaa gagaaagatt ggctactttg ttattcaaac atctgtacca 780 gcaagaactg tctttggagt aacaactgtg ctcaccatga caacattgag catcagtgcc 840 agaaactccc tccctaaggt ggcttatgca acagctatgg attggtttat tgccgtgtgc 900 tatgcctttg tgttctcagc tctgattgag tttgccacag taaactattt cactaagaga 960 ggttatgcat gggatggcaa aagtgtggtt ccagaaaagc caaagaaagt aaaggatcct 1020 cttattaaga aaaacaacac ttacgctcca acagcaacca gctacacccc taatttggcc 1080 aggggcgacc cgggcttagc caccattgct aaaagtgcaa ccatagaacc taaagaggtc 1140 aagcccgaaa caaaaccacc agaacccaag aaaaccttta acagtgtcag caaaattgac 1200 cgactgtcaa gaatagcctt cccgctgcta tttggaatct ttaacttagt ctactgggct 1260 acgtatttaa acagagagcc tcagctaaaa gcccccacac cacatcaata g 1311 <210> 2 <211> 456 <212> PRT <213> GABRA1 <400> 2 Met Arg Lys Ser Pro Gly Leu Ser Asp Cys Leu Trp Ala Trp Ile Leu   1 5 10 15 Leu Leu Ser Thr Leu Thr Gly Arg Ser Tyr Gly Gln Pro Ser Leu Gln              20 25 30 Asp Glu Leu Lys Asp Asn Thr Thr Val Val Phe Thr Arg Ile Leu Asp Arg          35 40 45 Leu Leu Asp Gly Tyr Asp Asn Arg Leu Arg Pro Gly Leu Gly Glu Arg      50 55 60 Val Thr Glu Val Lys Thr Asp Ile Phe Val Thr Ser Phe Gly Pro Val  65 70 75 80 Ser Asp His Asp Met Glu Tyr Thr Ile Asp Val Phe Phe Arg Gln Ser                  85 90 95 Trp Lys Asp Glu Arg Leu Lys Phe Lys Gly Pro Met Thr Val Leu Arg             100 105 110 Leu Asn Asn Leu Met Ala Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe         115 120 125 His Asn Gly Lys Lys Ser Val Ala His Asn Met Thr Met Pro Asn Lys     130 135 140 Leu Leu Arg Ile Thr Glu Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu 145 150 155 160 Thr Val Arg Ala Glu Cys Pro Met His Leu Glu Asp Phe Pro Met Asp                 165 170 175 Ala His Ala Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Thr Arg Ala             180 185 190 Glu Val Val Tyr Glu Trp Thr Arg Glu Pro Ala Arg Ser Val Val Val         195 200 205 Ala Glu Asp Gly Ser Arg Leu Asn Gln Tyr Asp Leu Leu Gly Gln Thr     210 215 220 Val Asp Ser Gly Ile Val Gln Ser Ser Thr Gly Glu Tyr Val Val Met 225 230 235 240 Thr Thr His Phe His Leu Lys Arg Lys Ile Gly Tyr Phe Val Ile Gln                 245 250 255 Thr Tyr Leu Pro Cys Ile Met Thr Val Ile Leu Ser Gln Val Ser Phe             260 265 270 Trp Leu Asn Arg Glu Ser Val Pro Ala Arg Thr Val Phe Gly Val Thr         275 280 285 Thr Val Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg Asn Ser Leu     290 295 300 Pro Lys Val Ala Tyr Ala Thr Ala Met Asp Trp Phe Ile Ala Val Cys 305 310 315 320 Tyr Ala Phe Val Phe Ser Ala Leu Ile Glu Phe Ala Thr Val Asn Tyr                 325 330 335 Phe Thr Lys Arg Gly Tyr Ala Trp Asp Gly Lys Ser Val Val Pro Glu             340 345 350 Lys Pro Lys Lys Val Lys Asp Pro Leu Ile Lys Lys Asn Asn Thr Tyr         355 360 365 Ala Pro Thr Ala Thr Ser Tyr Thr Pro Asn Leu Ala Arg Gly Asp Pro     370 375 380 Gly Leu Ala Thr Ile Ala Lys Ser Ala Thr Ile Glu Pro Lys Glu Val 385 390 395 400 Lys Pro Glu Thr Lys Pro Pro Glu Pro Lys Lys Thr Phe Asn Ser Val                 405 410 415 Ser Lys Ile Asp Arg Leu Ser Arg Ile Ala Phe Pro Leu Leu Phe Gly             420 425 430 Ile Phe Asn Leu Val Tyr Trp Ala Thr Tyr Leu Asn Arg Glu Pro Gln         435 440 445 Leu Lys Ala Pro Thr Pro His Gln     450 455 <210> 3 <211> 2000 <212> DNA <213> GABRA1 gene promoter <400> 3 tctctcatag agactcattt tgagaatcaa attgaattat tatgaaaatt acttttcaca 60 ttacttggca ccaagtaaac actctaagaa tattaaacac catagttatt atcatttaaa 120 aaaactgaca tcattatatg cacacatagt ttgaagtggt tcattctggc taaaacaaaa 180 cacaagaata tcaaaaatta tataaaatat tgaatataaa aacacaatat aaagtatttt 240 cttgtcatta atttattata ttttatggta gtatgtgcat atattaatga catatcagat 300 ggaacacaat aaacacaccc ataattagag tatcaattga tgttttggag tattaaaata 360 tcattatttt taaacactca caatttatga aaaatataga tgtcttaaag caaattacat 420 gtatgtgtgt atgtatacat acatgcatat acatattaca agctgatatt catagagaat 480 catctagtaa aatctacctt gtgagtatta cattttaaag agagaatcat agtataggta 540 tttccatttc catatacaca ctatagcccc caaattattt aatatttatt tctttaaatg 600 tttgaaaacg ctgtgcaatt gtatcctgtt tcatagtcac ataacatttt ttaatctgaa 660 gcagaatgta attggcattg ctctgagact attgttcaca tgatgaagct tcagtgaatt 720 tgtttgcaaa accaaaatca gtagagtgat ttagttgtgt ttcattagat aaaggatata 780 aatttatttt caatttgtag aagatcataa aaggacctgc aacagtttag ccaggagaac 840 actaaaaatt cagattttta taactgttga ctttcattaa aacaagacaa aactgtggaa 900 ttgcattgta gatgaagtaa aagaataatg ttttctaatc gttaactcag gatttcatta 960 ttaattataa atctcattaa attcagtttt attctccaaa ccactcaata aattaattaa 1020 tcaatggata agtaaataaa aattgggtaa tgacacatag ggaagctgtg atgtttcttc 1080 agcgaatatc tttaagaata cattggcatt tattatatct gaacataaca ggcaattaac 1140 tactgcccag aaacagggct atcttaacat aatcttcaaa tctctcttcc aaaagtttat 1200 ctctaaaatc acttcccagt tgacacttga ccacaaaact cagaaggtaa ccaagcccaa 1260 cccctaatgt ggtcttcagt tttatattcc ttattattgc aatcacctta tttaagtttt 1320 aatcattaag ctgattgcat tgctgcacaa ggtcaagaag cacttgtaga atcagaaacc 1380 ccaaagtggt tatggactgg ctactcagat ttttttcttc acttgctcag gctccgcccc 1440 atctgaaacg tcttctctgc taggaactca ctaggagaaa atgaccagtg agcttaccaa 1500 agctcagacc ttatccacca gaaacattgt atggggcacc tcaccttttc agcacctcct 1560 gccataacag gacggaagtg ctgaagggac agctcccagt gttttaggtc ttagtaagcg 1620 ctcccaggaa gacagcaccc atcccctctc tgctacaaac agccattcac aaaatccgag 1680 gaggaaacca agtcatccct gattccttgg aaatttagtt agactgatgt tgaaacacac 1740 ccccctttcc tagtgtataa ttatttgtgg gtggagagct accagggagg gtaacagaag 1800 atgccggaga agggggggga aaagtagatg cggatttcgt cctgacttct aaaaattcct 1860 cctctccctc tcccattttc ctaatccgag aatgatggag ctcgaggcaa aggaatgatt 1920 ccggaaatgg agatatgatt ctcaaaccta gaaatgatcg gagtgattta ttagttaaat 1980 attcttcgtc caggaaccca 2000 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 gggaagtaaa tttgggtgtg aa 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 ccccctctaa acacaaaatc tctt 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 6 attaagtgag tgagaggtag 20 <210> 7 <211> 222 <212> DNA <213> GABRA1 gene pyrosequencing part <400> 7 gggaagtaaa tttgggtgtg aaatttttag taaaggagta cggtagagtt tatgatggtt 60 tagattaagt gagtgagagg tagagcggag gacggttttt ttgttttggc ggcggttcgg 120 gattcgggat tcggtagatt cggcggttgg ttttagtttt tttacggatt ttttttttta 180 gatttttttt cgggttttaa gagattttgt gtttagaggg gg 222 <210> 8 <211> 3440 <212> DNA <213> cDNA clone (hMU002273) <400> 8 agcggagcgg gcgagcaagg gagcgagcag gacaggagcc tgatcccaca gctgctgctc 60 cagcccgcga tgaggaaaag tccaggtctg tctgactgtc tttgggcctg gatcctcctt 120 ctgagcacac tgactggaag aagctatgga cagccgtcat tacaagatga acttaaagac 180 aataccactg tcttcaccag gattttggac agactcctag atggttatga caatcgcctg 240 agaccaggat tgggagagcg tgtaaccgaa gtgaagactg atatcttcgt caccagtttc 300 ggacccgttt cagaccatga tatggaatat acaatagatg tatttttccg tcaaagctgg 360 aaggatgaaa ggttaaaatt taaaggacct atgacagtcc tccggttaaa taacctaatg 420 gcaagtaaaa tctggactcc ggacacattt ttccacaatg gaaagaagtc agtggcccac 480 aacatgacca tgcccaacaa actcctgcgg atcacagagg atggcacctt gctgtacacc 540 atgaggctga cagtgagagc tgaatgtccg atgcatttgg aggacttccc tatggatgcc 600 catgcttgcc cactaaaatt tggaagttat gcttatacaa gagcagaagt tgtttatgaa 660 tggaccagag agccagcacg ctcagtggtt gtagcagaag atggatcacg tctaaaccag 720 tatgaccttc ttggacaaac agtagactct ggaattgtcc agtcaagtac aggagaatat 780 gttgttatga ccactcattt ccacttgaag agaaagattg gctactttgt tattcaaaca 840 tacctgccat gcataatgac agtgattctc tcacaagtct ccttctggct caacagagag 900 tctgtaccag caagaactgt ctttggagta acaactgtgc tcaccatgac aacattgagc 960 atcagtgcca gaaactccct ccctaaggtg gcttatgcaa cagctatgga ttggtttatt 1020 gccgtgtgct atgcctttgt gttctcagct ctgattgagt ttgccacagt aaactatttc 1080 actaagagag gttatgcatg ggatggcaaa agtgtggttc cagaaaagcc aaagaaagta 1140 aaggatcctc ttattaagaa aaacaacact tacgctccaa cagcaaccag ctacacccct 1200 aatttggcca ggggcgaccc gggcttagcc accattgcta aaagtgcaac catagaacct 1260 aaagaggtca agcccgaaac aaaaccacca gaacccaaga aaacctttaa cagtgtcagc 1320 aaaattgacc gactgtcaag aatagccttc ccgctgctat ttggaatctt taacttagtc 1380 tactgggcta cgtatttaaa cagagagcct cagctaaaag cccccacacc acatcaatag 1440 atcttttact cacattctgt tgttcagtcc tctgcactgg gaatttattt atgttctcaa 1500 cgcagtaatt cccatctgct ttattgcctc tgtcttaaag aatttgaaag tttccttatt 1560 ttcataattc atttaagaac aagagacccc tgtctggcag tctggagcaa agcagactat 1620 gcagcttgga gacaggattc tgacagagca agcgaaagag caaagtcatg tcagaaggag 1680 acagaatgag agagaaaaga gggggaagat ggttcaaaga tacaagaaaa agtagaaaaa 1740 aaaataacac ttaactaaaa cccctaggtc atttgtagat atatatttcc aaatattcta 1800 aaaaagatac tgtatatgtc aaaaatattt ttatgtgaag gtgtttcaaa gggtaaatta 1860 taaatgtttc atgaagaaaa aattttaaaa atctacgtct ttattacaca aactatggtg 1920 tgcttatgtt tttgttttgc tttttaaact gatgtatagc tttaacattt tgtttccaaa 1980 gctgaagatc cccattcttt ctctttgaaa aaaaaaaagg cctaatgcat tattttgtca 2040 taaaatgcta ttttaaaatt catggaactt tcatacgtaa aggtgcagtt gctcattgta 2100 gagcacattt agtccaatga agataaatgc tttaaatagt ttacttcact ttcatctgag 2160 cttttaccac tagactcaag gaagaataat tttaacagac atgtatactc catagaaact 2220 aaattaaaat agtttaaaaa tattcccttt ttcaccctat tttcagatag cacatgagcc 2280 caacactcac ttaattctca ttatgaagat gtttttagag gggcaaaaat attttgcaag 2340 ctctggaatt gttgaatgta ttcttttata taactacatt aaaagcttta gattgaaatt 2400 tatgactagc aaacaaaaat agaatatata aacgatatat gtaaatatac agcatgagat 2460 tgtacatttt ttactttttt aaaattgtgt tcttaaaata ttgtgtaaga atcactgcac 2520 ttagctgttg gaatgttgtt aaatgctatg gaaatacatt tagaacctgc atttaagaac 2580 agaacagcaa gtatgaacca catggaactt aaaacatatg ggtgtgaagt ccacttatgt 2640 agacaaaact tataatttcc aaactgttgt ctagtataca gtgatcagtt gctctctgtt 2700 caagtcattc cacacatttc cctattttag gctattataa tatagaaaga aaatgggaag 2760 cattagttgg agctagaaaa tgaactgtat attattgcta tatttgctaa taccaactat 2820 ttcaataagt gttgtaccat atgtagcatt aaatataaaa tacataaaag aatgtacaga 2880 aaatagcttt tattgagtaa tattacattt catttatact gtagcaatat atttgtaggt 2940 atactatgta agggctttaa ataaaagagg tccattaata cttccttata aaaattctag 3000 tctgtttcat tactgcccag atgttttaga gataaatatt tatgcagaag gtatttttga 3060 agtctccttt tgtctgatag agtttaacag atatttaaat ttagtgctca gaatccacaa 3120 gtcacggtct aaacacactt agaatactac agcataaatc tgttagcatt attgccaaat 3180 aagacagttg ggatccaaac ccaagtcttg agcaatgttt ttctcaaaaa gctgctatcc 3240 aatgatatag gaaaatacat tgtgttttcc taaacacact tttcttttta aatgtgcttc 3300 attgtttgat ttggtcctgc ctaaatttca caagctaggc caatgaaggc tgaatcaaag 3360 acatttcatc caccaatatc atgtgtagat attatgtata gaaaataaaa taaattatgg 3420 ctccaaaaaa aaaaaaaaaa 3440 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 attagaattc gcgatgagga aaagt 25 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 taatctcgag gatctattga tgtggtg 27  

Claims (8)

An inhibitor of proliferation of colorectal cancer cells comprising a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ). The proliferation inhibitor of colorectal cancer cells according to claim 1, wherein the gene encoding gamma-aminobutyric acid A receptor alpha 1 is represented by SEQ ID NO: 1. A composition for inhibiting proliferation of colorectal cancer cells containing gamma-aminobutyric acid A receptor alpha 1 protein as an active ingredient. The composition for inhibiting proliferation of colorectal cancer cells according to claim 3, wherein the gamma-aminobutyric acid A receptor alpha 1 protein is represented by SEQ ID NO: 2. A therapeutic agent for colorectal cancer, comprising a gene carrier containing a gene encoding a gamma-aminobutyric acid A receptor alpha 1 ( GABRA1 ). The method of claim 5, wherein the gene encoding the gamma-aminobutyric acid A receptor alpha 1 is represented by SEQ ID NO: 2. A pharmaceutical composition for treating colorectal cancer, comprising a gamma-aminobutyric acid A receptor alpha 1 protein as an active ingredient. 8. The pharmaceutical composition of claim 7, wherein the gamma-aminobutyric acid A receptor alpha 1 protein is represented by SEQ ID NO: 2.

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