KR101128640B1 - Use of RPS 14 as a hepatocellular carcinomar diagnostic marker - Google Patents

Abstract

The present invention relates to the use of RPS 14 as a marker for diagnosing liver cancer. More specifically, the present invention relates to a marker for diagnosing liver cancer using the RPS 14 gene, a composition for diagnosing liver cancer, a kit, a microarray, and a method for diagnosing liver cancer, and to preventing liver cancer by inhibiting the expression of the RPS 14 gene or protein. The present invention relates to a method for screening a treatable substance and a composition for preventing or treating liver cancer comprising the substance. As the RPS 14 gene according to the present invention has been found to have an increased expression level in tissues or cells of liver cancer compared to tissues or cells of non-liver cancer, when the RPS 14 gene is used as a marker for diagnosing liver cancer, liver cancer is diagnosed quickly and accurately. And there is a predictable effect, only the blood of the patient can be an accurate diagnosis, and furthermore it can be used as a target for the development of liver cancer therapeutics.

RPS 14, Liver Cancer, Diagnostics, Markers, Compositions, Kits, Microarrays

Description

Use of RPS 14 as a hepatocellular carcinomar diagnostic marker

The present invention relates to a novel liver cancer diagnostic marker, liver cancer diagnostic composition, liver cancer diagnostic kit, microarray and liver cancer diagnostic method that can effectively diagnose and predict liver cancer early.

According to a recent report by the World Health Organization (WHO), cancer will be the leading cause of death for humanity in 2010, overcoming heart disease. In this regard, the IARC has seen a steady increase in the number of cancer patients worldwide. In 2008, the number of cancer patients worldwide reached 12 million, and cancer deaths reached 7 million.

Liver cancer is one of the most common and fatal cancers in the world, with 1 million patients dying from liver cancer each year, and the most common cancer in Asia, especially in Korea and Japan (Parkin, et al., Int . J. Cancer, 80, 827-841, 1999, Luo et al., Basic Biology and Clinical Sciences 1 st Ed ., Pp. 435-445, 1997, Chen et al., J. Gastroenterol . Hepatol . 12, S294-308, 1997). Generation of liver cancer is the exposure of the liver and hepatitis B infections, and aflatoxin of hepatitis C virus in tissue β1 is reported as the cause (Bosch, et al., Semin . Cancer Dis . 19, 271-285, 1999, Luo, et al., Basic Biology and Clinical Sciences 1 st Ed ., Pp. 435-445, 1997), several growth factors, such as transforming growth factor α (TGF-α) and TGF-β, have been reported to be involved in the mechanism of liver cancer, and RB is well known as a cancer suppressor. And p53 proteins have also been identified as candidate genes that play an important role in liver cancer formation (Collier, et al., Liver , 13, 151-155, 1993, Naka, et al., Anticancer Res . 18, 555-564, 1998). In addition, genetic mutations of DNA mutations and gene expression have been reported in patients with liver cancer (Park, et al., Cancer Res . 59, 307-310, 1999).

However, the exact cause of the pathogenesis of liver cancer is not known yet, and genes found to be related to the development of liver cancer do not accurately reflect the external and clinical characteristics of liver cancer patients.

In addition, despite intensive studies on the cause, diagnosis, and treatment of cancer over the past several years, there are no reliable treatments that can effectively cure cancer. Therefore, detecting cancer at an early stage and removing cancer cells or inhibiting growth by surgical or drug treatment may be the most effective method.

Recently, a diagnosis method using a tumor marker has been used to diagnose cancer early. This is because tumor markers are often overexpressed in certain cancer cells in the body, and as soon as it is confirmed that the tumor markers are expressed above a reference value, This is because the cancer of the organs can be diagnosed. In addition, the diagnosis of cancer using tumor markers is mostly performed by blood tests using antibodies, and some of them are tested using tissue extracts, urine, and stool. However, there are various types of tumor markers depending on where the cancer occurs, and there are no tumor markers that can identify all cancers occurring in humans, and many types of cancers do not have a tumor marker that can be diagnosed.

On the other hand, as a representative tumor marker of liver cancer, alpha-fetoprotein, that is, alpha-fetoprotein (AFP), is used. AFP is a glycoprotein abnormally produced when hepatocytes are transformed and dedifferentiated into cancer cells. It is used to detect primary liver cancer, hepatitis, cirrhosis and fetal cancer, and to observe the progress of treatment. Also recently, Des-gamma carboxyprothrombin (DCP), prothrombin induced by vitamin K absence-II (PIVKA-II), lens cularis agglutinin-reactive (AFP-L3), and GPC3 (glypican-3) have recently been diagnosed. There is research on whether it can be done.

However, the AFP liver cancer markers used in the prior art show low sensitivity and low specificity in early liver cancer diagnosis, and are also found in deterioration of cirrhosis and hepatitis, and there are many problems to specifically diagnose early liver cancer.

In addition, high frequency heat therapy or various radiotherapy methods have been developed for the treatment of liver cancer, but despite the development of such technology, long-term survival rate after liver cancer is very low and recurrence occurs frequently after surgery. However, the development of early biomarkers for liver cancer has become a clinically important issue since reports on the prognosis according to the time of diagnosis of liver cancer have a great influence on the survival rate after the onset of liver cancer. Therefore, it is urgent to develop new tumor markers that can effectively increase the diagnosis rate of early liver cancer.

The inventors of the present invention, while developing a new biomarker for early diagnosis of liver cancer, compared the expression of genes in liver cancer tissues and non-hepatic cancer tissues using reverse transcriptase polymerase chain reaction, and RPS 14 (ribosomal). protein 14) The present invention was completed by confirming that genes are differentially increased in liver cancer tissues compared to non-liver cancer tissues, and confirming that the genes can be used as diagnostic markers for early liver cancer.

Accordingly, an object of the present invention is to provide a marker for diagnosing liver cancer, which is composed of an RPS 14 (ribosomal protein S14) gene or a protein encoded from the gene.

In addition, another object of the present invention to provide a composition for diagnosing liver cancer comprising a substance for measuring the level of the RPS 14 (ribosomal protein S14) gene or the level of the RPS 14 protein.

In addition, another object of the present invention to provide a liver cancer diagnostic kit and liver cancer diagnostic microarray comprising the liver cancer diagnostic composition of the present invention.

In addition, another object of the present invention is to provide a method for predicting and diagnosing liver cancer comprising measuring the expression amount or protein amount of the RPS 14 gene.

Another object of the present invention is to provide a method for screening a substance capable of preventing or treating liver cancer.

Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer, which comprises a substance which inhibits the expression of the RPS 14 gene.

In order to achieve the object of the present invention as described above, the present invention provides a marker for diagnosing liver cancer consisting of a ribosomal protein S14 (RPS 14) gene or RPS 14 protein encoded from the gene.

In one embodiment of the present invention, the RPS 14 gene may be one having a nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides a composition for diagnosing liver cancer comprising a substance for measuring the level of RPS 14 (ribosomal protein S14) gene or the level of RPS 14 protein.

In one embodiment of the invention, the material for measuring the level of the RPS 14 gene may be a primer or probe that can amplify the RPS 14 gene.

In one embodiment of the invention, the substance for measuring the level of the RPS 14 protein may be an antibody that specifically recognizes the RPS 14 protein.

The present invention also provides a kit for diagnosing liver cancer comprising the composition for diagnosing liver cancer of the present invention.

In addition, the present invention provides a liver cancer diagnostic microarray comprising the composition for diagnosing liver cancer of the present invention.

In addition, the present invention,

(a) measuring the amount of protein or the amount of RPS 14 (ribosomal protein S14) gene present in the biological sample; And (b) it provides a method for predicting and diagnosing liver cancer comprising the step of comparing the measurement results of step (a) with the amount of expression or the amount of protein of the RPS 14 gene of the control sample.

In one embodiment of the invention, the biological sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.

In one embodiment of the present invention, the method of measuring the expression of the ribosomal protein S14 (RPS 14) gene or protein amount is reverse transcriptase-polymerase chain reaction (reverse transcriptase-polymerase chain reaction), real-time polymerase chain reaction ( Selection from a crowd consisting of real time-polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay It may be.

In addition, the present invention,

(a) contacting a sample to be analyzed with a cell comprising a ribosomal protein S14 (RPS 14) gene or a RPS 14 protein; (b) measuring the expression level of the RPS 14 gene, the amount of RPS 14 protein or the activity of RPS 14 protein; And (c) determining that the sample is a substance for preventing or treating liver cancer when the expression of RPS 14 gene, the amount of RPS 14 protein or the activity of RPS 14 protein is reduced as a result of the measurement in step (b). It provides a method for screening a substance for preventing or treating liver cancer.

In one embodiment of the present invention, the method for measuring the expression of the RPS 14 gene, the amount of RPS 14 protein or the activity of the RPS 14 protein is reverse transcriptase-polymerase chain reaction, real-time polymerase Real time-polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay It can be chosen from the crowd.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to the nucleotide sequence of the ribosomal protein S14 (RPS14) gene as an active ingredient. .

As the RPS 14 gene according to the present invention has been found to have an increased expression level in tissues or cells of liver cancer compared to tissues or cells of non-liver cancer, when the RPS 14 gene is used as a marker for diagnosing liver cancer, liver cancer is diagnosed quickly and accurately. And there is a predictable effect, only the blood of the patient can be an accurate diagnosis, and furthermore it can be used as a target for the development of liver cancer therapeutics.

The present invention is characterized by providing a novel marker for diagnosing liver cancer comprising a ribosomal protein S14 (RPS 14) gene or an RPS 14 protein encoded therefrom.

In the case of most markers used for diagnosing liver cancer, the rate of accurately diagnosing liver cancer is relatively low, and there is a problem in that the sensitivity and specificity of liver cancer are low because it does not distinguish clearly from other diseases except liver cancer. Therefore, the present inventors examined genes showing differences in expression in liver cancer tissues and non-liver cancer tissues in order to discover new liver cancer diagnostic markers. It was confirmed that overexpression (see Figure 2), and the nucleotide sequence of the RPS 14 gene is shown in SEQ ID NO: 1.

On the other hand, ribosomal protein (ribosomal protein) is a protein contained in ribosomal particles are known as a protein involved in the synthesis of proteins in the cytoplasm and endoplasmic reticulum. Ribosome proteins exist in almost every cell, from higher animals and plants to bacteria, and ribosome proteins are unique to chloroplasts and mitochondria, respectively, and are known to be involved in the formation and synthesis of various types of proteins and complexes. .

The present inventors have found that RPS 14 is based on the fact that the synthesis of protein is closely related to cell growth, and that the ribosome protein, RPS 14 (ribosomal protein S14), is overexpressed in liver cancer tissues compared to non-liver cancer tissues. It could be expected that the gene could be used as a biomarker to diagnose and predict the development of liver cancer.

In addition, the above-mentioned predictions were further confirmed through an embodiment of the present invention, that is, the amount of expression of RPS 14 gene and protein in the tissues and blood of chronic hepatitis patients, cirrhosis patients and hepatocellular carcinoma patients was normal. As a result, it was confirmed that the expression level of the RPS 14 gene was significantly increased in the liver cancer patients compared to the normal group (see Example 6).

Accordingly, the present invention provides a marker for diagnosing liver cancer comprising an RPS 14 (ribosomal protein S14) gene or an RPS 14 protein encoded from the gene.

In the present invention, the term "diagnosis" refers to confirming the pathological state, and for the purpose of the present invention, the diagnosis means confirming the occurrence of liver cancer by confirming the expression of a liver cancer diagnostic marker, In addition, the "diagnosis" in the present invention includes determining whether liver cancer diagnostic markers are expressed and the degree of expression, and determining whether liver cancer is developed, developed, and reduced.

In addition, the "diagnosis marker" refers to a substance capable of diagnosing cells of liver cancer from normal cells, and a polypeptide or nucleic acid (eg, increased or decreased in liver cancer cells compared to normal cells). mRNA and the like), lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. The marker for diagnosing liver cancer provided by the present invention may be an RPS 14 gene or protein whose expression level is increased in cells of liver cancer compared to normal cells, and preferably, the RPS 14 gene has a nucleotide sequence set forth in SEQ ID NO: 1.

In addition, the present invention provides a composition for diagnosing liver cancer comprising a substance measuring the level of the RPS 14 gene or the level of the RPS 14 protein.

In the present invention, the level of the RPS 14 gene, preferably means the mRNA level, that is, the amount of mRNA expressed in the RPS 14 gene, a material capable of measuring the level is a primer or probe specific to the RPS 14 gene It may include. In the present invention, the primer or probe specific for the RPS 14 gene may be a primer or probe capable of specifically amplifying the entirety of the RPS 14 gene or a specific region of the gene represented by SEQ ID NO: 1, and the primer or probe may be You can design it by any method known to

In the present invention, the term “primer” refers to a single which can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers. -Refers to stranded oligonucleotides. Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the RPS 14 gene, which is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. In addition, the primer according to the present invention is preferably used for gene amplification reaction, most preferably may be a primer pair consisting of SEQ ID NO: 2 and 3.

The “amplification reaction” refers to a reaction for amplifying a nucleic acid molecule, and amplification reactions of such genes are well known in the art, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). , Ligase chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.

In the present invention, the term “probe” 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, which is a natural It exists or artificially synthesized. The probe according to the invention may be single chain, preferably oligodioxyribonucleotides. Probes of the invention can include natural dNMPs (ie, dAMP, dGMP, dCMP and dTMP), nucleotide analogues 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, in the present invention, a substance capable of measuring the level of the RPS 14 protein may include “antibody” such as a polyclonal antibody, a monoclonal antibody, and a recombinant antibody capable of specifically binding to the RPS 14 protein. have.

In addition, in the present invention, as described above, since the RPS 14 protein has been identified as a marker protein for diagnosing liver cancer, a method for generating an antibody using the protein is well known in the art. It can be manufactured easily. For example, in the case of polyclonal antibodies, the RPS 14 antigen can be produced by methods well known in the art for injecting animals with blood and collecting blood from the animals to obtain serum comprising the antibody, and such polyclonal antibodies can be produced by goats, rabbits, It can be produced from any animal species host such as sheep, monkeys, horses, pigs, cattle, dogs and the like. In the case of monoclonal antibodies, the hybridoma method (Kohler et al., European Jounral of Immunology , 6, 511-519, 1976) or phage antibody libraries (Clackson et al, Nature , 352, 624-628, 1991, Marks et al, J. Mol . Biol ., 222: 58 , 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 antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

The present invention also provides a kit for diagnosing liver cancer comprising a composition for diagnosing liver cancer capable of measuring the expression level of an RPS 14 protein or a gene encoding the same.

Liver cancer diagnostic composition included in 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 an RPS 14 protein or a gene encoding the same, and the definition thereof is as described above.

If the kit for diagnosing liver cancer 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, and the liver cancer diagnostic kit 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. Furthermore, the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.

In addition, the present invention provides a liver cancer diagnostic microarray comprising a composition for diagnosing liver cancer capable of measuring the expression level of the RPS 14 protein or a gene encoding the same.

In the microarray of the present invention, primers, probes or antibodies capable of measuring the expression level of the RPS 14 protein or gene encoding it are used as a hybridizable array element and immobilized on a substrate. . Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method 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 (eg, 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 can be labeled (labeled), it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

In addition, the present invention can provide a method for diagnosing liver cancer by measuring the expression level of the RPS 14 gene or the level of RPS 14 protein, the method comprising the steps of: (a) the RPS 14 (ribosomal protein S14) gene present in a biological sample; Measuring the amount of expression or amount of protein; And (b) comparing the measurement result of step (a) with the expression amount or protein amount of the RPS 14 gene of the control sample. The method of measuring the expression level or RPS 14 protein level of the RPS 14 gene in the above may be carried out including a known process for separating mRNA or protein from a biological sample using a known technique.

In the present invention, the "biological sample" refers to a sample collected from a living body, wherein the expression level or protein level of the RPS 14 gene is different from the normal control group according to the incidence or progression of liver cancer. This may include, but is not limited to, tissues, cells, blood, serum, plasma, saliva and urine.

Preferably, the expression level of the RPS 14 gene is measured by measuring the level of mRNA. The method of measuring the level of mRNA may be reverse transcriptase chain reaction (RT-PCR), real-time reverse transcriptase chain reaction, RNase protection assay. , Northern blots and DNA chips, but is not limited thereto.

The measurement of the RPS 14 protein level may use an antibody, in which case the RPS 14 marker protein in the biological sample and the antibody specific thereto form a binding, ie, an antigen-antibody complex, and the amount of antigen-antibody complex formation. Can be measured quantitatively by the magnitude of the signal of the detection label. Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, 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 RPS 14 mRNA expression or protein in the control group and the amount of RPS 14 mRNA expression or protein in liver cancer patients or suspected liver cancer patients through the above-described detection methods, the degree of the expression amount By comparing with the control group, it is possible to predict and diagnose the incidence, progression or prognosis of liver cancer.

According to one embodiment of the present invention, blood of a liver disease patient including a liver cancer patient is obtained, serum is separated from the blood, and then an ELISA method using an antibody of RPS 14 is performed to obtain RPS 14 from each patient's sample. After measuring the amount of protein, the measurement was performed through a process of comparative analysis with the control.

The present invention further comprises the steps of (a) contacting a sample to be analyzed with a cell comprising a RPS 14 (ribosomal protein S14) gene or a RPS 14 protein; (b) measuring the expression level of the RPS 14 gene, the amount of RPS 14 protein or the activity of RPS 14 protein; And (c) determining that the sample is a substance for preventing or treating liver cancer when the expression of RPS 14 gene, the amount of RPS 14 protein or the activity of RPS 14 protein is reduced as a result of the measurement in step (b). It provides a method for screening a substance for preventing or treating liver cancer.

According to the method of the present invention, first, a sample to be analyzed may be contacted with a cell containing the RPS 14 gene or protein. Here, the "sample" refers to an unknown substance used in screening to test whether the expression of the RPS 14 gene, the amount of RPS 14 protein or the activity of the RPS 14 protein is affected. The sample may include, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts. Then, the expression level of the RPS 14 gene, the amount of RPS 14 protein or the activity of the RPS 14 protein in the cells treated with the sample can be measured, and as a result of the measurement, the amount of RPS 14 gene expression, the amount of RPS 14 protein or RPS 14 When it is determined that the activity of the protein is reduced, the sample may be determined as a substance capable of treating or preventing liver cancer.

The method of measuring the expression amount of the RPS 14 gene, the amount of RPS 14 protein or the activity of the RPS 14 protein in the above may be carried out through various methods known in the art, for example, but not limited thereto, reverse transcription Reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Radioimmunodiffusion and immunoprecipitation assays can be used.

In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising a substance that inhibits the expression of the RPS 14 gene or reduces the expression and activity of the RPS 14 protein as an active ingredient.

The pharmaceutical composition according to the present invention may include chemicals, nucleotides, antisenses, siRNA oligonucleotides or natural extracts as an active ingredient, preferably an antisense having a sequence complementary to the nucleotide sequence of the RPS 14 gene of the present invention or siRNA (small interference RNA) oligonucleotides may be included as an active ingredient.

In the present invention, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a 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 complementary to RPS 14 mRNA and capable of binding to RPS 14 mRNA, which translates into RPS 14 mRNA, translocation into the cytoplasm, and maturation. may inhibit essential activity on maturation or any other overall 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 comprising fat-soluble moieties and methods of preparation are already well known in the art (US 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.

Antisense oligonucleotides can be synthesized in vitro by conventional methods and administered in vivo or allow the antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro 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 origin is in the opposite direction of the recognition site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

As used herein, 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 an RPS 14 mRNA sequence) and an antisense strand (a sequence complementary to a DRG2 mRNA sequence) are positioned opposite to each other to form a double strand. The siRNA molecules of the invention may have a single chain structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but can be paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. Parts that do not achieve may be included. In addition, the siRNA terminal structure can be either blunt or cohesive ends as long as the expression of the RPS 14 gene can be suppressed by the RNAi effect, and the adhesive end structures are 3'-terminal protrusion structures and 5'-. 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 compositions of the present invention comprising gene specific siRNAs may include agents that promote intracellular influx of siRNAs. Agents that promote the influx of siRNA into the cell generally can be used agents that promote the influx of nucleic acids. Examples of these include the use of liposomes or the lipophilic of one of many sterols, including cholesterol, cholate and deoxycholic acid. It can be combined with a carrier. Poly-L-lysine, spermine, polysilazane, polyethylenimine, polydihydroimidazolenium, polyallylamine Cationic polymers such as chitosan), succinylated PLL, succinylated PEI, polyglutamic acid, and polyaspartic acid. anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, hyaluronic acid, etc. It is available.

In addition, when an antibody specific for RPS 14 protein is used as a substance to reduce the expression and activity of the RPS 14 protein, the antibody is directly coupled to an existing therapeutic agent or indirectly through a linker or the like (eg, a covalent bond). You can. Therapeutic agents that can be bound to antibodies include, but are not limited to, radionuclide such as 131I, 90Y, 105Rh, 47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123I, 111In, etc. ); Biological response variants or drugs such as methotrexate, adriamycin, and lympokine such as interferon; Toxins such as lysine, abrin, diphtheria and the like; Heterofunctional antibodies, ie antibodies that bind to other antibodies so that the complex binds to both cancer cells and agonist cells (eg, K cells such as T cells); and -May be associated with associated or non-complexed antibodies.

  The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier. 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, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise 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 may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form. 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.

Pharmaceutical compositions formulated in such a manner may be administered in an effective amount via a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The term “administration” introduces certain substances into a patient in any suitable manner. The route of administration of the substance can be administered via any general route as long as it can reach the target tissue.

In addition, the above "effective amount" refers to an amount that exhibits 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.

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

< Example  1>

Liver cancer tissue and Non-liver cancer  Gun from tissue RNA Separation of

The present inventors visited Kangnam St. Mary's Hospital, Catholic University Medical College to discover new markers for early diagnosis of liver cancer, and diagnosed the primary hepatocellular carcinoma and performed surgery around three patients with primary hepatocellular carcinoma tissue and liver cancer tissue. Sample tissues for three pairs of non-hepatic cancer tissues were obtained. The tissue obtained was used with the consent of the patients according to the “Bioethics and Safety Act” and Helsinki Declaration. The liver cancer tissues and the non-liver cancer tissues obtained above were each crushed in a mortar and mortar, and then mixed with 1 ml of Trizol reagent (Invitrogen), and allowed to stand at room temperature for 5 minutes. Thereafter, 0.2 ml of chloroform was added to the mixture, and the mixture was left to stand at room temperature for 2 minutes, and then centrifuged at 4 ° C and 12000 g for 15 minutes to transfer the supernatant to a new tube. And left at room temperature for 10 minutes. Then, again centrifuged for 10 minutes at 12000g 4 ° C to precipitate RNA and remove the supernatant. 1 ml of 75% ethanol was mixed well with the precipitated RNA, followed by further centrifugation at 12000 g for 4 minutes to remove the supernatant, and dried at room temperature for 10 minutes, and dissolved by adding 100ul of distilled water to the dried RNA. After that, 1ul was taken to measure absorbance using a spectrometer, and the purity of RNA extracted by electrophoresis of 1% agarose gel was observed.

< Example  2>

Reverse transcription Polymerase chain reaction  Used cDNA Synthesis of

CDNA was synthesized from RNA extracted by performing reverse transcriptase polymerase chain reaction on total RNA of liver cancer tissue and non-hepatic cancer tissue extracted in Example 1. In other words, the first strand cDNA was synthesized by performing reverse transcription at 42 ° C using dT-ACP that specifically binds 3ug of total RNA extracted in Example 1 to the poly A site of mRNA. The reverse transcription reaction was performed using 3ul of extracted RNA in 4ul of 5 × reaction buffer (Promega, USA), 5ul of dNTP (2mM each), 2ul of 10uM dT-ACP1 (5'-CGTGAATGCTGCGACTACGATIIIIIT (18) -3 '). A total of 20ul of reaction solution containing 0.5ul RNasinRNase inhibitor (40U / ul, promega), and 1ul of M-MLV (Moloney murine leukemia virus) transcriptase (200U / ul, promega) for 90 minutes at 42 ° C After the reaction, the synthesized cDNA was diluted with 180ul of distilled water and used in the following experiments.

< Example  3>

Differential manifestations Polymerase chain reaction  Liver Cancer Tissue Specific Expression Gene Analysis

CDNA synthesized in Example 2 was analyzed genes differentially expressed in hepatocellular carcinoma and non-hepatic carcinoma tissues by ACP-based PCR method using GeneFishing DEG kit (Cygen, Korea). To this end, a total of 20ul of the reaction solution containing 5ng cDNA synthesized in Example 2, 1ul of dT-ACP2 (10uM), 1ul of 10uM any ACP, 10ul of 2x master mix at 94 ℃ 1 minute, Double stranded cDNA was synthesized from the single stranded cDNA by reacting at 50 ° C. for 3 minutes and at 72 ° C. for 1 minute. Then, the synthesized double-stranded cDNA amplification reaction was repeated 40 times at 94 ℃, 40 seconds at 65 ℃, 40 seconds at 72 ℃ and 5 minutes at 72 ℃ 40 times, the reaction product is a 2% agarose gel Electrophoresis on the amplified product was confirmed.

As a result, newly expressed or amplified genes or missing or reduced genes were found in liver cancer tissues compared to non-liver cancer tissues, and 35 bands showing at least 1.5-fold difference in expression between liver cancer tissues and non-hepatic cancer tissues were found. It could be confirmed (result not shown).

< Example  4>

Liver cancer tissue and Non-liver cancer  Analysis of Genes Expressed in Tissue

 In Example 3, one band overexpressed in hepatocellular carcinoma tissues compared to non-hepatic carcinoma tissues was selected from among bands in which gene expression patterns were significantly different in liver cancer tissues and non-hepatic cancer tissues through electrophoresis, and then gels were used. Separation was performed using an extraction kit (Qiagen), and gene cloning was performed using the TOPO TA cloning vector (Invitrogen, USA) according to methods known by the manufacturer. After cloning, the expressed genes were separated using a plasmid purification kit, and then the amplified sequences were analyzed using the BigDye 3.1 kit (Applied Biosystem, USA) and the amplified sequences were analyzed using a blast program.

As a result, as shown in FIG. 2, the gene sequence of the gene for the band with significantly increased expression in the liver cancer tissues compared to the non-hepatic cancer tissues was found to be ribosomal protein S14 (RPS 14). In addition, the gene bank number of the gene was NM_001025070.1, and the base sequence of the gene thereof is set forth in SEQ ID NO: 1.

< Example  5>

real time Polymerase chain reaction  Used RPS  14 quantitative analysis

In Example 4, the real-time polymerase chain reaction was performed to confirm the difference in the expression rate of the ribosomal protein S14 (RPS 14) discovered as a gene with increased expression in liver cancer tissues compared to non-liver cancer tissues. . The reaction was carried out by reverse transcription reaction by adding the oligo-dT primer which is generally used for real-time polymerase chain reaction to 2ug of total RNA extracted in Example 1. Thereafter, 1/10 of the cDNA synthesized by the reverse transcription reaction was used as a template, and real time polymerase chain reaction was carried out using a primer pair of SEQ ID NOs: 2 and 3 capable of amplifying RPS 14 having the sequence described below. The expression level of 14 genes was quantitatively analyzed. At this time, the polymerase chain reaction was performed for 2 minutes at 50 ° C and 2 minutes at 95 ° C, followed by 45 times of 15 seconds at 95 ° C and 30 seconds at 59 ° C.

Primer sequence of SEQ ID NO: 2 (RPS14-F)

5'-ACCATATGCTGCTATGTTGGCTGC-3 '

Primer sequence of SEQ ID NO: 3 (RPS14-R)

5'-ATCCTCAATCCGCCCGATCTTCAT-3 '

As a result, as in the differential expression polymerase chain reaction, the expression level of RPS 14 was found to be overexpressed about two times or more in liver cancer tissues compared to non- liver cancer tissues.

Therefore, the results indicate that the present inventors can use RPS 14, which is overexpressed in liver cancer tissues, as a biomarker for diagnosing liver cancer, compared to non-liver cancer tissues.

< Example  6>

In the blood of patients with liver disease RPS  14 Expression amount  analysis

Furthermore, the present inventors investigated whether liver cancer can be diagnosed early by a method of confirming the expression level of RPS 14 in a clinical sample. The clinical sample was blood from chronic hepatitis patients, liver cirrhosis patients and patients with hepatocellular carcinoma at Kangnam St. Mary's Hospital, Catholic University Medical College, and blood from normal persons was used as a control. Serum was isolated from the blood obtained from each patient, and then the enzyme immunoassay (ELISA) known in the art using an antibody of RPS 14 (Abcam ab50390) was used to determine the expression of RPS 14 expressed in the serum of each patient. The amount of expression was compared and analyzed.

As a result, as shown in Figure 3, in chronic hepatitis patients, it was confirmed that the expression level of RPS 14 in the serum was expressed more than the control group normal group. In addition, the amount of RPS 14 expressed in serum was higher in patients with cirrhosis than in patients with chronic hepatitis, and in patients with hepatocellular carcinoma compared with patients with cirrhosis.

Therefore, from the above results, the present inventors were able to know that liver disease is a hepatocellular carcinoma, and as the symptoms worsen, the expression level of RPS 14 further increases. In addition, in chronic hepatitis patients, it was confirmed that RPS 14 of the present invention can be used as a marker for early detection of liver cancer through the fact that the expression level of RPS 14 is higher than that of normal people.

Furthermore, the present inventors have confirmed that the liver cancer diagnosis method of the present invention, which measures the expression level of RPS 14, can be sufficiently performed even with only blood or serum of a patient, so that the liver cancer diagnosis method according to the present invention is very simple for early liver cancer. It can be seen that it can accurately predict and diagnose.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

1 is a schematic diagram showing a process of discovering RPS 14, a diagnostic marker for liver cancer according to the present invention.

FIG. 2 shows a comparison of the expression levels of RPS 14 genes in liver cancer tissues and non-liver cancer tissues by performing polymerase chain reaction using RNA extracted from liver cancer tissues and non-liver cancer tissues as a template and primers specific for RPS 14. It is a photograph.

Figure 3 is a graph comparing the amount of expression of RPS 14 in the blood obtained from chronic hepatitis patients, liver cirrhosis patients, hepatocellular carcinoma patients and the control group using the ELISA method.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Use of RPS 14 as a hepatocellular carcinoma diagnostic marker <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 787 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (787) <223> RPS 14 <400> 1 ctccgccccc tcccactctc tctttccggt gtggagtctg gagacgacgt gcaggtagga 60 ggcccgggcg cgacaatcgg ggggcatcct gcggcgaggg gaccctgtgg ggcttgggac 120 gagagacggg ggtctttccg tgggaaccga gctaggtgcc gggcaagaga cgcgcggctg 180 gcccacctgg atcctggcca actcgggatt gagttcgttc ctggtctcag aaggcccgtt 240 ttgctttcag ggaggagctt gtgaaaaatg gcacctcgaa aggggaagga aaagaaggaa 300 gaacaggtca tcagcctcgg acctcaggtg gctgaaggag agaatgtatt tggtgtctgc 360 catatctttg catccttcaa tgacactttt gtccatgtca ctgatctttc tggcaaggaa 420 accatctgcc gtgtgactgg tgggatgaag gtaaaggcag accgagatga atcctcacca 480 tatgctgcta tgttggctgc ccaggatgtg gcccagaggt gcaaggagct gggtatcacc 540 gccctacaca tcaaactccg ggccacagga ggaaatagga ccaagacccc tggacctggg 600 gcccagtcgg ccctcagagc ccttgcccgc tcgggtatga agatcgggcg gattgaggat 660 gtcaccccca tcccctctga cagcactcgc aggaaggggg gtcgccgtgg tcgccgtctg 720 tgaacaagat tcctcaaaat attttctgtt aataaattgc cttcatgtaa actgtttcaa 780 aaaaaaa 787 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RPS 14 F primer <400> 2 accatatgct gctatgttgg ctgc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> RPS 14 R primer <400> 3 atcctcaatc cgcccgatct tcat 24  

Claims (11)

delete delete A composition for diagnosing primary hepatocellular carcinoma comprising a substance measuring the level of RPS 14 (ribosomal protein S14) protein present in blood or serum. delete The composition for diagnosing primary hepatocellular carcinoma of claim 3, wherein the substance for measuring the level of the RPS 14 protein is an antibody that specifically recognizes the RPS 14 protein. (a) measuring the amount of RPS 14 (ribosomal protein S14) protein present in blood or serum; And (b) providing information for diagnosing primary hepatocellular carcinoma comprising comparing the measurement result of step (a) with the amount of RPS 14 protein in the control sample. delete The method of claim 6, wherein the measurement is selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay. Information providing method for diagnosing primary hepatocellular carcinoma characterized in that. delete delete delete

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