JP5134859B2 - Method for determining risk of hepatocellular carcinoma - Google Patents

Description

  The present invention relates to a method for determining morbidity risk and / or prognosis of hepatocellular carcinoma using genes associated with hepatocellular carcinoma and polymorphisms thereof.

  Hepatocellular carcinoma is a malignant tumor that develops due to the transformation of hepatocytes and accounts for over 90% of primary liver cancer. It is known that hepatocellular carcinoma mostly develops against chronic hepatitis caused by viral infection or cirrhosis in which fibrosis has further progressed from chronic hepatitis. Among hepatoma patients, hepatitis C virus The proportion of people who are positive for (HCV) is about 70%, and the proportion of people who are positive for hepatitis B virus (HBV) is about 20%. Although the detailed mechanism by which these hepatitis viruses cause hepatocellular carcinoma has not yet been clarified, it is thought that hepatocellular carcinoma occurs in the process where persistent infection of the virus causes chronic hepatitis, cirrhosis, and the like.

  The prognosis for hepatocellular carcinoma is generally poor and there is currently no effective treatment. If the tumor is small and localized, long-term survival after surgery can be expected to some extent by surgical removal, but hepatocellular carcinoma is difficult to make a definitive diagnosis and is rarely detected at such an early stage. Often found at a much more advanced stage. Periodic testing is essential for early detection of hepatocellular carcinoma, but considering that the probability of hepatocellular carcinoma occurring from viral cirrhosis is around 5-7% per year, all patients with viral cirrhosis Requesting high-frequency inspection is difficult in terms of time and cost. Therefore, in order to efficiently prevent or detect early onset of hepatocellular carcinoma, an attempt to enclose a population with a particularly high risk of developing hepatocellular carcinoma among cirrhosis patients, particularly viral cirrhosis patients, has been made. Has been made.

  Among such attempts, the polymorphisms of genes associated with hepatocellular carcinoma have attracted particular attention. A gene may be mutated under the influence of ultraviolet rays, radiation, chemical substances, etc. If the mutation does not cause death or infertility, the mutation in an individual is inherited by its offspring. Among the mutations on the fixed gene in this way, those whose frequency in the species is 1% or more are genetically polymorphic, but when the mutation has some effect on the gene May affect the incidence of diseases involving the gene. Therefore, if a polymorphism highly related to hepatocellular carcinoma can be found, not only can a high-risk population be enclosed, but it will be possible to identify related genes and develop therapeutics for hepatocellular carcinoma. It becomes. In addition, the genetic polymorphism test can be performed on a sample that is relatively easy to obtain, such as blood or saliva, and has an established method, and thus has an advantage that it can be easily performed.

Examples of gene polymorphisms related to hepatocellular carcinoma reported so far include, for example, TGFβ1 (see Non-Patent Document 1), IL10 (see Non-Patent Document 2), UGT1A7 (see Non-Patent Document 3), IL1β ( Non-patent document 4)) and the like. However, no satisfactory polymorphism has yet been found as a marker for susceptibility to hepatocellular carcinoma, and genes that can be effectively used for the prevention or treatment of hepatocellular carcinoma have not been identified. It was.
Japanese Patent Laid-Open No. 10-57076 Kim YJ et al., Exp Mol Med. 2003, 35, 196-202 Shin HD et al., Hum Mol Genet. 2003, 12, 901-6 Wang Y. et al., Clin Cancer Res. 2004, 10, 2441-6 Hirankarn N. et al., World J Gastroenterol. 2006, 12, 776-9 Parker AR et al., Carcinogenesis. 2005, 26: 2010-8 Kim CJ et al., Oncogene. 2004, 23: 6820-2 Al-Tassan N. et al., Hum Genet. 2004, 114: 207-10 Zhang Y. et al., Cancer Epidemiol Biomarkers Prev. 2006, 15: 353-8 Wang J. et al., Hepatobiliary Pancreat Dis Int. 2005, 4: 398-402 Baudhuin LM et al., J Cancer Res Clin Oncol. 2006, 132: 159-62

  Therefore, an object of the present invention is to find a gene polymorphism associated with hepatocellular carcinoma, to determine the risk and prognosis of hepatocellular carcinoma using the polymorphism, The object is to provide a medicament for treating hepatocellular carcinoma by controlling expression.

In the course of diligent studies to solve the above-mentioned problems, the present inventors screened genetic polymorphisms for hepatocellular carcinoma patients suffering from hepatitis C and non-hepatocellular carcinoma patients, The present inventors have found that a single nucleotide polymorphism (SNP) in the MutYH gene is significantly more frequently observed in patients with hepatocellular carcinoma than in those without hepatocellular carcinoma.
That is, the present invention detects a MutYH-related indicator selected from the group consisting of polymorphisms related to MutYH gene, abnormal expression of MutYH gene, abnormal presence of MutYH protein, and abnormal function of MutYH protein. A method for determining the risk or prognosis of hepatocellular carcinoma.
The present invention also relates to the method, wherein the MutYH-related indicator is a polymorphism related to the MutYH gene.
Furthermore, the present invention relates to the above method, wherein the polymorphism is a polymorphism present at position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 and / or a haplotype block containing the polymorphism in the MutYH gene. .

Furthermore, the present invention relates to the above-described method, wherein in the MutYH gene, when the base species present at position 8314 of the base sequence represented by SEQ ID NO: 1 is A, it is determined that the risk of developing hepatocellular carcinoma is high. .
The present invention also relates to the above-described method, wherein in the MutYH gene, when the base species present at position 8314 of the base sequence represented by SEQ ID NO: 1 is G, it is determined that the risk of developing hepatocellular carcinoma is low.
The present invention further relates to the method, wherein the hepatocellular carcinoma is hepatocellular carcinoma caused by hepatitis C virus.
The present invention further relates to a marker for determining the risk of developing hepatocellular carcinoma, comprising a polynucleotide comprising a continuous base sequence of 15 to 1000 bases containing a polymorphism related to the MutYH gene.
The present invention also relates to a polynucleotide that allele-specifically hybridizes to a polymorphic site related to the MutYH gene, or an upstream sequence of the polymorphism containing a base that is one base upstream (5 ′ side) of the polymorphism. A polynucleotide that hybridizes in a sequence-specific manner with one end that hybridizes to the base that is one base upstream (5 ′ side) or one base downstream (3 ′ side) of the polymorphism A hepatocellular carcinoma comprising a polynucleotide that specifically hybridizes to a downstream sequence of the polymorphism containing a certain base, the one end of which hybridizes to a base that is one base downstream (3 ′ side). The present invention relates to a reagent for determining the risk of morbidity and / or prognosis.

Furthermore, the present invention relates to a) a nucleic acid having a base sequence represented by SEQ ID NO: 2 or a portion of the nucleic acid containing position 8314 of the base sequence and hybridizing to the nucleic acid portion, and the base sequence represented by SEQ ID NO: 1 Or a polynucleotide that does not hybridize to the nucleic acid portion containing position 8314 of the base sequence, b) a nucleic acid having the base sequence represented by SEQ ID NO: 1, or the nucleic acid portion containing position 8314 of the base sequence A nucleic acid that hybridizes and has a base sequence represented by SEQ ID NO: 2 or a polynucleotide that does not hybridize to the nucleic acid portion containing position 8314 of the base sequence; c) a base sequence represented by SEQ ID NO: 1 A polynucleotide that hybridizes to a contiguous portion of the nucleic acid comprising the base sequence containing position 8313 but not position 8314 of the base sequence, A nucleic acid having one end that hybridizes to the base at position 8313, and d) a nucleic acid having the base sequence represented by SEQ ID NO: 1, hybridizing to a continuous portion that includes position 8315 but does not include position 8314 The above-mentioned reagent comprising a polynucleotide selected from the group consisting of a polynucleotide whose one end hybridizes to the base at position 8315.
Furthermore, this invention relates to the kit for performing the said method containing the said reagent.
The present invention also relates to a composition for treating hepatocellular carcinoma comprising an agent that controls the abundance and / or function of MutYH protein.

  MutYH has been studied for its relevance to cancer because it is involved in the repair of damaged genes (see Patent Document 1), colorectal cancer (see Non-Patent Document 5), and stomach cancer (see Non-Patent Document 6). Although there are reports that suggest a relationship with some cancers, the relationship with lung cancer (see Non-Patent Document 7), breast cancer (see Non-Patent Document 8), prostate cancer, etc. has been denied, The situation was far from the original expectations. Regarding hepatocellular carcinoma, there is a report that a mutation was found in the coding region of MutYH gene in some hepatocellular carcinoma patients (see Non-Patent Document 9), but in this report, comparison with the control was made. In addition, there is no particular mention about the relationship with hepatocellular carcinoma, and another report denies the relationship between the amino acid mutation of MutYH protein and hepatocellular carcinoma (see Non-Patent Document 10). ). Thus, it was not possible for those skilled in the art to predict that the MutYH gene polymorphism has a high association with hepatocellular carcinoma.

By the determination method of the present invention, it becomes possible to easily and accurately determine the risk of suffering from the target hepatocellular carcinoma. As a result, for subjects who are determined to have a high risk of developing hepatocellular carcinoma, preventive measures can be taken and early detection can be performed even if hepatocellular carcinoma develops through thorough management screening. Become.
In addition, when the subject is an HCV-infected person, and particularly when he / she is suffering from hepatitis C, hepatocytes are compared with the case where he / she is not infected with HCV and the case where he / she is not suffering from hepatitis C. Since the risk of developing cancer is high, more accurate determination can be performed using the determination method of the present invention.
Furthermore, since the composition of the present invention treats hepatocellular carcinoma by a completely new approach to control the production and function of MutYH, high prophylactic / therapeutic results that could not be achieved with conventional pharmaceuticals are expected. In addition, a synergistic effect can be expected when used in combination with conventional medicines.

  The present invention includes a step of detecting an index selected from the group consisting of polymorphisms related to MutYH gene, abnormal expression of MutYH gene, abnormal presence of MutYH protein, and abnormal function of MutYH protein. The present invention relates to a method for determining the risk or prognosis of cell cancer.

  In the present invention, the MutYH gene is a gene encoding MutYH (MutY homolog), which is one of DNA repair enzymes, and is typically a sequence present in human chromosome 1p34.3-32.1. This refers to human MutYH having the base sequence represented by No. 1. MutYH is a homologue of the DNA repair enzyme MutY discovered in Escherichia coli, and is preserved in many biological species including rodents such as mice and mice, primates such as chimpanzees and rhesus monkeys, and cattle. It has been confirmed. The location and sequence of the MutYH gene in these species can be searched in public databases such as NCBI. For example, the GeneIDs in the human, mouse, rat, chimpanzee, rhesus monkey, and bovine NCBI databases are 4595, 70603, 170841, 456539, 705651, and 513051, respectively. However, the sequence of the MutYH gene in the present invention does not have to exactly match the sequence represented by SEQ ID NO: 1 or the sequence registered in the above database, and may occupy the same position on the chromosome. For example, it may contain one or more base mismatches. The MutYH gene includes a 5 ′ untranslated region, a 3 ′ untranslated region, an intron and the like in addition to a coding region (exon) encoding the MutYH protein.

The polymorphism in the present invention includes not only a polymorphism having a genetic meaning, that is, a change in a specific base existing at a frequency of 1% or more in a population on a gene, but also any other base change. included. Examples of the polymorphism in the present invention include, for example, VNTR polymorphism, STRP polymorphism, single nucleotide polymorphism (hereinafter also referred to as “SNP”), 1 to several tens of bases (sometimes several thousand bases) deleted, The polymorphism etc. which are substituted or added are also included. Furthermore, the number of polymorphisms to be detected is not limited to one and may be two or more.
In addition, the polymorphism in the present invention is encoded by a mutation that causes an amino acid mutation of MutYH protein, a mutation that changes the expression level of MutYH gene, a mutation that changes properties such as mRNA stability of MutYH gene, or a MutYH gene. It includes mutations of various properties such as mutations that change the activity of proteins. Specific examples of the polymorphism in the present invention include base addition, deletion, substitution, insertion and the like.

  The polymorphism in the present invention may be present not only in the exon region of the MutYH gene but also in any region including a 5 'untranslated region, a 3' untranslated region, and an intron region. Examples of such polymorphisms include polymorphisms on the MutYH gene registered in a public SNP database, such as the base sequence represented by SEQ ID NO: 1, positions 437, 555, 669, 816, 860. 989, 1032, 1053, 1053, 1303, 1456, 1699, 1808, 1823, 1825, 2436, 2445, 2446, 2453, 2540, 2780, 2819, 2922, 2923, 3329, 3456, 3752, 4199, 4199, 4547, 4684, 4726, 4888, 4939, 5494, 5612, 5646, 5671, 6596, 6713 , 6836, 6957, 7743, 8314, 8394, 8613, 8739, 9128, 9339, 9634, 9468, 9614, 9641, 10021, 10243, 10515, 10600 , 10671, 10754, 10945, 11161, 11324, 11799, 11799, 11826, 11842, and 12443. , Rs3219467, rs3219468 , Rs3219469, rs3219470, rs1140199, rs6696152, rs1140200, rs10548651, rs35518179, rs10638672, rs11403361, rs34686049, rs3219471, rs3219472, rs35804643, rs3219473, rs7522089, rs3219474, rs3219475, rs3219476, rs3219477 , Rs3219482, rs3219483, rs35203418, rs3219484, rs3219485, rs3219486, rs1140507, rs3219487, rs34612342, rs11545695, rs34126013, rs11211096, rs3219488, rs3219489, rs35352891, rs3219490, rs36053993, rs4660848, rs32194919rs , Rs10890325, rs34961970, rs3219496, rs3219497 and rs3216024 polymorphs).

In one embodiment of the determination method of the present invention, the polymorphism exists in an intron. Examples of such polymorphisms include rs2275602, rs3219467-83, rs3219485-8, rs3219490-3 and rs3219495. A preferred polymorphism in the present invention is a polymorphism (rs3219487) present at position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 in the MutYH gene.
In one embodiment of the determination method of the present invention, the polymorphism has a minor allele frequency of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8%. These are 9% or more, 10% or more, 11% or more, or 12% or more. Minor allele frequencies are listed in public databases such as NCBI.
A polymorphism of a gene can be referred to as a marker of a gene used to track whether an individual organism has a particular trait, for example, whether or not it has susceptibility. May occur more frequently than combinations. This is called linkage disequilibrium, and the range on the chromosome where this linkage disequilibrium is seen is called a haplotype block. This means, for example, that a polymorphism contained in the same haplotype block as the polymorphism associated with a certain disease is likely to have an association with the disease. Therefore, in the determination method of the present invention, not only polymorphisms on the MutYH gene but also any polymorphisms included in the same haplotype block as these polymorphisms are indicators for determining susceptibility to hepatocellular carcinoma and the like. Can be used as

  Haplotype blocks can be determined by various algorithms including diversity based algorithms, LD (linkage disequilibrium) based algorithms, information theory algorithms, etc. (Indap AR et al., BMC Bioinformatics. 2005). , 6: 303), there are also several haplotype block analysis software based on these. Examples of such software include, but are not limited to, SNPbrowser (registered trademark), HapBlock, MDBlocks, Haploview, HaploBlockFinder, LDMAP, and HaploBlock, many of which can be used free of charge. For analysis of haplotype blocks, not only gene polymorphism data collected in patient-controlled studies, but also existing gene polymorphism databases such as HapMap (see www.hapmap.org), Applied Biosystems LD Map database Etc. can be used.

  Therefore, the haplotype block in the determination method of the present invention can include any polymorphism on the MutYH gene among the haplotype blocks identified by the above-described arbitrary technique. Typical examples include those ranging from rs3219489 to rs1007860 including, for example, rs3219487, which is obtained by analyzing Japanese (JPT) data in the HapMap database using SNPbrowser (registered trademark) under default conditions. It is what was done. Therefore, any polymorphism included in the haplotype block, e.g., rs3219485, rs3219484, rs3219476, rs3219474, rs3219472, rs3219463, rs3811432, rs9429157, rs9429072, rs2298018, rs2153608, rs2153609, rs2185549, rs4660849, rs11586695, rs9429158, 943 , Rs9326141, rs12138495, rs2050884, rs4520450, rs6700975, rs7543428, rs6659464, rs884962, rs11211101, rs11800673, rs12025302, rs1417578, rs6670763, rs4660854, rs2487442, rs2487443, rs12567250, rs7532182, rs7532182, rs12 , Rs13375430, rs7536005, rs3790582, rs12061890, rs12139364, rs7531253, rs12046816, rs12080394, rs1771549, rs1691219, rs1691217, rs11579411, rs10493120, rs11211110, rs6661693, rs1006215, rs1006216, rs1007860, etc. It can be used as a polymorph.

  The gene contained in the specimen usually includes two types inherited from parents, with some exceptions such as germ cells that have undergone meiosis, so the polymorphism is also a genotype consisting of the same two alleles ( Homotype) and genotypes (heterotypes) composed of two different alleles. Thus, a polymorphism can be distinguished not only from a genetic location, but also from the perspective of whether it is homozygous or heterozygous, thereby changing its association with hepatocellular carcinoma. Homomorphic polymorphism often accompanies changes in protein phenotype, and is generally considered to be highly related to disease. However, heteromorphic polymorphism is also useful as a disease-related SNP. There is also. The polymorphism present at position 8314 of the base sequence represented by SEQ ID NO: 1 is highly related to the risk of developing hepatocellular carcinoma even if it is a heterozygous form.

As described above, since the base sequence of the MutYH gene contained in the specimen may be different from that registered in the database, the actual position of the polymorphism may be different from that described above. Corresponding positions in the specimen can be appropriately found by homology search based on the polymorphic sequence. For example, the polymorphism present at position 8314 of the base sequence represented by SEQ ID NO: 1 is a sequence of upstream (5 ′ side) and downstream (3 ′ side) continuous bases, for example, 15 to 20 or more. It can also be identified as a base sandwiched between a number of consecutive bases.
In addition, since genomic DNA usually has a double-stranded structure complementary to each other, in this specification, even if the DNA sequence in one strand is shown for convenience, it is a matter of course. However, it is also understood that a sequence complementary to the sequence is also disclosed. Therefore, if one DNA sequence is described, a sequence complementary to that sequence is obvious.

In the determination method of the present invention, abnormal expression of the MutYH gene is a suppression of at least one of various processes related to gene expression such as transcription, translation, post-translational modification of the MutYH gene compared to an individual having a normal MutYH gene. An abnormality in the amount of MutYH protein present in a subject means that the amount of MutYH protein present in the subject is a normal subject, ie, a subject with a low risk of developing hepatocellular carcinoma and / or a good prognosis. It means that it is significantly different. Further, the abnormal function of MutYH protein means that the activity of MutYH protein in a subject is significantly different from that of a normal subject, that is, a subject with a low risk of suffering from hepatocellular carcinoma and / or a good prognosis.
In the present invention, hepatocellular carcinoma refers to any cancer caused by hepatocellular carcinoma, and is not limited to hepatocellular carcinoma caused by hepatitis C virus, hepatocellular carcinoma caused by hepatitis B virus, Or hepatocellular carcinoma resulting from both hepatitis C virus and hepatitis B virus, or hepatocellular carcinoma resulting from alcoholic cirrhosis. In one embodiment of the present invention, the hepatocellular carcinoma is hepatocellular carcinoma resulting from hepatitis C virus.

  In the determination method of the present invention, the target to be determined may be any living individual, but is preferably an animal, more preferably a mammal, and even more preferably a human individual. In the present invention, the subject may be healthy or suffer from some kind of disease. In one embodiment of the determination method of the present invention, the subject is infected with hepatitis C virus, and in another embodiment, the subject suffers from hepatitis C. When the subject is a human individual, the race is not particularly limited, and may be any of white (Caucasian), yellow (Mongoloid), black (Negroid), southern (Australoid), and the like. In one embodiment of the invention, the subject race is a Mongoloid, typically a Japanese.

  The specimen used in the determination method of the present invention is not particularly limited as long as it contains the target DNA and / or RNA, preferably DNA, for detection of polymorphism and gene expression abnormality. For example, blood, saliva, oral cavity Includes various cells, body fluids, organs and the like collected by mucous membranes, hair, body hair, skin pieces, feces, urine, nails, surgery and biopsy. The specimen may be subjected to polymorphism detection as it is, but it is preferable to extract and purify DNA and / or RNA in advance. DNA and RNA extraction methods are well known in the art. For DNA extraction, for example, proteinase K / phenol extraction method, proteinase K / phenol / chloroform extraction method, alkali dissolution method, boiling method, etc. If RNA is extracted, for example, techniques such as guanidine / acidic phenol (AGPC) method and SDS-phenol method can be used. DNA and / or RNA may be amplified prior to detection to polymorphism. For amplification, any known method including a PCR method can be used. Typically, a part of a nucleic acid molecule containing the polymorphism is specifically amplified using an appropriate primer. By amplifying DNA or RNA, the detection operation becomes easy.

  The specimen for detecting an abnormal amount of MutYH protein or an abnormal function of MutYH protein is not particularly limited as long as it contains the target MutYH protein, particularly the target living cells. Including various cells and organs collected by surgery or biopsy. The specimen may be subjected to the detection operation as it is. However, if the MutYH protein is separated by, for example, disrupting the cells by sonication or the like and preparing a cell extract from the centrifuged supernatant, the detection operation is performed. It becomes easy. In addition, since MutYH protein loses its enzyme activity within a few minutes under normal conditions, the specimen is stored at a low temperature of −70 ° C. or lower, and at the time of dilution, REC (registered trademark) buffer (10 mM HEPES- It is preferable to use a medium such as KOH, pH 7.4, 100 mM KCl, 10 mM EDTA, 10% glycerol).

In the determination method of the present invention, the morbidity risk and / or prognosis of hepatocellular carcinoma is determined based on the detected index. That is, if an indicator highly related to the incidence and / or poor prognosis of hepatocellular carcinoma is detected, the disease is determined to be susceptible and / or poor prognosis, and the non-affected and / or good prognosis of hepatocellular carcinoma is determined. When a highly relevant index is detected, it is determined to be intractable and / or have a good prognosis. Here, indicators that are highly associated with the presence and / or poor prognosis of hepatocellular carcinoma or the non-prevalence and / or good prognosis of hepatocellular carcinoma are, for example, those events and statistical Indices with significant associations and / or statistically significant odds ratios. Having a statistically significant association means, for example, a risk factor of less than 10%, preferably less than 5%, more preferably less than 3%, even more preferably less than 1% in a chi-square (χ ² ) test That is, the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.03, and even more preferably less than 0.01, and a statistically significant odds ratio. Means 90%, preferably 95%, more preferably 97%, and even more preferably 99%, when the confidence interval does not include 1, the odds ratio included in this confidence interval.

  Specifically, for example, when the base species present at position 8314 of the base sequence represented by SEQ ID NO: 1 is A, it is determined that the patient is susceptible to hepatocellular carcinoma, that is, has a high morbidity risk, If it is G, it is determined that the patient is refractory to hepatocellular carcinoma, ie, the risk of morbidity is low. Further, for example, when the genotype of the base present at position 8314 of the base sequence represented by SEQ ID NO: 1 is A / A homotype or A / G heterotype, there is a risk of developing hepatocellular carcinoma. When it is determined to be high and is a homozygous type of G / G, it is determined that the risk of developing hepatocellular carcinoma is low. The correspondence between polymorphic alleles and / or genotypes other than the above and the risk and / or prognosis of hepatocellular carcinoma affects, for example, hepatocytes with and without hepatocellular carcinoma and / or with a good prognosis In patient-controlled studies comparing cancer patients with poor prognosis hepatocellular carcinoma patients, etc., it is possible to easily investigate by examining the above statistical relevance, and use the results obtained by such investigation Thus, determination of the risk and / or prognosis of hepatocellular carcinoma is also included in the determination method of the present invention.

According to the determination method of the present invention, for example, it is possible to determine the risk that a subject not infected with hepatitis C virus will suffer from hepatocellular carcinoma. It can also be determined whether the risk of suffering from hepatocellular carcinoma when infected with hepatitis C virus is high or low. In addition, if the subject is already infected with hepatitis C virus or suffers from hepatitis C, it is possible to determine whether the subject is more likely or less likely to develop hepatocellular carcinoma thereafter. Yes, it can be used to determine treatment policy.
The risk of suffering from hepatocellular carcinoma is currently not affected by hepatocellular carcinoma, but the subject is better at risk of suffering from hepatocellular carcinoma if the subject's current health is maintained in the future Can be judged on the basis of whether it is statistically higher or lower than when it is in a healthy state, so subjects infected with hepatitis C virus or hepatitis B virus Subjects with hepatitis C or hepatitis B are at higher risk of suffering from hepatocellular carcinoma than subjects with no hepatitis than those who are not infected In view of this, it is possible to determine the risk of suffering from hepatocellular carcinoma together with the result of the test for detecting the presence or absence of a polymorphism related to the MutYH gene by the determination method of the present invention.

  In the determination method of the present invention, the detection of the polymorphism is not limited, and the direct sequencing method, TaqMan (registered trademark) PCR method, invader method, restriction fragment length polymorphism (RFLP) method, PCR-SSCP method, allele Uses specific oligonucleotide (ASO) method, ARMS method, denaturing gradient gel electrophoresis (DGGE) method, detection method with enzymes such as RNase, S1 nuclease, MutY, MutS, etc., and mismatch cleavage with chemicals such as piperidine CCM (chemical cleavage of mismach) method, DOL method, MALDI-TOF / MS method, TDI method, padlock probe method, molecular beacon method, DASH (dynamic allele specific hybridization) method, UCAN method, DNA chip / DNA microarray method, ECA method, PINPOINT method, PROBE (primer oligo b ase extension) method, VSET (very short extension) method, Survivor assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, pyrosequencing method, SNP-IT method, melting curve analysis method, etc. Various methods can be used. Any of these methods are known to those skilled in the art, and specific procedures and reagents are described in many documents. Based on the position of the polymorphism of the invention and the base sequences before and after, it is possible to detect the polymorphism using these methods as appropriate. In addition, the polymorphism of the present invention may be detected by any method other than the above that can detect a predetermined polymorphism, and such a method is not known at present, but does not currently exist, but will be developed in the future. Also included. The following outlines some of the methods described above that are preferred in the determination method of the present invention.

The direct sequencing method is a method for detecting a polymorphism by determining the base sequence of a sample gene. For determination of the base sequence, any method such as dideoxy method, dye terminator method, die primer method, cycle sequence method, Maxam-Gilbert method and the like can be used without limitation. The sample gene may be amplified by an arbitrary method such as a PCR method before sequencing, and a double-stranded DNA may be made into a single strand as necessary. The direct sequence method is very simple because it can be performed automatically using any existing sequencer.
In the TaqMan (registered trademark) PCR method, a probe (TaqMan (registered trademark) probe) specific to a detection site hybridized with a sample nucleic acid and modified with a quencher (quencher) and a fluorescent dye (reporter) is used. This is a method using a reaction in which fluorescence is generated in the PCR reaction in the process of being decomposed along with the extension reaction by the nucleic acid polymerase. When there is no mismatch between the probe and the detection site, the above-described fluorescence is detected. However, when there is a mismatch, the extension reaction does not proceed and the fluorescence is not detected. Therefore, by using a probe specific to the allele to be detected, fluorescence is detected when a nucleic acid having the allele is present.

  This method is particularly advantageous in that multiplexing (multiplexing) is possible by using a plurality of probes having different labels. For example, by labeling the major allele and the minor allele with different labels, it is possible to simultaneously determine whether the polymorphism of the specimen is a homotype or a heterotype. That is, if only the major allele or minor allele label is detected, the major allele or minor allele is homozygous, and if both the major allele and minor allele are detected, the major allele and minor allele are heterozygous. . The fluorescent dye that can be used is not particularly limited as long as it can be detected by a measuring instrument. For example, any known fluorescent dye such as FAM, TET, JOE, VIC, Cy3, and Cy5 can be used. Moreover, as a quencher, known arbitrary things, such as TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3, can be used. Reagents and protocol collections used in TaqMan (registered trademark) PCR methods are available from Roche Molecular Systems, Inc., etc., and there are services for probe design and synthesis (for example, Custom TaqMan (registered trademark) Genomic Assays service) , Applied Biosystems).

  In the determination method of the present invention, abnormal expression of the MutYH gene is determined by, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, real-time PCR, in situ hybridization method, in vitro transcription method, etc. Any known gene expression analysis method such as can be used for detection. Abnormalities in the presence of MutYH protein can be detected using any known protein detection method such as immunoprecipitation, Western blotting, EIA, ELISA, RIA, immunohistochemistry, immunocytochemistry, etc. it can. Furthermore, abnormalities in the function of MutYH protein can be detected, for example, by evaluating adenine DNA glycosylase activity. Specifically, for example, a test protein is allowed to act on an oligonucleotide having an A / G mismatch (for example, A / GO Mismatch Mesophilic Oligonucleotide Set, R & D, Cat # 3800-100-A), and adenine DNA glycosylase activity is obtained. Such abnormalities are detected by comparison with normal MutYH protein.

  Since it is considered that a plurality of factors are involved in the onset and progression of hepatocellular carcinoma, in the determination method of the present invention, the above-mentioned MutYH-related index and other factors related to the onset and progression of hepatocellular carcinoma are used. In combination with an indicator, the risk of morbidity and prognosis of hepatocellular carcinoma can be determined. By doing so, it is possible to further increase the accuracy of the determination. As other indicators related to the onset and progression of hepatocellular carcinoma, any known indicator known in the art can be used, for example, without limitation, AFP, PIVKA-II, etc. And polymorphisms in genes such as TGFβ1, IL10, UGT1A7, and IL1β.

  The present invention also includes a polynucleotide comprising a continuous base sequence of 15 to 1000 bases, preferably 18 to 500 bases, more preferably 20 to 300 bases on the MutYH gene, or a complement thereof, including polymorphisms related to the MutYH gene. The present invention relates to a marker for determining the risk of developing hepatocellular carcinoma, including a chain. The polymorphism includes any polymorphism related to the MutYH gene, but is preferably a polymorphism present at position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 or a polymorphism contained in the same haplotype block, or Any polymorphism in the MutYH gene, more preferably the polymorphism present at the 8314th position of SEQ ID NO: 1. Since the above marker has a high correlation with the incidence of hepatocellular carcinoma, the risk of suffering from hepatocellular carcinoma in the subject can be determined by detecting this in the subject's body or in a sample collected from the subject. it can. For the detection of the marker, any method described above for detecting a polymorphism can be used.

The present invention also provides a polynucleotide that allele-specifically hybridizes to a polymorphic site related to the MutYH gene, or a downstream sequence of the polymorphism containing a base that is one base downstream (3 ′ side) of the polymorphism. A polynucleotide that hybridizes in a sequence-specific manner, one end of which hybridizes to the base that is one base downstream (3 ′ side) or one base upstream (5 ′ side) of the polymorphism A polynucleotide that hybridizes in a sequence-specific manner to an upstream sequence of the polymorphism containing a base, wherein one end of the polynucleotide hybridizes to a base that is one base upstream (5 ′ side). The present invention relates to a reagent for determining morbidity risk and / or prognosis.
Here, the polymorphic site related to the MutYH gene refers to the entire MutYH gene having the target polymorphism or a portion of the MutYH gene having an arbitrary length including the polymorphism. The part is preferably capable of causing specific hybridization, and has a length of, for example, 15 to 1000 bases, preferably 18 to 500 bases, more preferably 20 to 300 bases.

  A polynucleotide that hybridizes specifically to an allele hybridizes to a MutYH gene having a specific polymorphic allele of interest or a portion thereof, but hybridizes to a MutYH gene having other alleles or a portion thereof. Not polynucleotide. Specifically, for example, when the polymorphism is a polymorphism present at position 8314 of the base sequence represented by SEQ ID NO: 1, it hybridizes to the MutYH gene having a base type of position 8314 of A or a portion thereof. Means a polynucleotide that does not hybridize to the MutYH gene or its portion where the base species at position 8314 is other than A, particularly G. Such polynucleotides can be used as allele-specific probes in detection methods using allele-specific probes such as TaqMan (registered trademark) PCR method, invader method, and allele-specific oligonucleotide (ASO) method. .

  A polynucleotide that sequence-specifically hybridizes to a downstream sequence of the polymorphism including a base that is one base downstream (3 ′ side) of the polymorphism related to the MutYH gene, one end of which is one base downstream (3 What hybridizes to the base on the “side” includes the 8315th position of the base sequence when the polymorphism is located at position 8314 of the base sequence represented by SEQ ID NO: 1. A polynucleotide that hybridizes to a continuous portion that does not include a position and that hybridizes to the base at position 8315, for example, a polynucleotide having a base sequence of tatctcc at the 3 ′ end. And a polynucleotide that specifically hybridizes to an upstream sequence of the polymorphism including a base that is one base upstream (5 ′ side) of the polymorphism related to the MutYH gene, one end of which is one base upstream What hybridizes to the base on the (5 ′ side) includes the 8313th position of the base sequence when the polymorphism is the polymorphism present at the 8314th position of the base sequence represented by SEQ ID NO: 1. Is a polynucleotide that hybridizes to a continuous portion not including position 8314, one that hybridizes to the base at position 8313, for example, a polynucleotide having the base sequence of tatccttg at the 5 ′ end. In addition, since the base sequence represented by SEQ ID NO: 1 is the sense strand of the MutYH gene, for example, the 3 ′ end is ataggaac or the 5 ′ end is atagagg with respect to its complementary strand. Things. Such a polynucleotide can be used, for example, as a primer for a detection method using a primer extension method such as the PINPOINT method, the PROBE method, the VSET method, or the Survivor assay.

In one embodiment of the reagent of the present invention, a) a nucleic acid having a base sequence represented by SEQ ID NO: 2 or a portion of the nucleic acid containing position 8314 of the base sequence is hybridized and represented by SEQ ID NO: 1 A nucleic acid having a base sequence or a polynucleotide that does not hybridize to the portion of the nucleic acid comprising position 8314 of the base sequence; b) a nucleic acid having the base sequence represented by SEQ ID NO: 1 or the nucleic acid comprising position 8314 of the base sequence A nucleic acid having a base sequence represented by SEQ ID NO: 2 or a polynucleotide that does not hybridize to the nucleic acid part containing position 8314 of the base sequence; c) represented by SEQ ID NO: 1 A polynucleotide that hybridizes to a continuous portion of a nucleic acid having a base sequence that includes position 8313 but does not include position 8314 of the base sequence. One of which hybridizes to the base at position 8313, and d) a continuous portion of the nucleic acid having the base sequence represented by SEQ ID NO: 1 that includes position 8315 but does not include position 8314 And a polynucleotide selected from the group consisting of one end of which hybridizes to the base at position 8315.
Here, the “nucleic acid portion” refers to a portion having an arbitrary base length capable of causing specific hybridization, for example, 15 to 1000 base length, preferably 18 to 500 base length, more preferably 20 It is a part having a length of ˜300 bases.

  The type and length of the polynucleotide are not particularly limited as long as the polynucleotide can specifically hybridize with the polymorphism present in the 8314th position of SEQ ID NO: 1 or the polymorphism contained in the same haplotype block. Including various polymers such as RNA, DNA / RNA hybrid and PNA, the length may be 15 to 1000 bases, preferably 18 to 500 bases, more preferably 20 to 300 bases. The hybridization conditions are not particularly limited as long as the polynucleotide specifically hybridizes to the polymorphism. When the polymorphism is an SNP, since the sequence difference between alleles is small, it is easy to detect the polymorphism when the hybridization temperature is set near Tm (melting temperature). The design of polynucleotides having such properties is well known to those skilled in the art, and various contract services can be used. Such a polynucleotide can take the form of a probe for detecting a polymorphism or a primer for specifically amplifying a sequence containing or not containing a polymorphism, depending on the detection method.

  Various labels can be attached to the polynucleotide to facilitate detection. The label may be any substance known to those skilled in the art, for example, any radioisotope, a substance that binds to the labeling substance (eg, antibody, biotin, etc.), a fluorescent substance, a fluorophore, a chemiluminescent substance, and an enzyme. You can choose. The label may be provided in combination with a substance that masks the label, for example, a quencher that shields the fluorescent substance. The polynucleotide can be chemically synthesized by a conventional method as long as the base sequence is determined.

In addition to the polynucleotide itself, the reagent of the present invention may contain various buffers and media suitable for the polynucleotide. In this case, the polynucleotide may be present in the medium. . The reagent may be stored and transported in various containers such as tubes, ampoules, vials, and bottles. When the reagent contains a substance having photosensitivity such as a fluorescent substance, it is preferable to use a light-shielded container. The reagent of the present invention may be fixed to a solid phase such as a glass plate, nylon membrane, microbead, silicon chip, capillary. Such a solid phase reagent can be used in a detection method such as a DNA chip method.
In addition, the reagent of the present invention includes any substance other than the above customized for detecting a specific polymorphism of the MutYH gene.

  The present invention also relates to a kit for determining the risk and / or prognosis of hepatocellular carcinoma, comprising the reagent. In addition to the above reagents, this kit detects polymorphisms such as primers for amplifying DNA and / or RNA containing the desired polymorphism, various buffers, diluents, color formers, standards, and control substances. It can contain any material needed to do. Moreover, this kit may contain the description which described the procedure for performing the determination method of this invention.

The present invention also relates to a composition for treating hepatocellular carcinoma comprising an agent that controls the abundance and / or function of MutYH protein.
Here, the agent that controls the abundance of MutYH protein refers to any agent that increases, decreases or stabilizes the abundance of MutYH protein, preferably in the liver, more preferably in the hepatocytes. Without limitation, for example, an agent that releases MutYH protein itself or in the body, an agent that promotes or suppresses the production of MutYH protein by cells, an agent that suppresses or promotes the degradation of MutYH protein, and the like. In the present invention, among these, agents that increase the abundance of MutYH protein, such as exogenous MutYH protein, agents that promote MutYH protein production by cells, and / or agents that suppress degradation of MutYH protein are preferred.

  The MutYH protein itself refers to, for example, GenBank accession numbers AAM78555, AAH03178, CAI21720, CAI21718, CAI21714, CAI21713, CAI21719, CAI21717, CAI21716, Q9UIF7 (human), AAI28729, NP_579850, Q8R5G2 (rat), 513, 543, CAM In addition to those registered as Q99P21 (mouse), AAI05491, NP_001039600 (bovine), it refers to a functional equivalent having these functions, that is, adenine DNA glycosylase activity. Such functional equivalents include proteins containing one or more, preferably one or several amino acid differences from the registered MutYH protein. Whether or not it has an equivalent function is determined by, for example, allowing a test protein to act on an oligonucleotide having an A / G mismatch (for example, A / GO Mismatch Mesophilic Oligonucleotide Set, R & D, Cat # 3800-100-A), It can be easily confirmed by detecting whether or not the oligonucleotide is cleaved. The MutYH protein can be typically obtained by expressing a gene encoding the protein in bacteria such as Escherichia coli according to a conventional method and purifying the protein from the culture. Nucleic acid sequences encoding MutYH proteins are registered as, for example, GenBank accession numbers AF527839, BC003178 (human), BC128728, NM_133316 (rat), BC057942, NM_133250 (mouse), BC105490, NM_001046135 (bovine).

  The agent that releases MutYH protein in the body refers to any agent that can release the above-mentioned MutYH protein or a functional equivalent thereof in the body of the subject. For example, the MutYH protein is sustained and / or controlled. Preparations that are released in such a manner, cells that produce and secrete MutYH protein, substances that are converted to MutYH protein by chemical reaction, and the like. As cells that produce and secrete MutYH protein, any cells from bacterial cells to human cells have such ability, and any of these cells may be used. Preferably, cells having high ability to produce MutYH protein, For example, a cell of a bacterial strain having a high ability to produce MutYH protein, a cell modified to increase the ability to produce MutYH protein, and the like can be mentioned. Techniques for modifying a cell to increase production of a specific protein are well known, and examples include introduction of a MutYH protein gene operably linked to a promoter that enhances expression into the cell. In addition, self-derived cells that have less adverse effects on the living body such as rejection are preferred, and autologous hepatocytes that simultaneously supplement liver function are particularly preferred. Another example of a preferable cell is a cell capable of controlling proliferation and life and death. Methods for controlling cell growth and life / death by introducing a gene conferring sensitivity to a specific chemical substance are well known in the art. Therefore, a cell that is particularly preferable as an agent that releases MutYH protein in the body is an autologous hepatocyte that has a high ability to produce MutYH protein and can control proliferation and life and death.

The agent that promotes the production of MutYH protein by the cell is not limited, and for example, the MutYH gene itself, a vector containing the gene, an agent that stimulates the production of MutYH protein by the cell, and the promoter of MutYH protein in the cell are activated. And the like.
The MutYH gene includes not only the full-length MutYH gene on the chromosome but also any nucleic acid containing the coding region of MutYH protein. For example, (1) the nucleotide sequence represented by SEQ ID NO: 1 or GenBank Nucleic acid containing a sequence registered as session number AF527839, BC003178, BC128728, NM_133316, BC057942, NM_133250, BC105490 or NM_001046135, (2) encodes the same polypeptide as the nucleic acid (1) due to the degeneracy of the gene code A nucleic acid, (3) having one or more, preferably one or several mutations in the nucleic acid sequence of (1) or (2), but still having the same function as the polypeptide encoded by the nucleic acid A nucleic acid encoding a polypeptide having: and (4) a complementary strand of the nucleic acid of any one of (1) to (3) above, or a fragment thereof Hybridized with a condition, and includes nucleic acid such that encodes a polypeptide having the same function as the polypeptide nucleic acid encodes. Such MutYH gene can be integrated into the genome of a host cell as a naked nucleic acid and promote MutYH protein production by the cell.

The term “stringent conditions” as used herein is a well-known parameter in the art, and is a collection of standard protocols such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring. Harbor Press (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992).
The stringent conditions in the present invention are, for example, 3.5 × SSC (0.15 M sodium chloride / 0.15 M sodium citrate, pH 7) at 65 ° C., Ficoll 0.02%, polyvinylpyrrolidone 0.02% Hybridization using a hybridization buffer consisting of 0.02% bovine serum albumin, 25 mM NaH ₂ PO ₄ (pH 7), 0.05% SDS (sodium dodecyl sulfate), 2 mM EDTA (ethylenediaminetetraacetic acid). After hybridization, the membrane to which the DNA has been transferred is washed with 2 × SSC at room temperature and then with 0.1-0.5 × SSC / 0.1 × SDS at a temperature up to 68 ° C. Alternatively, stringent hybridization may be performed using a commercially available hybridization buffer such as ExpressHyb (registered trademark) Hybridization Solution (manufactured by Clontech) under the hybridization and washing conditions described by the manufacturer.
There are other conditions, reagents, etc. that can be used that result in the same degree of stringency, but those skilled in the art will be familiar with such conditions, so these are not specifically described herein. Not done. However, it is possible to manipulate the conditions so that a homologous or allele of the nucleic acid encoding the MutYH variant can be clearly identified.

  As the vector containing the MutYH gene, any known vector such as a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, or a virus vector can be used. The vector preferably includes at least a promoter that enhances expression of the MutYH gene. In this case, the MutYH gene is preferably operably linked to such a promoter. The expression that a gene is operably linked to a promoter means that the gene and the promoter are arranged so that the protein encoded by the gene is appropriately produced by the action of the promoter. The vector may or may not be replicable in the host cell, and transcription of the gene may be performed outside or inside the nucleus of the host cell. In the latter case, the MutYH gene may be integrated into the host cell genome.

Vectors may be targeted to them in order to make them reach the desired organ or cell efficiently. Accordingly, preferred vectors in the compositions of the present invention are those targeted to the liver and / or cells present in the liver, preferably hepatocytes. Any known technique can be used for targeting. For example, the vector can be specifically delivered to the liver by being carried on a carrier described in WO2006 / 068232.
Examples of agents that control the function of MutYH protein include agents that can introduce normal MutYH gene into individuals or cells that express abnormal MutYH protein, and modified MutYH gene with high substrate affinity and reaction rate. And agents that can introduce mutations into the MutYH gene, siRNAs that reduce the expression of the MutYH gene, and the like.

  The MutYH gene used in the composition of the present invention is preferably a MutYH gene that is difficult to develop hepatocellular carcinoma, in which the base type at position 8314 of the base sequence represented by SEQ ID NO: 1 is G. Introducing such a MutYH gene susceptible to hepatocellular carcinoma into the host cell genome is a MutYH gene susceptible to hepatocellular carcinoma, ie, the base type at position 8314 of the base sequence represented by SEQ ID NO: 1 is A. It is particularly preferable for a subject having the MutYH gene or the MutYH gene represented by SEQ ID NO: 2. The introduction of the refractory MutYH gene can be achieved by the MutYH gene itself or the MutYH gene carried on a liposome or a vector. Liposomes have an affinity for cell membranes and can be suitably used for gene transfer. Cationic liposomes are particularly useful in terms of introduction efficiency and the like, and various commercially available products can be used.

The composition of the present invention may further contain an agent useful for the treatment of hepatocellular carcinoma in addition to the agent for controlling the abundance or function of MutYH protein as described above.
As used herein, the term “treatment” is intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease. For example, when the disease is hepatocellular carcinoma, the term “treatment” can be used for various purposes, including delaying or stopping the progression of hepatocellular carcinoma, regression or disappearance of lesions, preventing the onset or recurrence of hepatocellular carcinoma, etc. Includes medically acceptable interventions.
Therefore, the above-mentioned agents are not limited, and include, for example, not only various antitumor agents but also agents for treating chronic hepatitis, liver fibrosis, cirrhosis and the like that can cause hepatocellular carcinoma. .

  Antitumor agents include, for example, ifosfamide, nimustine hydrochloride, cyclophosphamide, dacarbazine, melphalan, ranimustine and other alkylating agents, gemcitabine hydrochloride, enocitabine, cytarabine ocphosate, cytarabine preparations, tegafur uracil, tegafur gimeracil, Oteracyl potassium combination drug, doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, etc. Antitumor antibiotics such as C, etoposide, irinotecan hydrochloride, vinorelbine tartrate, docetaxel hydrate, paclita Cell, alkaloids such as vincristine sulfate, vindesine sulfate, vinblastine sulfate, hormone therapy agents such as anastrozole, tamoxifen citrate, toremifene citrate, bicalutamide, flutamide, estramustine phosphate, platinum complexes such as carboplatin, cisplatin, nedaplatin And angiogenesis inhibitors such as thalidomide, neobasstat, and bevacizumab, and L-asparaginase.

  Examples of the agent for treating chronic hepatitis, liver fibrosis, cirrhosis and the like are not limited, and include, for example, interferon preparations, therapeutic agents for viral hepatitis such as ribavirin, truncated TGFβ type II receptor, soluble TGFβ type II receptor, etc. TGFβ activity inhibitors, growth factor preparations such as HGF, MMP production promoters such as MMP gene-containing adenovirus vectors, PPARγ ligands, angiotensin II type I receptor antagonists, PDGF tyrosine kinase inhibitors, and sodium channels such as amiloride Production of extracellular matrix components such as hepatic stellate cell activation / proliferation inhibitors including inhibitors, apoptosis inducers such as compound 861 and gliotoxin, collagen, proteoglycan, tenastin, fibronectin, thrombospondin, osteopontin, osteonectin, and elastin A substance having an effect of suppressing the expression of a target molecule such as siRNA, ribozyme, antisense nucleic acid (including RNA, DNA, PNA, or a complex thereof) targeting these components, or Substances having a dominant negative effect such as dominant negative mutants, vectors expressing them, and drugs that suppress collagen production such as corticosteroids, penicillamine, colchicine, interferon γ, prostaglandin derivatives, etc. .

The compositions of the present invention may be used in a variety of routes including both oral and parenteral, such as, without limitation, oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular. , Transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine routes, and may be formulated into dosage forms suitable for each route of administration. Any known dosage form and formulation method can be adopted as appropriate (see, for example, Standard Pharmacology, Yoshiteru Watanabe, Nankodo, 2003, etc.).
For example, dosage forms suitable for oral administration include, but are not limited to, powders, granules, tablets, capsules, solutions, suspensions, emulsions, gels, syrups, etc. Suitable dosage forms include solution injections, suspension injections, emulsion injections, injections such as injections prepared at the time of use. Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions. Specifically, for example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, Appropriate combinations with preservatives, binders and the like can be formulated into suitable unit dosage forms. The amount of the active ingredient in these preparations can be appropriately set so that a dose effective for treatment can be supplied to the subject with the expected number of administrations.

Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. For example, alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80, HCO-50, etc. may be used in combination.
Examples of the oily liquid include sesame oil and soybean oil. As a solubilizing agent, benzyl benzoate, benzyl alcohol and the like may be used in combination. Moreover, you may mix | blend with buffering agents, such as a phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizers, such as benzyl alcohol, phenol, antioxidant, etc. The prepared injection solution is usually filled in a container such as an appropriate ampoule, vial, tube, bottle or pack.

  Administration into the body of a subject may be performed by any of the above routes, but parenteral administration is preferred, local administration or intravenous administration is more preferred, and portal vein administration is particularly preferred. The number of administration is preferably one time, but can be administered multiple times depending on the situation. Further, the administration time may be short or continuous. More specifically, the compositions of the present invention can be administered by injection or transdermally. Examples of administration by injection include, but are not limited to, intravenous injection, intraarterial injection, selective intraarterial injection, intraportal injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. I can't. In the case of intravenous injection, it is possible to administer in the manner of normal blood transfusion, and it is not necessary to operate the subject, and further no local anesthesia is required, so the burden on both the subject and the operator can be reduced. Moreover, it is advantageous in that the administration operation can be performed outside the operating room.

  The invention further relates to a method for treating hepatocellular carcinoma comprising administering to a subject an effective amount of a composition of the invention. In the treatment method of the present invention, administration of the composition of the present invention to a subject can be suitably performed according to, for example, the above-mentioned administration method. A doctor or veterinarian can appropriately modify the above administration method and administer the agent of the present invention to a subject. Here, the effective amount is an amount that reduces the onset of hepatocellular carcinoma, reduces symptoms, or prevents progression, and preferably prevents the onset of hepatocellular carcinoma or cures hepatocellular carcinoma. Amount. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by an in vitro test using cultured cells or the like, or a test in a model animal such as a mouse, rat, dog or pig, and such a test method is well known to those skilled in the art. . Examples of hepatocellular carcinoma models include hepatocarcinogenesis by administration of N, N-diethylnitrosamine (DEN), dimethylnitrosamine (DMN), methionine-choline deficient diet (MCDD), choline deficient amino acid (CDAA) diet, and the like. Can be mentioned.

The specific dose of the medicament to be administered in the treatment method of the present invention depends on various conditions relating to the subject requiring treatment, such as severity of symptoms, general health status of the subject, age, body weight, subject sex, diet, administration It may be determined in consideration of the timing and frequency of the drug, the medicine used in combination, the response to treatment, compliance with the treatment, etc., so it may differ from the general effective amount. These methods are still within the scope of the present invention.
Administration routes include various routes including both oral and parenteral, such as oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, Routes such as intranasal, intraperitoneal, intrapulmonary and intrauterine are included.
The frequency of administration varies depending on the properties of the pharmaceutical used and the conditions of the subject as described above. For example, many times a day (ie, 2, 3, 4 or 5 times a day), once a day, every few days (Ie every 2, 3, 4, 5, 6, 7 days, etc.) Every week, every few weeks (ie every 2, 3, 4 weeks, etc.).

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Extraction of genomic DNA Genomic DNA was extracted from peripheral blood mononuclear cells of 31 Japanese hepatitis C patients, including 13 hepatocellular carcinoma patients, as follows.
i) Separation of mononuclear cells 5 ml of peripheral blood was collected in an EDTA blood collection tube to which 1 ml of heparin had been added in advance. To remove contaminating red blood cells, transfer the entire amount of the collected peripheral blood to a 15 ml conical tube, add 5 ml of hydroxyethyl starch (6-HES, Lucer Morishita Co., Ltd.), mix by inverting 5 times, and then for 30 minutes at room temperature. The erythrocytes were precipitated by standing. The supernatant containing mononuclear cells was transferred to another new tube and centrifuged at 300 × g and 4 ° C. for 5 minutes to pellet the mononuclear cell fraction. To the obtained pellet, 500 μl of RBC lysis solution (Gentra) was added and allowed to stand at room temperature for 15 minutes to hemolyze the mixed RBC. After centrifugation at 300 × g and 4 ° C. for 5 minutes, the supernatant was discarded to obtain a cell pellet.

ii) Separation of genomic DNA After adding Cell Lysis solution (Gentra) to the cell pellet and lysing the cells at room temperature, 1 μl of 20 mg / ml RNAase was added, treated at room temperature for 15 minutes to degrade the RNA, and then the protein. 300 μl of precipitation solution (Gentra) was added and mixed by inversion to precipitate the protein. After centrifugation at 10,000 × g and 4 ° C. for 15 minutes, the protein was removed by precipitation. The supernatant was transferred to a new tube and 900 μl of 2-propanol was added and mixed by inversion. After visually confirming the precipitation of the genomic DNA, it was centrifuged at 2000 × g and 4 ° C. for 2 minutes to precipitate the genomic DNA. After removing the supernatant, 900 μl of 75% ethanol was added and centrifuged at 2000 × g and 4 ° C. for 2 minutes to remove the supernatant, and then the genomic DNA pellet was air-dried at room temperature for 10 minutes. Thereafter, 100 μl of 1 × TE (pH 8.0) was added, and the genomic DNA was dissolved overnight at room temperature.

Example 2 SNP analysis using Taqman (registered trademark) PCR SNP analysis of genomic DNA utilized a Custom TaqMan (registered trademark) Genomic Assays service provided by Applied Biosystems. Specifically, using MUTYH and GAPDH probes (5 ′ end labeled with FAM) / primer sets (Assay ID C___110818_10 and Hs99999905_m1 respectively), TaqMan PRISM (registered trademark) 7700 Sequence Detection System (PE Applied Biosystems) Real-time PCR was performed. The results are shown in FIG. The Ct value in FIG. 1 means the number of PCR cycles when the fluorescence intensity (ΔRn) reaches a threshold value. Therefore, the smaller the Ct value, the more corresponding nucleic acid is present in the sample.

As is clear from FIG. 1 and Table 1, a significant association was found between the MutYH gene polymorphism and hepatocellular carcinoma (p = 0.0003, χ ² test). In the group not affected with hepatocellular carcinoma, 94.4% of subjects were homozygous for major alleles, while minor alleles were homozygous for 0% and heterozygous for 5.6%, whereas hepatocellular carcinoma was affected. In the group, 15.3% were minor allele homozygous and 38.5% heterozygous, and the frequency of minor allele was significantly higher in the hepatocellular carcinoma affected group, The frequency of allele homotypes was found to be significantly higher.

Example 3 SNP confirmation using direct sequencing method i) Genomic PCR
100 ng of genomic DNA was used as a template, and the primer pair was sense: 5′-TGGTCAGTATACATCTCTCTGA-3 ′ (SEQ ID NO: 3), antisense: 5′-CTTGAGTCTTGCACTCCCAATCA-3 ′ (SEQ ID NO: 4), Advantage (registered trademark) GC 2 DNA was amplified using Kit (Takara-Clontech). The PCR reaction was performed for 37 cycles with denaturation: 95 ° C., 30 seconds, annealing: 60 ° C., 30 seconds, extension: 72 ° C., 2 minutes. PCR products were electrophoresed on a 2% agarose gel and then detected with ethidium bromide.

ii) Sequencing reaction The PCR product was electrophoresed in 2% agarose, and then the band was cut out and purified by GENECLEAN (registered trademark). To 100 ng PCR product, 8 μl of Big Dye (Applied Biosystem) and 1 μl of primer 10 pmol / μl used in the above PCR were added, and Milli-Q water (distilled water) was added to make a total volume of 20 μl. For PCR, a thermal cycler manufactured by Bio-Rad was used, and 35 cycles of 96 ° C., 10 seconds, 50 ° C., 5 seconds, 60 ° C., and 4 minutes were performed. In order to remove excess Big-Dye, the entire amount of the PCR product obtained by the sequencing reaction was applied to an Edge Gel Filtration Cartridge spin column (Edge BioSystems) and centrifuged at 3,000 × g for 2 minutes. The solution extracted through the column and into the tube is the desired product. After centrifugal drying under reduced pressure, 20 μl of HD (deionized formamide) was added, mixed well, and spun down. A heat shock was applied at 95 ° C. for 2 minutes, the mixture was quickly lifted on ice and cooled for 5 minutes or longer, then transferred to a dedicated column for sequencing reaction, and nucleotide sequence analysis was performed using ABI PRISM (registered trademark) 3100 Genetic Analyzer. The results confirmed the results of Taqman® PCR described in Example 2.

Example 4 Evaluation of MutYH Protein Activity in Subjects with Hepatocellular Carcinoma The difference in the activity of MutYH protein between subjects with and without hepatocellular carcinoma is evaluated as follows.
i) Preparation of cell extract From peripheral blood collected from each subject, nucleated mononuclear cells were separated by specific gravity centrifugation using Ficoll-Hypaque (specific gravity 1.077) and washed three times with PBS (−). To do. The obtained peripheral blood mononuclear cells 5 × 10 ⁶ were suspended in 250 μl of 1 × buffer (50 mM HEPES-KOH, pH 7.4, 200 mM NaCl, 2 mM β-mercaptoethanol, 0.1 mM EDTA, 10% glycerol). Cloudy and crushed by sonication. After centrifugation at 10,000 × g and 4 ° C. for 10 minutes, the supernatant is stored frozen at −80 ° C. as a cell extract. Since MutYH is inactivated in a few minutes under normal conditions, the cell extract is diluted with 1 × REC (registered trademark) buffer (10 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 mM EDTA, 10% glycerol). Dilute.

ii) Detection of adenine DNA glycosylase activity 4 pmol of A / GO Mismatch Mesophilic Oligonucleotide Set (R & D, annealed) containing mismatch oligo labeled with FAM and serially diluted recombinant MutYH protein (R & D) or the above cells Mix with the extract to make a total volume of 20 μl. Incubation at 37 ° C. for 1 hour causes the MutYH protein to cleave the FAM-labeled oligo DNA strand. Thereafter, 10 μl of 3 × Alkali Loading Buffer (0.3 M NaOH, 97% formamide, 0.2% bromophenol blue) is added and heated at 95 ° C. for 10 minutes before the reaction is rapidly cooled to 4 ° C. The double-stranded DNA is dissociated into single strands. The obtained sample is loaded on a 20% polyacrylamide gel at 10 μl / lane and subjected to electrophoresis to separate the cleaved and uncut oligos. By densitometric analysis, the ratio of cleaved oligo to uncleaved oligo is calculated, and the adenine DNA glycosylase activity of MutYH protein in the sample is evaluated. The higher the percentage of cleaved oligo, the higher the activity of MutYH protein.

Example 5 Evaluation of MutYH Expression in Subjects with Hepatocellular Carcinoma The difference in expression of MutYH between subjects with and without hepatocellular carcinoma is evaluated as follows. Nucleated mononuclear cells are separated from peripheral blood collected from each subject by specific gravity centrifugation using Ficoll-Hypaque (specific gravity 1.077). This was washed 3 times with PBS (−), divided into 1 × 10 ⁷ pieces, and NP-40 lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, Dissolve using protease inhibitors cocktail (Roche). Each sample was boiled for 5 minutes after adding sample buffer (250 mM Tris-HCl, pH 6.8, 8.4% SDS, 10% glycerol, 0.006% bromophenol blue), and a 4/20% polyacrylamide gradient gel. (Multigel (registered trademark), Dai-ichi Chemical Co., Ltd.) is run at 40 mA for 60 minutes (SDS-PAGE). Transfer to a nylon membrane (Immobilon (registered trademark), Millipore) for 45 minutes at a constant current of 200 mA using a semi-driving apparatus, and block overnight at 4 ° C. with 10% skim milk. 1 μg / ml anti-human MutYH polyclonal antibody (Calbiochem) is added, and after reacting at room temperature for 1 hour, the plate is washed 3 times with PBS (−) containing 0.05% Tween-20. A peroxidase-labeled goat anti-rabbit IgG antibody (Dako) is reacted as a secondary antibody with ECL (registered trademark) plus (Amersham Biosciences) and exposed to a film (Eastman Kodak). By analyzing the degree of exposure, the difference in relative abundance of MutYH protein between hepatoma affected and unaffected subjects is detected.

Example 6 Inhibition of Hepatocellular Carcinoma by Enhancing MutYH Expression After subcloning the MutYH cDNA by RT-PCR, it is cloned into an adeno-associated virus vector plasmid (Strategene) to obtain a MutYH gene-carrying plasmid (AAV-MutYH). AAV293 cells made 70% confluent in a 10cm dish (1% gelatin-coated dish) were passaged with 0.2% EDTA PBS and centrifuged at 300 × g for 3 minutes at 4 ° C. As a virus producing cell Use. AAV-MutYH plasmid, helper plasmid and RC plasmid were co-transfected into AAV293 cells by calcium phosphate method kit (Invitrogen), and then incubated in a humidified CO ₂ incubator overnight at 37 ° C., and the medium was changed the next day. To do. After 4 days, all of the supernatant and cells were collected, and freeze-thawing was repeated three times with dry ice ethanol, and then the supernatant centrifuged at 10,000 × g for 15 minutes was dispensed into a container to obtain an AAV-MutYH virus stock. Store at -80 ° C.
MutYH gene introduction into the liver of a hepatocellular carcinoma model animal is performed by thawing the cryopreserved AAV-MutYH virus and injecting the portal vein into the portal vein or intravenously through the peripheral vein. The inhibitory effect on hepatocellular carcinoma by introduction of MutYH gene is evaluated in comparison with control animals using tumor weight, hepatocellular carcinoma markers such as AFP and PIVKA-II, and / or survival rate as indices.

It is a figure which shows the result of having analyzed the genotype frequency of the polymorphism of MutYH gene in a hepatocellular carcinoma affected subject and a hepatocellular carcinoma nonaffected subject by Taqman (trademark) PCR method. In the figure, ○ indicates subjects with hepatocellular carcinoma, and ■ indicates subjects without hepatocellular carcinoma.

Claims (8)

Oite to MutYH gene polymorphism, and / or including the step of detecting a polymorphism included in the haplotype block comprising the same, morbidity squirrel hepatocellular cancer present at position 8314 of the nucleotide sequence shown in SEQ ID NO: 1 How to judge In MutYH gene, nucleotide species present in position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 in the case of A, morbidity risk of hepatocellular carcinoma is determined to be high, the method of claim 1. In MutYH genes, when the base species present at position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 is G, morbidity risk of hepatocellular carcinoma is determined to be lower, the method according to claim 1. The method according to any one of claims 1 to 3 , wherein the hepatocellular carcinoma is hepatocellular carcinoma caused by hepatitis C virus. Of the base sequence of MutYH gene , a continuous base sequence of 15 to 1000 bases including a polymorphism present at position 8314 of the base sequence represented by SEQ ID NO: 1 and / or a polymorphism contained in a haplotype block containing the polymorphism A marker for determining the risk of developing hepatocellular carcinoma, comprising a polynucleotide comprising: Definitive in MutYH gene polymorphisms, and / or allele-specific hybridizing to a site containing a polymorphism included in the haplotype block comprising the same that occurs at position 8314 of the nucleotide sequence represented by SEQ ID NO: 1 15 to 1000 bases of the polynucleotide, or a 15 to 1000 bases of a polynucleotide sequence specifically hybridize to upstream sequences of the multi-type comprising a base on the one base upstream (5 'side) of the multi-type, the those 5 'end is the one base upstream (5' hybridizes to bases on the side), or, sequence downstream sequence of polymorphism comprising a base at the polymorphic one bases downstream (3 'side) a polynucleotide which specifically hybridizes to 15 to 1000 bases, including those that 3 'end is the one bases downstream of (3' hybridizes to the nucleotide on the side), hepatocytes Reagents for determining the morbidity risk. a) a nucleic acid having a base sequence represented by SEQ ID NO: 2 or a nucleic acid having a base sequence represented by SEQ ID NO: 1 that hybridizes to the nucleic acid part containing the nucleic acid sequence containing position 8314 of the base sequence A polynucleotide of 15 to 1000 bases that does not hybridize to the nucleic acid part containing position 8314 of b), b) a nucleic acid having the base sequence represented by SEQ ID NO: 1 or the nucleic acid part containing position 8314 of the base sequence A 15-1000 base polynucleotide that is soy and does not hybridize to the nucleic acid having the base sequence represented by SEQ ID NO: 2 or the nucleic acid portion containing position 8314 of the base sequence; c) represented by SEQ ID NO: 1 that in a nucleic acid having a nucleotide sequence, including position 8313 of the base sequence hybridizes to a contiguous portion including no position 8314 1 A 1000 base polynucleotide itself to 5 'end hybridizes to the nucleotide at position 8313, and, d) in a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1, 8315-position of the base sequence a 15 to 1000 bases of the polynucleotide but which hybridize to contiguous portions containing no position 8314 containing the polynucleotides its 3 'end is one that hybridizes to 8315 position of the base, which is selected from the group consisting of The reagent of Claim 6 containing this. The kit for performing the method of Claims 1-4 containing the reagent of Claim 6 or 7 .

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