WO2006085407A1 - Procede de criblage d'un gene associe au taux de vhc - Google Patents

Procede de criblage d'un gene associe au taux de vhc Download PDF

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WO2006085407A1
WO2006085407A1 PCT/JP2005/018573 JP2005018573W WO2006085407A1 WO 2006085407 A1 WO2006085407 A1 WO 2006085407A1 JP 2005018573 W JP2005018573 W JP 2005018573W WO 2006085407 A1 WO2006085407 A1 WO 2006085407A1
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gene
genes
virus group
low
virus
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PCT/JP2005/018573
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Japanese (ja)
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Mariko Esumi
Tadatoshi Takayama
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Nihon University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D

Definitions

  • the present invention relates to a method for screening genes whose expression is increased in the high virus group of HCV RNA and genes whose expression is increased in the low virus group.
  • Hepatitis virus is a major cause of liver disease.
  • 80% of chronic liver diseases are caused by hepatitis c virus (HCV) infection.
  • 70-80% of HCV infections do not end with transient infections, but persistent infections are established. After that, it progresses to hepatocellular carcinoma over 20-30 years after chronic hepatitis and cirrhosis. Therefore, radical cure at the stage of chronic hepatitis C, which is a high-risk group for developing hepatocellular carcinoma, is desirable.
  • IFN interferon
  • IFN interferon
  • Hepatitis C in hepatitis C is caused by a host immune response that attempts to eliminate HCV. However, due to insufficient immune response, it is considered that HCV cannot be completely eliminated, leading to persistent infection. Even if HCV cannot be eliminated completely, if the viral load can be reduced, it should be possible to stop the progression of the disease. In general, HCV has a low ability to grow, but there are cases in which the amount of liver virus is 1000 times higher. It is not clear what causes this difference in the amount of liver virus. Disclosure of the invention
  • HCV hepatitis C virus
  • the present inventor selected a plurality of cases of high viral load and low viral load from human chronic liver inflammation cases, and the difference in gene expression between these two groups occurred. It was investigated by two methods. (1) When a virus infects cells, IFN is induced as a mechanism of virus elimination. The IFN induces various antiviral molecules and goes toward virus elimination. In order to investigate whether the strength of this host defense response is a factor that creates a high or low level of liver HCV, it is necessary to induce apoptosis through IFN downstream genes and two groups between two groups with different viral loads. The amount of Bcl2-associated X protein (BAX) involved was compared.
  • BAX Bcl2-associated X protein
  • the present invention is as follows.
  • a method for screening a gene whose expression is enhanced in a high virus group tissue containing a large amount of HCV comprising:
  • liver tissue derived from liver tissue derived from liver tissue with a value of 300 units or less divided by the 18S rRNA quantitative value per 50ng of liver tissue-derived cDNA was selected as the low virus group tissue.
  • liver tissue of 30000 units or more as a high virus group tissue Selecting a liver tissue of 30000 units or more as a high virus group tissue
  • liver tissue-derived cDNA (a) Divide the number of HCV copies per 50 ng of liver tissue-derived cDNA by the 18S rRNA quantitative value Select a liver tissue with a value of 300 units or less as a low virus group tissue, and select a liver tissue with a value of 30000 units or more as a high virus group tissue,
  • a test agent for a pathological condition associated with viral load comprising at least one gene selected from the following genes (a) to (:
  • a diagnostic agent for a disease state associated with the amount of virus comprising at least one gene selected from the following genes (a) to (! 1):
  • test drug described in any one of (7) to (10), which is in the form of a microarray. 'Brief description of the drawings
  • Fig. 1 is a diagram showing the mechanism of interferon action.
  • IFN interferon
  • ISGF transcription factor IFN-stimulated gene factor
  • ISG IFN-stimulated gene
  • MxA binds to viral RNA and inhibits RNA replication.
  • OAS and PKR suppress virus growth by shutting off host cell reactions.
  • p53 induces apoptosis of host cells via Bcl2_associated X protein (BAX) and suppresses viral growth. Control.
  • BAX Bcl2_associated X protein
  • the HCV protein counteracts the host's defense against viral infection with multiple mechanisms of inhibition. .
  • FIG. 2 shows the results of quantification of liver HCV RNA.
  • Low virus group (Low) 15 cases chronic hepatitis 9 cases, cirrhosis 6 cases
  • high virus group (High) 19 cases chronic hepatitis 9 cases, cirrhosis 10 cases.
  • the HCV genotype is shown below the graph bar. All cases not listed are lb type. “+ 2a” means double infection of type lb and type 2a. Black bars are for chronic hepatitis, hatched bars are for cirrhosis, and “” is used for oligonucleotide microarray analysis. Shows the column.
  • FIG. 3 is a diagram showing genes whose expression levels are different between the high virus group (High) and the low virus group (Low). Expression levels were compared using 14 cases of high virus group (5 cases of chronic hepatitis and 9 cases of cirrhosis) and 11 cases of low virus group (6 cases of chronic hepatitis and 5 cases of cirrhosis). Significant difference was tested by Mann Whitney U test (p 0.05). The horizontal line shows the median value of chronic hepatitis cases with significant differences. “ ⁇ ” indicates the gene expression level derived from patients with chronic hepatitis, and “ ⁇ ” indicates the gene expression level derived from patients with cirrhosis.
  • FIG. 4 is a diagram showing how to obtain genes having different expression levels between the high virus group and the low virus group.
  • 8 microarrays using 4 cases of the chronic hepatitis high virus group and 4 cases of the low virus group we searched for genes that were more than twice as differential in expression as compared between the two groups.
  • Each microarray used has 54,675 probes covering more than 47,000 genes.
  • Probes with expression expressed in at least one microarray were selected (28,505), and three types of parametric tests were performed by comparison between the two groups. The number of probes with significantly different expression levels was determined.
  • probes with a difference of more than 2 times between the two groups were selected, and probes with the expression of expression in the four groups with higher expression levels were selected.
  • the genes with high expression in the high virus group were selected as high virus genes, and those with high expression in the low virus group were selected as low virus genes.
  • Fig. 5 is a diagram showing a Condition tree by clustering analysis. Clustering of 8 microarrays was performed using 117 genes (A) that differed in expression level between the two groups and 28,505 probes (B) expressed in the liver. For the microarra, HI, H2, H3, and H4 were assigned in descending order of the viral load in the high virus group, and LI, L2, L3, and L4 were assigned in the low virus group in ascending order.
  • FIG. 6 shows the results of quantification of the expression of the endogenous control gene.
  • A The signals of GAPDH and RPL 34 were compared using 8 microarrays. The signal value after per gene normalization was used.
  • B The amounts of 18S rRNA, RPL 34, and GAPDH in 34 cases of liver cDNA were quantified using real-time PCR. The expression level was expressed as a corrected value with the median of each gene as 1. “ ⁇ ” indicates the low virus group, and “ ⁇ ” indicates the high virus group.
  • FIG. 7 is a diagram showing the results of quantification of the expression of high virus genes and low virus genes by real-time PGR.
  • Three high viral genes (A, B, C) and low viral genes (D, E, F) were quantified by real-time PGR.
  • Chronic hepatitis high virus group (High) 9 cases and low The virus group (Low) was compared with 9 cases.
  • Each of the 4 cases used in the microarray is indicated by a gray circle.
  • the gene expression level was corrected with 18S rRNA. Significant difference was tested by Mann Whitney U test (p ⁇ 0.05).
  • the horizontal line shows the median value of 9 cases of chronic hepatitis.
  • FIG. 8 is a diagram showing the position and classification of 117 probes on the gene structure.
  • a box ( ⁇ ) and a white arrow indicate a gene consisting of 5 exons and their direction.
  • the black arrow indicates the position and direction of the probe used for the microarray.
  • (5) shows the case where the transcript is proved in the region where the gene has not been identified.
  • the numbers on the right indicate the number of genes for each of the 78 high virus genes and 39 low virus genes.
  • FIG. 9 is a diagram showing an outline of analysis of gene expression level in chronic hepatitis.
  • FIG. 10 is a diagram showing an outline of analysis of gene expression levels in cirrhosis.
  • Figure 11 shows the results of analyzing the presence or absence of genes commonly expressed in chronic hepatitis and cirrhosis.
  • Figure 12 shows the results of clustering analysis.
  • Figure 13 shows the location and classification of the probes used in the microarray on the gene structure.
  • Fig. 14 shows the results of verification of the expression of high viral genes in chronic hepatitis.
  • Fig. 15 shows the results of verification of the expression of high viral genes in chronic hepatitis.
  • Fig. 16 shows the results of verification of the expression of low viral genes in cirrhosis.
  • FIG. 17 is a diagram showing the quantitative results of HCV levels in cancerous and non-cancerous parts.
  • FIG. 18 shows the results of measuring the expression level of receptor-related genes.
  • FIG. 19 shows the results of measuring the expression level of receptor-related genes.
  • the present inventor selected the target non-cancerous tissue of type C hepatocellular carcinoma into a high virus group with a high amount of HCV RNA and a low virus group with a low amount of HCV RNA.
  • the present invention provides a screening method for genes whose expression is enhanced in a high virus group having a high amount of HCV RNA, and a screening method for a gene whose expression is enhanced in a low virus group having a low amount of HCV RNA. Is.
  • the present invention also provides a diagnostic agent for a disease state associated with viral load, including a gene whose expression is increased in a high virus group or a low virus group. 2. High virus group and low virus group
  • the target for crystallization is classified into a high virus group or a low virus group according to the amount of HCV RNA.
  • the target tissue of the method of the present invention is a non-cancerous tissue of a patient with type C hepatocellular carcinoma. It may also be a tissue with chronic hepatitis or cirrhosis infected with hepatitis C virus. Tissues can be frozen in liquid nitrogen and stored at -80 ° C if the method of the present invention is not performed immediately after collection.
  • RNA is extracted from the tissue.
  • a method for extracting RNA from tissue can be appropriately selected by those skilled in the art.
  • rizol Invitrogen
  • rizol can be used.
  • the amount of HCV RNA can be measured by real time PCR.
  • real time PGR Rotor-Gene 3000 (Corbett Research, Mortalke, Australia) and ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster, CA) can be used.
  • Measurement of the amount of HCV RNA can be performed by synthesizing cDNA from the RNA obtained in 2. (1) by reverse transcriptase and using, for example, 50 ng of the obtained cDNA. At that time, as a primer, for example
  • 0.3 ⁇ ⁇ ⁇ may be used.
  • the amount of HCV RNA is represented by “tmit”. “Unit” means the value obtained by converting the amount of plasmid DNA used to create the calibration curve into the number of copies, calculating the number of HCV RNA copies per 50 ng of liver tissue-derived cDNA from the calibration curve, and dividing by the 18S rRNA quantitative value. To do.
  • 18S rRNA quantification value refers to 18S rRNA in cDNA (standard sample) derived from a liver. This is the amount of standard sample cDNA equivalent to 0.25 ng of liver tissue-derived cDNA when a standard curve is prepared by measuring real-time PCR. The amount of standard sample cDNA corresponding to 0.25 ng of cDNA derived from liver tissue can be obtained from a calibration curve. '
  • the “low virus group” means a case where the amount of 110 ⁇ 11 ⁇ is 300 1111 or less.
  • the “high virus group” means a case where the amount of HCV RNA is 30000 units or more.
  • the method of the present invention is characterized in that the analysis target is selected into the high virus group and the low virus group according to the amount of HCV RNA.
  • a high virus group and a low virus group can be selected by HCV genotype. That is, by nested PCR method, la type, lb type, 2a type,
  • the HCV genotype By performing 4 HCV genotype-specific PCR of type 2b, the HCV genotype can be clarified, and the expression level of the host gene can be analyzed focusing on the specific genotype of HCV.
  • an oligonucleotide microarray or real-time PCR can be used to measure the expression level of a gene.
  • the genes whose expression is enhanced in the high virus group or low virus group are further selected from these genes by real-time PCR. Can also be selected.
  • biotin-labeled cRNA is first synthesized from the total RNA obtained in 2. (1).
  • the synthesis can be performed, for example, by partially modifying the manual of Affymetrix Gene Chip expression analysis (see Example 2 (1)).
  • the obtained cRNA is appropriately purified and fragmented for use as a target gene sample. Purification and fragmentation can be easily performed by those skilled in the art.
  • the microarray is preferably a commercially available product such as Human Genome U133 Plus 2.0 array (Affymetrix), but is not limited thereto.
  • the prepared cRNA fragment is hybridized to the array as a target gene sample.
  • Devices such as Fluidics Station 450 (Asymetrix) can also be used for hybridization, washing, and staining.
  • a scanner for example, Scanner 3000 (Affymetrix)
  • software is preferably used, and examples of the software include Gene Spring version 7 (Silicon Genetics, Redwood, CA).
  • the contingency table test uses a two- test or Fisher's exact test.
  • the Marm-Whitney U test is preferably used for the significant difference test between the two groups.
  • a method for selecting a gene whose expression is increased in the high virus group and a method for selecting a gene whose expression is increased in the low virus group are shown below.
  • the gene corresponding to this probe is a gene expressed in the liver of a patient infected with HCV. '
  • probe means a part of a gene set in a microarray.
  • the “gene corresponding to the probe” means the gene that is the source of the probe.
  • a parametric test can then be performed to extract genes that have significant differences in expression between the two groups, the high virus group and the low virus group. For example, Student's t test assuming that the variance is the same in the two groups, Welch's t test assuming that the variance is not equal, or to estimate the population variance as accurately as possible from a small number of replicates, so it will converge when increasing replicate.
  • One example is the cross-gene error model parametric test that predicts the wax standard deviation.
  • one type of test or a plurality of types of test may be used, but it is preferable to perform a plurality of tests. When multiple tests are performed, it is preferable to prepare a Venn diagram in order to examine duplication of probes extracted by these tests.
  • the degree of expression enhancement between the two groups is more than doubled, that is, between the two groups.
  • Probes that differ by more than 2 times in the expression level in The difference in the degree of expression enhancement is preferably 2 times or more, more preferably 2.5 times or more, and further preferably 3 times or more.
  • the gene corresponding to the probe whose expression is increased in the high virus group is a gene whose expression is increased in the high virus group (hereinafter also referred to as “high virus gene”).
  • the gene corresponding to the probe whose expression is up-regulated is a gene whose expression is up-regulated in the low virus group (hereinafter also referred to as “low virus gene”).
  • probes can be extracted by focusing on the present flag in all of the plurality of microarrays or in all the microarrays of the high expression group.
  • polyA + RNA for example, rRNA
  • the present inventor was able to select a total of 117 genes, 78 (Table 5) as high viral genes and 39 (Table 6A) as low viral genes by the above method (Examples). 2). Of these, the genes that differed more than 2.5 times between the two groups are shown in Tables 3 and 4.
  • each of the high virus group and low virus group was classified into four groups by classifying into chronic hepatitis and cirrhosis, and as a result of analyzing the gene expression of each group, 66 genes were found as chronic hepatitis high virus genes (Table 10). ), 21 hepatitis low virus genes (Table 11), 27 cirrhosis high virus genes (Table 12), and 17 cirrhosis low virus genes (Table 13) (Example 5).
  • RNA obtained in 2. (1) above is appropriately treated with DNasel, purified, random primer and reverse transcriptase. And synthesize cDNA.
  • DNasel purified, random primer and reverse transcriptase.
  • synthesize cDNA For example, AMV reverse transcriptase XL (Life Sciences, Gaithersurg, MD) 3 ⁇ 4i! / I can do it.
  • Oligo (dT) primer can be used instead of random primer.
  • Measurement of gene expression by real-time PCR should be performed using commercially available equipment such as Rotor-Gene 3000 (Corbett Research, Moi'talke, Australia) or ABI Prism 7000 Sequence jDetection System (Applied Biosystems, Foster, CA). Can do.
  • the reaction is performed, for example, in a 25 1 reaction solution containing 10 ng of cDNA, SYBR Green PCR Master Mix (Applied Biosystems), 0.5 M of various gene primers, 95 ° C., 10 min preheat, and 95. C 15 sec, 60 ° C 60 sec can be performed for 45 cycles. At this time, housekeeping genes such as 18S rRNA can be quantified using, for example, 0.25 ng of cDNA. Those skilled in the art can appropriately design primers used for the reaction using software. The URL of typical software “primer 3” is shown below. (http://frodo.wi.mit.edu/cgi-bm/primer3/prirner3_www.cgi;
  • a calibration curve is prepared using a certain liver cDNA as a standard sample for quantification and used for quantification of various genes.
  • the liver cDNA showing the highest expression in each gene can be used as a standard sample, and the amount of the cDNA can be used as a quantitative value. If a sample of the sample cDNA to be measured is subjected to real-time PCR of gene X and the sample can be evaluated as 5 ng from the calibration curve, the sample should have the same amount as gene X mRNA contained in 5 ng of the standard sample cDNA. Gene X mRNA will be present.
  • the expression level of each gene is obtained by dividing the quantitative expression value of each gene in the measurement target sample obtained from the calibration curve by the endogenous control gene expression quantitative value (for example, the quantitative value of 18s rRNA in the measurement target sample). Relative value.
  • 18S rRNA glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L34 (RPL34) can be used as the “endogenous control gene”, but the expression level is small. 18S rRNA is preferred.
  • the expression level of each gene in the host can be a value corrected with 18S rRNA.
  • the expression level of the low virus group is compared with the expression level of the high virus group.
  • a gene having a value obtained by dividing the median expression level of the high virus group by the median value of the low virus group is 2 or more, preferably 2.5 or more, more preferably 3 or more is selected as the high virus gene. be able to.
  • a gene of 2 or more, preferably 2.5 or more, more preferably 3 or more is selected as the low virus gene. can do.
  • High virus genes are considered to include genes that are induced beyond the ability of the host virus defense function of the high virus group to eliminate HCV. Therefore, there may be factors in the high viral genes that are expected to work in favor of growth on the HCV side.
  • the low viral genes there may be genes involved in creating an environment where HCV is difficult to propagate. That is, if the expression of a low viral gene screened by the present invention is enhanced, the growth of hepatitis C virus can be suppressed. It is expected to lead to the development of hepatitis C virus growth inhibitor and hepatitis c virus growth suppression method characterized by enhancing the expression of low viral genes.
  • the high virus gene and the low virus gene can be further classified into those derived from chronic hepatitis and those derived from cirrhosis, and the expression level can be analyzed.
  • patients can be classified into chronic hepatitis cases and cirrhosis cases as patient background factors, and comparison between the two groups can be performed for each item (sex, age, stage, etc.) using the viral load as an index.
  • the high virus gene and the low virus gene may be further classified into those derived from chronic hepatitis and those derived from cirrhosis, and after classification of the disease state (chronic hepatitis and cirrhosis), Classify viral genes and low viral genes
  • the order is not limited to the order. That is, in the gene expression analysis in the present invention, the gene group is a virus gene group in chronic hepatitis (CHH group), a low virus gene group in chronic hepatitis (CHL group), or a high virus gene group in cirrhosis (LCH group). As long as it is classified into 4 types of low viral gene group (LCL group) in cirrhosis and cirrhosis, the order of classification is not limited.
  • CHH group chronic hepatitis
  • CHL group chronic hepatitis
  • LCH group high virus gene group in cirrhosis
  • SEQ ID NO: 1 7 A gene that hybridizes under stringent conditions to a base sequence that is complementary to any of the base sequences represented by ⁇ 2 37 and that is highly expressed in the high virus group of chronic hepatitis
  • stringent conditions are washing conditions when nucleic acids are hybridized with each other, and are defined by the salt concentration and temperature of the buffer.
  • conditions of 37-52 ° C with concentrations of 0.5-2 X SSC and 0.1% SDS can be mentioned, and more stringent conditions include, for example, 65 ° C with 2 X SSC and 0.1% SDS.
  • the conditions include conditions such as 42 ° C at 0.5 X SSC and 0.1% SDS.
  • a person skilled in the art can set appropriate conditions by taking into consideration various conditions such as the concentration and length of other probes and reaction time in addition to the conditions such as the salt concentration and temperature of the buffer. it can.
  • the expression “enhanced expression” of a gene refers to a case where quantitative analysis is performed by real-time PCR, and the obtained quantitative value is higher than that of a group having different viral load to be compared.
  • Each gene belonging to the CHH gene group, the CHL gene group, the LCH gene group or the LCL gene group analyzed in the present invention becomes a viral load marker in chronic hepatitis for the CHH gene group and the CHL gene group.
  • Genes and LCL gene cluster can be a marker of viral load in cirrhosis. Therefore, by analyzing the expression levels of these genes from liver tissue obtained from patients, etc., how much virus is proliferating in which disease, how much hepatic function is maintained, and what kind of antiviral It can be judged whether a reaction can be induced.
  • the gene analyzed in the present invention is hybridized with a gene obtained from a liver tissue of a patient or the like, and is detected by hydration to determine which pathological condition is related to viral load. Judgment can be made. Hybridization conditions and labeling methods are well known to those skilled in the art, and any method other than those described herein can be employed.
  • the above gene can be used as a diagnostic agent for pathological conditions related to viral load.
  • gene expression level analysis can be performed easily and comprehensively.
  • the gene can be used in the form of a kit together with a buffer (for example, Tris buffer), a labeling reagent (for example, a fluorescent labeling reagent) and the like.
  • a buffer for example, Tris buffer
  • a labeling reagent for example, a fluorescent labeling reagent
  • a microarray carrying the above genes can also be included in the kit.
  • Non-cancerous tissues were isolated from cancer excision specimens of 59 patients with type C hepatocellular carcinoma, immediately frozen in liquid nitrogen and stored at -80 ° C. The use of specimens for research was obtained with the consent of each patient.
  • RNA 6000 nano assay chip from Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, Calif.) To evaluate the total RNA quality. DNase I treatment was performed to remove DNA mixed in the extracted RNA.
  • Total DNase I (Takara, Shiga, Japan) 10 units against 20 g of RNA, calorie-free, reacted at 37 ° C for 20 min in 50 ⁇ 1, and purified with rizol 9 RNA after DNase I treatment 10
  • random primer and 25 units of AMV reverse transcriptase XL (Life Sciences, Gaithersurg, MD) were added to synthesize cDNA in 1001.
  • Rotor-Gene 3000 Corbett Research, Mortalke, Australia
  • ⁇ Prism 7000 Sequence Detection System (Applied Biosystems, Foster, CA) were used for expression quantification by real-time PCR in the Examples.
  • HCV RNA was quantified using 50 ng of cDNA, and 18S rRNA was quantified using 0,25 ng of cDNA.
  • the HCV primer was used at 0.3 M.
  • plasmid DNA standard plasmid DNA
  • liver cDNA showing the highest expression for each gene was used as a standard sample
  • the cDNA was used to create a calibration curve for quantitative value calculation.
  • the quantitative amount of HCV RNA was calculated by converting the amount of standard plasmid DNA into the number of copies of the virus, obtaining the number of copies of HCV RNA per 50 ng of cDNA, and dividing the value by the quantitative value of 18S rRNA as “unit”.
  • endogenous control genes 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ribosomal protein L34 (RPL34) were used as endogenous control genes.
  • the expression level of each gene is determined by dividing the quantitative expression value of each gene by the quantitative expression value of the endogenous control gene. .wi. mit.edu/cgi-bin/primer3/primer3—use www.cgi)! I'm sorry. (table 1).
  • HCV quantitation HCV GACCAAGCTCAAACTCACTC 3 GCACGAGACAGGCTGTGATA 4
  • IFN-related genes PKR ACAATTGGCCGCTAAACTTG 16 GCGAGTGTGCTGGTCACTAA 17 p53 AACAACACCAGCTCCTCTCC 18 AACAACACCAGCTCCTCTCC 19
  • AW612461 a TGTGTAAGGCACAGGGTTTT 32 CAGCTGACTGTGGAAGGGTA 33
  • Microarray Low 154 a CACCTTGGATGACGAAACAA 42 GAGTTTCTGGGAAGGCAAAA 43
  • ENC1 GAAATCATTCCCAAGGCTGA 46 CTTTCGAGACCCCATTTTCA 47
  • HCV genotype-specific PCRs of la type, lb type, 2a type, and 2b type were performed by Okamoto's nested PCR method.
  • the first round PCR was performed using a consensus primer of 35 cycles in 20 ⁇ 1.
  • 35 cycles were carried out in a second front PCR 20 ⁇ 1 with a common forward primer and an additional U reverse primer.
  • the primer sequences used are shown in Table 1.
  • the PCR product 51 was subjected to 3% agarose gel electrophoresis, and the HCV genotype was determined from the size of the PCR product because the la type was 49 bp, the lb type was 144 bp, the 2a type was 174 bp, and the 2b type was 123 bp. (4) Selection of target cases
  • the quantification value was distributed in the range of 0 to 372,068 units. Cases with a viral load of 300 units or less were classified as a low virus group, and 30000 units or more were classified as a high virus group.
  • the low virus group was 15 cases, of which 9 were chronic hepatitis (CH) and 6 were cirrhosis (LC).
  • the high virus group consisted of 19 cases, consisting of 9 cases of chronic hepatitis and 10 cases of cirrhosis (Fig. 2).
  • CH chronic hepatitis
  • LC cirrhosis
  • High high virus group
  • Low low virus group
  • ICG-R15 indocyanine green IV rate after 15 minutes
  • Alb serum albumin
  • AST asparate aminotransferase
  • ALT alanine aminotransferase
  • T.bil total bilirubin
  • Table 2 in 34 cases (CH + LC), the percentage of cirrhosis (LC), sex ratio, age distribution, and hepatoma progression rate Divided by difference There was no difference between the two groups. Blood test results indicating liver function (“Liver function”) also show mild liver damage, but there was no significant difference between the two groups. Based on the above, using these 34 cases, it was considered possible to analyze the level of gene expression in livers that differ 1000-fold in viral load.
  • biotin-labeled cRNA was synthesized from total RNA as follows.
  • the manual of Affymetrix Gene Chip expression analysis was partially modified and performed as follows. First, 10 g of total RNA was used in the presence of RNase inhibitor42. First strand cDNA was synthesized at C and 2 hr. After synthesizing the second strand cDNA according to the manual, incubate the 43 1 reaction solution containing the following composition based on MEGAscript T7 kit (Ambion, Austin, TX) for 9 hr at 37 ° C. In vitro transcription was performed, and biotirrcRNA 3 ⁇ 4r was synthesized.
  • the expression of OAS, MxA, and BAX was significantly increased in the high virus group compared with 11 cases of chronic hepatitis ( ⁇ 0.05). There was no significant difference between the two groups in 14 cirrhosis cases or 25 cases in total. In addition, no significant difference was observed when comparing the 9 cases of HCV genotype lb alone. Thus, a significant difference was observed only in chronic hepatitis cases.
  • Fig. 3 show the measurement results of the expression level when 18S rRNA is used as the endogenous control gene.
  • 18S i'RNA Each gene expression level was evaluated as a trawl gene (described later).
  • Figure 8 Expression analysis by microarray was performed using the 8 cases indicated by “T” (4 cases of high virus group and 4 cases of low virus group in chronic hepatitis cases). Human Genome U133 Plus 2.0 airay (Affymetrix) was used, and 54,675 probes (probe) corresponding to more than 47,000 transcripts of humans were targeted. Figure 4 shows how to obtain genes that are significantly different in expression between 4 high virus groups and 4 low virus groups.
  • Fig. 4 (a) This is the gene expressed in the liver (specifically, the liver of chronic hepatitis).
  • three types of parametric tests were performed using 28,505 probes to extract genes with significant differences in expression between the two groups, the high virus group and the low virus group (Fig. 4 (b)). Student's t test, which is assumed to have the same variance in the two groups, extracted 1,710 probes, and Welch's t test, which assumes that the variances are not equal, extracted 1,327 probes.
  • FIG. 5A A clustering analysis of eight microarrays using these 117 genes was performed to create a Condition tree (Fig. 5A). Unlike the Condition tree with 28,505 probes (Fig. 5B), it was shown that the gene list can distinguish cases between the high virus group and the low virus group.
  • Tables 3 and 4 show the genes that were more than 2.5 times different between the two groups among 78 high virus genes and 39 low virus genes (Table 3) (Table 4).
  • proteasome proteasome (prosome, macropain)
  • alpha type, 8 (1) O is described in the gene whose expression was quantified by a real-time PCR.
  • the difference captured here is considered to be a transcript of a gene having a high homology with this probe.
  • Table 78 shows all 78 high virus genes and Table 6 shows all 39 low virus genes.
  • Example 2 found by comparison of 8 cases of microarray In order to investigate whether it was indeed correlated with the difference in HCV levels, we performed expression analysis by real-time PCR using 34 cases of liver cDNA. .
  • the housekeeping gene which is said to be expressed at a constant level per cell, is used as a control gene for evaluating the expression level of the gene.
  • j3 -actin and GAPDH genes are typical genes. However, when examining tissues and cells of various pathologies and conditions, it cannot be said that these genes are expressed constantly. Recently, 18S i'RNA has been used. In this example, we independently examined 8 microarrays of genes that are constantly expressed in the target hepatitis tissue.
  • the HG U133 Plus2.0 array used here lists 100 genes as control genes among the probes on it. Of these, 91 genes had present flags in all 8 microarrays. From these, we searched for genes that showed no difference in expression level among the 8 microarrays and that showed the same expression level as GAPDH. As a result, ribosomal protein L34 (RPL34) was selected. Compared to the GAPDH expression signal on the same microarray, RPL34 certainly had less variation in expression level (Figure 6A).
  • 18S rRNA cannot be evaluated by microarray analysis. Therefore, in order to clarify which of RPL34 and 18S rRNA is suitable as a control gene, the expression levels were compared by real-time PCR in all 34 cases.
  • Figure 6B shows a comparison of the expression levels of the 3 genes including GAPDH. Comparing the expression level of cDNA—quantitatively, the variation in the expression level of 18S rRNA was the smallest compared to PRL34 and GAPDH. Therefore, in this example, the following gene expression was evaluated using 18S rRNA as an endogenous control gene.
  • the comparison between the two groups was divided into 8 cases of chronic hepatitis (CH) and 18 cases of total chronic hepatitis (CH) and 16 cases of total cirrhosis (LC) used for microarray analysis.
  • As the expression level of the gene values corrected with 18S rRNA were used.
  • High virus group The number of low virus group is shown in parentheses. The result of 8 high virus genes is shown in the “High” column of Table 7, and the result of 5 low virus genes is shown in the “: Low” column of Table 7.
  • Fold change indicates the value when compared with the median of each group. For example, in the case of a high virus gene, the value obtained by dividing the median value of the high virus group of the gene by the median value of the low virus group is shown. In addition, the Mann Whitney U test was performed between the two groups, and the values of genes that had a significant difference (P ⁇ 0.05) were surrounded by a broken line. The reversible changes that showed a significant difference are enclosed in a solid line.
  • Figure 7 shows typical comparison results for chronic hepatitis.
  • the high virus genes shown in A to C of FIG. 7 were genes that clearly showed a significant difference in expression level between the virus group and the low virus group.
  • low virus genes showed similar results to microarray expression in 2 out of 5 genes, but after all, there was only 1 significant difference in chronic hepatitis (Table 7).
  • This heritable SELE showed a different tendency even in cirrhosis (Table 7).
  • the results of the above two genes SELE and FLJ461542 are shown in D and 3 ⁇ 4 of FIG.
  • N80145 was recognized as an opposite significant difference in chronic hepatitis, ie, a high viral gene (F).
  • the 117 genes that differed in expression extracted with Oligonucleotide microarra were classified according to the functions of known genes, unknown genes, and known genes.
  • (A) Structural classification Figure 8 shows the structural classification of 117 probes based on the latest gene information. There are five categories of gene classifications. .
  • (1) means the transcript of the relevant gene.
  • (2) may be a alternatively spliced transcript of the gene of interest or a new transcript.
  • (3) may be a transcript as an interfering RNA or a new transcript.
  • (4) may be a transcript of the gene of interest or a new transcript.
  • (5) is considered an unknown transcript.
  • INF-inducible genes were found as differential genes. Of the 239 genes known as INF-inducible genes, only 2 were among the high viral genes. This indicates that IFN is not working so strongly, even in high virus cases. In chronic hepatitis in which persistent infection was established, it was suggested that host genes other than IFN are involved in viral load control.
  • low viral genes there were genes with serine protease inhibitory active regions and genes related to the plugeothesome. This gene is expected to inhibit viral growth.
  • genes involved in inflammation other than high viral genes were also included.
  • more than half of the low-viral genes were unknown genes whose functions were unknown.
  • transcripts that act as HCV interfering RNA were not included, or homology with the HCV 9.5 kb sequence was examined. However, there was no such sequence in the low viral gene.
  • Low viral genes may have genes involved in creating an environment in which HCV is difficult to grow.
  • HCV ' was quantified, and the number of cases was increased from Example 1 and 2 and divided into two pathological conditions, and the viral load was related.
  • the genes to be studied were examined. First, the amount of liver virus was quantified, and cases were selected. The method is shown below. The methods for total RNA extraction and real-time RT-PCR are the same as in Example 1. '
  • the amount of virus was determined by quantifying the virus gene. As materials, non-cancerous tissues of 59 cases of hepatocellular carcinoma were used, and I and II with the lowest hepatocellular carcinoma stage were selected as much as possible.
  • LC shows a case where the non-cancerous tissue has progressed to cirrhosis.
  • A indicates the case used for microarray analysis. Liver HCV levels ranged from 4 to 480000 units. Among them, 20 cases of 30000 units or more were assigned to the high virus group, and 15 cases of 300 units or less were assigned to the low virus group.
  • CH and LC showed different gene expression patterns. Therefore, in this example, genes related to viral load were examined by dividing into two disease states. did. Patient background factors were compared between the selected high virus group and low virus group. The patient background factors were divided into 18 cases of chronic hepatitis and 17 cases of cirrhosis, and each item was compared between two groups according to viral load. Table of significant difference test values
  • HCV RNA d (unit) 69,900 ⁇ 8,700 63 ⁇ 15 118,000 ⁇ 39,00C 158 ⁇ 42
  • the latest version of the oligonucleotide microarray was used to comprehensively examine the total mRNA expressed in liver tissue for differences in gene expression.
  • the GeneChip used has 54675 probes corresponding to about 47000 genes that are considered to be all human genes.
  • 5 cases of high virus group of chronic hepatitis and 5 cases of low virus group were newly added, and 7 cases of cirrhosis high virus group and 3 cases of low virus group were newly added.
  • a total of 20 microarrays were used, 10 for each disease state, and comparisons were made between two groups, 5: 5 for chronic hepatitis and 7: 3 for cirrhosis, to identify genes with differential expression.
  • Fig. 9 is a schematic diagram of a method for determining genes with different expression levels using microarray analysis results.
  • analysis was performed in 4 cases each for the high virus group and low virus group, but in this example, analysis was performed for 5 cases in each group.
  • Tables 10 to 13 list the chronic hepatitis high virus gene, chronic hepatitis low virus gene, cirrhosis high virus gene, and cirrhosis low virus gene, respectively.
  • ⁇ ⁇ A is the exon sequence of the gene
  • B is the gene.
  • Introri is the sequence in the same direction as the gene
  • C is the gene.
  • ⁇ ⁇ The sequence is in the opposite direction to the gene in intron
  • D is the sequence in the gene The contiguous arrangement in the same direction, E, indicates the sequence where the gene is not.
  • CHH-2 LCH-3 (Table 12) Table 1 1. Low viral genes in chronic hepatitis (CHL 21)
  • b A gene extracted by oligonucleotide microarray analysis in 8 cases of chronic hepatitis.
  • clustering analysis was performed to determine whether the expression pattern of the extracted gene is effective as a gene that can distinguish the difference in viral load.
  • the chronic hepatitis gene 87 gene was clustered into 66 high virus genes and 21 low virus genes, and 10 cases of chronic hepatitis were classified into low virus (L) and high virus (H) groups. It was shown that the cluster can be classified neatly (Fig. 12).
  • analysis of 10 cases of cirrhosis with 44 cirrhosis genes revealed a clean cluster classification of 2 groups of 3 and 7 cases.
  • the cluster classification of 10 cases could not be performed correctly when the cirrhosis cases were analyzed with the chronic hepatitis gene and when the chronic hepatitis cases were analyzed with the cirrhosis gene. Therefore, the genes related to viral load were shown to be different between chronic hepatitis and cirrhosis. '' Example 7
  • Table 14 shows the primer sequences used.
  • HCV type 2 accounts for about half of the low virus group. 4 of 9 cases with chronic hepatitis and 3 out of 6 cases with cirrhosis.
  • the low virus group was divided by HCV genotype and compared with the high virus group. Since there are only a few cases of genotype dependence, we narrowed down to only those that were graved on a boxplot. Some genes are analyzed by analyzing the cirrhosis high virus group in 9 cases. The ratio of the current dose in the high / less group and the low virus group was measured by the Mann-Whitney.U.test. The results of analysis for each of chronic hepatitis and cirrhosis are shown as' P values. 'P Blank indicates no measurement. The low virus group was divided by HCV genotype, and a comparison between the two groups was also performed.
  • the left panel compares the low virus group with high viral cancers divided into genotype 1 and type 2.
  • the right panel summarizes the entire low virus group compared to the high virus group.
  • the figure is a box-and-whisker plot, showing the distribution of measured values in each group. From the bottom, the lower end of the whiskers represents the 10th percentile, the lower end of the box represents the 25th percentile, the middle line of the box represents the 50th percentile, the upper end of the box represents the 75th percentile, and the upper end of the whiskers represents the 90th percentile.
  • the margin P value is The result of comparison between two groups of the amount of virus was based on the Mann- Whitney U test.
  • the left panel shows the results of dividing the low virus group into HCV genotypes 1 and 2 and comparing it to the high virus group.
  • OASL is a high virus gene commonly found in chronic hepatitis and cirrhosis. This gene was verified only in chronic hepatitis. SNAI2 was also verified as a chronic hepatitis high virus gene. All genes showed low expression in the low virus group regardless of the HCV genotype.
  • Figure 15 is an example of the chronic hepatitis high virus (CHH) gene.
  • CXCL6 expression was suppressed only in the HCV genotype 1b low virus group.
  • AK025967 was a low viral gene in cirrhosis.
  • Figure 16 shows an example of the cirrhosis low virus (LCL) gene.
  • LCL cirrhosis low virus
  • HCV is a single-stranded RNA virus with a positive strand RNA of approximately 9.6 kb. There are many genotypes in the HCV genome and so far it has been divided into more than 6 genotypes. HCV is a spherical virus with a diameter of 50-60 nm and has a structure in which the core particles are enveloped.
  • HCV adsorbs to hepatocytes via a receptor, enters the cytoplasm by endocytosis, and then sheds and releases RNA.
  • This RNA immediately binds to the ribosome as mRNA and a precursor protein consisting of about 3000 amino acids is translated.
  • Precursor proteins translated on the endoplasmic reticulum membrane are produced as three structural proteins and seven nonstructural proteins by cell signalases and two proteases encoded by the virus itself.
  • the synthesized viral RNA-dependent RNA polymerase causes the viral RNA to replicate one strand from the + strand and then the + strand.
  • the + strand RNA is taken up by the core protein, and particle formation occurs while covering the endoplasmic reticulum with an envelope.
  • Virus particles are thought to pass through the Golgi apparatus, reach the cell membrane, and be released out of the hepatocytes. In these processes, cell adsorption and invasion can be considered as the first step to control the viral load. Therefore, in this example, it was examined whether the difference in viral load was related to the expression of viral receptors and endocytosis-related genes. '
  • the method was as follows. First, the amount of visceral virus was quantified, and cases were selected. The method is shown below. The methods for total RNA extraction and real-time RT-PCR are the same as in Example 1.
  • the amount of virus was determined by quantifying the virus gene. As materials, I and II with the lowest hepatocellular carcinoma stage were selected as much as possible, using cancerous and non-cancerous tissues of 50 hepatocellular carcinoma cases.
  • Fig. 17 is a graph showing the results of liver HCV RNA quantification, with the ordinate indicating the amount of HCV in the cancerous part and the horizontal axis indicating the amount of HCV in the non-cancerous part.
  • the solid line shows the case where the amount of the cancerous part (T) is 1 with respect to the non-cancerous part (NT), and the dotted line shows 1 node 4 (T / NT, below the solid line) and 4 times (on the solid line).
  • Non-cancerous HCV is widely distributed from tens to hundreds of thousands.
  • HCV in the cancer area was the same amount or decreased, and it was found that 29 cases (58%) out of 50 cases were less than 1/4. In more than half of cases in the cancer area, it can be said that HCV is difficult to infect or increase. Therefore, regarding the difference in viral load, we examined not only differences among non-cancerous members but also differences among cancerous members.
  • CD81 and heparan sulfate are known as receptors that recognize HCV envelope proteins.
  • heparan sulfate three molecules of glycosyltransferase involved in heparan sulfate synthesis were measured.
  • Receptors that recognize the sugar chains of HCV envelope proteins include the C-type lectins DC-SIGN, L-SIGN, ASGR, and MBL2, and mannose and galactose are the recognition molecules. Since HCV particles bind to LDL and HDL in blood to form a complex, it is suggested that these receptors, LDLR and SCARB1, act as HCV receptors. SCARB1 is also thought to recognize E2 protein directly.
  • Endocytosis-related molecules include clathrin proteins and adapter proteins.
  • an adapter protein that exists in the vicinity of the receptor in the cell membrane, gathers around the receptor, which triggers the heavy chain (Clathrin) C) Trimer and light chain (Clathrin A)
  • the clathrin consisting of trimer comes together. By gathering a lot of clathrin, a clathrin-coated pit is created and a endome is formed.
  • TLR3, TICAM1, DDX58 Toll-like receptor-related signal molecules
  • Table 16 shows the primer sequences of each gene.
  • Table 17 shows the results of quantification by PCR.
  • Table 16 HCV receptor candidates and viral entry-related genes and their PCR primer sequences
  • **: The cDNA used for PGR is 25 ng. The other is 10ng.
  • NL low virus group
  • NH high virus group
  • Table 18 shows a comparison of background factors between the two groups.
  • HCV genotype (lb: lb + 2a: 2a: 2b) FE 6: 0: 2: 2 11: 3: 0: 0 0.0455 6: 0: 2: 2 6: 1: 0: 0 5: 2 6: 1
  • Count Average soil standard error, 'ICG, Indocyanine green 15 minutes after intravenous injection; Alb, serum albmin; AST, aspartate aminotransferase! AL ⁇ T, alanin aminotransferase; T.bill, total bilirubin; AFP, PIV, protein induced by vitamin K absence; HCVRNA, normalized by 18S rRNA; MW, Mann-Whitney's U test; FE, Fisher's sexact probability test
  • L-SIGN receptor gene
  • This high viral gene may contain factors that are expected to work favorably for HCV growth, so it is possible to suppress the expression of high viral genes. Can be inhibited.
  • This low viral gene may have a 3 ⁇ 4 gene that is involved in creating a circle in which HCV is difficult to grow.
  • HCV proliferation can be suppressed by enhancing the expression of the low virus gene.

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Abstract

La présente invention concerne un procédé de criblage d'un gène dont l'expression augmente dans un tissu à titre viral élevé contenant une quantité importante de VHC, comprenant les étapes suivantes : (a) sélection d'un tissu hépatique pour lequel la valeur obtenue en divisant le nombre de copies de VHC pour 50 ng d'ADNc dérivés du tissu hépatique par la valeur quantitative d'ARNr 18S, n'est pas supérieure à 300 unités s'il s'agit d'un tissu à titre viral faible et à 30000 unités s'il s'agit d'un tissu à titre viral élevé; (b) mesure du taux d'expression génique dans le tissu à titre viral faible et dans le tissu à titre viral élevé; et (c) sélection d'un gène dont l'expression augmente davantage dans le tissu à titre viral élevé que dans le tissu à titre viral faible.
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WO2011034449A1 (fr) * 2009-09-16 2011-03-24 Massey University Polypeptides de fusion et leurs utilisations
JP2012515534A (ja) * 2009-01-21 2012-07-12 バーテックス ファーマシューティカルズ インコーポレイテッド Ｃ型肝炎ウイルス核酸を増幅する方法

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JP2012515534A (ja) * 2009-01-21 2012-07-12 バーテックス ファーマシューティカルズ インコーポレイテッド Ｃ型肝炎ウイルス核酸を増幅する方法
WO2011010168A1 (fr) * 2009-07-24 2011-01-27 Agri-Food Biosciences Institute Oligonucléotides pour détecter un astrovirus de poulet
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WO2011034449A1 (fr) * 2009-09-16 2011-03-24 Massey University Polypeptides de fusion et leurs utilisations

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