JP2008295301A - Method for analyzing tumorigenic risk - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means capable of adequately analyzing a tumorigenic risk utilizing a biological index. <P>SOLUTION: A method for analyzing the tumorigenic risk includes the steps which include (a) a step of measuring expression levels of two or more indicator genes selected from a cell growth-promoting gene cluster, a cell growth-inhibiting gene cluster, an apoptosis-promoting gene cluster, an apoptosis-inhibiting gene cluster, a cell differentiation-promoting gene cluster and a cell differentiation-inhibiting gene cluster in a test tissue collected from a non-tumorous part of a specimen; (b) a step of measuring the expression level of a housekeeping gene in the same tissue; (c) a step of correcting the measured values for the expression level of each of the indicator genes, obtained in step (a) with the measured values for the expression level of the housekeeping gene obtained in step (b) and employing the corrected values as a tumorigenic index related to each of the indicator genes; (d) a step of comparing the tumorigenic index of each of the indicator genes, obtained in step (c) with the preset standard value and evaluating the tumorigenic index; and (e) a step of combining two or more evaluations for each of the indicator genes obtained in step (d). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a technique for measuring the expression level of a gene in a test tissue collected from a non-tumor part and analyzing the risk of tumor development.

  In recent years, the mortality rate due to cancer has continued to be a high value, and the mortality rate of colorectal cancer is particularly high. Therefore, research for early detection of cancer or prevention of cancer has been actively conducted.

  Many cancers are thought to go from premature cancer to advanced cancer through precancerous lesions, and cancer screening aims to find a treatable early cancer. In order to find early cancer, frequent examinations are performed. However, examinations for finding cancer early are often painful and dangerous, and require a great deal of cost and time. For example, colorectal cancer is a cancer that can be expected to be almost completely cured by early detection and early treatment. Colonoscopy, which is a test for it, is an extremely burdensome test. Specifically, it is necessary to take a large amount of a laxative from the day before the examination, remove all the contents of the large intestine, and then insert an examination tool from the anus to the large intestine to the cecum. The inspection takes 30 minutes or more, and several days are constrained if the rest is included. Inspection costs also cost tens of thousands of yen. As described above, the inspection requires a considerable burden on the subject and cannot be performed unnecessarily. However, since almost no appropriate risk assessment has been found, at present, most people are recommended to test once a year. Also, examinations need to be performed by skilled doctors, but there are still very few skilled doctors.

  Thus, in the conventional method, the burden on the subject is large, and skill is required for the inspection. For this reason, an index that can appropriately determine the inspection interval is extremely awaited.

  In particular, the incidence of colorectal cancer in Japan is increasing rapidly, but the increase in the incidence cannot be suppressed by early detection and treatment, and it is important to prevent carcinogenesis. If an abnormality is detected by endoscopy, a biopsy is performed and the presence of cancer or precancerous lesions can be confirmed, but the future cancer incidence cannot be determined. In addition, it is efficient and ethically desirable to prevent carcinogenesis for people at high risk, but few appropriate indicators of risk have been found so far.

  In addition, in the prevention and treatment of carcinogenesis, if there is an indicator to confirm whether the risk of carcinogenesis has decreased due to changes in lifestyle habits or internal use of substances that prevent carcinogenesis, development of effective carcinogenesis prevention methods and efficient Practice of carcinogenesis prevention becomes possible. However, few such indicators have been found yet.

  The present invention is an analysis means that uses a biological index to grasp the risk of tumor development with high accuracy, and enables an efficient and appropriate implementation of a test for early detection of a tumor, or prevention or treatment of a tumor. Is the main issue.

  As a result of intensive studies with the main purpose of achieving the above-mentioned problems, the present inventor examined the expression level of specific genes and combined them with an evaluation of tumor development risk as an index to generate tumors. The present inventors have found that the risk can be analyzed with high accuracy and efficiency, and have further intensively studied to complete the present invention.

  That is, the present invention relates to the following matters.

Item 1 (a) Cell proliferation promoting gene group, cell growth inhibiting gene group, apoptosis promoting gene group, apoptosis inhibiting gene group, cell differentiation promoting gene group, and cell differentiation inhibition in a test tissue collected from a non-tumor part of a specimen Measuring the expression level of two or more indicator genes selected from the gene group,
(B) measuring the expression level of a housekeeping gene in the same tissue;
(C) Tumor incidence index for each index gene obtained by correcting the measured value of the expression level of each indicator gene obtained in step (a) with the measured value of the expression level of housekeeping gene obtained in step (b) The process of
(D) evaluating the tumor occurrence index of each index gene obtained in step (c) by comparing with a preset reference value; and
(E) having a step of combining two or more evaluations of each indicator gene obtained in step (d),
The cell proliferation promoting gene group is a gene group comprising a PCNA gene, a CyclinD1 gene, a cdk2 gene, a farnesyl transferase gene, a Ki67 gene, and an EGF receptor gene,
The cell growth inhibitory gene group is a gene group including TGFβ gene, p21 gene, and INK4 gene,
The apoptosis promoting gene group is a group comprising Bax gene, caspase gene, and Fas gene,
The apoptosis suppressing gene group is a group containing Bcl-2 gene, IAP gene, and survivin gene, and the cell differentiation promoting gene group is a group containing mucin gene, Cdx-1 gene, intestinal alkaline phosphatase gene, and CD10 gene And
A method for analyzing a tumor development risk, wherein the differentiation-suppressing gene group includes a Cdx-2 gene.

One of the preferred embodiments is
Item 2 (a) Cell proliferation promoting gene group, cell growth inhibiting gene group, apoptosis promoting gene group, apoptosis inhibiting gene group, cell differentiation promoting gene group, and cell differentiation inhibition in a test tissue collected from a non-tumor part of a specimen Measuring the expression level of two or more indicator genes selected from the gene group,
(B) measuring the expression level of a housekeeping gene in the same tissue;
(C) Tumor incidence index for each index gene obtained by correcting the measured value of the expression level of each indicator gene obtained in step (a) with the measured value of the expression level of housekeeping gene obtained in step (b) The process of
(D-1) Expression when the expression level of the indicator gene is high as a risk factor (+) when the expression level is low as a risk factor (-), with tumor occurrence as a result (+) without occurrence as a result (-) A step of setting a value that maximizes the sum of sensitivity and specificity as a reference value for the indicator gene,
(D-2) For each indicator gene, the tumor occurrence index of the gene in step (c) is compared with the reference value of the gene in step (d-1),
(D-3a) When the index gene is a cell growth promoting gene, a cell differentiation suppressing gene, or an apoptosis suppressing gene, the occurrence risk increasing factor when the tumor occurrence index is a reference value or more, and the occurrence risk decreasing factor when the index gene is less than the reference value And
(D-3b) When the indicator gene is a cell growth inhibitory gene, a cell differentiation promoting gene, or an apoptosis promoting gene, the tumor occurrence index is less than the reference value when the tumor occurrence index is less than the reference value, and the occurrence risk decreasing factor when the index gene is greater than the reference value Evaluate and
(E) The method according to item 1, comprising a step of combining two or more evaluations of each indicator gene obtained in steps (d-3a) and (d-3b).

Another preferred embodiment is as follows:
Item 3 (a) Cell growth promoting gene group, cell growth inhibiting gene group, apoptosis promoting gene group, apoptosis inhibiting gene group, cell differentiation promoting gene group, and cell differentiation inhibiting in a test tissue collected from a non-tumor part of a specimen Measuring the expression level of two or more indicator genes selected from the gene group,
(B) measuring the expression level of a housekeeping gene in the same tissue;
(C) Tumor incidence index for each index gene obtained by correcting the measured value of the expression level of each indicator gene obtained in step (a) with the measured value of the expression level of housekeeping gene obtained in step (b) The process of
(D-1) A plurality of reference values are set in several stages depending on the expression level of the indicator gene,
(D-2) For each index gene, the tumor occurrence index in the step (c) is compared with the reference value in the step (d-1), and the one with a high expression level is assigned a high score,
(D-3a) When the indicator gene is a cell proliferation promoting gene, a cell differentiation suppressing gene, or an apoptosis suppressing gene, the score of (d-2) is evaluated as an addition factor,
(D-3b) When the indicator gene is a cell growth inhibitory gene, a cell differentiation promoting gene, or an apoptosis promoting gene, the score of (d-2) is evaluated as a subtraction factor,
(E) Item 1 having a step of scoring a combination of two or more evaluations of each indicator gene by adding the evaluation obtained in step (d-3a) and subtracting the evaluation obtained in (d-3b) It is the method of description.

  Item 4: The method according to any one of Items 1 to 3, which is a method for analyzing the risk of tumor development in the large intestine, wherein the non-tumor part of the specimen is normal large intestinal mucosa.

In other words, the step (a) includes a cell growth promoting gene group, a cell growth inhibiting gene group, an apoptosis promoting gene group, an apoptosis inhibiting gene group, a cell differentiation promoting gene group, and a cell in a test tissue collected from a normal large intestine mucosa. It is a step of measuring the expression level of two or more indicator genes selected from the differentiation suppression gene group.
Item 4. The method for analyzing colorectal tumor development risk according to any one of Items 1 to 3.

  Item 5: The method according to any one of Items 1 to 4, wherein the housekeeping gene is a GADPH (glyceralde-hydo-3-phosphate dehydrogenase) gene.

  In other words, the method according to any one of Items 1 to 4, wherein the step (b) is a step of measuring the expression level of the GADPH gene in the same tissue.

  Item 6: The method according to any one of Items 1 to 5, wherein the indicator gene is two or more indicator genes selected from a cell growth promoting gene group and an apoptosis promoting gene group.

  In other words, the step (a) is a step of measuring the expression level of two or more indicator genes selected from a cell proliferation promoting gene group and an apoptosis promoting gene group in a test tissue collected from a non-tumor part of a specimen. It is the method in any one of -5.

  One of the preferred embodiments is that in step (a), two or more genes selected from a cell growth promoting gene group and one or more genes selected from an apoptosis promoting gene group in a test tissue collected from a non-tumor part of a specimen. Item 6. The method according to any one of Items 1 to 5, which is a step of measuring the expression level of the indicator gene.

  Item 7: The method according to any one of Items 1 to 6, wherein the cell growth promoting gene group is a group consisting of a PCNA gene and a CyclinD1 gene.

  Item 8: The method according to any one of Items 1 to 7, wherein the apoptosis-promoting gene group is a group consisting of a Bax gene.

  In an example of a preferred embodiment, the step (a) is a step of measuring the expression level of two or more indicator genes selected from a cell proliferation promoting gene group and an apoptosis promoting gene group in a test tissue collected from a non-tumor part of a specimen. The method according to any one of Items 1 to 7, wherein the cell growth promoting gene group is a group consisting of a PCNA gene and a CyclinD1 gene, and the apoptosis promoting gene group is a group consisting of a Bax gene. In other words, the step (a) is a step of measuring the expression level of two or more indicator genes selected from the group consisting of PCNA gene, CyclinD1 gene and Bax gene in the test tissue collected from the non-tumor part of the specimen. It is the method in any one of 1-7.

  Hereinafter, the present invention will be described in more detail.

  In addition, in this specification, a tumor refers to neoplasms, such as an adenoma which is cancer and a precancerous lesion.

1. Object The method for analyzing tumor risk of the present invention is performed using a test tissue collected from a non-tumor part of a specimen.

  The non-tumor part refers to a part that is not a tumor, that is, a part that is not cancer or adenoma.

  The non-tumor site is preferably a tissue that looks macroscopically normal.

  The type of the test tissue is not particularly limited as long as the expression level of the gene can be measured, and includes all cells and tissues serving as a carcinogenic matrix. Specific examples include mucosal cells.

  The type of test tissue can be appropriately set according to the type of tumor, cancer, or adenoma to be analyzed.

  For example, in the case of a method for analyzing the risk of tumor development in the large intestine, the test tissue can be cells collected from normal large intestine mucosa.

  Collection of the test tissue can be appropriately performed according to a known method used as a biopsy.

2. Indicative gene In the present invention, a gene used as an indicator is a gene that is considered to be associated with cell proliferation, apoptosis and / or cell differentiation.

  In other words, in the present invention, the indicator gene belongs to a cell growth promoting gene group, a cell growth inhibiting gene group, an apoptosis promoting gene group, an apoptosis inhibiting gene group, a cell differentiation promoting gene group, and / or a cell differentiation inhibiting gene group. It is a gene.

  In the present specification, a group means a unit of a group of one or a plurality of genes.

2-1. Cell proliferation promoting / suppressing gene group The cell proliferation promoting gene group includes PCNA gene, CyclinD1 gene, cdk2, farnesyl transferase gene, Ki67 gene, EGF receptor gene and the like.

  The cell growth promoting gene group is a group set by appropriately selecting from cell growth promoting genes having a function of promoting cell growth.

  For example, the CyclinD1 gene is a gene that is related to cell cycle control and whose expression is induced by proliferation signals. The PCNA gene was discovered as a gene encoding an antigen found in the nuclei of proliferating cells, and is involved in DNA replication and cell cycle progression.

  In one preferred embodiment, the cell growth promoting gene group is a group consisting of a PCNA gene and a CyclinD1 gene.

  The cell growth inhibitory gene group includes TGFβ gene, p21 gene, INK4 gene and the like. The cell growth suppression gene group is a group that is appropriately selected and set from cell growth suppression genes having a function of suppressing cell growth.

2-2. Apoptosis promotion / suppression gene group The apoptosis promotion gene group includes Bax gene, caspase gene, Fas gene and the like.

  The apoptosis promoting gene group is a group set by appropriately selecting from apoptosis promoting genes having a function of promoting apoptosis. For example, the Bax gene is a gene that promotes apoptosis and inhibits the anti-apoptotic activity of Bcl-2.

  In addition, the apoptosis suppressing gene group includes Bcl-2 gene, IAP gene, survivin gene and the like.

  The apoptosis suppressing gene group is a group set by appropriately selecting from apoptosis suppressing genes having a function of suppressing apoptosis. For example, the Bcl-2 gene is a gene having apoptosis inhibitory activity.

2-3. Cell differentiation promoting / suppressing gene group The cell differentiation promoting gene group includes mucin gene, Cdx-1 gene, intestinal alkaline phosphatase gene, CD10 gene and the like.

  The cell differentiation promoting gene group is a group set by appropriately selecting from cell differentiation promoting genes having a function of promoting cell differentiation.

  The cell differentiation suppressing gene group includes Cdx-2 gene and the like.

  The cell differentiation suppressing gene group is a group set by appropriately selecting from cell differentiation suppressing genes having a function of suppressing cell differentiation.

3. Housekeeping gene The housekeeping gene refers to a gene that encodes a protein necessary for the general function of a cell, and a constant amount is always constitutively expressed in any cell.

  The type of housekeeping gene can be set as appropriate, and examples thereof include GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2-microglobulin, HPRT 1 (hypoxanthine phosphoribosyltransferase 1) and the like.

4). Methods for measuring gene expression levels There are no limitations on the methods and methods for measuring the expression levels of indicator genes and housekeeping genes, and all known methods are used, including real-time PCR (RT-PCR). can do.

  More specifically, the expression level of a gene can be measured by a method for detecting a transcript of a gene in a test tissue, a method for detecting the translation product, or the like.

  The method of detecting transcripts is to measure the expression level of the gene by detecting mRNA, which is the transcript of the gene. For example, RT-PCR, Northern blot analysis, RT-LAMP method, in situ high Hybridization methods and other various methods can be used.

  The method for detecting the translation is to measure the expression level of the gene by detecting the protein that is the translation of the gene. For example, Western blot analysis using a specific antibody against the protein, immunohistochemical staining, etc. The immunological assay method can be used.

5. Tumor occurrence index The tumor occurrence index is obtained by correcting the measured value of the expression level of the index gene with the measured value of the expression level of the housekeeping gene.

  For example, when measuring the expression level of a gene by PCR, the tumor occurrence index of each gene is obtained by subtracting the Ct of the index gene from the Ct of the housekeeping gene using the PCR cycle number threshold cycle (Ct) as ΔCt Can be expressed.

In other words, ΔCt can be expressed by the following formula:
ΔCt = housekeeping gene Ct−index gene Ct
The tumor development index obtained for each index gene can be used as an appropriate factor for evaluating the risk of tumor development by comparing with a reference value set in advance in relation to the presence or absence of tumor development.

6). Evaluation
6-1. Reference Value Calculation Method The reference value used in the present invention can be appropriately set based on factors such as the presence or absence of tumor development and the gene expression level.

  For example, in one of the preferred embodiments, the reference value is calculated by examining the relationship between the expression level of the indicator gene as a risk factor and the presence or absence of tumor as a result (endpoint). be able to.

  Specifically, in one of the preferred embodiments, when the expression level of the indicator gene is high, the risk factor (+) is low, and when the expression level is low, the risk factor is (−), and the presence of tumor occurrence is the result (+) no tumor occurrence. As a result (−), a value that maximizes the sum of sensitivity and specificity of the expression level can be used as a reference value for the indicator gene.

  Here, the sensitivity means a ratio at which a factor showing a high tumor occurrence value by the expression level correctly determines the presence of tumor occurrence.

  Specificity means the rate at which a factor that shows a low tumor incidence depending on the expression level correctly determines the absence of tumor development.

  The reference value obtained in this way can be appropriately set depending on the type of housekeeping gene.

  In one preferred embodiment, the reference value can be set in several stages according to the degree of tumor occurrence.

6-2. Comparison of tumor incidence index and reference value, and evaluation of tumor occurrence for each indicator gene By comparing the tumor occurrence index obtained above and the reference value, evaluate the relationship between tumor occurrence for each indicator gene Can do.

  The comparison method and the evaluation method can be appropriately set according to a reference value setting method and the like.

  Specifically, the following modes can be considered.

(Aspect 1)
When the expression level of the indicator gene is high, the risk factor (+) is low. The risk factor (-) is low. The tumor occurrence is the result (+). Is calculated as a reference value for each indicator gene.

  Next, the reference value obtained and the tumor occurrence index determined by the above-described method are compared for each index gene.

  In comparison, if the indicator gene is a cell growth promoting gene, a cell differentiation inhibitor gene, or an apoptosis inhibitor gene, if the tumor occurrence index is above the reference value, the risk is increased, and if it is less than the reference value, the risk is reduced Evaluate as a factor.

  In addition, when the indicator gene is a cell growth inhibitory gene, a cell differentiation promoting gene, or an apoptosis promoting gene, when the tumor occurrence index is less than the reference value, the occurrence risk is evaluated as an occurrence risk increasing factor, and when it is above the reference value, the occurrence risk reducing factor is evaluated. .

  More specifically, for example, for the CyclinD1 gene, when the reference value is −2.51, it is evaluated as an occurrence risk lowering factor if ΔCt is less than −2.51, and an occurrence risk increasing factor if it is −2.51 or more.

  For example, for the PCNA gene, when the reference value is −1.27, if the ΔCt is less than −1.27, it is evaluated as an occurrence risk lowering factor, and if it is −1.27 or more, it is evaluated as an occurrence risk increasing factor.

  For the Bax gene, when the reference value is −2.30, it is evaluated as an occurrence risk increasing factor if ΔCt is less than −2.30, and an occurrence risk decreasing factor if it is −2.30 or more.

  The risk of cancer can be analyzed by combining two or more evaluations regarding the increase / decrease in the risk of occurrence obtained for each indicator gene. For example, it can be analyzed that the risk of tumor development tends to be higher as the increase factor is smaller and the decrease factor is smaller. On the other hand, it can be analyzed that the lower the decrease factor and the higher the increase factor, the lower the risk of tumor development.

(Aspect 2)
Divide into multiple groups based on the relationship between the level of expression of the indicator gene and the degree of tumor development. The lowest expression level is 1, and the number increases in order as the expression level increases. .

  For comparison, if the indicator gene is a cell growth promoting gene, cell differentiation inhibiting gene, or apoptosis inhibiting gene, the number added is added, and the indicator gene is a cell proliferation inhibiting gene, cell differentiation promoting gene, apoptosis promoting gene The score is obtained by subtracting the assigned number. This score can be used to analyze the risk of cancer development. For example, it can be analyzed that the higher the score, the higher the risk of tumor development. Conversely, it can be analyzed that the lower the score, the lower the risk of tumor development.

7.2 Combination of Evaluations of Two or More In the present invention, two or more evaluations of each index gene obtained in the above manner are combined to analyze the risk of tumor development.

  For example, the evaluation of the indicator gene is divided into two groups, an increase factor and a decrease factor, and analysis is performed by combining them. Further, for example, when the evaluation of the indicator gene is evaluated by scores, the scores are combined and scored, and the degree of occurrence risk is analyzed with the obtained scores.

  By combining two or more evaluations, the accuracy of analysis with respect to the risk of tumor development is significantly increased.

  The combination may be a combination of evaluations regarding two or more indicator genes selected from the same group, or a combination of evaluations of one or more indicator genes selected from one group and one or more indicator genes selected from another group. May be.

  Specifically, it may be a combination of evaluations using two or more indicator genes selected from the cell growth promoting gene group, and one or more selected from one or more indicator genes selected from the cell growth promoting gene group and an apoptosis promoting gene group. It is also possible to use a combination of evaluations based on the indicator genes.

  More specifically, it may be a combination of evaluation using the CyclinD1 gene and evaluation of the PCNA gene, or may be a combination of evaluation using the PCNA gene and evaluation using the Bax gene. Moreover, the combination of evaluation by CyclinD1 gene, PCNA gene, and Bax gene may be sufficient.

  In particular, the combination preferably includes an evaluation with two or more indicator genes selected from the same group. For example, evaluation of two or more genes selected from a cell growth promoting gene group may be combined with evaluation of one or more genes selected from another group, for example, an apoptosis promoting gene group, to evaluate the risk of tumor development. preferable.

  By performing analysis using a combination including two or more evaluations of genes belonging to the same group, it is possible to remarkably increase the analysis accuracy of the tumor development risk.

  Analysis of the risk of tumor development by the combination of indicator genes can be performed, for example, as follows.

(Analysis example 1)
Proliferation ↓ ・ Apoptosis ↑ ・ Effective preventive treatment Proliferation ↓ ・ Apoptosis ↓ ・ Mucosa is calm Proliferation ↑ ・ Apoptoup ↑ ・ Mucosal hyperproliferation (Inflammation etc.)
Proliferation ↑ / Apoptosis ↓ ... Highest risk of carcinogenesis

  Moreover, the analysis of the risk of developing a tumor including a combination of evaluations of two or more indicator genes selected from the same group can be performed, for example, as follows.

(Analysis example 2)
Proliferation ↓, Proliferation ↓, Apoptosis ↑ ... Effective preventive treatment Proliferation ↑, Proliferation ↓, Apoptosis ↑ (Intermediate state)
Proliferation ↓ ・ Proliferation ↑ ・ Apoptosis ↑ (Intermediate state)
Proliferation ↓ ・ Proliferation ↓ ・ Apoptosis ↓ ・ A state where cell dynamics are calm Proliferation ↑ ・ Proliferation ↓ ・ Apoptosis ↓ ・ ・ (Intermediate state)
Proliferation ↓ ・ Proliferation ↑ ・ Apoptosis ↓ ・ ・ (Intermediate state)
Proliferation ↑, Proliferation ↑, Apoptosis ↑, Increased mucosal cell proliferation Proliferation ↑, Proliferation ↑, Apoptosis ↓, Highest risk of carcinogenesis

  In the analysis examples 1 and 2, proliferation ↓ indicates that the tumor growth index of the cell proliferation promoting gene is less than the reference value (development risk reduction factor). Proliferation ↑ indicates that the tumor growth index of the cell proliferation promoting gene is greater than or equal to a reference value (risk increase factor).

  Apoptosis ↑ indicates that the tumor development index of the apoptosis promoting gene is greater than or equal to a reference value (development risk reduction factor). Apoptosis ↓ indicates that the tumor development index of the pro-apoptotic gene is less than the reference value (risk increase factor).

  Thus, by combining the evaluation based on the expression level of the gene, it becomes possible to analyze the tumor development risk with high accuracy.

  In the analysis method of the present invention, within the range where the effects of the present invention can be achieved, the evaluation of genes other than the above index genes can be added as appropriate to analyze the risk of tumor development. Furthermore, the analysis can be performed including environmental factors and other factors such as the age, sex, or medical history of the subject as long as the effects of the present invention are achieved.

8). Tumor development risk analysis and use thereof The method of the present invention can be used to set an appropriate examination interval and examination method for early detection of a tumor or cancer. In addition, based on the analysis results, guidance for cancer prevention can be provided, and measures and treatment policies can be set at an early stage.

  For example, according to the analysis result of the tumor development risk obtained by the present invention, the examination interval is shortened for a person with a high risk of occurrence, and the examination interval is lengthened for a person with a low risk of occurrence. Can be set. Furthermore, when the risk of occurrence is high, not only the test interval can be shortened, but also active carcinogenesis can be prevented by changing lifestyle habits or taking a carcinogenic agent.

  In addition, when the risk of occurrence is low, anxiety about cancer can be reduced and it can be used as a means for providing useful information to the subject.

  Carcinogenesis prevention is effective and ethically desirable for people with a high risk of occurrence, but it is possible to practice effective prevention for appropriate subjects. It can also be used for configuration.

  Furthermore, the method of the present invention can be used for the development of an efficient carcinogenesis prevention method and the practice of carcinogenesis prevention. For example, the method of the present invention can also be used to confirm whether or not the risk of carcinogenesis has decreased due to changes in lifestyle habits or internal use of a carcinogenic substance.

The method for analyzing tumor development risk of the present invention can be applied not only to colorectal cancer but also to solid cancers such as esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, lung cancer, laryngeal pharyngeal cancer, breast cancer and the like. It can also be applied to blood tumors such as leukemia.
Specifically, it can be suitably used as a method for analyzing the risk of occurrence in organs such as colorectal cancer, gastric cancer, and liver cancer that can be easily collected.

  In particular, the analysis method of the present invention can be suitably used as a method for analyzing the risk of developing colorectal cancer.

  The present invention provides a method capable of appropriately evaluating the risk of tumor development, particularly the risk of colorectal tumor development, with high accuracy.

  The present invention makes it possible to analyze the risk of tumor development with high accuracy by combining two or more indices.

  In particular, in the present invention, it has been clarified that the analysis accuracy of the tumor development risk can be remarkably improved by combining them even with an index showing the same function, for example, an index related to the same cell growth promotion. .

  Furthermore, in the present invention, it has also been clarified that the analysis accuracy can be further improved by evaluating and combining the indices.

  According to the present invention, it is possible to appropriately grasp the risk of tumor development, and thus, an examination for early cancer detection, which has conventionally been required to be frequently performed, is carried out at an appropriate interval to an appropriate subject. It becomes possible. Specifically, efficient inspection can be performed by shortening the interval between examinations for people with a high risk of occurrence and increasing the interval for those with low risk.

  Thereby, the pain and danger of the person to be inspected can be reduced, and a great amount of cost and time for the inspection can be reduced.

  Further, by knowing the degree of risk, when the risk is low, the concern about cancer can be reduced. In addition, when the risk is high, it is possible not only to shorten the examination interval, but also to actively prevent cancer by changing lifestyle habits or taking a carcinogenic agent.

  Carcinogenesis prevention should be performed efficiently and ethically for people with a high risk of occurrence, but by understanding the risk of occurrence, effective carcinogenesis prevention will be practiced for appropriate subjects. It becomes possible.

  Further, the conventional method requires detailed examination for inspection, but according to the present invention, it is possible to easily obtain a highly accurate analysis result.

  Furthermore, by using the present invention, it is possible to confirm whether or not the risk of carcinogenesis has decreased due to lifestyle changes or internal use of substances that prevent carcinogenesis. It can also be used for effective practice.

  As described above, the present invention provides means capable of appropriately grasping the risk of tumor development with high accuracy, and can greatly contribute to prevention or early treatment of cancer, reduction of morbidity, and the like.

  Hereinafter, examples will be shown to describe the present invention more specifically, but the present invention is not limited to the examples.

1. Subjects: We used some of the patients who had undergone colon cancer removal endoscopically from 1993 to 1997 at Osaka Prefectural Center for Adult Diseases and participated in a clinical trial for preventing colon cancer.

Participants in clinical trials are 40 to 69 years old at the time of clinical trial participation, all colons are observed, and two or more histologically diagnosed colorectal adenomas (adenomas / early cancers), all of them He had a history of resection, no history of intestinal resection (except for appendectomy), no current cancer, and all of these conditions.

In addition, among those who participated in these clinical trials, those who obtained the consent of the study on the collection of the sigmoid colonic mucosa at the time of endoscopy in the 4th year were considered for this study. .

The subjects were 170 males and 42 females, a total of 212 (average age 55.0 ± 0.4 years; 40-66 years).

2. material
(A) Normal colonic mucosa cells collected at the time of colonoscopy at the end of the 4th year, which is the end of the test tissue test, were used as materials. The biopsy site is the sigmoid colon (20cm mouth side of the anal edge), taken from the site where no lesion is found endoscopically. If there is a neoplastic lesion, it should be at least 5cm away from the neoplastic lesion Biopsy was performed at the site. The colonic mucosa was collected and immediately frozen in liquid nitrogen and stored at -80 ° C.

(B) Indicator gene
CyclinD1 gene, PCNA (proliferating cell nuclear antigen) gene, and Bax gene were measured.

(C) Housekeeping gene The glyceralde-hydo-3-phosphate dehydregenase (GAPDH) gene was used as the housekeeping gene.

3. Method The expression level of each gene was measured by real-time PCR for mRNA expression. RNA was extracted from colonic mucosal cells collected by the above method, cDNA (complementary deoxyribonucleic acid) was synthesized by reverse transcription, and mRNA was quantified by real-time PCR.

(A) RNA extraction and cDNA synthesis 350 μl of TRIzol Reagent (GIBCO BRL, Rockville, USA) was added to the sample, the tissue was ground, 100 μl of chloroform was added, and then RNA was extracted by centrifugation at 10000 rpm at 4 ° C. for 5 minutes. . The total RNA amount was adjusted to 1.5 μg, and then RNA was washed with 80% ethanol and dried, dissolved in 12.5 μl of DEPC (diethylpyrocarbonate) -treated water, heated at 95 ° C for 2 minutes, and then iced. After cooling and breaking the RNA higher order structure, 10.5 μl of a mixed solution containing reverse transcriptase was added and incubated at 42 ° C. for 60 minutes. This mixture consists of 4.5 μl 5 × buffer (containing 0.1 M DTT), 1 μl oligo dT (Amarham, Hokkaidou, Japan), 3 μl 2.5 M dNTP mixture (TAKARA, Shiga, Japan), 1 μl Rnasin ( Promega, WI, USA) 1 μl of AMV Reverse Transcriptase (Promega, WI, USA).

  After synthesizing cDNA, the enzyme was completely inactivated by incubation at 65 ° C. for 10 minutes.

(B) Cell culture colorectal cancer cell line HCT-116 is DMEM (Nihonseiyaku, Tokyo, Japan) + 10% FBS (fetal bovine serum, Hyclone, Logan, USA) + penicillin-streptomycin (GIBCO BRL, Rockville, USA) ) Was cultured in a 25 cm 2 flask (IWAKI, Funabasi, Japan) with 10% CO ₂ . From these HCT-116 cells, RNA was extracted by the same method as described above to synthesize cDNA. The cDNA obtained from HCT-116 was used as a standard for real-time PCR.

(C) Real-time PCR (quantitative PCR) method
The PCR reaction solution was 12.5 μl of 2 × QuantiTect SYBR Green PCR Master Mix (Qiagen, Tokyo, Japan), 0.25 μl of each primer, 1.5 μl of sample cDNA, and 10.5 μl of Rnase-removed water to a total volume of 25 μl. . Real-time PCR was performed using the ABI 5700 Sequence Detection System with the following protocol:
50 ° C for 2 minutes, 95 ° C for 15 minutes, then 50 cycles, 95 ° C for 15 seconds, 60 ° C for 45 seconds, 72 ° C for 15 seconds.

  As an internal control, glyceralde-hydo-3-phosphate dehydregenase (GAPDH) was used. The standard curve was confirmed by measuring a dilution of cDNA extracted from HCT-116 for each plate of GAPDH and the gene to be measured, and checking the correlation coefficient. The dilution series was 100 ng, 33 ng, 10 ng, 3.3 ng, and 1 ng.

  The evaluation method of the real-time PCR method detects the fluorescence of SYBR Green when the amplification product reaches a certain amount in the PCR reaction. The number of PCR cycles when this fluorescence rises is called the threshold cycle (Ct), and this number was used for evaluation.

ΔCt was calculated for each indicator gene with the following formula:
△ Ct = (GAPDH Ct value)-(Indicator gene Ct value)
The obtained ΔCt value was used as a tumor occurrence index of each index gene.

(D) Establishment of measurement system In order to establish a measurement system for an indicator gene, the base sequence of mRNA of each indicator gene was searched from NCBI, and primers were designed based on the base sequence. PCR was performed using this primer and the cDNA obtained from HCT-116, and it was confirmed that a specific band was detected. Then, a dilution test was performed using HCT-116 cDNA, and concentration-dependent analysis was performed using real-time PCR. It was confirmed whether fluorescence could be detected, and an actual sample was measured.

(E) Statistical analysis data was statistically analyzed using StatView, and the test method was significant difference test using Mann-Whitney U test.

4). Tumor incidence evaluation based on each index gene by comparing tumor incidence index and reference value, and examination of combination
(A) Calculation of reference value
For each indicator gene of Cyclin D1 gene, PCNA gene, and Bax gene, if the expression level of the indicator gene is high, the risk factor (+) is the risk factor (-). As points, sensitivity and specificity were obtained and an ROC curve was drawn.

  Each ROC curve is shown in FIGS. FIG. 1 shows the ROC curve of the Cyclin D1 gene, FIG. 2 shows the ROC curve of the PCNA gene, and FIG. 3 shows the ROC curve of the Bax gene.

  Further, for each index gene, a value that maximizes the sum of sensitivity and specificity was determined as a reference value for the index gene.

  The reference values were −2.51 for the CyclinD1 gene, −1.27 for the PCNA gene, and −2.30 for the Bax gene.

  An ROC curve was also drawn for the combination value obtained by adding the measured expression value of the PCNA gene to the measured expression value of the Cyclin D1 gene and subtracting the measured expression value of Bax. FIG. 4 shows the ROC curve according to the combination value.

  Also, in the case of the combination value, a value that maximizes the sum of sensitivity and specificity was determined as a reference value. The reference value was -0.26.

  As shown by the comparison of the ROC curves in FIGS. 1 to 4, the sensitivity and specificity by the combination were clearly improved as compared with the case where the indicator gene was examined alone.

(B) Correlation of index genes FIGS. 5 to 7 show the results of examining the correlation by the distribution of ΔCt of each index gene. FIG.
The figure which investigated the correlation of CyclinD1 gene and PCNA gene is shown. FIG. 6 shows a drawing in which the correlation between the PCNA gene and the Bax gene was examined. FIG. 7 shows a drawing in which the correlation between the CyclinD1 gene and the Bax gene was examined.

  In the results of FIG. 5, FIG. 6, and FIG. 7, no correlation between the indicator genes was observed.

  In particular, as shown in FIG. 5, it was revealed that there was no correlation between CyclinD1 gene and PCNA gene, which are the same cell growth promoting genes.

(C) Examination by comparison of tumor occurrence index and reference value For each index gene, the tumor occurrence index obtained in 3 above was compared with the reference value obtained in 4 (a) above, and the relationship with tumor occurrence was examined. Further, in the case where ΔCt of each index gene was combined, the same examination was performed in comparison with the reference value obtained in 4 (a) above. The results are shown in Table 1.

  In addition, RR in Table 1 indicates a relative risk (relative ratio).

As shown in Table 1, RR was improved by performing analysis by combining indices.
(D) Examination by combination of two groups Each indicator gene was evaluated based on an increase or decrease in development risk, and these were combined to examine the relationship with tumor development. The results are shown in Table 2 and FIG.

  In the column of CYD in Table 2, low indicates an evaluation when the tumor incidence index of the Cyclin D1 gene is less than the reference value (development risk reduction factor), and high indicates that the tumor occurrence index of the Cyclin D1 gene is greater than the reference value The evaluation (risk increase factor) is shown.

  In the PCNA column, low indicates an evaluation when the PCNA gene tumor incidence index is less than the standard value (factor of risk reduction), and high indicates an evaluation when the PCNA gene tumor incidence index is greater than the standard value (incidence risk). Increase factor).

  In the Bax column, high indicates an evaluation when the tumor incidence index of the Bax gene is greater than or equal to a reference value (development risk reduction factor), and low indicates an evaluation when the tumor incidence index of the Bax gene is less than the reference value (occurrence risk) Increase factor).

  Moreover, RR shows a relative risk (relative ratio).

  As shown in Table 2 and FIG. 8, using the cell growth promoting gene and the apoptosis promoting gene as an indicator gene, the expression level of all the indicator genes is low, and the situation where the cell dynamics of the mucosa is calm is set to 1. It was found that RR decreased to 0.47 when the gene increased, and RR increased to 3.21 when the cell growth promoting gene increased.

Results 1) From the above examination results, it was found that the analysis accuracy was remarkably improved by combining two or more indices. In particular, it was found that high accuracy can be obtained by analysis including two indices belonging to the same group. There is almost no correlation between the two indicators of cell proliferation, and the factors that affect them are also considered to be different.

  2) As shown in Table 2, assuming that the expression level of all the indicator genes is low and the cell dynamics are calm, the risk of tumor development decreases to 0.47 when the pro-apoptotic gene increases, and cell proliferation It was found that the risk of tumor development increased to 3.21 when the promoter gene increased. There has never been an inspection method for finding such a large difference, and the usefulness of the analysis method of the present invention has been confirmed.

  3) As shown in Table 1, RR is certainly improved by the analysis using the value obtained by simply adding and subtracting the measured values, but each index is theoretically grouped as shown in Table 2. The combined analysis revealed that the RR was further improved.

FIG. 1 is a drawing showing an ROC curve based on a measured value of Cyclin D1 gene expression level. FIG. 2 is a drawing showing an ROC curve based on the measured value of the PCNA gene expression level. FIG. 3 is an ROC curve based on the measured value of the Bax gene expression level. FIG. 4 is an ROC curve based on values obtained by combining measured values of expression levels of the Cyclin D1 gene, the PCNA gene, and the Bax gene. FIG. 5 shows the correlation between CyclinD1 gene and PCNA gene. FIG. 6 is a diagram showing the correlation between the PCNA gene and the Bax gene. FIG. 7 is a diagram showing the correlation between the CyclinD1 gene and the Bax gene. FIG. 8 is a drawing showing the results of combining the evaluation of CyclinD1 gene, PCNA gene and Bax gene and examining the relationship with tumor development.

Claims (8)

(A) Cell growth promoting gene group, cell growth inhibiting gene group, apoptosis promoting gene group, apoptosis inhibiting gene group, cell differentiation promoting gene group, and cell differentiation inhibiting gene group in a test tissue collected from a non-tumor part of a specimen Measuring the expression level of two or more indicator genes selected from
(B) measuring the expression level of a housekeeping gene in the same tissue;
(C) Tumor incidence index for each index gene obtained by correcting the measured value of the expression level of each indicator gene obtained in step (a) with the measured value of the expression level of housekeeping gene obtained in step (b) The process of
(D) evaluating the tumor occurrence index of each index gene obtained in step (c) by comparing with a preset reference value; and
(E) having a step of combining two or more evaluations of each indicator gene obtained in step (d),
The cell proliferation promoting gene group is a gene group including a PCNA gene, a CyclinD1 gene, a cdk2 gene, a farnesyl transferase gene, a Ki67 gene, and an EGF receptor gene.
The cell growth inhibitory gene group is a gene group including TGFβ gene, p21 gene, and INK4 gene,
The apoptosis promoting gene group is a group comprising Bax gene, caspase gene, and Fas gene,
The apoptosis suppressing gene group is a group containing Bcl-2 gene, IAP gene, and survivin gene, and the cell differentiation promoting gene group is a group containing mucin gene, Cdx-1 gene, intestinal alkaline phosphatase gene, and CD10 gene And the differentiation suppression gene group includes a Cdx-2 gene. In the step (d), the following (d-1): When the expression level of the indicator gene is high, the risk factor (+) is when the expression level is low. As a result (-), a step of setting a value that maximizes the sum of sensitivity and specificity of the expression level as a reference value for the gene,
(D-2) For each indicator gene, the tumor occurrence index of the gene in step (c) is compared with the reference value of the gene in step (d-1),
(D-3a) When the index gene is a cell growth promoting gene, a cell differentiation suppressing gene, or an apoptosis suppressing gene, when the tumor occurrence index is greater than or equal to a reference value, an occurrence risk increasing factor, and when it is less than the reference value, the occurrence risk is decreased Evaluate as a factor,
(D-3b) When the indicator gene is a cell growth inhibitory gene, a cell differentiation promoting gene, or an apoptosis promoting gene, the occurrence risk increasing factor when the tumor occurrence index is less than the reference value, and the occurrence risk decreasing factor when the index gene is greater than the reference value The method according to claim 1, wherein In step (d), the following (d-1) sets a plurality of reference values in several stages according to the expression level of the indicator gene,
(D-2) For each index gene, the tumor incidence index in the step (c) is compared with the reference value in the step (d-1), and the one with a high expression level is assigned a high score,
(D-3a) When the indicator gene is a cell proliferation promoting gene, a cell differentiation suppressing gene, or an apoptosis suppressing gene, the score of (d-2) is evaluated as an addition factor,
(D-3b) The evaluation method according to claim 1, wherein when the index gene is a cell growth inhibitory gene, a cell differentiation promoting gene, or an apoptosis promoting gene, the score of (d-2) is a step of evaluating as a subtraction factor. . The method according to any one of claims 1 to 3, which is a method for analyzing the risk of tumor development in the large intestine, wherein the non-tumor part of the specimen is normal large intestinal mucosa. The method according to any one of claims 1 to 4, wherein the housekeeping gene is a GADPH (glyceralde-hydo-3-phosphate dehydrogenase) gene. The method according to any one of claims 1 to 5, wherein the indicator gene is two or more indicator genes selected from a cell growth promoting gene group and an apoptosis promoting gene group. The method according to any one of claims 1 to 6, wherein the cell growth promoting gene group is a group consisting of a PCNA gene and a CyclinD1 gene. The method according to any one of claims 1 to 7, wherein the apoptosis-promoting gene group is a group consisting of Bax genes.

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