Method of detecting colon cancer marker

The present invention relates to a method for detecting a tumor marker for colorectal cancer diagnosis, which comprises a step of extracting RNA from a biological sample, which does not include a means for separating cell components from the biological sample.

The number of deaths from colorectal cancer is increasing. Colorectal cancer deaths are the fourth most common cancer death in men and the second most common cancer death in women (1999 cancer death statistics). In addition, in the 2015 cancer patient estimation, it is estimated that both men and women will be ranked first, and comprehensive colorectal cancer measures including secondary prevention are required. It is one of the most effective methods. For mass screening of cancer, it is important to have a simple and non-invasive detection method. The only non-invasive method currently available is a stool test for the presence or absence of occult blood, a fecal occult blood test, which is widely used as a standard method for mass screening of colorectal cancer. However, the fecal occult blood test has low sensitivity and specificity (sensitivity 30-90%, specificity 70-98%) because the appearance of hemoglobin in feces is not tumor-specific, and is therefore false negative. There is a drawback that there are not a few false positives. In addition, for the diagnosis of colorectal cancer, after screening by immunological fecal occult blood method or at the same time, total colonoscopy or enema examination and sigmoid colonoscopy are used in combination. However, it has the disadvantage that it takes a lot of time and effort. As an alternative method for fecal occult blood test, methods using DNA such as detection of K-ras, p-53, APC gene mutation and microsatellite instability in feces have been reported (Sidransky et al. (D). .Sidransky, et al.), Science, Vol. 256, April 3, 1992, pp. 102-105; Don et al. (SM Dong, et al.), Journal of the National -Cancer Institute (Journal of the National Cancer Institute), Vol. 93, No. 11, June 11, 2001, pp. 858-865; Travelso et al. (G. Traverso, et al.), The The New England Journal of Medicine Medicine), Vol. 346, No. 5, January 31, 2002, pp. 311-320; Travelso et al. (G. Traverso, et al.), The Lncet, Vol. 359. , February 2, 2002, pp. 403-404). The method using these DNAs is a non-invasive method that can capture direct changes in cancer cells, has a feature of high specificity, and is considered to be a promising method. It has the disadvantages of being less sensitive than some fecal occult blood tests and taking a considerable amount of time and effort. In addition, as an alternative method for further fecal occult blood test, a method for detecting mRNA such as protein kinase C (PKC) in feces has been developed in order to detect gene expression more directly (Davidson et al. (Written by Davidson et al.). LA Davidson, et al.), Carcinogenesis, Vol. 19, No. 2, 1998, pp. 253 to 257; by Alexander and RF Raicht, Digestive Desiges and. Science (Digestive) Diseases and Sciences), Vol. 43, No. 12, 1998, pp. 2652 to pp. 2658; by Tamaki et al. (T. Yamao et al.), Gastroenterology, Vol. 114, No. 6. , 1998, pp. 1198 to pp. 1205). However, even with the above method using RNA, RNA could not be easily and efficiently extracted from a small amount of stool, and sensitivity exceeding the fecal occult blood method could not be obtained. A method for qualitatively and quantitatively detecting RNA by combining the PCR method with reverse transcriptase reaction (RT) is known. This RT-PCR method is superior to the Northern blotting method in that it can detect trace molecules, and is superior to the in situ hybridization method in terms of speed and ease of procedure. However, RNA is always at risk of degradation by RNA-degrading enzymes (RNases), which are more unstable than DNA, are ubiquitous in all biological samples, and are extremely stable. Therefore, strict control is required to prevent RNase contamination in the RT-PCR method as well as in the RNA purification process and after purification. Therefore, when extracting RNA from a biologically extremely crude sample called feces, a step of separating cell fractions in advance was required in order to eliminate the influence of RNase. Therefore, it is impossible to directly detect RNA in feces in which a huge amount of RNase derived from an extremely large amount of microorganisms is present, and at least for the removal of exogenous RNase derived from microorganisms, etc., the cell fraction. Separation was considered essential. However, the present inventor has surprisingly found that the above problems can be solved by homogenizing a frozen biological sample in the presence of an RNA-degrading enzyme inhibitor. Was completed.

Therefore, an object of the present invention is to provide a method for detecting a tumor marker for non-invasive and simple diagnosis of colorectal cancer, which has a sensitivity and specificity exceeding that of a conventional fecal occult blood test. The present invention comprises a method for detecting a tumor marker for colorectal cancer diagnosis, which comprises the following steps: a) homogenizing the collected biological sample in the presence of an RNA degrading enzyme inhibitor to prepare a suspension. It is a method for preparing a sample for extracting RNA used in the above, and is characterized in that it does not include a step of separating cell components from a biological sample. Here, the collected biological sample is preferably frozen. The present invention is the above method, wherein the RNA-degrading enzyme inhibitor is guanidine thiocyanate. The present invention is also the method described above, wherein the biological sample is feces. Further, in the present invention, in addition to the steps of the above method, the following steps: b) step of extracting RNA from the sample for extracting the obtained RNA, c) reverse transcription of the extracted RNA to obtain cDNA. A method for detecting a tumor marker for diagnosing colon cancer, which comprises a step of obtaining, d) a step of amplifying the obtained cDNA, and e) a step of detecting the amplified cDNA. The present invention is a method for detecting a tumor marker for diagnosing colorectal cancer, wherein the tumor marker is COX-2. The present invention also presents on tumor markers for the diagnosis of colorectal cancer, including means: a) means for homogenizing the collected biological sample in the presence of an RNA degrading enzyme inhibitor to prepare a suspension. It is a kit for preparing a sample for extracting RNA used in a detection method, and is characterized by not including a means for separating cell components from a biological sample. The kit of the present invention also preferably comprises means of freezing the collected biological sample. Further, the present invention is the above-mentioned kit in which the RNA-degrading enzyme inhibitor is guanidine thiocyanate. The present invention is also the kit described above, wherein the biological sample is feces. Further, the present invention further comprises the following means: b) means for extracting RNA from a sample for extracting the obtained RNA, c) means for reverse transcribing the extracted RNA to obtain cDNA, d) obtained. A kit for detecting tumor markers for diagnosing colorectal cancer, which comprises means for amplifying the expanded cDNA and e) for detecting the amplified cDNA. Further, the present invention is the above-mentioned kit in which the tumor marker is COX-2.

FIG. 1 shows the electrophoresis results of Example 2. Lane 1 is total RNA extracted from human feces by the method of Alexander et al. Lane 2 is total RNA extracted from human feces by the method of the invention. Lane 3 is total RNA extracted from human colorectal cancer tissue. Lane M is a molecular weight marker.

Examples of the RNA-degrading enzyme inhibitor of the present invention include guanidine thiocyanate, Isogene, Ultraspec II (registered trademark) and the like. Examples of the biological sample of the present invention include animal and plant tissues, body fluids, excrement and the like, preferably feces, more preferably human feces. The biological sample of the present invention can be used as it is or, optionally frozen. As the freezing method, any conventional technique can be used, and a method using liquid nitrogen is preferable. The freezing temperature is -1 to -196 ° C, preferably -20 to -196 ° C, preferably -75 to -196 ° C, and more preferably -110 to -196 ° C. Most preferably it is -196 ° C. The frozen sample may be stored frozen. The storage temperature is -75 to -196 ° C, preferably -110 to -196 ° C, and more preferably -196 ° C. The storage period is 1 day to 10 years, preferably 1 day to 3 years, and more preferably 1 day to 1 year. Tumor markers used in the present invention include COX-2, matrix metalloproteinase (MMP), c-met, CD44 variants, EGF-R, EF-1, Wnt-2, Bradion, SKP2, KPC-1, KPC-2, PRL-3, Angiogenin, Integrin, Snail, Dysadherin and the like can be mentioned, but COX-2 is preferable. The above steps c) to e) are called the RT-PCR method, and are performed according to the description of, for example, Takao Sekiya et al., Forefront of PCR method, 1997, Kyoritsu Shuppan, pp. 187 to 196. Can be done. For the extraction of RNA from the suspension, a conventionally known method can be used, and for example, a commercially available kit such as RNeasy Mini (QIAGEN) or RNA Extraction Kit (Pharmacia Biotech) can be used. In the present invention, reverse transcription is referred to as reverse transcriptase (Reverse). Transcriptase) is used to convert RNA into complementary DNA (cDNA). Reverse transcription reaction is usually buffer, MgCl₂It is carried out using a solution containing salts such as KCl and dithiothreitol (DTT), primers, deoxyribonucleotides, RNase inhibitors and reverse transcriptase. The above salts can be tried by changing to other salts as appropriate. Further, proteins such as gelatin and albumin, surfactants and the like can also be added. PCR is usually used for the amplification of cDNA following the reverse transcription. The PCR reaction solution is usually a buffer, MgCl.₂And salts such as KCl, primers, deoxyribonucleotides, and thermostable polymerases. The above salts can be tried by changing to other salts as appropriate. Further, proteins such as gelatin and albumin, dimethyl sulfoxide, surfactants and the like can also be added. For the amplification of cDNA, the LAMP method (Japanese Patent No. 3313358) or the ICAN method (Japanese Patent Laid-Open No. 2001-136965) can also be used. In the present invention, the primer refers to an oligonucleotide that acts as a synthesis starting point during cDNA synthesis or nucleic acid amplification. The primer is preferably single-stranded, but Japanese strands can also be used. When the primer is double-stranded, it is desirable to make it single-stranded prior to the amplification reaction. Primers can be synthesized according to known methods and can also be isolated from an organism. The reverse transcriptase used in the reverse transcription reaction means an enzyme capable of reverse transcribing RNA into cDNA. Reverse transcriptases include RAV (Rous associated virus) and AMV (Avian myeloblastosis). There are reverse transcriptases derived from retroviruses such as virus) and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus), but the present invention is not limited thereto. Examples of the thermostable polymerase used for PCR include, but are not limited to, Taq polymerase. As a method for detecting the amplified DNA, electrophoresis using an agarose gel can be used, but the method is not limited thereto. The kit of the present invention may also include instructions describing the method of the present invention.

The following examples illustrate the invention, but do not limit the invention. 30 patients who were admitted to the Department of Internal Medicine, Hamamatsu Medical University for scrutiny and treatment and whose presence of colorectal cancer was confirmed by colonoscopy, and no tumor or inflammatory changes in the large intestine (non-colon disease) Twenty-two patients were included. Informed consent was obtained from all patients. Feces were collected in 5 ml tubes as soon as possible after collection, and about 1 g each was separated, frozen in liquid nitrogen, and stored at -80 ° C. For comparison, human hemoglobin (Hb) in stool was measured by immunological fecal occult blood test for each sample. Tissues were frozen at -80 ° C after freezing the biopsy material of the cancerous and normal areas with liquid nitrogen during endoscopy received prior to treatment. Then, homogenization was performed using a homogenizer, a guanidine salt and phenol, and total RNA was extracted with chloroform and ethanol. 1 μg of the obtained RNA was reverse transcribed using Riverscript II (registered trademark) (reaction solution volume 20 μl, Wako Pure Drug) to obtain cDNA, and a part of it was Gene. Amplified by Nestide PCR using Taq (Wako Pure Drug). The obtained PCR was electrophoresed on a 4% agarose gel and stained with ethidium bromide. The primers used were random primers for reverse transcription, CEA was reported to Gerhard (JJCO, 1994) for PCR, and COX-2 was originally designed. did. PCR was performed in 20 cycles in the first round and 25 cycles in the second round. The primers used are shown below. &lt;CEA&gt;<img file="JP4134047B2_D0001.tif" />&lt;COX-2&gt;<img file="JP4134047B2_D0002.tif" /> Results We attempted to detect CEA and COX-2 from feces in 30 cases of colorectal cancer (3 cases of early stage cancer and 27 cases of advanced cancer) and 22 cases of control group, and obtained the following results. CEA was detected in all 30 cases of colorectal cancer and in 21 of 22 cases in the control group. It was also found that RNA that can be amplified by RT-PCR can be extracted from both. COX-2 was found in 27 of 30 colorectal cancers (cecum 2/2, ascending colon 3/5, descending colon 1/1, sigmoid colon 7/7, rectum 12/13, early cancer 2/3, It was detected in advanced cancer 25/27), but none of the 22 control groups was detected (sensitivity 90%, specificity 100%). The immunological fecal occult blood test was positive in 23 of 28 cases of colorectal cancer and 3 of 22 of the control group (sensitivity 82.1%, specificity 86.3%). Of the three COX-2 negative colorectal cancer cases, one had a positive immunological fecal occult blood test and two had a negative. COX-2 was detected in 3 of 5 colorectal cancers with a negative immunological fecal occult blood test.

The distribution of total RNA and molecular weight obtained from human feces was compared between the method of the present invention and Alexander's method (Non-Patent Document 6). As a target, total RNA was extracted from human colon cancer tissue using a commercially available RNA extractant (Isogen, Wako Pure Drug). The same amount of total RNA extracted from each sample was electrophoresed on an agarose gel. The two major bands found in lane 3 (RNA derived from human colon cancer tissue) show 28S and 18S rRNA. In addition, the smear-like portion indicates that various high molecular weight RNAs are contained in the obtained total RNA. The two major bands found in lane 2 (facal-derived RNA by the method of the invention) show 23S and 16S rRNA from gut microbiota. Further, since a smear-like portion is observed as in lane 3, it is considered that various high molecular weight RNAs are contained in the total RNA obtained from feces by the method of the present invention. In contrast, in lane 1, no bands or smears were observed, indicating that the sample extract did not contain high molecular weight RNA. In fact, the sample in lane 2 gave the desired product by RT-PCR, but the sample in lane 1 did not give the PCR product. From the results of this study, it was clarified that RNA extracted from human feces by the method of the present invention can be amplified by RT-PCR. In addition, the detection of COX-2 in feces by RT-PCR proved to be superior to the conventional immunological fecal occult blood method because it has 90% sensitivity and 100% specificity. Was done. Further, the method of the present invention requires less feces and higher detection sensitivity than the reported detection of APC, K-ras and p53 gene mutations, so that the time required for detection and the detection sensitivity are high. It can save a lot of labor. Whereas the prior art fecal occult blood method targets the general and indirect event of "bleeding" from a lesion, the method of the present invention is specific and direct, with the expression of the carcinogenic marker COX-2. The data obtained by the method of the present invention can provide a higher quality diagnosis because it targets various events. Therefore, the method of the present invention is clinically extremely useful as a non-invasive screening method for novel colorectal cancer with high specificity and sensitivity.