JPWO2007132864A1 - Primer, tumor marker, and cancer detection method - Google Patents

Abstract

The primer of the present invention that provides a primer useful for early diagnosis of cancer and prognosis such as micrometastasis, and a method for detecting cancer using a gene that is specifically expressed in cancer cells encodes the Eras gene Oligonucleotides capable of hybridizing to the nucleic acid MRNA amplified with such a primer is used as a tumor marker. Or this invention is a detection method of cancer which detects the expression of the Eras gene in the extract | collected biological sample.

Description

  The present invention relates to a primer, a tumor marker, and a method for detecting cancer.

A tumor marker is a substance detected from collected biomaterials, and is useful for early detection of cancer and prognosis such as micrometastasis. In particular, due to advances in analysis technology due to advances in molecular cell biology in recent years, the importance of prognosis such as early detection of cancer and micrometastasis has increased. For example, detection of mRNA of a tumor-specific gene present in lymph nodes or blood by a real-time reverse transcription polymerase chain reaction (RT-PCR) method is performed. If this method is used, even if one cancer cell is contained in 10 ⁶ cells, it can be detected. Known tumor-specific genes used in this method include carcinoembryonic antigen (CEA) gene and sugar chain antigen (CA19-9) gene.

  However, the above genes are also expressed in normal cells. For this reason, if the expression of these genes is used as a marker, false positives may occur, and there is a problem in accuracy. For example, there are not a few cases of peritoneal recurrence among those in which cancer cells are not recognized by abdominal cytology (CY0). There are reports that the presence or absence of cancer cells present in minute amounts in the peritoneal washing cells is identified by the expression of cytokeratin 20 (CK20) and CEA mRNA and correlates with prognosis and recurrence rate. There are some reports that CEA mRNA is also expressed in mesothelial cells, and the determination of false positives is a future subject. Therefore, in order to use as a highly accurate tumor marker, it is important to select genes that are specifically expressed in cancer cells.

  In addition, a gene that is specific and frequently found in gastric cancer has not been identified. For this reason, at present, the diagnosis using the PCR method has not led to a definitive diagnosis of the presence of a minute amount of tumor cells. Therefore, identification of a gene specific to gastric cancer and development of a highly sensitive detection method are expected.

Embryonic stem cells (hereinafter referred to as “ES cells”) have pluripotency capable of differentiating into all types of cells and tumor-forming ability that forms tumors when transplanted. The Eras gene was cloned as one of the causative genes of tumor formation in ES cells (see, for example, Non-Patent Document 1). The Eras gene has homology with other ras genes such as Kras and Hras. In addition, the Eras gene is an oncogene involved in a proliferation signal, and is considered to be expressed only in the embryonic period in humans. Eras gene expression is thought to be regulated at epigenetic levels such as DNA methylation and deacetylation with cell differentiation.
Nature (2003) 423 (6939), 541-545.

  The present invention has been made in view of the above problems, and its purpose is to use a gene specifically expressed in cancer cells, a primer useful for early detection of cancer, prognosis such as micrometastasis, tumor marker, And providing a method for detecting cancer.

As a result of intensive studies to solve the above problems, the present inventors have found that the Eras gene is specifically expressed in cancer cells and is not expressed at all in normal tissues. In addition, the expression of the Eras gene is detected by molecular cell biological techniques. As a result, even if one cancer cell is contained in 10 ⁶ cells, it can be detected. Therefore, it is useful for early detection of cancer and prognosis diagnosis such as micrometastasis.

  For example, cancer lymph node metastasis is believed to occur first in the sentinel lymph node. A sentinel lymph node is a lymph node that cancer cells reach first on the lymph flow. Using the method of the present invention, it is possible to identify sentinel lymph nodes and diagnose micrometastasis. If the presence or absence of micrometastasis can be confirmed, it will be easier to determine whether or not to perform a reduction surgery that omits lymph node dissection.

Further, in the cancer diagnostic method of the present invention, even if one cancer cell is contained in 10 ⁶ cells, cancer can be detected. In addition, it is possible to accurately diagnose the presence or absence of cancer cells present in minute amounts in the peritoneal washing cells. If micrometastasis can be detected early, treatment can be started early.
That is, the present invention is as follows.

  The present invention is a primer characterized by comprising an oligonucleotide capable of hybridizing to a nucleic acid encoding Eras.

  Furthermore, the primer is a primer set in which an oligonucleotide that can hybridize to the nucleic acid comprises a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2, and a primer set of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4. One primer set selected from a primer set consisting of the primer of SEQ ID NO: 5 and the primer of SEQ ID NO: 6 and a primer set consisting of the primer of SEQ ID NO: 7 and the primer of SEQ ID NO: 8 is preferable.

  The tumor marker of the present invention is an Eras gene mRNA amplified using the above primers.

  The present invention is a cancer detection method for detecting the expression of an Eras gene in a collected biological sample.

  In the detection method, the expression level of the Eras gene is detected by detecting the expression of the mRNA of the Eras gene.

  The cancer detection method of the present invention is preferably to detect the tumor marker.

  Since the present invention uses an Eras gene that is specifically expressed in cancer cells and not expressed in normal cells, and detects the Eras gene by molecular cell biological techniques, it is highly accurate, such as early detection of cancer and micrometastasis. Provided are primers useful for prognosis, tumor markers, and methods for detecting cancer.

FIG. 1 is a diagram showing RT-PCR results of Eras gene expression using a plurality of prepared primers. FIG. 2 is a diagram showing RT-PCR results of Eras gene expression in normal cells and cancer cell lines. FIG. 3 shows the results of RT-PCR of Eras gene expression in the cancerous part and normal part of the resected stomach. FIG. 4 is a diagram showing RT-PCR results of Eras gene expression in the cancerous part and normal part of the resected colon.

  As described above, in the cancer detection method of the present invention, the expression of the Eras gene in the collected biological sample is detected. The present invention is described in detail below. The base sequence of the exon part of the Eras gene is shown in SEQ ID NO: 9.

  In the present invention, “collected biological sample” includes (a) tissue, (b) culture of the collected tissue, (c) ascites, (d) tissue extract, (e) sputum, and (f) urine. (G) feces or (h) blood. Tissue can be collected by biopsy. Usable tissues or organs include, for example, brain, pituitary gland, spinal cord, salivary gland, thyroid, lung, breast, skin, skeletal muscle, heart, liver, spleen, adrenal gland, pancreas, stomach, small intestine, large intestine, rectum, bladder , Prostate, testicles, ovary, placenta, uterus, bone marrow, terminal mononuclear cells and the like.

  The Eras gene is composed of one exon. Therefore, no splicing occurs when the Eras gene is expressed. As a result, the obtained mRNA is stable with less occurrence of deletion, substitution, addition and the like.

  “Eras gene expression” refers to measuring the expression level of the Eras gene in cancerous cells. The measurement of the expression level does not necessarily require the expression level to be quantified, but is sufficient to detect the expression of the Eras gene.

  Methods for measuring the expression level of the Eras gene in cancerous cells include, for example, reverse transcription polymerase chain reaction (RT-PCR) method, LAMP method (Loop-Mediated Isolation Amplification), nucleic acid sequence amplification method (NASBA) method A known method such as a transcription-mediated amplification (TMA) method can be used.

  RT-PCR is performed as follows, for example. First, a single-stranded DNA (cDNA) complementary to this mRNA is synthesized by reverse transcriptase with respect to Eras mRNA, which is an Eras gene transcript in the collected biological sample. Next, the obtained cDNA or a fragment thereof is PCR amplified using a primer that can be specifically amplified. The obtained DNA is subjected to detection such as electrophoresis. For RT-PCR, a generally known method may be used.

In the present invention, primers that amplify DNA play an important role in Eras detection. As primers for amplifying DNA, the following primers of SEQ ID NOs: 1 to 8 are used. Specifically, (1) the primer of SEQ ID NO: 1 is a sense primer, the primer of SEQ ID NO: 2 is a primer set consisting of an antisense primer of SEQ ID NO: 1, and (2) the primer of SEQ ID NO: 3 is a sense primer, sequence The primer of number 4 is a primer set consisting of the antisense primer of SEQ ID NO: 3, (3) the primer of SEQ ID NO: 5 is a sense primer, the primer of SEQ ID NO: 6 is a primer set consisting of the antisense primer of SEQ ID NO: 5 (4) The primer of SEQ ID NO: 7 is a sense primer, and the primer of SEQ ID NO: 8 is a primer set consisting of an antisense primer of SEQ ID NO: 7. Preferred is the primer set of SEQ ID NOs: 7 and 8 in (4).

  As long as the primer includes the site shown in Table 1 on the target gene Eras, the length of the base sequence may be set long up to about 30, and other base sequences may be included on the 5 ′ end side. May be. If the length of the whole primer is within the above range and the base sequence is complementary, extending to the 5 'side or 3' side will not have a significant effect on the amplification of the target gene. is there. On the other hand, other base sequences may be included on the 5 ′ end side, the base sequence on the 3 ′ end side is important in the specific amplification reaction of PCR, and the base sequence on the 5 ′ end side is slightly different. This is because the adverse effect on the PCR reaction itself is small.

  These primer sets can be designed based on the nucleotide sequence of the cDNA of SEQ ID NO: 1 and prepared through synthesis and purification steps. In addition, the following can be pointed out as points to keep in mind when designing the primer. The size (number of bases) of the primer is 15-40 bases, preferably 15-30 bases in consideration of satisfying specific annealing with the template DNA. However, when LA (long accumulate) PCR is performed, at least 30 bases are effective. In order not to anneal one pair or one pair (two strands) of the sense strand (5 ′ end side) and the antisense strand (3 ′ end side) to each other, avoid complementary sequences between both primers and In order to prevent the formation of hairpin structures, self-complementary sequences should also be avoided. Further, in order to ensure stable binding with the template DNA, the GC content is set to about 50% so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on Tm (melting temperature), in order to obtain highly specific PCR products, primers having Tm values of 55 to 65 ° C. that are close to each other are selected. It is also necessary to pay attention to adjusting the final concentration of primers used in PCR to be about 0.1 to about 1 μM. Also, commercially available software for primer design, such as Oligo ™ [National Bioscience Inc. (Manufactured by (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)], etc. can also be used.

  The mRNA of the Eras gene amplified using the above primers can be used as a tumor marker.

  The method for quantifying the PCR amplified fragment is not particularly limited. For quantification of the PCR amplified fragment, for example, a quantification method using a radioisotope (RI), a quantification method using a fluorescent dye, or the like can be used. .

The quantification method using RI, for example, the PCR amplified fragment the previously added as a substrate, incorporated into PCR amplified fragment (1) nucleotides RI labeled reaction solution (such as ³² P-labeled dCTP such) RI After labeling and separating the PCR-amplified fragment by electrophoresis, etc., a method of quantifying the PCR-amplified fragment by measuring radioactivity, (2) RI-labeling the PCR-amplified fragment by using an RI-labeled primer, and PCR amplification After separating the fragments by electrophoresis or the like, a method of quantifying the PCR amplified fragments by measuring radioactivity, (3) after electrophoresis of the PCR amplified fragments, blotting on a membrane, hybridizing with a RI-labeled probe, Examples thereof include a method for quantifying a PCR amplified fragment by measuring radioactivity. Radioactivity can be measured using, for example, a liquid scintillation counter, an X-ray film, an imaging plate, or the like.

  As a quantification method using a fluorescent dye, (1) a PCR dye fragment is stained with a fluorescent dye (for example, ethidium bromide (EtBr), SYBR Green I, PicoGreen, etc.) that intercalates with double-stranded DNA, and excited. Method of quantifying PCR amplified fragments by measuring fluorescence intensity emitted by light irradiation, (2) PCR amplified fragments labeled with fluorescent dyes using primers labeled with fluorescent dyes, electrophoresis of PCR amplified fragments, etc. (3) Quantitative RT-PCR by using a TaqMan (registered trademark) probe modified with a fluorescent dye in addition to the primer for amplification. And a method for quantifying PCR amplified fragments by increasing the specificity and sensitivity of gene detection. . The fluorescence intensity can be measured using, for example, a CCD camera, a fluorescence scanner, a spectrofluorometer, or the like.

  In the cancer detection method of the present invention, the expression of the Eras gene is expressed as canceration and cancer progress, and the presence or absence of cancer cells in the sample is diagnosed using the expression of the Eras gene in the collected biological sample as an index. To do. That is, when the Eras gene is expressed in the biological sample, it is diagnosed that cancer cells are present in the biological sample.

  The cancer diagnosis method of the present invention can be widely used for general diagnosis of cancer. Examples of the cancer that can be diagnosed by the cancer diagnosis method of the present invention include liver cancer, stomach cancer, lung cancer, colon cancer, breast cancer, and the like. In particular, since the Eras gene used in the present invention is not expressed in normal cells, it is useful for early diagnosis of cancer and detection of micrometastasis.

  The primer of the present invention is extremely useful for detecting the expression of such an Eras gene.

  Moreover, if mRNA of the Eras gene amplified using the primer of the present invention is used as a tumor marker, it can be used as a tumor marker for effective cancer detection. Moreover, if a cancer detection kit containing the primer of the present invention is used, early diagnosis of cancer and detection of micrometastasis are easy.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this Example.

[Example 1]
(Create primer)
4 types of primer sets sandwiching a part of the exon region of Eras (1) The primer of SEQ ID NO: 1 is a sense primer, the primer of SEQ ID NO: 2 is a primer set consisting of an antisense primer of SEQ ID NO: 1, (2) SEQ ID NO: 3 The primer of SEQ ID NO: 4 is a primer set consisting of an antisense primer of SEQ ID NO: 3, (3) the primer of SEQ ID NO: 5 is a sense primer, the primer of SEQ ID NO: 6 is from an antisense primer of SEQ ID NO: 5 (4) A primer set consisting of a sense primer as a primer of SEQ ID NO: 7 and a primer set of an antisense primer as SEQ ID NO: 8 as a primer of SEQ ID NO: 8 was prepared.

(1) Preparation of total RNA 1 ml of TRIZOL Reagent (manufactured by Gibco-BRL) is added to 1 g of wet weight of human gastric cancer cells and normal tissues, and homogenized with a Polytron homogenizer while cooling with ice for 5 minutes at room temperature. I left it alone. 200 μl of chloroform (manufactured by Kanto Chemical Co., Inc.) was added, and the mixture was vigorously stirred up and down for 15 seconds, and left at room temperature for 5 minutes. Subsequently, after centrifugation at 12,000 rpm for 10 minutes at 4 ° C., the aqueous layer was recovered in a new 1.5 ml centrifuge tube. Furthermore, 500 ml of 2-propanol (manufactured by Kanto Chemical Co., Inc.) was added, pipetted, and allowed to stand at room temperature for 10 minutes. After centrifugation at 15,000 rpm for 10 minutes at 4 ° C., the supernatant was removed to obtain a pellet. The obtained pellet was vortexed by adding 75% cooled ethanol. It was dissolved with 50 μl of DEPC-treated sterilized distilled water (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain total RNA. For normal tissues, the same operation as described above was performed to prepare total RNA.

(2) Preparation of cDNA A mixed solution of 10 μg of total RNA prepared from stomach cancer tissue and normal RNA prepared from normal tissue in (1) above, 0.5 μl of RNAse inhibitor, and 10.5 μl of DEPC-treated sterile distilled water was 70 ° C. After heating for 5 minutes, it was cooled on ice for 1 minute. T7- (dT) 24 primer (manufactured by Amersham) 11 μl of a mixed solution containing 100 pmol was heated at 70 ° C. for 10 minutes and then cooled on ice to prepare RNA sample primer Mix. After cooling, this RNA sample primer mix was mixed with oligo (dT) Primer 0.5 μg (manufactured by Invitrogen), 10 mM dNTPmix 1 μl (manufactured by AB), M-MLV-Reverse Transscriptase 1 μl (manufactured by Invitrogen) 4 μL was mixed and incubated at 37 ° C. for 50 minutes. Thereafter, the mixture was heated at 70 ° C. for 15 minutes and then cooled on ice for 2 minutes to prepare cDNA. On the other hand, for total RNA prepared from normal tissue, a cDNA solution was prepared by performing the same operation as described above.

(Primer test)
PCR was performed as follows using the prepared cDNA as a template, and the expression of mRNA of the Eras gene in each primer set was examined. That is, 1.5 μl of the cDNA, 10 pmol of primer 1 consisting of the base sequence shown in SEQ ID NO: 1, 10 pmol of primer 2 consisting of the base sequence shown in SEQ ID NO: 2, 10 × PCR buffer 5 μl, 50 mM MgCl 2 μl, 10 mM dNTP Mix 0 50 μl of a reaction solution containing 5 μl, Taq polymerase 0.2 μl, and DEPC-treated sterile distilled water 40 μl was prepared, pre-denatured at 94 ° C. for 10 minutes using PTC200 (MJ Research), then 94 ° C., 50 After denaturation by incubating for 2 seconds, annealing was performed at 59 ° C. for 50 seconds, followed by 35 cycles of 72 ° C. for 1 minute, followed by incubation for 10 minutes at 72 ° C. PCR was performed. (2) The primer of SEQ ID NO: 3 is a sense primer, the primer of SEQ ID NO: 4 is a primer set comprising the antisense primer of SEQ ID NO: 3, (3) the primer of SEQ ID NO: 5 is a sense primer, and the primer of SEQ ID NO: 6 is SEQ ID NO: The primer set consisting of 5 antisense primers, (4) the primer set of SEQ ID NO: 7 was the sense primer, and the primer set of SEQ ID NO: 8 was the same as the primer set consisting of the antisense primer of SEQ ID NO: 7. The expression of the Eras gene in each primer set was examined from the amplified DNA. Expression was confirmed by UV electrophoresis on 2% agarose gel with ethidium bromide. As a reference gene, a primer for human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used. The results are shown in FIG. FIG. 1 is a diagram showing RT-PCR results of Eras gene expression using a plurality of prepared primers.

  In FIG. 1, each lane of Eras1-Eras4 shows the presence of the Eras gene amplified by each primer set of (1)-(4), respectively. The whitest colored portion in each lane in FIG. 1 shows the expression of the Eras gene. From FIG. 1, it was found that each of the primer sets (1) to (4) can detect the expression of the Eras gene. Moreover, it turned out that the primer set of (4) is the most suitable for detecting the expression of Eras gene among the primer sets of (1)-(4). The following experiment was performed using the primer set of (4).

(Example 2)
(Evaluation of Eras gene expression in normal cells)
In the same manner as in Example 1 above, total RNA was prepared from gastric cancer cell line (OCUM-2MD3) and human normal tissues (lymphocytes, mesothelial cells, fibroblast cell lines (NF24, NF25, NF26)). The cDNA was prepared. Next, the expression of the Eras gene in these cDNAs was examined using the primer set (4). Also in this example, a primer for human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene. The results are shown in FIG. FIG. 2 is a diagram showing RT-PCR results of Eras gene expression in normal cells and cancer cell lines. As apparent from FIG. 2, expression of the Eras gene was observed only in the gastric cancer cell line (OCUM-2MD3). It was also found that the Eras gene was not expressed in normal cells. From this, it was found that the Eras gene amplified using the primer set of this example can be used as a tumor marker.

(Evaluation of Eras gene expression in clinical specimens)
After obtaining informed consent in accordance with the guidelines approved by the Osaka City University Ethics Committee, the cancer and normal areas of the resected stomach and resected colon of patients who underwent surgery in 2004-2006 at Osaka City University Hospital In the same manner as in Example 1 above, total RNA was prepared and cDNA was prepared. Next, the expression of the Eras gene in these cDNAs was examined using the primer set (4). FIG. 3 shows the results of the cancerous part and normal part of the resected stomach, and FIG. 4 shows the results of the cancerous part and normal part of the resected colon. FIG. 3 is a diagram showing the expression of the Eras gene for each of the cancer part and the normal part of the resected stomach for each cancer type, progress, and differentiation degree in the cancer part and the normal part. FIG. 4 is a diagram showing the expression of the Eras gene for each of the cancerous part and normal part of the resected large intestine for each cancer type and progress in the cancerous part and the normal part.

  3 and 4, T: tumor (tumor), N: normal (normal), differentiation degree [po. Poorly differentiated adenocarcinoma (undifferentiated adenocarcinoma), tub. Tubular adenocarcinoma (tubular adenocarcinoma), sig. Signet ring cell carcinoma (signature ring cell carcinoma)], progress [sm; Tela submucose (submucosa), mp; Tunica muscarisi propria (muscle layer), ss; Tela subserosa, submembrane (Serosa exposure)], degree of differentiation [Mod; moderately differentiated adenocarcinoma (moderately differentiated adenocarcinoma), Wel; well differentiated adenocarcinomas (well-differentiated adenocarcinoma)]. In addition, there are cases in which the progress is shown in capital letters in FIG. 3, but the meaning is the same. The same number attached to tub means a different part of the same patient.

  As is apparent from FIGS. 3 and 4, the Eras gene was expressed in all cancer parts regardless of the type of cancer, the degree of advancement, and the degree of differentiation (100%; gastric cancer 20/20, colon cancer 2/2). . On the other hand, no expression was observed in the normal part (0%; gastric cancer 0/20, colon cancer 0/2).

  From the above, it was found that the cancer detection method of the present invention exerts a high detection rate when used for actual cancer diagnosis.

Claims (6)

  A primer comprising an oligonucleotide capable of hybridizing to a nucleic acid encoding an Eras gene.   The oligonucleotide capable of hybridizing to the nucleic acid is a primer set consisting of a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2, a primer set consisting of a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4, a primer of SEQ ID NO: 5 The primer according to claim 3, which is one primer set selected from a primer set consisting of a primer of SEQ ID NO: 6 and a primer set consisting of a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8.   A tumor marker, which is mRNA of the Eras gene, amplified using the primer according to claim 3 or 4.   A method for detecting cancer, comprising detecting the expression of an Eras gene in a collected biological sample.   The method for detecting cancer according to claim 1, wherein the detection of the expression level of the Eras gene detects the expression of mRNA of the Eras gene.   The method for detecting cancer according to claim 4, wherein the tumor marker according to claim 3 is detected.

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