Method for assessing risk of patient suffering from gastric cancer after sterilization of helicobacter pylori, polynucleotide and kit

Abstract

The present invention provides a method for assessing the risk of suffering from gastric cancer in a patient from which Helicobacter pylori has been sterilized, the method comprising a step for detecting the expression of the KK-LC-1 gene in a gastric tissue sample derived from the patient before Helicobacter pylori has been sterilized, the expression of the KK-LC-1 gene is positive, and it is indicated that the patient has the risk of suffering from the gastric cancer after the helicobacter pylori is sterilized.

Description

Technical Field

[0001] The present invention relates to a method, polynucleotide and kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori sterilization. This application claims priority based on Japanese Patent Application No. 2022-140097 filed in Japan on September 2, 2022, the contents of which are incorporated herein by reference. Background Art

[0002] It is well known that Helicobacter pylori infection is one of the causes of gastric cancer. Currently, when Helicobacter pylori infection is discovered, the insurance measure is to eliminate the bacteria by taking it orally. It is reported that the risk of gastric cancer in cases where Helicobacter pylori has been eliminated has been reduced to 1 / 3 compared to the non-elimination group. On the other hand, the results show that there are cases where cancer is still developed even after Helicobacter pylori has been eliminated. At present, there is no technology to evaluate the risk of gastric cancer after Helicobacter pylori elimination. In other words, even if the elimination is successful, the subject patient still has the same concerns about cancer as before the elimination. Furthermore, since there is no risk indicator for cancer after elimination, all cases where the elimination is successful require regular physical examinations, and medical economy and medical resources cannot be reduced. The risk indicators mentioned above are unmet medical needs that both the medical field and patients strongly desire.

[0003] However, cancer / testis antigen is a general term for proteins that are not observed to be expressed except in cancer cells and testes. Kita Kyushu lung cancer antigen 1 (KK-LC-1) is a type of cancer / testis antigen. So far, the inventors have clarified that KK-LC-1 can be used as a marker expressed in various cancers (for example, refer to Patent Document 1).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 6028253 Summary of the invention

[0007] Problems to be solved by the invention

[0008] The object of the present invention is to provide a technology for evaluating the risk of gastric cancer in a patient after Helicobacter pylori has been eliminated.

[0009] Technical means to solve problems

[0010] The present invention includes the following aspects.

[0011] [1] A method for evaluating the risk of gastric cancer in a patient after Helicobacter pylori eradication, the method comprising: a step of detecting the expression of the KK-LC-1 gene in a gastric tissue sample from the patient before Helicobacter pylori eradication, wherein a positive expression of the KK-LC-1 gene indicates that the patient is at risk of developing gastric cancer after Helicobacter pylori eradication.

[0012] [2] The method according to [1], wherein the expression of the KK-LC-1 gene is detected by end-point RT-PCR.

[0013] [3] The method according to [2], wherein in the above-mentioned end-point RT-PCR, a primer consisting of the base sequence recorded in SEQ ID NO: 1 and a primer consisting of the base sequence recorded in SEQ ID NO: 2 are used.

[0014] [4] The method according to [1], wherein the expression of the KK-LC-1 gene is detected by real-time RT-PCR.

[0015] [5] The method according to [4], wherein in the above-mentioned real-time RT-PCR, a qPCR probe is used, wherein the above-mentioned qPCR probe has a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, the base length is 15 to 50 bases, and has 1 or 2 base mutations relative to the base sequence recorded in sequence number 3.

[0016] [6] The method according to [4] or [5], wherein the real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, wherein the single-stranded nucleic acid fragment has a base sequence comprising the bases at positions 322 and 323, or the bases at positions 1096 and 1097 in the base sequence recorded in sequence number 4, and the base length is 20 to 50 bases.

[0017] [7] The method according to [1], wherein the expression of the KK-LC-1 gene is detected at the protein level.

[0018] [8] A kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori eradication, the kit comprising a primer consisting of the base sequence recorded in SEQ ID NO: 1 and a primer consisting of the base sequence recorded in SEQ ID NO: 2.

[0019] [9] The kit according to [8] further comprises a qPCR probe, wherein the qPCR probe has a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, the base length is 15 to 50 bases, and has 1 or 2 base mutations relative to the base sequence recorded in sequence number 3.

[0020]

[10] A kit according to [8] or [9], further comprising a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, wherein the single-stranded nucleic acid fragment has a base sequence comprising the 322nd and 323rd bases, or the 1096th and 1097th bases in the base sequence recorded in SEQ ID NO: 4, and the base length is 20 to 50 bases.

[0021]

[11] A kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori eradication, the kit comprising a substance that specifically binds to KK-LC-1 protein.

[0022]

[12] A polynucleotide having a base sequence comprising the 195th and 196th bases in the base sequence recorded in SEQ ID NO: 3, having a base length of 15 to 50 bases, and having a mutation of 1 or 2 bases relative to the base sequence recorded in SEQ ID NO: 3.

[0023]

[13] A polynucleotide having a base sequence comprising the 322nd and 323rd bases, or the 1096th and 1097th bases in the base sequence described in SEQ ID NO: 4, wherein the base length is 20 to 50 bases.

[0024]

[14] A method for detecting the expression of the KK-LC-1 gene in a biological sample, wherein the detection is performed by real-time RT-PCR, wherein a qPCR probe is used in the real-time RT-PCR and the real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, wherein the single-stranded nucleic acid fragment has a base sequence comprising the bases at positions 322 and 323, or the bases at positions 1096 and 1097 in the base sequence recorded in sequence number 4, and the base length is 20 to 50 bases.

[0025]

[15] The method according to

[14] , wherein the qPCR probe has a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, the base length is 15 to 50 bases, and has 1 or 2 base mutations relative to the base sequence recorded in sequence number 3.

[0026] [Effects of the invention]

[0027] According to the present invention, a technique for evaluating the risk of gastric cancer in a patient after Helicobacter pylori has been eliminated can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the 5-year cumulative incidence rate of gastric cancer in patients in the KK-LC-1 positive group and negative group in Experimental Example 1.

[0029] Figure 2 This is a graph showing the 5-year cumulative incidence rate of gastric cancer among patients in the KK-LC-1 positive group and negative group in Experimental Example 2.

[0030] Figure 3 This is a graph showing the results of real-time PCR in Experimental Example 3.

[0031] Figure 4 This is a graph showing the results of real-time PCR in Experimental Example 3.

[0032] Figure 5 This is a graph showing the results of real-time PCR in Experimental Example 3.

[0033] Figure 6 This is a graph showing the results of real-time PCR in Experimental Example 3.

[0034] Figure 7 This is a graph showing the results of real-time PCR in Experimental Example 4.

[0035] Figure 8 This is a graph showing the results of real-time PCR in Experimental Example 4.

[0036] Fig. 9 This is a graph showing the results of real-time PCR in Experimental Example 5.

[0037] Fig.10 This is a graph showing the results of real-time PCR in Experimental Example 6.

[0038] Fig.11 This is a graph showing the results of real-time PCR in Experimental Example 6. DETAILED DESCRIPTION

[0039] [Method for assessing the risk of gastric cancer in patients who have been freed of Helicobacter pylori]

[0040] In one embodiment, the present invention provides a method for assessing the risk of gastric cancer in a patient after Helicobacter pylori eradication, the method comprising: a step of detecting the expression of the KK-LC-1 gene in a gastric tissue sample from the patient before Helicobacter pylori eradication, wherein a positive expression of the KK-LC-1 gene indicates that the patient is at risk of developing gastric cancer after Helicobacter pylori eradication.

[0041] The inventors found that if the expression of KK-LC-1 gene is positive in the gastric tissue sample from the patient before the Helicobacter pylori eradication, the patient is at risk of gastric cancer after the Helicobacter pylori eradication, thereby completing the present invention. The NCBI accession number of the cDNA of the human KK-LC-1 gene is NM_001017978.4. In addition, the NCBI accession number of the genomic DNA of the human KK-LC-1 gene is NC_000023.11. In addition, the NCBI accession number of the human KK-LC-1 protein is NP_001017978.1.

[0042] As shown in the examples described below, the method of this embodiment can assess the risk of gastric cancer in a patient after H. pylori eradication. Currently, there is no technology for assessing the risk of gastric cancer in a patient after H. pylori eradication.

[0043] As a gastric tissue sample, a gastric tissue sample collected when confirming Helicobacter pylori infection can be appropriately used. If the expression of the KK-LC-1 gene is positive, it means that the patient is still at risk of gastric cancer even after Helicobacter pylori is eradicated. In this case, measures such as increasing the frequency of gastric cancer examinations for the patient can be taken.

[0044] When the expression of KK-LC-1 gene is negative, it means that the risk of gastric cancer in patients after Helicobacter pylori eradication is low. In this case, the frequency of gastric cancer examinations for the patient can be reduced, which can save limited medical resources. In addition, it can also alleviate the anxiety of patients who are worried about gastric cancer and reduce the psychological burden of patients.

[0045] In the method of this embodiment, the expression of the KK-LC-1 gene can be detected by end-point RT-PCR. In this case, it is preferred to use a primer consisting of the base sequence described in SEQ ID NO: 1 and a primer consisting of the base sequence described in SEQ ID NO: 2. Here, end-point RT-PCR refers to a normal RT-PCR other than real-time RT-PCR.

[0046] In the method of this embodiment, the expression of KK-LC-1 gene can be detected by real-time RT-PCR. As shown in the examples described below, the detection of the expression of KK-LC-1 gene by real-time RT-PCR can more accurately assess the risk of gastric cancer in patients after Helicobacter pylori eradication compared with endpoint RT-PCR.

[0047] In real-time RT-PCR, qPCR probes are preferably used. qPCR probes are also called TaqMan (registered trademark) probes, which are oligonucleotides modified with fluorescent substances and quenching substances. The qPCR probe specifically hybridizes with the template DNA in the annealing step in the PCR reaction. For qPCR probes, since there is a quenching substance on the probe, the generation of fluorescence can be suppressed even when irradiated with excitation light.

[0048] In the subsequent extension reaction step, the qPCR probe hybridized with the model is degraded by the 5'→3' exonuclease activity of Taq DNA polymerase. As a result, the fluorescent substance and the quenching substance are released, and the inhibition by the quenching substance is released, and fluorescence is emitted by irradiation with excitation light.

[0049] In real-time RT-PCR using a qPCR probe, nucleic acid is quantified by utilizing the linear relationship between the cycle number at which the PCR amplification product, that is, the fluorescence intensity reaches a certain level and the initial amount of target DNA.

[0050] In real-time RT-PCR, for example, the difference between the Ct value of β-actin and the Ct value of KK-LC-1 gene can be calculated as ΔCt, and in this case, for example, a ΔCt of -2 or more can be classified as a high expression group, a ΔCt of -5 or more and less than -2 can be classified as a medium expression group, and a ΔCt of less than -5 can be classified as a low expression group.

[0051] The NCBI accession number of the cDNA of the human β-actin gene is NM_001101.5. In addition, the NCBI accession number of the human β-actin gene is NP_001092.1.

[0052] As shown in the examples described below, the cumulative rate of gastric cancer in the five-year period was 40% in the KK-LC-1 high expression group, and 5% in the KK-LC-1 medium expression group. In contrast, the cumulative rate of gastric cancer in the five-year period was 0% in the KK-LC-1 low expression group. Therefore, when the Ct value of β-actin is used as a reference, it can be said that the smaller the ΔCt, the lower the risk of gastric cancer in patients after Helicobacter pylori eradication.

[0053] In the method of this embodiment, the qPCR probe preferably has a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, with a base length of 15 to 50 bases, and has 1 or 2 base mutations relative to the base sequence recorded in sequence number 3.

[0054] The genomic DNA encoding KK-LC-1 has a first exon, an intron, and a second exon. SEQ ID NO: 4 shows the base sequence of the genomic DNA of KK-LC-1.

[0055] The base sequence recorded in sequence number 3 is the base sequence of the cDNA of the KK-LC-1 gene. The 195th base in sequence number 3 corresponds to the last base of the first exon of KK-LC-1. In addition, the 196th base in sequence number 3 corresponds to the first base of the second exon of KK-LC-1.

[0056] In the method of this embodiment, the qPCR probe preferably has a base sequence including the 195th and 196th bases in the base sequence described in SEQ ID NO: 3. That is, the qPCR probe is preferably a probe that hybridizes with the junction of the first exon and the second exon in the cDNA of the KK-LC-1 gene.

[0057] Such a qPCR probe reduces hybridization with genomic DNA of the KK-LC-1 gene while maintaining the hybridization ability with the cDNA of the KK-LC-1 gene. As a result, the cDNA of the KK-LC-1 gene can be detected more accurately.

[0058] The base length of the qPCR probe is not particularly limited as long as the Tm value of the qPCR probe can be appropriately set, and may be about 15 to 50 bases, for example, 15 to 40 bases, or about 15 to 30 bases.

[0059] qPCR probes can be modified with a minor groove binder (MGB). By modifying with MGB, the Tm value can be increased. Therefore, when the qPCR probe is modified with MGB, the base length of the qPCR probe can also be designed to be shorter.

[0060] The MGB is not particularly limited as long as it increases the Tm value, and examples thereof include dihydropyrroloindole tripeptide (CDPI3) and Hoechst 33258.

[0061] In the method of the present embodiment, the qPCR probe preferably has a mutation of one or two bases relative to the base sequence of the cDNA of the KK-LC-1 gene (SEQ ID NO: 3).

[0062] The base sequence of the second exon contains a portion similar to the base sequence of the intron of the KK-LC-1 gene. Therefore, the qPCR probe that hybridizes with the junction of the first exon and the second exon in the cDNA of the KK-LC-1 gene may also hybridize with the genomic DNA of the KK-LC-1 gene.

[0063] In contrast, as shown in the examples described below, if the qPCR probe has a mutation of 1 or 2 bases relative to the base sequence of the cDNA of the KK-LC-1 gene (sequence number 3), the hybridization ability to the cDNA of the KK-LC-1 gene is maintained while the hybridization with the genomic DNA of the KK-LC-1 gene is reduced. As a result, the cDNA of the KK-LC-1 gene can be detected more accurately. As such a qPCR probe, for example, a qPCR probe having a base sequence recorded in any one of sequence numbers 5 to 10 can be cited. Among them, a qPCR probe having a base sequence recorded in any one of sequence numbers 7 to 10 is preferred.

[0064] In the method of the present embodiment, the real-time RT-PCR can also be performed in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification. For the single-stranded nucleic acid fragment, preferably: having a base sequence of the 322nd and 323rd bases or the 1096th and 1097th bases in the base sequence (SEQ ID NO. 4) of the genomic DNA containing KK-LC-1, the base length is 20 to 50 bases. For the base length of the single-stranded nucleic acid fragment, as long as the Tm value of the single-stranded nucleic acid fragment can be appropriately set, there is no particular limitation, it can be about 20 to 50 bases, for example, 20 to 40 bases, for example, 25 to 35 bases, for example, 20 to 34 bases, or for example, 20 to 31 bases.

[0065] The 1096th base in SEQ ID NO. 4 corresponds to the first base in the intron of KK-LC-1. The 1097th base in SEQ ID NO. 4 corresponds to the last base in the first exon of KK-LC-1.

[0066] Therefore, a single-stranded nucleic acid fragment having a base sequence including bases 1096 and 1097 in SEQ ID NO: 4 hybridizes at the junction of the first exon and intron of the genomic DNA of KK-LC-1, and can inhibit the hybridization of the qPCR probe with the genomic DNA of KK-LC-1. As a result, the cDNA of the KK-LC-1 gene can be detected more accurately.

[0067] The 322nd base in SEQ ID NO. 4 corresponds to the 1st base in the second exon of KK-LC-1. In addition, the 323rd base in SEQ ID NO. 4 corresponds to the last base in the intron of KK-LC-1.

[0068] Therefore, a single-stranded nucleic acid fragment having a base sequence including bases at positions 322 and 323 in SEQ ID NO: 4 hybridizes at the junction of the intron and the second exon of the genomic DNA of KK-LC-1, and can inhibit the hybridization of the qPCR probe with the genomic DNA of KK-LC-1. As a result, the cDNA of the KK-LC-1 gene can be detected more accurately. As such a single-stranded nucleic acid fragment, for example, a single-stranded nucleic acid fragment having a base sequence described in any one of SEQ ID NOs: 11 to 13 can be cited.

[0069] In the method of this embodiment, the expression of the KK-LC-1 gene can be detected at the protein level. In this case, the KK-LC-1 protein in the gastric tissue sample can be detected by ELISA, Western blotting, etc. using a specific binding substance for KK-LC-1. The specific binding substance is described below.

[0070] [Kit for assessing the risk of gastric cancer in patients who have been sterilized with Helicobacter pylori]

[0071] In one embodiment, the present invention provides a kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori sterilization, the kit comprising a primer consisting of the base sequence recorded in sequence number 1 and a primer consisting of the base sequence recorded in sequence number 2.

[0072] The kit of this embodiment further includes a qPCR probe having a base sequence including the 195th and 196th bases in the base sequence recorded in SEQ ID NO: 3, a base length of 15 to 50 bases, and may have a mutation of 1 or 2 bases relative to the base sequence recorded in SEQ ID NO: 3. The qPCR probe is the same as the above-mentioned qPCR probe.

[0073] The kit of this embodiment further comprises a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, wherein the single-stranded nucleic acid fragment has a base sequence that includes the 322nd and 323rd bases, or the 1096th and 1097th bases in the base sequence recorded in SEQ ID NO: 4, and the base length may be 20 to 50 bases. The single-stranded nucleic acid fragment that inhibits genomic DNA amplification is the same as the single-stranded nucleic acid fragment described above.

[0074] The kit of this embodiment may include a specific binding substance for KK-LC-1 protein. As a specific binding substance for KK-LC-1 protein, an antibody for KK-LC-1, an aptamer for KK-LC-1, etc. can be cited. The antibody can be an antibody fragment. As an antibody fragment, F(ab')2, Fab', Fab, Fv, scFv, etc. can be cited. In addition, as an aptamer, a nucleic acid aptamer, a peptide aptamer, etc. can be cited.

[0075] Using these kits, the above-mentioned method for assessing the risk of developing gastric cancer can be appropriately carried out.

[0076] [Polynucleotide]

[0077] In one embodiment, the present invention provides a polynucleotide having a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, with a base length of 15 to 50 bases, and having a mutation of 1 or 2 bases relative to the base sequence recorded in sequence number 3.

[0078] The polynucleotide of the present embodiment is preferably modified with a fluorescent substance and a quencher substance to constitute a qPCR probe.

[0079] As the fluorescent substance, substances generally used for qPCR probes can be used without particular limitation. Specific fluorescent substances include, for example, fluorescein, Alexa488, ATTO542, Alexa647, FAM, Cy5, and Cy3.

[0080] As the quenching substance, substances commonly used for qPCR probes can be used without particular limitation. Specific quenching substances include, for example, Black Hole Quencher (BHQ) (registered trademark) -1, BHQ (registered trademark) -2, BHQ (registered trademark) -3, Iowa Black FQ, Iowa Black RQ, etc. As the quenching substance, a substance that can quench the fluorescence of the fluorescent substance used is selected.

[0081] The polynucleotide of this embodiment can be suitably used as the above-mentioned qPCR probe. By performing real-time RT-PCR using the polynucleotide of this embodiment as a qPCR probe, cDNA of the KK-LC-1 gene can be detected more accurately.

[0082] In another embodiment, the present invention provides a polynucleotide having a base sequence comprising bases at positions 322 and 323, or bases at positions 1096 and 1097 in the base sequence described in SEQ ID NO: 4, and having a base length of 20 to 50 bases. The polynucleotide of this embodiment can be suitably used as the above-mentioned single-stranded nucleic acid fragment for inhibiting genomic DNA amplification.

[0083] More specific examples of the single-stranded nucleic acid fragment include a single-stranded nucleic acid fragment having a base sequence as described in any one of SEQ ID NOs: 11 to 13. As described above, by performing real-time RT-PCR in the presence of the single-stranded nucleic acid fragment of the present embodiment, cDNA of the KK-LC-1 gene can be detected more accurately.

[0084] [Method for detecting the expression of KK-LC-1 gene in a biological sample]

[0085] In one embodiment, the present invention provides a method for detecting the expression of the KK-LC-1 gene in a biological sample, wherein the detection is performed by real-time RT-PCR, wherein the real-time RT-PCR is performed using a qPCR probe in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, wherein the single-stranded nucleic acid fragment has a base sequence comprising the 322nd and 323rd bases, or the 1096th and 1097th bases in the base sequence described in SEQ ID NO: 4, and the base length is 20 to 50 bases. The single-stranded nucleic acid fragment is the same as the single-stranded nucleic acid fragment described above.

[0086] As described above, the single-stranded nucleic acid fragment hybridizes at the junction of the first exon and intron of the genomic DNA of KK-LC-1, and can inhibit the hybridization of the qPCR probe with the genomic DNA of KK-LC-1. As a result, as shown in the examples described later, the cDNA of the KK-LC-1 gene can be detected more accurately. Here, more accurate detection means that false positive detection can be suppressed compared to the case where the above-mentioned single-stranded nucleic acid fragment is not used.

[0087] In the method of this embodiment, examples of biological samples include tissues, serum, plasma, cultured cells, cell culture supernatants, and the like derived from humans or non-human animals.

[0088] In the method of the present embodiment, the qPCR probe has a base sequence comprising the 195th and 196th bases in the base sequence recorded in sequence number 3, preferably a base length of 15 to 50 bases, and has a mutation of 1 or 2 bases relative to the base sequence recorded in sequence number 3. As described above, this qPCR probe maintains hybridization ability against the cDNA of the KK-LC-1 gene while reducing hybridization of the genomic DNA of the KK-LC-1 gene. As a result, the cDNA of the KK-LC-1 gene can be detected more accurately. As described above, as such a qPCR probe, for example, a qPCR probe having a base sequence recorded in any one of sequence numbers 5 to 10 can be cited. Among them, a qPCR probe having a base sequence recorded in any one of sequence numbers 7 to 10 is preferred.

[0089] Example

[0090] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0091] [Experimental Example 1]

[0092] (Study based on end-point RT-PCR)

[0093] In 111 patients who had been eradicated of Helicobacter pylori, gastric tissue samples collected before eradication were used for RT-PCR to detect the expression of the KK-LC-1 gene.

[0094] In RT-PCR, primers consisting of the base sequence recorded in sequence number 1 and primers consisting of the base sequence recorded in sequence number 2 were used. After 40 cycles of PCR reaction, agarose gel electrophoresis was performed, and the case where a visible band was detected was judged as positive, and the case where no visible band was detected was judged as negative. In addition, a 5-year follow-up survey was conducted on the occurrence of gastric cancer in each patient after Helicobacter pylori was eradicated.

[0095] Figure 1 This is a graph showing the cumulative rate of gastric cancer in the KK-LC-1 positive group and the KK-LC-1 negative group over a five-year period. The results show that the cumulative rate of gastric cancer in the KK-LC-1 positive group was 43%. In contrast, the cumulative rate of gastric cancer in the KK-LC-1 negative group was 1%.

[0096] This result indicates that when the expression of the KK-LC-1 gene is positive in the gastric tissue sample of a patient before Helicobacter pylori eradication, the patient is at risk of developing gastric cancer after eradication.

[0097] [Experimental Example 2]

[0098] (Real-time RT-PCR based study)

[0099] In Experimental Example 1, cancer cases were also observed in the KK-LC-1 negative group. Therefore, the expression of KK-LC-1 gene was detected using real-time RT-PCR with higher sensitivity.

[0100] In 96 patients who had been eradicated of Helicobacter pylori, gastric tissue samples collected before eradication were used for real-time RT-PCR to detect the expression of the KK-LC-1 gene.

[0101] In real-time RT-PCR, primers consisting of the base sequence recorded in sequence number 1 and primers consisting of the base sequence recorded in sequence number 2 were used, and further, a qPCR probe (named "MGB") consisting of the base sequence recorded in sequence number 8 was used. MGB-D is a qPCR probe whose 5' end is modified with a fluorescent substance and whose 3' end is modified with a quenching substance and a minor groove binder (Minor Groove Binder, MGB). MGB is a substance that increases the Tm value.

[0102] The difference between the Ct value of β-actin and the Ct value of KK-LC-1 gene was calculated and set as ΔCt. According to the ΔCt value, the KK-LC-1 high expression group, the KK-LC-1 medium expression group, and the KK-LC-1 low expression group were classified. Specifically, the case where ΔCt was -2 or more was classified as the high expression group. In addition, the case where ΔCt was -5 or more and less than -2 was classified as the medium expression group. In addition, the case where ΔCt was less than -5 was classified as the low expression group.

[0103] Figure 2 It is a graph showing the proportion of patients in each group who have suffered from gastric cancer in 5 years. As a result, in the KK-LC-1 high expression group, the proportion of gastric cancer in 5 years was 40%, and in the KK-LC-1 medium expression group, the proportion of gastric cancer in 5 years was 5%. In contrast, in the KK-LC-1 low expression group, the proportion of gastric cancer in 5 years was 0%. This result shows that the detection sensitivity of KK-LC-1 is increased by high-sensitivity real-time RT-PCR. It also shows that the expression level of the KK-LC-1 gene in the gastric tissue sample of the patient before the Helicobacter pylori eradication is related, and the patient has a risk of gastric cancer after the eradication. It also shows that when the expression level of the KK-LC-1 gene in the gastric tissue sample of the patient before the Helicobacter pylori eradication is low (when ΔCt is less than -5), it can be judged that the patient has a low risk of gastric cancer after the eradication.

[0104] [Experimental Example 3]

[0105] (qPCR probe study 1)

[0106] Plasmids containing genomic DNA and cDNA of the KK-LC-1 gene were amplified by real-time PCR using primers consisting of the base sequence described in SEQ ID NO: 1 and primers consisting of the base sequence described in SEQ ID NO: 2, and further using the qPCR probes shown in Table 1 below. The concentration of the qPCR probe in the real-time PCR reaction solution was 0.25 μM.

[0107] [Table 1]

[0108] name Serial Number The existence of MGB The number of mutations Base length MGB-A 5 have 4 17 MGB-B 6 have 3 19 MGB-C 7 have 2 18 MGB-D 8 have 2 18 Non-MGB-E 9 none 2 22 Non-MGB-F 10 none 1 22

[0109] Table 1 shows the presence or absence of MGB (Minor Groove Binder), the number of mutations, and the base length for each qPCR probe. The qPCR probes shown in Table 1 all have a c The base sequence of the 195th and 196th bases in the base sequence of the DNA (sequence number 3). Therefore, the qPCR probes shown in Table 1 all hybridize at the junction of the first exon and the second exon in the cDNA of the KK-LC-1 gene. In addition, the qPCR probes shown in Table 1 have 1 to 4 base mutations relative to the base sequence of the cDNA of the KK-LC-1 gene (sequence number 3). In Table 1, the number of mutations refers to the number of mutations relative to sequence number 3.

[0110] In addition, for comparison, a commercially available qPCR probe for the cDNA of the KK-LC-1 gene (named "Hs02386421_g1", Thermo Fisher Scientific) was used. Hs02386421_g1 is a qPCR probe modified with a fluorescent substance at the 5' end and a quenching substance and MGB at the 3' end. Although the manufacturer has not disclosed the base sequence of Hs02386421g1, no mutation has been introduced into the base sequence of the cDNA of the KK-LC-1 gene.

[0111] Figure 3 It means that the KK-LC-1 gene will be included c Figure 1 shows the results of real-time PCR using a DNA plasmid as a model. The qPCR probe must react with the cDNA of the KK-LC-1 gene. Figure 3 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the cycle number. In addition, the figure shows a cell line containing the KK-LC-1 gene used as a model. c The amount of plasmid DNA.

[0112] The results showed that MGB-A and MGB-B did not interact with the KK-LC-1 gene. cDNA reaction. In contrast, MGB-C and MGB-D showed no significant effect on the KK-LC-1 gene. c DNA showed reactivity.

[0113] Figure 4 It is a figure which shows the result of real-time PCR using genomic DNA as a model. Figure 4 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the number of cycles. In addition, in the figure, the amount of genomic DNA used as a model is shown. As genomic DNA, genomic DNA of H-111-TC, a human gastric cancer cell line, was used. Preferably, the qPCR probe does not react with the genomic DNA.

[0114] As a result, it was confirmed that a commercially available qPCR probe (named "Hs02386421_g1") for cDNA of the KK-LC-1 gene also reacted with genomic DNA. On the other hand, it was confirmed that MGB-C and MGB-D did not react with genomic DNA.

[0115] Figure 5 It is a figure which shows the result of real-time PCR using cDNA as a model. Figure 5 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the cycle number. In addition, the figure shows the amount of the plasmid containing the cDNA of the KK-LC-1 gene used as a model. The qPCR probe must react with the cDNA of the KK-LC-1 gene.

[0116] The results show that from Figure 5 From the left, Non-MGB-E and Non-MGB-F reacted with the cDNA of the KK-LC-1 gene at the same Ct value as MGB-D. Figure 5 From the right, Non-MGB-E and Non-MGB-F show higher sensitivity compared to MGB-D.

[0117] Figure 6 It is a figure which shows the result of real-time PCR using genomic DNA as a model. Figure 6 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the number of cycles. In addition, in the figure, the amount of genomic DNA used as a model is shown. As genomic DNA, genomic DNA of H-111-TC, a human gastric cancer cell line, was used. Preferably, the qPCR probe does not react with the genomic DNA.

[0118] The results showed that MGB-D and Non-MGB-E also reacted with genomic DNA when 10 ng of genomic DNA was used, while Non-MGB-F showed low reactivity with genomic DNA.

[0119] [Experimental Example 4]

[0120] (qPCR probe study 2)

[0121] A plasmid containing genomic DNA and cDNA of the KK-LC-1 gene was amplified by real-time PCR using primers consisting of the base sequence described in SEQ ID NO: 1, primers consisting of the base sequence described in SEQ ID NO: 2, MGB-D as the qPCR probe shown in Table 1, and single-stranded nucleic acid fragments shown in Table 2. The concentration of the qPCR probe in the real-time PCR reaction solution was 0.25 μM. In addition, the concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction solution was 1.0 μM.

[0122] [Table 2]

[0123] name Serial Number Base length B lock1 11 31 Block2 12 31 Block 3 13 27

[0124] In Table 2, the base length is shown for each single-stranded nucleic acid fragment. The single-stranded nucleic acid fragments shown in Table 2 are single-stranded nucleic acid fragments that inhibit genomic DNA amplification. The single-stranded nucleic acid fragments shown in Table 2 all have a base sequence containing the 322nd and 323rd bases in the base sequence (SEQ ID NO. 4) of the genomic DNA of KK-LC-1. Therefore, the single-stranded nucleic acid fragments shown in Table 2 all hybridize at the junction of the intron and the second exon in the genomic DNA of KK-LC-1.

[0125] Figure 7 It is a figure which shows the result of real-time PCR using genomic DNA as a model. Figure 7 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the number of cycles. In addition, in the figure, the amount of genomic DNA used as a model is shown. As genomic DNA, genomic DNA of H-111-TC, a human gastric cancer cell line, was used. Preferably, the qPCR probe does not react with the genomic DNA.

[0126] The results confirmed that MGB-D also reacted with genomic DNA when 25 ng of genomic DNA was used. On the other hand, it was found that the reaction of MGB-D with genomic DNA was inhibited when MGB-D was co-present with any of Block 1, Block 2, and Block 3.

[0127] Figure 8 It is a figure which shows the result of real-time PCR using cDNA as a model. Figure 8 In the graph, the ordinate represents the fluorescence intensity (relative value) and the abscissa represents the cycle number. In addition, the figure shows the amount of plasmid containing cDNA of KK-LC-1 gene used as a model. c DNA reaction.

[0128] The results show that the reactivity of MGB-D is not adversely affected even when MGB-D is coexistent with any of Block 1, Block 2, and Block 3. Figure 8 As shown on the right, when MGB-D coexists with any of Block1, Block2, and Block3, the expression of KK-LC-1 gene c The detection sensitivity of DNA increases.

[0129] [Experimental Example 5]

[0130] (qPCR probe study 3)

[0131] A plasmid containing genomic DNA and cDNA of the KK-LC-1 gene was amplified by real-time PCR using primers consisting of the base sequence recorded in sequence number 1, primers consisting of the base sequence recorded in sequence number 2, Non-MGB-E or Non-MGB-F as qPCR probes shown in Table 1, and Block 1 as a single-stranded nucleic acid fragment shown in Table 2. The concentration of the qPCR probe in the real-time PCR reaction solution was 0.25 μM. In addition, the concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction solution was 1.0 μM. In addition, for comparison, a group using MGB-D as a qPCR probe was also prepared.

[0132] Fig. 9 It is a figure which shows the result of real-time PCR using cDNA as a model. Fig. 9 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the cycle number. In addition, the figure shows the amount of the plasmid containing the cDNA of the KK-LC-1 gene used as a model. The qPCR probe must react with the cDNA of the KK-LC-1 gene.

[0133] The results showed that even when Block1 coexists with Non-MGB-E or Non-MGB-F, it does not have an adverse effect on the reactivity.

[0134] The above results indicate that the qPCR probes shown in Table 1 or the combination of the qPCR probes shown in Table 1 and the single-stranded nucleic acid fragments shown in Table 2 are suitable for detecting the expression of the KK-LC-1 gene.

[0135] [Experimental Example 6]

[0136] (qPCR probe study 4)

[0137] Genomic DNA was amplified by real-time PCR using primers consisting of the base sequence recorded in sequence number 1, primers consisting of the base sequence recorded in sequence number 2, MGB-D, Non-MGB-E, and Non-MGB-F as qPCR probes shown in Table 1 above, and Block 1 as a single-stranded nucleic acid fragment shown in Table 2 above.

[0138] In addition, for comparison, a commercially available qPCR probe for the cDNA of the KK-LC-1 gene (named "Hs02386421_g1", Thermo Fisher Scientific) was also used. Hs02386421_g1 is a qPCR probe modified with a fluorescent substance at the 5' end and a quenching substance and MGB at the 3' end. Although the manufacturer has not disclosed the base sequence of Hs02386421_g1, no mutation has been introduced into the base sequence of the cDNA of the KK-LC-1 gene.

[0139] The concentration of the qPCR probe in the real-time PCR reaction solution was 0.25 μM. The concentration of the single-stranded nucleic acid fragment in the real-time PCR reaction solution was 1.0 μM.

[0140] Fig.10 and Fig.11 It is a figure which shows the result of real-time PCR using genomic DNA as a model. Fig.10 and Fig.11 In the figure, the ordinate represents the fluorescence intensity (relative value), and the abscissa represents the number of cycles. In addition, in the figure, the amount of genomic DNA used as a model is shown. As genomic DNA, genomic DNA of H-111-TC, a human gastric cancer cell line, was used. Preferably, the qPCR probe does not react with the genomic DNA.

[0141] As a result, it was confirmed that Hs02386421_g1, MGB-D, Non-MGB-E, and Non-MGB-F also reacted with genomic DNA. On the other hand, it was revealed that when Block1 was coexisted with these qPCR probes, the reaction with genomic DNA was suppressed.

[0142] [Experimental Example 7]

[0143] (Study using clinical test samples)

[0144] Cancer tissue samples from breast cancer patients and cancer tissue samples from colorectal cancer patients were subjected to endpoint RT-PCR and real-time RT-PCR, respectively, to detect the expression of KK-LC-1 gene.

[0145] In the end-point RT-PCR, primers consisting of the base sequence described in SEQ ID NO: 1 and primers consisting of the base sequence described in SEQ ID NO: 2 were used. After 40 cycles of PCR reaction, agarose gel electrophoresis was performed, and the case where a visible band was detected was judged as positive, and the case where no visible band was detected was judged as negative.

[0146] In real-time RT-PCR, primers consisting of the base sequence described in SEQ ID NO: 1 and primers consisting of the base sequence described in SEQ ID NO: 2, and MGB-D as a qPCR probe shown in Table 1 were used. In addition, real-time RT-PCR was performed in the presence or absence of Block 1 as a single-stranded nucleic acid fragment shown in Table 2. The difference between the Ct value of β-actin and the Ct value of the KK-LC-1 gene was determined and defined as ΔCt.

[0147] The results using cancer tissue samples from breast cancer patients are shown in Table 3 below, and the results using cancer tissue samples from colorectal cancer patients are shown in Table 4 below. As a result, except for the test sample No. 7 from a colorectal cancer patient, the expression of the KK-LC-1 gene was detected in the real-time RT-PCR even when it was below the detection limit in the endpoint RT-PCR. In the test sample No. 7 from a colorectal cancer patient, the expression of the KK-LC-1 gene was not detected in both the endpoint RT-PCR and the real-time RT-PCR.

[0148] In the case of the absence of Block 1, the expression of the KK-LC-1 gene was detected in the test sample Nos. 8 and 9 from breast cancer patients and the test sample Nos. 3, 5, 6, and 8 from colorectal cancer patients, but the expression of the KK-LC-1 gene was not detected in the presence of Block 1. It is believed that this result is a false positive result of the reaction of the qPCR probe with genomic DNA in the absence of Block 1. This indicates that the false positive can be suppressed in the presence of Block 1, and the accurate expression of the KK-LC-1 gene can be detected.

[0149] [Table 3]

[0150] (Breast Cancer)

[0151]

[0152] [Table 4]

[0153] (Colorectal Cancer)

[0154]

[0155] Industrial Applicability

[0156] According to the present invention, it is possible to provide a technique for evaluating the risk of gastric cancer in a patient who has been freed of Helicobacter pylori.

Claims

1. A method for assessing the risk of gastric cancer in a patient after Helicobacter pylori sterilization, the method comprising: A step of detecting the expression of KK-LC-1 gene in a gastric tissue sample from the patient before Helicobacter pylori eradication, wherein a positive expression of KK-LC-1 gene indicates that the patient has a risk of developing gastric cancer after Helicobacter pylori eradication.

2. The method according to claim 1, wherein: The expression of KK-LC-1 gene was detected by end-point RT-PCR.

3. The method according to claim 2, wherein: In the endpoint RT-PCR, a primer consisting of the base sequence described in SEQ ID NO: 1 and a primer consisting of the base sequence described in SEQ ID NO: 2 are used.

4. The method according to claim 1, wherein: The expression of KK-LC-1 gene was detected by real-time RT-PCR.

5. The method according to claim 4, wherein: In the real-time RT-PCR, a qPCR probe is used, wherein the qPCR probe has a base sequence including bases at positions 195 and 196 in the base sequence described in SEQ ID NO: 3, The base length is 15 to 50 bases. The nucleotide sequence of SEQ ID NO: 3 has one or two base mutations.

6. The method according to claim 4 or 5, wherein: The real-time RT-PCR is performed in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, The single-stranded nucleic acid fragment has a base sequence including bases at positions 322 and 323, or bases at positions 1096 and 1097, in the base sequence described in SEQ ID NO:

4. The base length is 20 to 50 bases.

7. The method according to claim 1, wherein: The expression of KK-LC-1 gene was detected at the protein level.

8. A kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori has been sterilized, the kit comprising a primer consisting of the base sequence recorded in SEQ ID NO: 1 and a primer consisting of the base sequence recorded in SEQ ID NO:

2.

9. The kit according to claim 8, wherein Also contains qPCR probes, The qPCR probe has a base sequence including bases at positions 195 and 196 in the base sequence described in SEQ ID NO: 3, The base length is 15 to 50 bases. The nucleotide sequence of SEQ ID NO: 3 has one or two base mutations.

10. The kit according to claim 8 or 9, wherein It also contains a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, The single-stranded nucleic acid fragment has a base sequence including bases at positions 322 and 323, or bases at positions 1096 and 1097, in the base sequence described in SEQ ID NO:

4. The base length is 20 to 50 bases.

11. A kit for evaluating the risk of gastric cancer in a patient after Helicobacter pylori has been eliminated, the kit comprising a specific binding substance for KK-LC-1 protein.

12. A polynucleotide, characterized in that A base sequence comprising the 195th and 196th bases in the base sequence recorded in SEQ ID NO: 3, a base length of 15 to 50 bases, and a mutation of 1 or 2 bases relative to the base sequence recorded in SEQ ID NO:

3.

13. A polynucleotide, characterized in that having a base sequence including the 322nd and 323rd bases, or the 1096th and 1097th bases in the base sequence described in SEQ ID NO: 4, The base length is 20 to 50 bases.

14. A method for detecting the expression of KK-LC-1 gene in a biological sample, wherein the detection is performed by real-time RT-PCR, wherein the real-time RT-PCR is performed using a qPCR probe in the presence of a single-stranded nucleic acid fragment that inhibits genomic DNA amplification, The single-stranded nucleic acid fragment has a base sequence including bases at positions 322 and 323, or bases at positions 1096 and 1097, in the base sequence described in SEQ ID NO:

4. The base length is 20 to 50 bases.

15. The method according to claim 14, wherein: The qPCR probe has a base sequence including bases at positions 195 and 196 in the base sequence described in SEQ ID NO: 3, The base length is 15 to 50 bases. The nucleotide sequence of SEQ ID NO: 3 has one or two base mutations.

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