CN114150060A - Molecular marker and kit for diagnosing digestive system tumor - Google Patents
Molecular marker and kit for diagnosing digestive system tumor Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses a molecular marker and a kit for diagnosing digestive system tumor, in particular to application of L3MBTL4 gene promoter region DNA methylation as a molecular marker in preparation of a product for diagnosing digestive system tumor. According to the invention, hypermethylation of the L3MBTL4 promoter region is frequently found in pancreatic cancer, liver cancer, gastric cancer and esophageal cancer for the first time by an MSP method, L3MBTL4 is found to participate in an NHEJ pathway of DNA damage repair, and L3MBTL4 methylation is a synergistic lethal marker for killing tumor cells by combining CHK1 inhibitor with cisplatin and other therapeutic drugs. The invention also provides a primer and a kit for detecting the methylation state of the L3MBTL4 gene promoter region. The primer and the kit for detecting the hypermethylation state of the L3MBTL4 gene promoter region can be used as powerful means for diagnosing pancreatic cancer, liver cancer, stomach cancer and esophageal cancer, observing curative effect, judging prognosis, detecting minimal residual disease and the like, and have the advantages of simple and convenient operation, good stability and profound clinical significance and popularization.
Description
Technical Field
The invention relates to the field of biotechnology. More particularly, it relates to a molecular marker and a kit for diagnosing digestive system tumor.
Background
Pancreatic cancer is a highly malignant cancer of the digestive tract, with a 5-year survival rate of less than 10%, and is called "king of cancer". In recent years, the incidence and mortality of pancreatic cancer have been on the rise, and it is expected that this will become the second lethal tumor in the world by 2030. The 2017 year data of cancer center in China show that pancreatic cancer is in the 7 th position of the incidence rate of malignant tumor of male in China, the 11 th position of female in China, and is located in the 6 th position of the mortality rate related to malignant tumor. Pancreatic cancer is insidious, has atypical early symptoms, progresses to late stage at the time of diagnosis, and loses the operation chance. The risk of postoperative recurrence is also high in patients with resectable pancreatic cancer, and local recurrence or distant metastasis occurs in the early postoperative period in some patients. Domestic data show that for 3279 patients after pancreatic cancer resection in 2016 + 2019, the recurrence rate within 9 months is 45.87% (Zhaoyiping, Chinese guidelines for pancreatic cancer diagnosis and treatment (2021); Chinese journal of practical surgery, 2021.7). Groot et al analyzed 957Patients after surgery for Pancreatic cancer, 51.5% of Patients had local Recurrence or distant metastasis within one year after surgery (Groot, v.p., et al, Defining and Predicting Early recovery in 957Patients With selected surgery dual anticancer. ann Surg,2019.269(6): p.1154-1162.).
Primary liver cancer is one of the common malignant tumors worldwide, and the incidence rate of the primary liver cancer is ranked 6 th among the worldwide malignant tumors in 2018, and is the cause of death of the 4 th tumor. Hepatocellular carcinoma, called liver cancer for short, is found in 75-85% of cases of primary liver cancer. China is a big liver cancer country, about half of new cases occur in China all over the world, and 30-40 million people die of liver cancer every year. In China, chronic hepatitis B-cirrhosis is the leading cause of liver cancer. The liver has no pain nerve, no obvious symptoms exist in the early canceration stage, liver pain, anorexia, epigastric discomfort, hypodynamia and the like appear in a few patients, and most liver cancer patients lose the best treatment opportunity after being diagnosed in the middle and late stage.
Gastric cancer is the most common digestive tract malignant tumor in China, the prognosis is poor, the overall survival rate in 5 years is 35.1%, and the health of people is seriously harmed. According to statistics, the new cases and the death cases of the stomach cancer are respectively the fifth and the fourth in 2020, wherein 43.9 percent of the cases and 48.6 percent of the death cases of the stomach cancer occur in China. Helicobacter pylori infection is the most important risk factor for gastric cancer. Most of patients with gastric cancer have atypical early symptoms, such as abdominal pain, hypodynamia, emaciation and the like, and most of the patients have advanced to the late stage when the diagnosis is confirmed.
Esophageal cancer is one of the high-grade malignant tumors in China, and ranks the fourth place in death reasons of malignant tumors in China. In 2020, the number of the esophageal cancer patients reaches 32 thousands and the number of the esophageal cancer patients death 30 thousands, which account for about 55 percent of the whole world. Esophageal cancer has obvious regional and population distribution, and China is mainly distributed in Taihang mountainous areas, which indicates that the environment is a risk factor for the onset of esophageal cancer. Esophageal cancer is classified into squamous cell carcinoma and adenocarcinoma, and about 90% of esophageal cancers in China are squamous cell carcinoma. Surgery is the most effective treatment for esophageal cancer, but most diagnoses are made in the middle and late stages, and the chance of surgery is lost due to the atypical early symptoms. Therefore, finding a highly sensitive and specific early diagnosis method and finding a treatable target is of great significance for improving the therapeutic effect and prognosis of the above-mentioned malignant tumor.
With the advent of the post-genomic era, Epigenetics (Epigenetics) has become the leading edge of life sciences research. In the field of digestive tumors, epigenetic studies have focused on DNA methylation and histone acetylation. DNA methylation modification is a main mode in genome epigenetic regulation, the occurrence and development of human tumors are related to DNA methylation abnormality, and hypermethylation of CpG islands in gene promoter regions can cause the expression of cancer suppressor genes to be silenced, thereby causing tumorigenesis. DNA methylation refers to the transfer of a methyl group to carbon atom number 5 of a CG dinucleotide cytosine to form a 5 methylcytosine, using s-adenosylmethionine as a methyl donor, under the action of a methyltransferase. DNA methylation abnormality is an early and frequent event in the canceration process of cells, so that methylation of specific genes can be used as a molecular marker for early diagnosis of cancer, a therapeutic target and a prognostic judgment means.
The method aims to screen out the diagnosis molecular markers with strong sensitivity and high specificity for pancreatic cancer, liver cancer, gastric cancer or esophageal cancer, search for an effective detection method and establish an effective monitoring means, so that early diagnosis and early treatment are realized, and important clinical application value is realized.
Disclosure of Invention
The first purpose of the invention is to provide a molecular marker for diagnosing digestive system tumors, which has the characteristics of strong sensitivity and high specificity, and the application of the molecular marker in preparing products for diagnosing pancreatic cancer, liver cancer, gastric cancer or esophageal cancer.
The second purpose of the invention is to provide an application of the substance for detecting the molecular marker in preparing products for diagnosing digestive system tumors.
The third purpose of the invention is to provide a kit for diagnosing digestive system tumor based on the molecular marker.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a DNA fragment of a promoter region of an L3MBTL4 gene as a molecular marker in preparation of a product for diagnosing digestive system tumors, wherein the digestive system tumors are pancreatic cancer, liver cancer, stomach cancer or esophageal cancer.
According to a specific embodiment of the invention, the nucleotide sequence of the L3MBTL4 gene promoter region DNA fragment as a molecular marker is shown as SEQ ID No.1 (original DNA sequence before sulfuration treatment), and comprises at least one methylation site.
The MBT domain is a domain that binds to histone tail methylated lysine (as shown in fig. 3), and during development binds to the promoter region of the target gene to inhibit its transcription. L3MBTL4 is a member of this family. Based on similar mechanisms of tumors and development processes, expression conditions of different members of an MBT gene family are screened in cell lines of different tumors, expression deficiency of L3MBTL4 in various tumor cells is found, expression regulation mechanisms of the tumor cells are analyzed, and frequent methylation of an L3MBTL4 promoter region is detected in pancreatic cancer, liver cancer, gastric cancer and esophageal cancer by an MSP method. Therefore, the hypermethylation state of the promoter region of the gene is inferred to be a tumor molecular marker, and the L3MBTL4 gene is a new tumor-related cancer suppressor candidate gene. Based on the fact that the methylation state of the L3MBTL4 gene promoter region is determined by detecting a large number of clinical tumor (pancreatic cancer, liver cancer, gastric cancer and esophageal cancer) tissues and normal pancreas, liver, stomach and esophageal specimens by applying an MSP method. In addition, the invention discovers for the first time that L3MBTL4 participates in NHEJ pathway of DNA damage repair, and L3MBTL4 methylation is a synergistic lethal marker for killing tumor cells by combining CHK1 inhibitor with cisplatin and other therapeutic drugs.
The invention also provides application of a substance for detecting the methylation state of the DNA fragment of the promoter region of the L3MBTL4 gene in preparing a product for diagnosing digestive system tumors, wherein the digestive system tumors mainly comprise pancreatic cancer, liver cancer, gastric cancer or esophageal cancer.
Alternatively, the nucleotide sequence of the L3MBTL4 gene promoter region DNA fragment is shown in SEQ ID No.1 (original DNA sequence before sulfuration treatment), and comprises at least one methylation site.
The invention also provides a kit for diagnosing digestive system tumors, which comprises a substance for detecting the methylation state of the L3MBTL4 gene promoter region, wherein the digestive system tumors are mainly pancreatic cancer, liver cancer, stomach cancer or esophageal cancer.
Further, the substance for detecting the methylation state of the L3MBTL4 gene promoter region aims at the DNA sequence shown as SEQ ID No.1 of the nucleotide sequence of the L3MBTL4 gene promoter region.
Further, the substance comprises a methylated primer pair and an unmethylated primer pair.
Preferably, the upstream primer of the methylation primer pair is 5 'TTTTAGGGAACGAATATAGAGAAGGAC 3' as shown in SEQ ID No.2, and the downstream primer is 5 'CCACACCCCGAAAAACCGAATAACG 3' as shown in SEQ ID No. 3; the upstream primer of the unmethylated primer pair is 5 'TTTTTTAGGGAATGAATATAGAGAAGGAT 3' as shown in SEQ ID No.4, and the downstream primer is 5 'ATTCCACACCCCAAAAAACCAAATAACA 3' as shown in SEQ ID No. 5.
MSP amplification is carried out by using the methylation primer pair and DNA subjected to vulcanization modification as a template, and if methylation exists in an L3MBTL4 gene promoter region, a 117bp fragment can be obtained; if the L3MBTL4 gene is normal, i.e., unmethylated, no amplification product is produced. In this regard, the primer pair is referred to herein as a methylated primer pair. The Tm value of the upstream primer of the methylated primer pair is 58.6, the GC content of the methylated primer pair is 37.0 percent, the Tm value of the downstream primer is 64.7, and the GC content of the methylated primer pair is 52.0 percent.
MSP amplification is carried out by using the unmethylated primer pair and DNA subjected to sulfide modification as a template, and a 122bp fragment is obtained if the L3MBTL4 gene is not methylated. Based on this, the present application refers to this primer pair as an unmethylated primer pair. The upstream primer of the unmethylated primer pair has a Tm value of 57.1 and a GC content of 27.6%, and the downstream primer has a Tm value of 62.6 and a GC content of 35.7%.
In the present invention, amplification with methylated primer pairs results in amplification products, whereas amplification with unmethylated primers results in no amplification products being completely methylated; amplifying by using methylation primers and non-methylation primers, wherein amplification products are obtained and are partially methylated; amplification with unmethylated primers with amplification product and with methylated primers with no amplification product is methylation-free. Both partial and complete methylation are judged as methylation.
Further, the kit also comprises a methylated positive control PCR template and an unmethylated positive control PCR template.
Wherein the methylation positive control PCR template is tumor cell DNA with 100% methylation of a L3MBTL4 gene promoter region after sulfuration modification, such as pancreatic cancer, liver cancer, gastric cancer or esophageal cancer cells; the unmethylated positive control PCR template is vulcanized and modified L3MBTL4 gene promoter area 100% unmethylated normal lymphocyte or normal tissue cell DNA, such as normal pancreas, liver, stomach or esophagus tissue cell.
Further, the kit also comprises reagents required by MS-PCR reaction, such as 10 xMSP buffer solution, 20mM dNTP, Hot start taq enzyme, deionized water and the like.
Further, when diagnosing tumors of the digestive system, particularly pancreatic cancer, liver cancer, stomach cancer or esophageal cancer, using the kit of the present invention, MS-PCR methods known in the art can be used.
Preferably, the reaction conditions are as follows:
pre-denaturation at 95 ℃ for 5min, followed by cycles of denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 40s for 35 cycles, after which extension at 72 ℃ is continued for 5 min.
The invention has the following beneficial effects:
by using the molecular marker, a detection substance (primer) and a kit to detect pancreatic cancer, liver cancer, gastric cancer and esophageal cancer tissue cells, the L3MBTL4 gene promoter region in tumor cells is in a hypermethylation state, the methylation rates are respectively about 48.8%, 53.3%, 50.0% and 21.4% in pancreatic cancer, liver cancer, gastric cancer and esophageal cancer, and the L3MBTL4 gene promoter region in normal tissue cells is in a non-methylation state, which indicates that the methylation state of the gene promoter region has specificity to pancreatic cancer, liver cancer, gastric cancer and esophageal cancer; meanwhile, by applying the primer, the kit, the reaction system and the reaction conditions, the sensitivity for detecting pancreatic cancer, liver cancer, gastric cancer and esophageal cancer cells can reach 0.1 percent, namely 1 cancer cell in 1000 cells can be detected, which shows that the methylation state of the L3MBTL4 gene promoter region can be used as a new molecular marker for pancreatic cancer, liver cancer, gastric cancer and esophageal cancer. According to the invention, the L3MBTL4 gene is selected as a target gene for the first time, and the sensitivity of pancreatic cancer cells to a CHK1 inhibitor (MK8776) is discussed, and the result shows that under the induction of a cisplatin drug, the IC50 value of L3MBTL4 methylated pancreatic cancer cells (MiaPaCa2) is 1.24 +/-0.03 mu M, the IC50 value of L3MBTL4 unmethylated pancreatic cancer cells (CFpac1) is 7.28 +/-0.15 mu M, and the L3MBTL4 methylated pancreatic cancer cells are more sensitive to MK8776 (P < 0.05). L3MBTL4 was found to be involved in the NHEJ pathway of DNA damage repair, and L3MBTL4 methylation is a synergistic lethal marker for tumor cells killed by CHK1 inhibitors in combination with chemotherapeutic drugs such as cisplatin. Therefore, the application of the molecular marker, the primer or the kit for detecting the hypermethylation state of the L3MBTL4 gene promoter region can be used as a powerful means for diagnosis, curative effect observation, prognosis judgment and the like of pancreatic cancer, liver cancer, gastric cancer and esophageal cancer, and the kit is simple and convenient to operate, good in stability and has profound clinical significance and popularization.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of amplification products of MSP using DNA of pancreatic cancer, liver cancer, stomach cancer and esophageal cancer tissues after sulfurization modification and DNA of normal pancreatic cancer, liver cancer, stomach cancer and esophageal cancer tissues after sulfurization modification as templates and using methylation primer pair and non-methylation primer pair respectively; wherein the content of the first and second substances,
PC1-PC5 is pancreatic cancer, HCC1-HCC5 is liver cancer, GC1-GC5 is stomach cancer, EC1-EC5 is esophageal cancer, NP1-NP2 is normal pancreas, NH1 and NH2 are normal liver, NG1 and NG2 are normal stomach, and NE1 and NE2 are normal esophagus. Also IVD-labeled is a methylation positive control, NL-labeled is a non-methylation positive control, H2O negative control (deionized water).
FIG. 2 is a diagram showing the results of agarose gel electrophoresis of amplification products obtained by mixing DNA of a pancreatic cancer cell line MiaPaCa2(L3MBTL4 gene promoter region 100% methylated) with DNA of a normal lymphocyte (L3MBTL4 gene promoter region 100% unmethylated) in proportion, performing a sulfuration modification, and thereafter performing amplification with a methylated primer; wherein the content of the first and second substances,
group 1: 100% MiaPaCa2 cellular DNA + 0% normal cellular DNA;
group 2: 50% MiaPaCa2 cellular DNA + 50% normal cellular DNA;
group 3: 5% MiaPaCa2 cellular DNA + 95% normal cellular DNA;
group 4: 1% MiaPaCa2 cellular DNA + 99% normal cellular DNA;
group 5: 0.1% MiaPaCa2 cellular DNA + 99.9% normal cellular DNA;
group 6: 0% MiaPaCa2 cellular DNA + 100% normal cellular DNA.
FIG. 3 shows a schematic MBT domain of L3MBTL 4.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
1. Preparation of template (extraction of genomic DNA and modification by sulfurization)
Preparation of DNA: pancreatic cancer, liver cancer, stomach cancer, esophageal cancer and normal tissue specimens are obtained. In this example, 5 pancreatic cancers (PC1-PC5), 5 liver cancers (HCC1-HCC5), 5 stomach cancers (GC1-GC5), 5 esophageal cancers (EC1-EC5), normal pancreas (NP1-NP2), normal liver (NH1-NH2), normal stomach (NG1-NG2) and normal esophagus (NE1-NE2) were extracted by phenol-chloroform extraction, and the content and purity of genomic DNA were determined by measuring absorbance (A) with an ultraviolet spectrophotometer.
Modification by sulfite: the method reported by reference to Herman (J.G.Herman, J.R.Graff, S.Myohannen, B.D.Nelkin and S.B.Baylin, Methylation-specific PCR: a novel PCR assay for Methylation status of CpG islands, Proc.Natl.Acad.Sci.USA 93 (1996)), 9821-. The prepared genome DNA is diluted, the DNA concentration is measured by using an ultraviolet spectrophotometer, 2 mu g of DNA is accurately taken, deionized water is added to the final volume of 50 mu L, 2M NaOH5.5 mu L prepared freshly is added, and the mixture is incubated for 15min at 37 ℃. Then, 30. mu.L of freshly prepared 10mM hydroquinone and 520. mu.L of 3M sodium bisulfite were added and mixed well (to which freshly prepared 2M NaOH had been added, calculated by adding 740. mu.L of 2M NaOH to 10ml of 3M sodium bisulfite), and incubated at 50 ℃ for 16 hours, after which the DNA was purified and recovered using a DNA purification kit (product of Promega corporation), the DNA was eluted using 50. mu.L of deionized water, 5.5. mu.L of 3M NaOH was added thereto to terminate the sulfuration modification, 1. mu.L of glycogen and 17. mu. L7.5M ammonium acetate were added, the DNA was precipitated using 3-fold volume of absolute ethanol, and finally the sulfuration-modified DNA was redissolved in 20. mu.L of deionized water to obtain a DNA template, which was immediately used or stored at-20 ℃.
2. MSP amplification
A PCR reaction system for amplification by using a methylated primer pair comprises the following components in a total volume of 25 mu L:
template (DNA after sulfuration modification): 2 mu L of the solution;
methylation primer pair (50 pmol/. mu.L):
upstream primer (5 'TTTTAGGGAACGAATATAGAGAAGGAC 3') for: 0.5 mu L of the suspension liquid is prepared,
downstream primer (5 'CCACACCCCGAAAAACCGAATAACG 3') for: 0.5 mu L;
2.5. mu.L of 10 XSSP buffer;
25mMdNTPs：1.25μL；
hot Start Taq enzyme: 0.5 mu L;
ddH2O：17.75μL。
a PCR reaction system for amplification with unmethylated primer pairs, in a total volume of 25 μ L, comprising:
the difference between the PCR reaction system and the PCR reaction system for amplifying the methylated primer pair is that: primers are different, in this system are unmethylated primers (50 pmol/. mu.L):
upstream primer (5 'TTTTTTAGGGAATGAATATAGAGAAGGAT 3') for: 0.5 mu L of the suspension liquid is prepared,
downstream primer (5 'ATTCCACACCCCAAAAAACCAAATAACA 3'): 0.5 mu L;
the rest is the same as that in the PCR reaction system for amplifying the methylated primer pair.
The 28 prepared vulcanized DNA templates were subjected to MSP reaction using the MSP reaction system set up above: pancreatic cancer 5 cases (PC1-PC5), liver cancer 5 cases (HCC1-HCC5), stomach cancer 5 cases (GC1-GC5), esophageal cancer 5 cases (EC1-EC5), normal pancreas (NP1-NP2), normal liver (NH1-NH2), normal stomach (NG1-NG2), normal esophagus (NE1-NE2), and a methylation positive control (IVD) and a non-methylation control (NL-normal peripheral blood DNA) are respectively amplified, and deionized water is used as a negative control, and the amplification program is as follows: pre-denaturation at 95 ℃ for 5min, followed by cycles of denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 40s for 35 cycles, after which extension at 72 ℃ is continued for 5 min.
Detection of PCR reaction product
The PCR product was subjected to 2% agarose gel electrophoresis, examined by an ultraviolet transmission analyzer and photographed.
4. Results
The results are shown in FIG. 1, and the amplification products of pancreatic cancer (PC1, PC3, PC5), liver cancer (HCC1, HCC2, HCC5), gastric cancer (GC1, GC2, GC3) and esophageal cancer (EC3) are obtained by using methylation primers for amplification; and normal pancreas (NP1-NP2), liver (NH1-NH2), stomach (NG1-NG2), esophagus (NE1-NE2), pancreatic cancer (PC2, PC4), liver cancer (HCC3, HCC4), gastric cancer (GC4, GC5), esophageal cancer (EC1, EC2, EC4, EC5) have no amplification products. And the non-methylated primers are used for amplification, and all sample tissues have amplification products. Therefore, pancreatic cancer (PC1, PC3, PC5), liver cancer (HCC1, HCC2, HCC5), gastric cancer (GC1, GC2, GC3), esophageal cancer EC3 are partially methylated; normal pancreas (NP1-NP2), liver (NH1-NH2), stomach (NG1-NG2), esophagus (NE1-NE2), pancreatic cancer (PC2, PC4), liver cancer (HCC3, HCC4), gastric cancer (GC4, GC5), esophageal cancer (EC1, EC2, EC4, EC5) are methylation-free. Both partial and complete methylation are judged as methylation.
EXAMPLE 2 clinical specimen testing
160 clinical samples of pancreatic cancer, 15 liver cancer, 16 stomach cancer and 14 esophageal cancers were taken. MSP amplification is carried out, the preparation of a template, a PCR amplification system and conditions, and the detection of an amplification product are the same as those in the first embodiment, and the detection results are shown in the following table:
| classification | Number of examples | Number of M (methylated) cases | Number of U (non-methylated) instances | Positive for methylationRate of change |
| Pancreatic cancer | 160 | 78 | 82 | 48.8％ |
| Liver cancer | 15 | 8 | 7 | 53.3％ |
| Stomach cancer | 16 | 8 | 8 | 50.0％ |
| Esophageal cancer | 14 | 3 | 11 | 21.4％ |
Example 3 sensitivity test
Pancreatic cancer MiaPaCa2 cell line DNA (L3MBTL4 gene promoter region 100% methylated) and normal lymphocyte DNA (L3MBTL4 gene promoter region 100% unmethylated) were mixed in proportion and subjected to sulfurization modification (as in example one) followed by MSP. The PCR product was subjected to 2% agarose gel electrophoresis, measured by an ultraviolet transmission analyzer and photographed.
Grouping: group 1: 100% MiaPaCa2 cellular DNA + 0% normal cellular DNA;
group 2: 50% MiaPaCa2 cellular DNA + 50% normal cellular DNA;
group 3: 5% MiaPaCa2 cellular DNA + 95% normal cellular DNA;
group 4: 1% MiaPaCa2 cellular DNA + 99% normal cellular DNA;
group 5: 0.1% MiaPaCa2 cellular DNA + 99.9% normal cellular DNA;
group 6: 0% MiaPaCa2 cellular DNA + 100% normal cellular DNA.
The results are shown in FIG. 2: 1 pancreatic cancer cell in 1000 normal cells can be detected, namely the primer and the reaction system of the invention are used for detecting the methylation state of the pancreatic cancer cell L3MBTL4 gene promoter region under the condition, the sensitivity can reach 0.1 percent, and the sensitivity is higher.
Example 4 detection of a synthetic lethal marker for tumor cell killing by CHK1 inhibitor (MK8776) in combination with cisplatin and other therapeutic agents
Pancreatic cancer cells MiaPaCa2(L3MBTL4 gene promoter region 100% methylated) and CFpac1(L3MBTL4 gene promoter region 100% unmethylated) were subjected to drug susceptibility experiments, and sensitivity of the cells to CHK1 inhibitor (MK8776), i.e. IC50 value (median lethal dose of the cells), was calculated by setting different MK8776 concentration gradients MiaPaCa2(0, 0.5, 1, 2, 4, 8, 16, 32 μ M) and CFpac1(0, 0.5, 1, 2, 4, 8, 16, 32, 64 μ M) and detecting absorbance values at 490nm wavelength using MTT experiments after 48 hours of treatment. The results of the measurements are shown in the following table:
| pancreatic cells | IC50(μM) |
| MiaPaCa2 | 1.24±0.03 |
| CFpac1 | 7.28±0.15 |
Example 5 kit composition for detecting pancreatic cancer, liver cancer, gastric cancer and esophageal cancer cells
The kit for detecting pancreatic cancer, liver cancer, gastric cancer and esophageal cancer cells comprises the following components, wherein the dosage of MSP for 1 time is as follows:
1. methylated primer pair at a concentration of 50 pmol/. mu.L: the upstream primer with the nucleotide sequence shown as SEQ ID No.2 and the downstream primer with the nucleotide sequence shown as SEQ ID No.3 are respectively 0.5 mu L.
2. Unmethylated primer pair concentration 50 pmol/. mu.L: the upstream primer with the nucleotide sequence shown as SEQ ID No.4 and the downstream primer with the nucleotide sequence shown as SEQ ID No.5 are respectively 0.5 mu L.
3. 2 parts of reaction system, which is respectively matched with a methylated primer pair and an unmethylated primer pair for use, wherein each part of reaction system comprises:
(1)10 × MSP buffer (buffer): 2.5 mu L;
(2)25mMdNTPs：1.25μL；
(3) hot start Taq enzyme: 0.5 mu L;
(4)ddH2O：17.75μL。
a kit may comprise the above components in multiple MSP doses, e.g., 25, 50, 100 etc., the specific amounts of each component being determined as appropriate.
To prevent the occurrence of false positive and false negative of MSP amplification products, the kit may further comprise:
a. methylation positive control PCR template: after being treated by methyltransferase, the promoter region of the L3MBTL4 gene after sulfuration modification is tumor tissue DNA with 100 percent methylation, and MSP is carried out for 1 time, and the dosage is as follows: 2 μ L.
b. Unmethylated positive control PCR template: the promoter region of the L3MBTL4 gene after sulfuration modification is 100 percent of unmethylated normal lymphocyte DNA, and the dosage of MSP is 1 time: 2 μ L.
c. Double negative system control: ddH2O, to assess whether contamination of the system with PCR products, e.g. ddH2O inspectionThe result of the test is double negative (M and U are negative), and the system result is credible.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications belonging to the technical solutions of the present invention are within the scope of the present invention.
SEQUENCE LISTING
<110> first medical center of general hospital of people liberation force of China
<120> molecular marker and kit for diagnosing digestive system tumor
<130> JLP21I1337
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1584
<212> DNA
<213> L3MBTL4 gene promoter region DNA fragment
<400> 1
cgtgagtagc aaagtcaaga gcaggaaagg ggagtggggg aggttgcaaa atcggagcga 60
gcagcaggac cgagagggat ccggaggaga gagccttcag acggtgaacc ccaagagctg 120
ggatgaagca tgggtgagct actgggactt cgctgccagg gagcaagctg gcaggcagga 180
agtagccaag atttggggga atccttccca gggaacgaat acagagaagg acgcatctga 240
aaattccgcc gcagccacct ccctcccatg ggaaagcccc gacctcggcg ggcccagcgc 300
cacccggctc cccggggtgt ggaacactga gcgaggcagg tgcggcggga gcatgcgcgc 360
ggggcgggca gggggcggct cccgcgcccc caccccgcgc gtgcaccctc gcgcccctcc 420
cgccgggcac cgcggggcag cggcgccgca gcctgggctg gcggaacctc gcgctccgac 480
tcgccccccg cctgtagctc ggtcggcgtt gcgggcaggc gccgctccgc tgggggtaag 540
aaggacgccg ccgcccgcgt gggatggtcg cgagcccgcg tgggtgccca cggccgccgc 600
cgcgcggggt ggccccatcc ccgggcaggt agaggggcct gcacgactcc cgcggcgagg 660
gccgggccgg tgggggcggt ggcggttggg gatagccccg gtcccggccc aggctctttc 720
tctttctctt tcgccgccgg ggcgcgcgcc gggctccccg cgagaggcgc tgcctcactt 780
gtcgcctact ttggacgggc gcggccgccg cagagcccgg cgcctcggtc agccgcgagg 840
gcagggtcgg ggtgccaggc gcggctacct gggcgccctg gagccagcgc agcctgggac 900
cgggggagcg ggacgggacg ggactcctga gggcccccag gcagtcctcc ggccatcgag 960
cgcccggcgg tgggcatttg gcttttgttt cctctggggc taacgccccc gtctgcccgc 1020
gtgtccccct cagccgacaa gtgtcgtgtg cccggacggg cgagcgctgc ctccctccac 1080
ccgctttctg cccttgtctt cctctgcgag ttctgcccag cctcgggagc gggagcccag 1140
ggagtgcgcg cagggcgggc gggcgcctgg ccagcctggg acggcggctc tcaggctcct 1200
gccaccgcct ggaactgcgg ggggcgcgcc ggccacagtc gccctcgcct gctctccggg 1260
ccttcttggg cccgcttggt cccaggtgga cgcgagtcga gctgggcaca cctgggccag 1320
cgccgcactt acctgcggga ttggtgagag cgttttcgcg aggtcgcaaa ttccgggtgg 1380
gtattgtccc ctgcttttga tctcccttag tcactggtta aagtgcgaag tgtgctttat 1440
cttacatatg cctttagagg gacattctta atgcgccaat gccccgaggt ggaactaaat 1500
aaagggaagg cctggtcatt gttactgttg agactgctgt acttaacagt cagaggaagg 1560
cagctggtca ccacgaccca ctcg 1584
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttttagggaa cgaatataga gaaggac 27
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccacaccccg aaaaaccgaa taacg 25
<210> 4
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttttttaggg aatgaatata gagaaggat 29
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
attccacacc ccaaaaaacc aaataaca 28
Claims (10)
- The application of the L3MBTL4 gene promoter region DNA fragment as a molecular marker in the preparation of products for diagnosing digestive system tumors is characterized in that the digestive system tumors are pancreatic cancer, liver cancer, gastric cancer or esophageal cancer.
- 2. The use according to claim 1, wherein the L3MBTL4 gene promoter region DNA fragment has the nucleotide sequence shown in SEQ ID No.1 and comprises at least one methylation site.
- 3. The application of the substance for detecting the methylation state of the DNA fragment of the promoter region of the L3MBTL4 gene in the preparation of products for diagnosing digestive system tumors is characterized in that the digestive system tumors are pancreatic cancer, liver cancer, stomach cancer or esophageal cancer.
- 4. The use according to claim 3, wherein the L3MBTL4 gene promoter region DNA fragment has the nucleotide sequence shown in SEQ ID No.1 and comprises at least one methylation site.
- 5. A kit for diagnosing digestive system tumors, comprising: comprises a substance for detecting the methylation state of an L3MBTL4 gene promoter region, and the digestive system tumor is pancreatic cancer, liver cancer, gastric cancer or esophageal cancer.
- 6. The kit according to claim 5, wherein the substance for detecting the methylation state of the promoter region of the L3MBTL4 gene is directed against a DNA sequence shown in SEQ ID No.1 as the nucleotide sequence of the promoter region of the L3MBTL4 gene.
- 7. The kit of claim 6, wherein the substance comprises a methylated primer pair and an unmethylated primer pair; preferably, the nucleotide sequence of the upstream primer of the methylation primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 3; the upstream primer nucleotide sequence of the non-methylated primer pair is shown as SEQ ID No.4, and the downstream primer nucleotide sequence is shown as SEQ ID No. 5.
- 8. The kit of claim 6, further comprising a methylated positive control PCR template and an unmethylated positive control PCR template.
- 9. The kit of claim 8, wherein the methylation positive control PCR template is tumor cell DNA with 100% methylation in the promoter region of L3MBTL4 gene after sulfuration modification; the unmethylated positive control PCR template is vulcanized and modified normal lymphocyte or normal tissue cell DNA with 100 percent unmethylated in L3MBTL4 gene promoter area.
- 10. The kit of claim 6, wherein the kit further comprises reagents required for the MS-PCR reaction.
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