CN111793693A - A combined detection kit for tumor suppressor gene methylation and its application - Google Patents

Abstract

The invention discloses application of a methylation detection reagent of at least one gene selected from LRRC3B, PENK and RASSF1 genes in preparing a kit for diagnosing whether a subject has colorectal cancer or predicting whether the subject has the risk of having the colorectal cancer or judging the prognosis of the colorectal cancer, and belongs to the technical field of biological detection. The invention can realize prevention, early diagnosis, clinical treatment and prognosis evaluation of colorectal cancer by jointly detecting the methylation degree of the promoters of three cancer suppressor genes of LRRC3B, PENK and RASSF 1.

Description

A combined detection kit for tumor suppressor gene methylation and its application

technical field

The invention relates to the technical field of biological detection, in particular to a combined detection kit for tumor suppressor gene methylation and its application.

Background technique

The incidence of colorectal carcinoma (CRC) ranks third in China, and it is the fourth leading cause of cancer death. Colorectal cancer is a malignant tumor that can be prevented, diagnosed and treated early. "Three early" (early detection, early diagnosis, and early treatment) is the most effective way to improve the treatment effect of colorectal cancer. Clinical data show that the 5-year survival rate of early-stage cancer patients is as high as 90%. However, due to the lack of specific clinical manifestations in the early stage of colorectal cancer and the lack of high-sensitivity and specific early diagnosis techniques, most patients are diagnosed with advanced cancer and lose the best time for treatment, resulting in a very low 5-year survival rate after surgery. (below 20%).

Early diagnosis of tumors usually depends on two key factors: the high specific sensitivity of the examination indicators and the non-invasive and easy implementation of the detection methods. Faecal occult blood testing (FOBT) is currently the most widely used method for colorectal cancer screening, which provides an important diagnostic value for the detection of colorectal cancer. However, FOBT is greatly affected by diet and drugs, has poor specificity, and cannot distinguish polyps from cancer. Imaging examinations such as barium enema (double contrast barium enema, DCBE) are simple, easy and less painful, but cannot assess the depth and extent of invasion of colorectal cancer. Colonoscopy (CS) is the gold standard for the diagnosis of colorectal cancer. It can excise and biopsy the diseased part, but it is an invasive operation and is not suitable for early screening. However, the current tumor markers such as CEA and CA199 used in the classic screening of colorectal cancer have poor specificity and sensitivity.

Therefore, it is of great significance to find and develop economical, simple, high-sensitivity and specific biomarkers for early screening and diagnosis of colorectal cancer.

SUMMARY OF THE INVENTION

In order to overcome the defects in the prior art, the inventor's previous research found that LRRC3B, PENK and RASSF1 are highly expressed in most normal tissues, but abnormally low in colorectal cancer patients, and are negatively correlated with tumor stage. Further studies showed that serum free LRRC3B, PENK and RASSF1 three tumor suppressor gene promoter methylation levels can predict the incidence of colorectal cancer. Thus, the present invention has been completed.

The first aspect of the present invention provides a methylation detection reagent for at least one gene selected from the LRRC3B, PENK and RASSF1 genes in preparation for diagnosing whether a subject has colorectal cancer or for predicting whether a subject has a disease Rectal cancer risk or use in a kit for judging the prognosis of colorectal cancer.

In some embodiments of the present invention, the methylation detection reagent is used to detect the promoter methylation status of the gene.

In some specific embodiments of the present invention, the methylation detection reagent includes specific primers for detecting the methylation status of the gene promoter.

In some preferred embodiments of the present invention, specific primers for detecting the methylation status of the LRRC3B gene promoter include an upstream primer having the nucleotide sequence shown in SEQ ID NO.1 and an upstream primer having the nucleotide sequence shown in SEQ ID NO.2 The downstream primer of the nucleotide sequence; the specific primer for detecting the unmethylated state of the LRRC3B gene promoter includes the upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and the nucleoside with the nucleotide sequence shown in SEQ ID NO.4 Downstream primers for acid sequences.

In some preferred embodiments of the present invention, the specific primers used to detect the methylation state of the PENK gene promoter include an upstream primer having the nucleotide sequence shown in SEQ ID NO.5 and an upstream primer having the nucleotide sequence shown in SEQ ID NO.6 The downstream primer of the nucleotide sequence; the specific primer for detecting the unmethylated state of the PENK gene promoter includes the upstream primer with the nucleotide sequence shown in SEQ ID NO.7 and the nucleoside shown in SEQ ID NO.8 Downstream primers for acid sequences.

In some preferred embodiments of the present invention, specific primers for detecting the methylation state of the RASSF1 gene promoter include an upstream primer having the nucleotide sequence shown in SEQ ID NO.9 and an upstream primer having the nucleotide sequence shown in SEQ ID NO.10 The downstream primer of the nucleotide sequence; the specific primer for detecting the unmethylated state of the RASSF1 gene promoter includes the upstream primer with the nucleotide sequence shown in SEQ ID NO.11 and the nucleoside shown in SEQ ID NO.12 Downstream primers for acid sequences.

In some embodiments of the present invention, the methylation detection reagent further comprises a reagent for extracting sample DNA.

In the present invention, the sample is whole blood or plasma, and the DNA is plasma cell-free DNA.

The second aspect of the present invention provides the use of a promoter methylation detection reagent of at least one gene selected from the LRRC3B, PENK and RASSF1 genes in the preparation of a kit suitable for the following method, the method comprising:

S1, extract the subject sample DNA,

S2, utilize the detection reagent to detect the promoter methylation state of the gene,

S3, according to the methylation state detected by S2, diagnosing whether the subject has colorectal cancer or predicting whether the subject has the risk of developing colorectal cancer or judging the prognosis of colorectal cancer.

In some embodiments of the invention, the subject is diagnosed with colorectal cancer, or predicted to have colorectal cancer, when the methylation level of at least one of the LRRC3B, PENK, and RASSF1 genes is higher than normal in the subject Risk of colorectal cancer, or poor prognosis of colorectal cancer.

A third aspect of the present invention provides a kit for diagnosing whether a subject has colorectal cancer or for predicting whether a subject has a risk of developing colorectal cancer or for judging the prognosis of colorectal cancer, comprising a kit selected from the group consisting of A reagent for detecting the methylation state of the promoter of at least one of the LRRC3B, PENK and RASSF1 genes, wherein the specific primer for detecting the methylation state of the promoter of the LRRC3B gene comprises a nucleotide with the nucleotide shown in SEQ ID NO.1 The upstream primer of the sequence and the downstream primer with the nucleotide sequence shown in SEQ ID NO.2; the specific primer for detecting the non-methylation state of the LRRC3B gene promoter includes the nucleotide sequence shown in SEQ ID NO.3 The upstream primer and the downstream primer with the nucleotide sequence shown in SEQ ID NO.4. The specific primers used to detect the methylation state of PENK gene promoter include the upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.6; for detection The specific primers for the unmethylated state of the PENK gene promoter include the upstream primer having the nucleotide sequence shown in SEQ ID NO.7 and the downstream primer having the nucleotide sequence shown in SEQ ID NO.8. The specific primers used to detect the methylation state of the RASSF1 gene promoter include the upstream primer with the nucleotide sequence shown in SEQ ID NO.9 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.10; for detection The specific primers for the unmethylated state of the RASSF1 gene promoter include the upstream primer with the nucleotide sequence shown in SEQ ID NO.11 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.12.

The beneficial effects of the present invention

The present invention has the following beneficial effects relative to the prior art:

Combined detection of LRRC3B, PENK and RASSF1 can be used as biomarkers for early screening, diagnosis, efficacy evaluation and prognosis judgment of colorectal cancer, and the combined detection kit based on it can be used for early screening, diagnosis, curative effect of colorectal cancer Surveillance and prognostic evaluation.

The detection method of the invention is convenient, quick, and inexpensive, and is suitable for popularization and application.

Compared with the classical tumor markers, ctDNA has higher accuracy and specificity, and is a novel tumor marker with non-invasive and wide clinical application prospects, which can be qualitatively, quantitatively and tracked. Disappearance, spread and recurrence of tumors.

In addition, DNA methylation is a more frequent early event in tumorigenesis. As an early diagnosis and screening of tumors, it has incomparable advantages of RNA, protein and gene mutations:

1) DNA samples are extremely stable, unlike RNA and proteins, and are greatly affected by the environment;

2) The sensitivity of methylation-specific PCR (MSP), the method is simple to operate, does not require expensive equipment, and is more feasible than quantitative detection methods (the inevitable presence of sequences derived from normal cells in clinical samples will seriously interfere with tumor cells. The detection of characteristic genetic biomarkers is an obstacle to the early diagnosis of tumors by quantitative detection methods; to explore the role of methylation of selected tumor suppressor genes in the occurrence and development of breast cancer, the experimental method is simple and feasible;

3) Methylation of tumor suppressor gene promoters mostly occurs in CpG islands, the location is more limited than gene mutation, and it is easy to design probes and primers;

4) Multiple pairs of primers can be designed for joint detection at the same time;

5) Suitable for various body fluid detection.

Description of drawings

Figure 1 shows the methylation status of LRRC3B, PENK and RASSF1 promoters detected by Methtarget.

Figure 2 shows LRRC3B of human colon cancer tissue samples and paired normal colon tissue samples in the MethHC DNA methylation database (http://methhc.mbc.nctu.edu.tw/php/index.php) obtained from the TCGA dataset , PENK and RASSF1 expression levels and promoter relative methylation levels.

Figure 3 shows a representative graph of the methylation status of LRRC3B, PENK and RASSF1 promoters in colon cancer patient tissues that were clinicopathologically positive for MSP assay.

Figure 4 shows a representative graph of the methylation status of plasma cell-free DNA LRRC3B, PENK and RASSF1 promoters detected by plasma cell-free DNA methylation capture technology.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

Example

The following examples are used herein to demonstrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred solutions for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the public references herein and the materials to which they refer are incorporated by reference .

Those skilled in the art will recognize, or be aware of through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are to be included in the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory equipment unless otherwise specified; the test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

Example 1

1. Tissue, Cell and Plasma Cell-Free DNA Extraction

1.1 Plasma cell-free DNA extraction (magnetic bead method)

(1) Add 750 μl lysis buffer, 50 μl proteinase k, and 10 μl RNaseA to 1 mL of plasma, mix well, and incubate at room temperature for 20 minutes;

(2) Add 50 μl of magnetic beads, invert and mix, and incubate at room temperature for 10 minutes;

(3) Place on the magnetic stand for 3 minutes, discard the supernatant;

(4) Wash twice with 500 μl of washing buffer, using a magnetic stand;

(5) 50 μl of elution buffer to elute DNA, and measure the concentration.

1.2 Tissue and cell DNA extraction (QIAamp kit)

(1) If the sample is tissue, pour liquid nitrogen into the mortar, quickly pour the tissue into the mortar, grind the tissue vigorously, and quickly move the ground tissue powder to an enzyme-free EP tube; if the sample is a cell, PBS 1mL Wash the cells, add 1 ml of trypsin for 5 min; centrifuge at 800 rpm for 5 min, discard the supernatant.

(2) Add 180 μL Buffer ATL, mix well, and suck it into an EP tube;

(3) Add 20 μL proteinase K, mix well, and incubate at 55°C for 30 minutes until the cells are completely lysed;

(4) Add 20mg/μL RNase A 20μL, shake and mix for 15s, and incubate at room temperature for 2-5min;

(5) Add 200 μL Buffer AL, mix for 15s, and incubate at 70°C for 10min;

(6) Add 200 μL of absolute ethanol and mix for 15s;

(7) The liquid is sucked into the cannula and centrifuged at 8000rpm for 1min;

(8) Discard the supernatant, add 500 μL Buffer AW1, and centrifuge at 8000 rpm for 1 min;

(9) Discard the supernatant, add 500 μL Buffer AW2, and centrifuge at 12,000 rpm for 3 min;

(10) Discard the next clearing, and leave it at 12000rpm for 1min;

(11) Transfer the column to a new EP tube, add 30 μL of Buffer AE/tri-distilled water, and let stand for 3 minutes at room temperature;

(12) Centrifuge at 8000 rpm for 1 min, and measure the concentration of DNA in the EP tube.

2. DNA bisulfite modification and methylation detection

2.1 DNA bisulfite modification (EZ DNA methylation-Gold Kit)

Prepare CT Conversion Reagent and add in sequence according to the system:

Dissolve at room temperature and shake for 10 min.

Bisulfite treatment of DNA

(1) Add 24 mL of absolute ethanol to M-Wash Buffer for subsequent use;

(2) Add 20μL (0.2-0.5ug) DNA to 130μL CT Conversion Reagent;

(3) Constant temperature incubation mixture: 98°C, 10min; 64°C, 2.5h; 4°C, 20h;

(4) Suction the liquid into the casing column, add 600 μL of M-Binding Buffer, mix well, centrifuge at 12000rpm for 30s, and discard the liquid;

(5) Add 200 μL of M-Wash Buffer, and centrifuge at 12000 rpm for 30 s;

(6) Add 200 μL M-Desulphonation Buffer, place at room temperature for 20 min, and centrifuge at 12000 rpm for 30 s;

(7) Add 200 μL of M-Wash Buffer, centrifuge at 12000rpm for 30s; repeat once;

(8) Move the column into a new EP tube, add 10 μL of M-Elution Buffer to the column, and centrifuge at 12000rpm for 30s;

(9) Store at -20°C.

2.2 Methtarget screening potential methylation sites as diagnostic biomarkers for colorectal cancer

To identify meaningful molecular markers in colon cancer, the inventors utilized the TCGA dataset, MethHC online analysis (http://MethHC.mbc.nctu.edu.tw) and kaplan-Meier Plotter (http://kmplot. com/analysis/) prognostic analysis to screen potential methylation sites as diagnostic biomarkers for colon cancer. The 80 genes screened were analyzed for CpG islands, using multiple target region methylation enrichment sequencing, and the hypermethylated genes in colon cancer were screened according to FDR<0.05 and β absolute value difference>0.2. The results showed that the promoter methylation sites of the three genes in colon cancer tissue were significantly higher than those in normal colon tissue (as shown in Figure 1) .

Expression of LRRC3B, PENK and RASSF1 in human colon cancer tissue samples and paired normal colon tissue samples from the MethHC DNA methylation database (http://methhc.mbc.nctu.edu.tw/php/index.php) obtained from the TCGA dataset and promoter relative methylation levels. The results showed that the expression levels of LRRC3B, PENK and RASSF1 in colon cancer were significantly lower than those in adjacent normal tissues, while their corresponding gene promoter methylation levels were significantly increased (the results are shown in Figure 2).

2.3 MSP detection of LRRC3B, PENK and RASSF1 promoter methylation

The present invention designs specific primers according to the gene sequences of LRRC3B, PENK and RASSF1 promoters, and uses MSP technology to detect methylation detection.

Specific primers for rapid identification of methylation status, including:

Primer 1: 5'-TCGATTGTATTTTTGGGTAAGC-3' (SEQ ID No. 1);

Primer 2: 5'-TAACGCGACTAACGTCTTCG-3' (SEQ ID No. 2);

Primer 3: 5'-GTTGATTGTATTTTTGGGTAAGT-3' (SEQ ID No. 3);

Primer 4: 5'-ACATAACACAACTAACATCTTCA-3' (SEQ ID No. 4).

Primer 5: 5'-TACGTTAGTTTCGCGTCGAC-3' (SEQ ID No. 5);

Primer 6: 5'-CGTATAAAAACCACCGAACG-3' (SEQ ID No. 6);

Primer 7: 5'-GTTTTATGTTAGTTTTGTGTTGAT-3' (SEQ ID No. 7);

Primer 8: 5'-ACCATATAAAAAACCACCAAACA-3' (SEQ ID No. 8);

Primer 9: 5'-TAGGATTTAGATTGGGCGGC-3' (SEQ ID No. 1);

Primer 10: 5'-AAAAAAACTCTACGAAAACGCG-3' (SEQ ID No. 2);

Primer 11: 5'-TTTAGGATTTAGATTGGGTGGT-3' (SEQ ID No. 3);

Primer 12: 5'-CAAAAAAAACTCTACAAAAACACA-3' (SEQ ID No. 4).

2.3.1 Methylation-specific PCR (MSP)

(1) Prepare a 25 μL reaction system:

(2) Mixing, instant separation;

(3) On the PCR machine: 95°C for 10 minutes; 95°C for 30s, 60°C for 30s, 72°C for 30s, cycle times 42 times; 72°C for 5 minutes.

The inventors selected 67 colon cancer tissues and 22 normal colon tissues (confirmed by pathologists as pathologically normal adjacent to the cancer) to detect the methylation of the promoters of the three genes. MSP results showed that three genes, LRRC3B, PENK and RASSF1, were hypermethylated in colon cancer tissue (Figure 3), with methylation rates of 82.8%, 80.6% and 64.2%, respectively. The combined detection of the three reached 98.5%.

2.4 Plasma cell-free DNA methylation capture technology

Cell-free DNA (cfDNA) is believed to be released by apoptotic cells or necrotic tumor cells and secreted and released into the blood circulation by macrophages or other scavenger cells after being phagocytosed at a distance. Molecular genetic markers consistent with the primary tumor tissue. Circulating tumor DNA comes from tumor cells, accounting for about 0.01%-1% of plasma free DNA, with a short half-life (about 2 hours), which can accurately reflect the current status of tumors. In order to clarify the sensitivity of these genes in cell-free DNA, the inventors used shingled methylation capture probe methylation enrichment sequencing to remove the methylation level in cell-free DNA according to the FDR<0.05 and the absolute difference of β>0.2. Genes at hypermethylated sites in colon cancer were identified above those genes that were methylated in tissues.

The methylation status of the plasma cell-free DNA LRRC3B, PENK and RASSF1 promoters detected by the plasma cell-free DNA methylation capture technology is shown in Figure 4. The results show that the promoter methylation levels of the three genes in the plasma cell-free DNA of colon cancer patients Significantly higher than normal healthy human plasma cell-free DNA.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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Claims (10)

1. Use of a methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes in the manufacture of a kit for diagnosing whether a subject has colorectal cancer or for predicting whether a subject is at risk of having colorectal cancer or for determining a prognosis for colorectal cancer.

2. The use of claim 1, wherein the methylation detection reagent is used to detect the promoter methylation status of the gene.

3. The use of claim 2, wherein the methylation detection reagent comprises a specific primer for detecting the methylation status of the gene promoter.

4. The use of claim 3, wherein the specific primers for detecting the methylation state of the LRRC3B gene promoter comprise an upstream primer having a nucleotide sequence shown by SEQ ID number 1 and a downstream primer having a nucleotide sequence shown by SEQ ID number 2; the specific primers for detecting the unmethylated state of the LRRC3B gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID number 3 and a downstream primer with a nucleotide sequence shown in SEQ ID number 4.

5. The use as claimed in claim 3, wherein the specific primers for detecting the methylation status of the PENK gene promoter comprise an upstream primer having a nucleotide sequence shown by SEQ ID number 5 and a downstream primer having a nucleotide sequence shown by SEQ ID number 6; the specific primers for detecting the non-methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 7 and a downstream primer with a nucleotide sequence shown by SEQ ID number 8.

6. The use according to claim 3, wherein the specific primers for detecting the methylation state of the RASSF1 gene promoter comprise an upstream primer having the nucleotide sequence shown by SEQ ID number 9 and a downstream primer having the nucleotide sequence shown by SEQ ID number 10; the specific primers for detecting the unmethylated state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID number 11 and a downstream primer with a nucleotide sequence shown in SEQ ID number 12.

7. The use of any one of claims 1-6, wherein the methylation detection reagent further comprises a reagent for extracting DNA from a sample.

8. The use of any one of claims 1 to 5, wherein the sample is whole blood or plasma and the DNA is plasma-free DNA.

9. Use of a promoter methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes in the preparation of a kit suitable for use in a method comprising:

s1, extracting DNA of the sample of the subject,

s2, detecting the promoter methylation state of the gene by using the detection reagent,

s3, diagnosing whether the subject has colorectal cancer or predicting whether the subject is at risk of having colorectal cancer or judging the prognosis of colorectal cancer according to the methylation state detected by the S2.

10. A kit for diagnosing whether a subject has colorectal cancer or predicting whether a subject has a risk of having colorectal cancer or for judging a prognosis of colorectal cancer, comprising a promoter methylation state detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes, wherein a specific primer for detecting the methylation state of the promoter of LRRC3B gene comprises an upstream primer having a nucleotide sequence represented by SEQ ID number 1 and a downstream primer having a nucleotide sequence represented by SEQ ID No. 2; the specific primers for detecting the methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 5 and a downstream primer with a nucleotide sequence shown by SEQ ID number 6; the specific primers for detecting the methylation state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 9 and a downstream primer with a nucleotide sequence shown by SEQ ID number 10.

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