DNA methylation biomarker for liver cancer detection and application of DNA methylation biomarker

Abstract

The invention provides a DNA methylation biomarker for liver cancer detection and application of the DNA methylation biomarker, and belongs to the technical field of biology. The biomarker disclosed by the invention is selected from any one of the following methylation sites: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112 and cg26472334, and the biomarker disclosed by the invention can be used for detecting the methylation sites of the cg13597025 and the cg00033450. The biomarker provided by the invention can be used for noninvasive detection of liver cancer, and has important significance for early screening of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to DNA methylation biomarkers for liver cancer detection and their applications. Background Art

[0002] Liver cancer is a malignant tumor that occurs in the liver. The early detection of liver cancer mainly relies on imaging examinations such as B-ultrasound and magnetic resonance. However, the misdiagnosis rate and missed diagnosis rate are both relatively high, and there is an urgent need to develop a non-invasive liquid biopsy method to assist in the early detection and screening of liver cancer. In the research of Yang Binghui (1999) et al. in "Screening and Early Diagnosis of Liver Cancer", only alpha-fetoprotein (AFP) is used to assist its early diagnosis, but the sensitivity and specificity are both relatively low. The sensitivity is only about 60-70%, and the specificity is only about 70-80%.

[0003] DNA methylation plays an important role in cell differentiation and disease occurrence by regulating gene transcription and expression. In the past few decades, the research on methylation in the gene promoter region has made rapid progress, and has successfully entered clinical transformation and application, and its clinical application value in tumor risk screening and early diagnosis has been initially revealed. The prior art CN113234817A discloses a biomarker for detecting early liver cancer using the methylation level of CpG sites, mainly including the following methylation sites: cg14171514, cg07721852, cg05166871, cg18087306, cg05213896, cg18772205. Without traditional liver biopsy, CT, MRI and other imaging examinations, the detection level is highly reliable, and a new method for non-invasive detection of the human liver has been developed. However, this method needs to comprehensively consider the methylation levels of 6 sites, and the experimental process is time-consuming and laborious. Summary of the Invention

[0004] In order to search for DNA methylation sites closely related to malignant tumors such as liver cancer, and develop early diagnosis strategies for malignant tumors such as liver cancer based on these DNA methylation sites will have significant economic and social benefits. The present invention provides a DNA methylation biomarker for liver cancer detection and its applications. The specific technical solutions are as follows:

[0005] On the one hand, the present invention provides an application of a methylation site as a biomarker in a liver cancer detection product, and the methylation site includes at least one methylation site selected from the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112 and cg26472334.

[0006] Specifically, the methylation site is selected from any one of the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

[0007] Specifically, the product includes, but is not limited to: kits, chips, or test strips.

[0008] Specifically, the kit is achieved by detecting the methylation level of the aforementioned biomarker in a sample to be tested, and the detection methods include: bisulfite sequencing PCR, methylation-specific PCR, pyrosequencing, Sanger sequencing, high-throughput sequencing, third-generation sequencing, or single-molecule sequencing.

[0009] Specifically, when the methylation level of cg13597025 is greater than 0.718346, cg00033450 is greater than 0.565371, cg20335273 is greater than 0.631323, cg06121113 is greater than 0.663489, cg27629428 is greater than 0.693474, cg02307184 is greater than 0.162977, cg15602112 is greater than 0.554076, and cg26472334 is greater than 0.296116, it indicates liver cancer.

[0010] In some embodiments, the test samples of the kit include any one or more of tissue, whole blood, plasma, and serum.

[0011] Specifically, the kit includes reagents for detecting the methylation level of the biomarker, and may also include any one or more of: sample collection reagents, sample preservation reagents, and sample pretreatment reagents.

[0012] More specifically, the reagent for detecting the methylation level of the biomarker includes amplification primers and an amplification reaction system; the sample pretreatment reagent includes a DNA extraction reagent and a methylation reagent.

[0013] On the other hand, the present invention provides a kit for detecting liver cancer, which includes reagents for detecting the methylation level of methylation sites serving as biomarkers, and the methylation sites include at least one methylation site selected from the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

[0014] On the other hand, the present invention provides a computer system for diagnosing liver cancer, which includes a detection device, a calculation device, and an output device, wherein:

[0015] The detection device includes a sampler and a detector, and the detector is used to detect the methylation level of any methylation biomarker in the sample;

[0016] The biomarker includes at least one methylation site selected from the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides new methylation sites, which can be used as biomarkers for liver cancer detection and have good discrimination ability. Description of the Drawings

[0019] Figure 1 For the methylation levels of the 8 methylation sites in normal human blood and liver cancer patient blood in Example 1, each point represents a mixture of 10 samples.

[0020] Figure 2 For the methylation levels of the 8 methylation sites in hepatitis B carrier blood and liver cancer patient blood in Example 2, each point represents a mixture of 10 samples. Detailed Embodiments

[0021] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0022] The important reagents or materials involved in the embodiments are shown in Table 1:

[0023] Table 1 Sources of Reagents or Materials

[0024]

[0025] Example 1 Detection of Methylation Sites by Whole Genome Methylation Chip

[0026] Training set information:

[0027] 60 cases of normal human blood, 60 cases of liver cancer patients.

[0028] The selection criteria for the liver cancer patients are as follows: primary hepatocellular carcinoma diagnosed pathologically, without a history of previous malignant tumors or severe medical diseases, and without receiving any form of anti-tumor treatment before surgery.

[0029] The normal subjects are healthy individuals with normal physical examinations.

[0030] 1.1 DNA extraction

[0031] Extract human DNA from the serum samples of the subjects. Specifically, use a DNA kit and follow the experimental steps in the kit's instruction manual.

[0032] 1.2 Methylation treatment of human DNA

[0033] Use a DNA methylation kit (EZ DNA Methylation Kit) for treatment. The specific experimental steps are as follows:

[0034] (1) Use a pipette to transfer 500 ng of the DNA sample to a PCR reaction tube. Dilute the sample with less than 45 μL with NucLease-FreeWater.

[0035] (2) Add 5 μL of M-Dilution Buffer to each PCR reaction tube containing 45 μL of the sample. Vortex for 15 s, then place the PCR reaction tube on a PCR instrument and incubate at 37 °C for 15 min.

[0036] (3) Then add 100 μL of CT Conversion Reagent and vortex for 30 seconds. Transfer 75 μL of the liquid to a new PCR reaction tube and centrifuge briefly. Place it in a PCR instrument and run 16 cycles of 95 °C for 30 s and 50 °C for 60 minutes.

[0037] (4) Assemble the Zymo-SpinTM extraction column with the collection tube. Add 400 μL of M-BindingBuffer to each extraction column, and transfer the reaction solution from step (3) to the extraction column. Shake well 10 times and incubate at room temperature for 1 min.

[0038] (5) Centrifuge the extraction column at 16100×g for 1 min, pour out the liquid in the collection plate, and recycle the collection tube. Then add 100 μL of M-Wash Buffer, incubate at room temperature for 1 min, centrifuge at 16100×g for 1 min, pour out the liquid in the collection tube, and recycle the collection tube.

[0039] (6) Add 200 μL of M-Desulphonation Buffer, incubate at room temperature for 20 min, centrifuge at 16,100×g for 1 min, pour out the liquid in the collection tube, and reuse the collection tube; add M-Wash Buffer to a new collection column, centrifuge at 16,100×g for 1 min, and discard the collection plate and the liquid in it.

[0040] (7) Replace with a new sample collection tube, add 11 μL of M-Elution Bufer, centrifuge at 16,100×g for 1 min, and collect the liquid in the sample collection tube.

[0041] (8) Quantify the purified sample using nano.

[0042] 1.3 DNA Methylation Chip Hybridization Detection

[0043] The DNA methylation chip used in this example is the Illumina Infinium MethylationEPIC BeadChip (935K DNA methylation chip). Amplify, fragment, precipitate, resuspend, hybridize, and extend according to the instructions to finally obtain the raw chip data and chip scan images.

[0044] (1) Add 20 μL of MA1 to the wells of the MSA4 plate respectively, then add 4 μL of the sample, record the sample number corresponding to the well on the MSA4 plate on lab tracking from, and then add 4 μL of 0.1 M NaoH to the MSA4 plate; oscillate and mix well, centrifuge at 280×g for 1 min on the centrifuge, and let stand for 10 min.

[0045] (2) Dispense 68 μL of RPM and 5 μL of MSM into each well of the MSA1 plate containing the sample, mix well, and centrifuge at 280×g for 1 min; incubate overnight at 37 °C for 22 h in a hybridization oven.

[0046] (3) Take out the MSA1 and centrifuge at 50×g for 1 min on the centrifuge, add 50 μL of FMS to the MSA4 plate and centrifuge at 50×g for 1 min, then incubate at 37 °C for 1 h; add 100 μL of PM1 to the MSA4 plate and incubate at 37 °C for 5 min, after centrifugation, add 300 μL of isopropanol, incubate in a 4 °C refrigerator for 30 min, then centrifuge at 3000×g for 20 min, discard the supernatant and dry.

[0047] (4) Add 46 μL of RA1 to the MSA4 plate, cover the MSA4 with metal foil and seal it, place it in a 48 °C hybridization oven and incubate for 1 h, and centrifuge at 280×g for 30 seconds in the centrifuge.

[0048] (5) Open the hybridization cassette, place the denatured MSA4, 26 μL of the sample and the chip, and then incubate at 48 °C for 24 h; then wash and scan the chip.

[0049] Through data analysis, 8 sites with large differences in methylation levels in the sample were screened out, namely cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112 and cg26472334. The detection results of the levels of 8 methylation sites in the training set samples are as Figure 1 shown. There are obvious differences in the levels of each methylation site between healthy people and liver cancer patients, and liver cancer patients and healthy people can be accurately distinguished.

[0050] Example 2 Verification of 8 methylation sites

[0051] Verification set sample information:

[0052] Blood of 60 hepatitis B carriers and 60 liver cancer patients.

[0053] The selection criteria for the hepatitis B carriers are: positive hepatitis B surface antigen.

[0054] The selection criteria for the liver cancer patients are: primary hepatocellular carcinoma diagnosed pathologically, without a history of previous malignant tumors and severe medical diseases, and no prior anti-tumor treatment before surgery.

[0055] Subsequently, the accuracy of determining liver cancer patients by testing 8 methylation sites in the population with hepatitis B and liver cancer patients was tested. The results are as Figure 2 shown. It can be seen that all 8 methylation sites can effectively distinguish liver cancer patients from hepatitis B carriers.

Claims

1. Use of a methylation site as a biomarker in the preparation of a liver cancer detection product, characterized in that, The methylation sites include at least one of cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

2. The application according to claim 1, wherein The methylation site is selected from any one of cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

3. The application according to claim 1, wherein The product is used to detect the methylation level of the biomarker described in claim 1 in a sample to be tested, and the detection methods include: bisulfite sequencing PCR, methylation-specific PCR, pyrosequencing, Sanger sequencing, high-throughput sequencing, third-generation sequencing, or single-molecule sequencing.

4. The application according to any one of claims 1 to 3, characterized in that The product described above includes: a kit, a chip, or a test strip.

5. The application according to claim 4, characterized in that The methylation level is as follows: cg13597025 greater than 0.718346, cg00033450 greater than 0.565371, cg20335273 greater than 0.631323, cg06121113 greater than 0.663489, cg27629428 greater than 0.693474, cg02307184 greater than 0.162977, cg15602112 greater than 0.554076, cg26472334 greater than 0.296116 indicates liver cancer.

6. The application according to claim 4, wherein The detection samples of the kit include: tissues, whole blood, plasma, and serum.

7. The application according to claim 1, characterized in that, The kit includes reagents for detecting the methylation level of the biomarker, and also includes at least one of: sample collection reagents, sample preservation reagents, and sample pretreatment reagents.

8. The application according to claim 7, wherein The reagents for detecting the methylation level of the biomarker include amplification primers and an amplification reaction system; the sample pretreatment reagents include DNA extraction reagents and methylation reagents.

9. A kit for diagnosing or assisting in the diagnosis of liver cancer, characterized in that, It includes reagents for detecting the methylation level of methylation sites serving as biomarkers, and the methylation sites include at least one methylation site selected from the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

10. A computer system for diagnosing liver cancer, characterized in that, The computer system includes a detection device, a computing device, and an output device; The detection device includes a sampler and a detector, and the detector is used to detect the methylation level of any methylation biomarker in the sample; The biomarker includes at least one methylation site selected from the following: cg13597025, cg00033450, cg20335273, cg06121113, cg27629428, cg02307184, cg15602112, and cg26472334.

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