CN114921555A - DNA methylation biomarker for early liver cancer detection, application and kit - Google Patents

Abstract

The invention discloses a DNA methylation biomarker for early liver cancer detection, application and a kit, and belongs to the technical field of biological detection. The kit can be used for obtaining the DNA methylation marker as an early liver cancer diagnosis marker by researching the methylation difference of DNA methylation modification on specific CpG sites of plasma cfDNA in people with liver cancer patients at different periods. The invention discloses two specific methylation markers related to liver cancer occurrence, which can obviously improve the detection rate of early liver cancer and are suitable for large-scale popularization and application in view of the non-invasiveness of plasma detection.

Description

DNA methylation biomarker for early liver cancer detection, application and kit

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a DNA methylation biomarker for early liver cancer detection, application and a kit.

Background

Liver cancer is the sixth and third most common malignant disease of morbidity and mortality worldwide, and more than 83 thousands of people die from liver cancer in 2020. According to data published by the world health organization international cancer research institution in 2020, liver cancer is second to lung cancer, colorectal cancer, gastric cancer and breast cancer, which are the fifth cancer of Chinese morbidity, and the mortality rate is behind lung cancer, and the rank is the second. Liver cancer causes endless pain to individuals and also causes heavy economic burden to individuals and society.

It is well known that early detection and early treatment of cancer can greatly improve the survival of patients. The relative 5-year survival rate of liver cancer is only 12.1%, but if the early diagnosis is treated effectively, a relatively good prognosis can be achieved, especially the 5-year survival rate of small liver cancer within 5 centimeters can reach more than 80%. At present, the screening effect of liver cancer is not ideal, the detection rate and the early diagnosis rate are relatively low, and how to discover cancer early becomes the focus of attention in the medical field, so that the development of a novel convenient early cancer screening method has great significance for individuals and society.

DNA methylation is closely associated with tumor development and changes in DNA methylation status are prevalent in tumor cells, characterized by decreased overall methylation levels and increased local methylation levels. Also, methylation modification plays a crucial role in the development of early stage tumors. Compared with other blood tumor markers, the blood cfDNA methylation stability is better, and the blood cfDNA methylation stability can be stably detected. Therefore, the detection of the methylation of the blood cfDNA at a specific site can be used for conjecturing whether the liver cancer in a body has canceration or not, so that the aim of early screening and early diagnosis of the liver cancer is fulfilled.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a DNA methylation biomarker for early liver cancer detection, application and a kit.

The technical scheme adopted by the invention is as follows: a DNA methylation biomarker for early liver cancer detection, the methylation biomarker comprising cg 2404194 and/or cg20170223 in the human genome;

the DNA methylation marker of the cg 245632 refers to CpG sites between chr6:31527895 and 31527945 in the human genome;

the DNA methylation marker of the cg20170223 refers to CpG sites between chr20:34356175-34356225 in the human genome.

Preferably, the human genome is the hg19 human genome.

An application of the DNA methylation biomarker for early liver cancer detection in a liver cancer detection product.

A kit for early liver cancer detection, said kit comprising said DNA methylation biomarker;

and probes that also include methylated biomarkers.

Preferably, the probe is a hybridization capture DNA sequence fragment containing the methylation biomarker.

Preferably, the kit adopts the following platform for detection: PCR amplification, digital PCR, fluorescent quantitative PCR, methylation chip method, liquid chip method, bisulfite sequencing, first generation sequencing, second generation sequencing, third generation sequencing or combinations thereof.

Preferably, the kit employs a second generation sequencing method.

The detection process of the kit comprises the following steps:

step 1, extracting cfDNA of a detection sample, carrying out enzyme conversion treatment on the NEB EM-seq kit, and building a methylated DNA library;

step 2, using the liver cancer prediction panel to carry out targeted hybridization and capture a library;

step 3, performing second-generation sequencing after quantifying the target hybridization capture library;

and 4, taking the data off the machine, and performing quality control and pretreatment on the sequencing data to obtain real data.

And 5, carrying out data analysis on the methylated biomarkers and the methylated biomarkers combination.

The invention has the beneficial effects that:

the invention provides a kit for detecting early liver cancer, which is obtained by researching the methylation difference of DNA methylation modification on specific CpG sites of plasma cfDNA in people with liver cancer patients at different periods, and a DNA methylation marker can be used as an early liver cancer diagnosis marker.

The invention discloses two specific methylation markers related to liver cancer occurrence, which can obviously improve the detection rate of early liver cancer and are suitable for large-scale popularization and application in view of the non-invasiveness of plasma detection.

Drawings

FIG. 1 is a flow chart of the development of two methylation markers for detecting liver cancer;

FIG. 2 is a schematic of the ROAUC for the independent prediction of methylated biomarker cg 245694 in plasma samples;

FIG. 3 is a schematic representation of the ROAUC independently predicted for the methylated biomarker cg20170223 in TCGA plasma samples;

FIG. 4 is a graphical representation of the ROAUC predicted for the combination of two methylation marker combinations in a plasma sample.

Detailed Description

The present invention is further illustrated below with reference to specific examples. It will be appreciated by those skilled in the art that the following examples, which are set forth to illustrate the present invention, are intended to be part of the present invention, but not to be construed as limiting the scope of the present invention. The reagents used are all conventional products which are commercially available.

The experimental procedures not specified in the examples of the invention described below are generally carried out using conventional experimental conditions, or according to standard conditions specified by the manufacturer. The various reagents used in the examples are commercially available.

Technical and scientific terms used herein are the same as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the invention discloses at least one or a combination of two plasma cfDNA gene methylation markers for liver cancer detection and application. The gene fragment specifically comprises the following gene fragments with obvious differential methylation in the blood plasma of the liver cancer patient: cg 245639, cg 20170223.

In order to solve the problems of low content of plasma free cfDNA and damage of bisulfite to DNA, the invention adopts NEBNext enzymetic Methyl-seq kit to convert cytosine into uracil.

The liver cancer is divided into stages I-IV according to an AJCC TNM international standard staging system, and the early stage of the liver cancer comprises stage I.

In one embodiment of the invention, the screening of cfDNA methylated liver cancer markers and the detection of liver cancer comprise the following steps: as shown in fig. 1:

step 1, excavating a liver cancer specific DNA methylation marker according to a TCGA public database;

step 2, designing and synthesizing a capture probe of the liver cancer specific methylation marker;

step 3, extracting plasma cfDNA, performing enzyme conversion and establishing a methylated DNA library;

step 4, capturing a methylated DNA library by the liver cancer specific panel in a targeted manner;

step 5, performing second-generation sequencing after library quantification;

step 6, calculating the methylation level of the target site after performing quality control and pretreatment on sequencing data;

and 7, analyzing and screening the methylation markers with consistent methylation difference in the tissues of the liver cancer and the blood plasma, and constructing and verifying an algorithm model for liver cancer detection.

Experimental example 1 method for detecting plasma cfDNA methylation marker for liver cancer detection

1. Blood cf DNA extraction: the specific operation steps of the blood cf DNA extraction are carried out according to the operation instruction of the free DNA extraction kit (suction filtration method) in combination with a vacuum suction filtration pump: the blood was lysed with proteinase K and lysis buffer, DNA bound using a vacuum pump and DNA binding column, washed with washing solution and absolute ethanol, and finally eluted with elution buffer.

2. Fragment screening of cfDNA using magnetic beads:

adding SPRIselect magnetic beads which are 0.75 time of cfDNA products into cfDNA, uniformly mixing by vortex, and incubating for 5 minutes at room temperature; placing the solution on a magnetic frame, and sucking supernatant into a new 1.5mL centrifuge tube for later use after the solution is clarified;

adding SPRISELect magnetic beads which are 1.05 times of cfDNA products into the supernatant of the previous step, blowing, uniformly mixing, incubating at room temperature for 5 minutes, placing on a magnetic frame, and removing the supernatant after the solution is clarified;

thirdly, washing with newly prepared 80 percent ethanol, removing supernatant, and repeating the step once; air-drying, and eluting with elution buffer.

3. Preparing quality control products: 10 μ L of 0.1 ng/. mu.L CpG methylated pUC19 and 10 μ L of 2 ng/. mu.L unmethylated lambda DNA were added to each sample; two quality control products need to be broken in advance by a Covaris M220 ultrasonic breaking instrument and screened to be about 200 bp.

4. Library construction of cfDNA: the library was constructed using NEBNext enzymic Methyl-Seq kit.

4.1 end repair and 3' end addition of "A"

4.1.1 taking 50ng of a sample to be tested, diluting the sample to 50 mu L by using NF water, and then adding the following reagents for reaction.

Components	Volume (μ L)
		Control DNA working solution	20
End repairing reaction liquid	5
		End repairing enzyme mixture	5

4.1.2 placing in a PCR apparatus to carry out the reaction according to the following procedure

4.2 Joint connection

4.2.1 reaction of the end-repairing product with the following reagents

Components	Volume (mu L)
		EM-seq connection joint solution	5
Connection enhancing fluid	5
		Connecting mixed liquid	50

4.2.2 placing the mixture in a PCR instrument, and setting a PCR reaction program according to the following conditions:

60 ℃ for 60min, the hot lid is closed.

4.2.3 linker ligation product purification

A. And (4) balancing the purified magnetic beads for 30min at room temperature, and fully and uniformly mixing the purified magnetic beads on a vortex instrument.

B. According to the weight ratio of absolute ethyl alcohol: preparing 80% ethanol according to the proportion of 8:2 for later use.

C. Adding 55 mu L of purified magnetic beads into the connection products respectively, blowing, uniformly mixing, incubating at room temperature for 5 minutes, placing on a magnetic frame, and removing the supernatant after the solution is clarified; washing with newly prepared 80% ethanol, discarding the supernatant, and repeating the steps once; air-dried, eluted with 15. mu.L of elution buffer, and then 14. mu.L of supernatant was aspirated for the next reaction.

4.3 methylation: oxidation reaction

4.3.1 preparation of buffer:

a. add 400ul E1 buffer to 100ul E1 buffer, mix well, label the preparation date.

b, 10 mu.L of 500mM Fe (II) is added into 1249 mu.L of nuclease-free water, and the diluted solution is used immediately and is ready to use and cannot be stored.

c dilution of stop buffer with nuclease free water at a ratio of 1: 10.

4.3.2 to 14. mu.L of the purified ligation product, the following reagents were added and mixed, and then to the purified ligation product to which oxidase had been added, 10. mu.L of diluted Fe (II) was added and mixed and centrifuged.

4.3.3 placing on a PCR instrument, and reacting according to the following conditions: 37 ℃ for 1h, and the temperature of a hot cover is not less than 45 ℃.

4.3.4 after the reaction is completed, 1. mu.L of diluted reaction stop solution is added to the product, and the mixture is placed on a PCR instrument and reacted according to the following conditions: 37 ℃ for 30min, and the temperature of a hot cover is not less than 45 ℃.

4.3.5 Oxidation reaction product purification

A. Balancing the purified magnetic beads for 30min at room temperature, and fully and uniformly mixing the purified magnetic beads on a vortex instrument;

B. according to the weight ratio of absolute ethyl alcohol: preparing 80% ethanol according to the proportion of 8:2 for later use;

C. respectively adding 45 mu L of NEB Next Sample purified magnetic beads into the connection products, blowing, uniformly mixing, incubating for 5 minutes at room temperature, placing on a magnetic frame, and removing the supernatant after the solution is clarified; washing with newly prepared 80% ethanol, discarding the supernatant, and repeating the steps once; air dried, eluted with 9.5. mu.L of elution buffer, and 8. mu.L of supernatant was aspirated for the next reaction.

4.4 methylation: cytosine deamination

4.4.1 denaturation:

a, preheating a PCR instrument to 85 ℃ in advance, and opening a hot cover;

b, adding 2 mu L formamide into 8 mu L of the purified oxidation reaction product, carrying out vortex oscillation, uniformly mixing, and carrying out instantaneous centrifugation.

4.4.2 adding the following reagents to the denatured product to carry out the reaction

Components	Volume (μ L)
		Nuclease-free water	34
E2 reaction solution	5
		Bovine serum albumin	0.5
E2	0.5

4.4.3 placing on a PCR instrument, and reacting according to the following conditions: 37 ℃ for 3h, and the hot cover temperature is ≧ 45 ℃.

4.4.4 purification of Cytosine deamination products

a, balancing purified magnetic beads for 30min at room temperature, and fully and uniformly mixing the purified magnetic beads on a vortex instrument;

b according to absolute ethyl alcohol: preparing 80% ethanol for later use according to the proportion of nuclease-free water to 8: 2;

c, respectively adding 50 mu L of NEB Next Sample purified magnetic beads into the connection products, blowing, uniformly mixing, incubating for 5 minutes at room temperature, placing on a magnetic frame, and removing the supernatant after the solution is clarified; washing with newly prepared 80% ethanol, discarding the supernatant, and repeating the steps once; air dried, eluted with 11. mu.L of elution buffer, and 10. mu.L of supernatant was aspirated for the next reaction.

4.5PCR amplification and purification

4.5.1 adding the following reagents to the above purified product to carry out a reaction

Components	Volume (mu L)
		Primer and method for producing the same	2.5
Enzyme mixture	12.5

4.5.2 placing on a PCR instrument, and reacting according to the following conditions

4.5.3 purification of PCR amplification products

a, balancing purified magnetic beads for 30min at room temperature, and fully and uniformly mixing the purified magnetic beads on a vortex instrument;

b according to absolute ethyl alcohol: preparing 80% ethanol at a ratio of 8:2 for later use;

c, respectively adding 22.5 mu L of NEB purified magnetic beads into the connection products, blowing, uniformly mixing, incubating for 5 minutes at room temperature, placing on a magnetic frame, and removing the supernatant after the solution is clarified; washing with newly prepared 80% ethanol, discarding the supernatant, and repeating the steps once; air-drying, eluting with 15 μ L of elution buffer, sucking 14 μ L of supernatant, and performing quality inspection.

5. Elution of hybridization

5.1 hybridization:

a, taking 10ng of each library to be hybridized, calculating the library volume required by combined hybridization, and mixing 12 libraries together;

b, adding the following reagents into the library mixed solution for reaction;

c, opening a vacuum concentrator, setting a V-AQ mode, and concentrating into dry powder at normal temperature;

d, immediately adding 20 mu L of hybridization mixed solution into the concentrated dry powder, uniformly mixing, standing at room temperature for 5 minutes, adding 30 mu L of hybridization enhancement solution, uniformly mixing and centrifuging;

e, placing the mixture on a PCR instrument, and carrying out reaction according to the following conditions.

TABLE 1 reagent composition Table

Components	Volume (μ L)
		Liver cancer probe	2
Internal reference probe	2
		Universal sealing liquid	8
Closed reaction solution	5

TABLE 2 PCR reaction conditions Table

5.2 Capture elution

5.2.1 buffer preheating:

preheating the rapid binding solution and the rapid washing solution 2 at 48 ℃ until the precipitate is dissolved, preheating the rapid washing solution 1 at 63 ℃ until the precipitate is dissolved, and balancing the streptavidin magnetic beads at room temperature for at least 30 min.

5.2.2 magnetic bead Capture

a, vortexing, shaking and uniformly mixing the streptavidin magnetic beads which are balanced to room temperature;

b, taking 70 mu L of streptavidin magnetic beads, and adding the streptavidin magnetic beads into a 1.5mL centrifuge tube;

c, adding 20 mu L of binding buffer solution, blowing, uniformly mixing, placing on a magnetic frame after instantaneous centrifugation, standing for 1min, and removing supernatant;

d, repeating the steps b and c twice, and carrying out streptavidin magnetic bead cleaning for three times;

e adding 20 mu L of binding buffer solution to resuspend streptavidin magnetic beads;

f transfer 20. mu.L of resuspended streptavidin magnetic beads to the hybridization reaction tube and then transfer the entire liquid to a 1.5mL centrifuge tube.

g, uniformly mixing the materials on a uniformly mixing instrument for 30 minutes at room temperature without vortex;

and h, after the uniform mixing is finished, performing instantaneous centrifugation, placing on a magnetic frame, standing for 1 minute, and removing the supernatant.

5.2.3 elution

a, adding 200 mu L of quick washing buffer solution 1 preheated at 54 ℃, uniformly blowing and stirring, and placing in a constant-temperature metal bath for incubation at 54 ℃ for 5 min;

b, after the incubation is finished, performing instantaneous centrifugation, transferring all liquid into a new 1.5mL centrifuge tube, and removing the non-specific capture fragment bound on the surface of the centrifuge tube;

c, placing the mixture on a magnetic frame, standing for 1 minute, and discarding the supernatant;

d, adding 200 mu L of washing buffer solution 1 preheated at 63 ℃, uniformly blowing and stirring, and placing in a constant-temperature metal bath for incubation at 54 ℃ for 5 minutes;

e, after the incubation is finished, performing instantaneous centrifugation, placing on a magnetic frame, standing for 1min, and removing a supernatant;

f, adding 200 mu L of washing buffer solution 2 preheated at 54 ℃, uniformly blowing and beating, and placing in a constant-temperature metal bath for incubation at 54 ℃ for 5 minutes;

g, after incubation is finished, performing instantaneous centrifugation, placing on a magnetic frame, standing for 1 minute, and removing a supernatant;

h, repeating the steps a to g for two times, and cleaning for three times;

i instantaneous centrifugation, 10. mu.L pipette tip to discard residual supernatant, immediately adding 45. mu.L nuclease-free water, blowing and mixing, and incubating on ice.

5.3 PCR enrichment of Capture products

5.3.1 reaction was carried out by adding 22.5. mu.L of the capture product (streptavidin magnetic bead suspension) to the following reagents.

Components	Volume (μ L)
		Amplification primers	2.5
Taq U + enzyme reaction solution	25

Placing the mixture on a PCR instrument to perform reaction under the following conditions

5.3.2 PCR product purification

a, balancing DNA purified magnetic beads for 30min at room temperature, and fully and uniformly mixing on a vortex instrument;

b according to absolute ethyl alcohol: preparing 80% ethanol at a ratio of 8:2 for later use;

c, respectively adding 90 mu L of DNA purified magnetic beads into the connection products, blowing, uniformly mixing, incubating for 5 minutes at room temperature, placing on a magnetic frame, and after the solution is clarified, discarding the supernatant; washing with newly prepared 80% ethanol, discarding the supernatant, and repeating the steps once; air-drying, eluting with 32 μ L elution buffer, sucking 30 μ L supernatant for quality control, and performing sequencing on the machine.

6 analysis of sequencing data:

raw data from off-line first low quality sequences (phred33 score < (20)) were filtered using fastQC, as well as linker sequences and polyA/T sequences in read. And (3) replying the filtered high-quality reads to the hg19 human genome by using BSMAP, and screening the reads with higher replying quality. Picard was used to remove PCR repeats followed by selection of reads that were attached back to the genomic region of interest (the region corresponding to the DNA methylation marker) by samtools and calculation of the methylation level of each DNA methylation marker.

The cfDNA fragmentation markers calculate the ratio of the number of cfDNA fragments ranging in length from 80bp to 150bp to the number of cfDNA fragments ranging in length from 150bp to 200bp within the gene region covered by each probe.

Experimental example 2:

this example verifies two DNA methylation markers disclosed in the present invention for liver cancer detection based on 245 benign liver disease plasma and 248 liver cancer plasma samples.

As shown in fig. 2 and 3, two methylation markers independently distinguished AUC values of liver cancer plasma.

As shown in FIG. 2, the AUC value of the model constructed by using the methylation marker cg 245633, which distinguishes benign liver disease plasma from liver cancer plasma, is 0.862.

As shown in FIG. 3, the AUC value for differentiating benign liver disease plasma from liver cancer plasma by using the model constructed by the methylation marker cg 245639 is 0.854.

The above shows that both methylation markers have certain efficacy for predicting liver cancer.

Further, as shown in FIG. 4, the model constructed by combining two methylation markers has the best predictive performance, with an AUC value of 0.947.

In conclusion, the invention discloses two specific methylation markers related to liver cancer occurrence, which can obviously improve the detection rate of early liver cancer, and is suitable for large-scale popularization and application in view of the non-invasiveness of plasma detection.

The present invention is not limited to the above alternative embodiments, and any other products in various forms can be obtained by the present invention, and the present invention is within the protection scope of the present invention. The above embodiments should not be construed as limiting the scope of the present invention, and it will be understood by those skilled in the art that modifications may be made to the technical solutions described in the above embodiments, or equivalent substitutions may be made to some or all of the technical features thereof, without departing from the scope of the present invention, and at the same time, such modifications or substitutions may not make the essence of the corresponding technical solutions depart from the scope of the embodiments of the present invention.

Claims (7)

1. A DNA methylation biomarker for early stage liver cancer detection, wherein the methylation biomarker comprises cg 2404194 and/or cg20170223 in the human genome;

the DNA methylation marker of cg 245604194 refers to CpG sites between chr6:31527895 and 31527945 in the human genome;

the DNA methylation marker of the cg20170223 refers to CpG sites between chr20:34356175-34356225 in the human genome.

2. The DNA methylation biomarker for early stage liver cancer detection according to claim 1, wherein the human genome is hg19 human genome.

3. Use of the DNA methylation biomarker for early liver cancer detection according to claim 1 or 2 in a liver cancer detection product.

4. A kit for early liver cancer detection, comprising the DNA methylation biomarker of claim 1 or 2;

and probes that also include methylated biomarkers.

5. The kit for detecting early liver cancer according to claim 4, wherein the probe is a DNA sequence fragment for capturing the methylated biomarker according to claim 1 by hybridization.

6. The kit for detecting early liver cancer according to claim 5, wherein the kit is used for detection by adopting the following platforms: PCR amplification, digital PCR, fluorescent quantitative PCR, methylation chip method, liquid chip method, bisulfite sequencing, first generation sequencing, second generation sequencing, third generation sequencing or combinations thereof.

7. The kit for detecting early liver cancer according to claim 6, wherein the kit adopts a next generation sequencing method.

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