CN115651982A

CN115651982A - Lung cancer methylation gene marker and application thereof

Info

Publication number: CN115651982A
Application number: CN202211108555.9A
Authority: CN
Inventors: 樊鸿宇; 闵敬丽; 陆军; 张林锋; 罗剑
Original assignee: Shanghai Yichuang Yunkang Biotechnology Co ltd
Current assignee: Shanghai Yichuang Yunkang Biotechnology Co ltd
Priority date: 2022-09-13
Filing date: 2022-09-13
Publication date: 2023-01-31

Abstract

The invention discloses a lung cancer methylation gene marker and application thereof. The gene marker is CSF2RB gene and/or DUSP5 gene. According to the invention, through research on methylation detection of CSF2RB gene and DUSP5 gene, primers and probes of the two genes are explored and optimized, and CSF2RB gene and DUSP5 gene methylation fragments with extremely low concentrations in a plasma sample can be detected, so that the sensitivity of diagnosis of a lung cancer patient is improved, and meanwhile, the specificity is high.

Description

Lung cancer methylation gene marker and application thereof

Technical Field

The invention belongs to the technical field of lung cancer detection, and particularly relates to a lung cancer methylation gene marker and application thereof.

Background

Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lung, and one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. The etiology of lung cancer is not completely clear up to now, and a large amount of data show that a large amount of smoking for a long time has a very close relationship with the occurrence of lung cancer.

The clinical manifestations of lung cancer are complex, and the presence, severity and early and late occurrence of symptoms and signs depend on the tumor occurrence location, pathological type, metastasis and complications, and differences in the degree of response and tolerance of patients, the early symptoms of lung cancer are mild and even no discomfort, the central symptoms of lung cancer appear early and severe, and the peripheral symptoms of lung cancer appear late and mild and even no symptoms, and are usually found during physical examination, and the symptoms of lung cancer are roughly classified as: local symptoms, systemic symptoms, extrapulmonary symptoms, infiltrates and metastases. Because the clinical symptoms of lung cancer are relatively complex, the lung cancer is diagnosed by adopting a traditional medical observation mode with larger error.

DNA methylation is a common epigenetic modification mode in human body, can regulate and control the expression condition of a modified gene, is considered to have close relation with the occurrence of tumor, and can complete early screening, auxiliary diagnosis, treatment and prognosis of the tumor by detecting the methylation state of a tumor-related gene. A number of lung cancer methylation genes have been screened, for example patent 201510203539.1 discloses a method and kit for diagnosing methylation of the human SHOX2 gene and the human RASSF1A gene; patent 202011112826.9 discloses PTGER4, TAC1, HOXA7 and SCT as diagnostic lung cancer-associated methylated gene combinations in plasma; patent 201810886500.8 is used for identifying lung nodule and/or lung cancer status, and application thereof, and discloses APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX, SOX17 and ZAR1 as markers for lung cancer detection. Although the prior art finds a plurality of lung cancer methylation markers, the detection accuracy of most of the markers is not strong, and new methylation markers still need to be developed so as to expand the selection of the markers and improve the detection accuracy.

Disclosure of Invention

The invention aims to provide a lung cancer methylation gene marker and application thereof.

A lung cancer methylation gene marker, wherein the gene marker is CSF2RB gene and/or DUSP5 gene.

A detection kit for methylation of a CSF2RB gene and/or a DUSP5 gene, comprising primers and probes for the CSF2RB gene and the DUSP5 gene; the sequences of the primer and the probe aiming at the CSF2RB gene are shown in a sequence table SEQ ID NO: 1. the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3 is shown in the figure; the sequences of the primer and the probe aiming at the DUSP5 gene are shown in a sequence table SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: and 6.

The kit also comprises a primer and a probe aiming at the reference gene GAPDH; the sequences of the primer and the probe aiming at the reference gene GAPDH are shown in a sequence table SEQ ID NO: 7. SEQ ID NO:8 and SEQ ID NO: shown at 9.

The kit also comprises a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent comprises a nucleic acid cracking adsorption solution, a proteinase K solution, magnetic beads, a washing solution and an eluent.

The nucleic acid lysis adsorption solution comprises the following components: 2M guanidine hydrochloride, 15% Triton X-100, 30mM Tris-HCl, 2% SDS, 3M guanidine isothiocyanate, 15% absolute ethanol, 15% isopropanol, 35% glycerol, purified water.

The proteinase K solution consists of the following components: 30mg/ml proteinase K, 50mM Tris-HCl, 40mM potassium chloride, 50mM sodium chloride, 20mM calcium chloride, 15% Triton X-100, 40% glycerol, purified water.

The kit further comprises a sulfite conversion reagent; the sulfite conversion reagent comprises conversion solution, binding solution, desulfonation solution, magnetic beads, washing solution and eluent.

The conversion solution consists of the following components: 50% ammonium bisulfite, 15% ammonium sulfite, 20% sodium bisulfite, 4M sodium hydroxide, purified water.

The binding liquid consists of the following components: 15% TritonX-100, 30mM Tris-HCl, 5M guanidinium isothiocyanate, 3M sodium chloride, 15% absolute ethanol, 15% isopropanol, 40% glycerol, purified water.

The desulfurated liquid consists of the following components: 1M sodium hydroxide, 50mM Tris-HCl, 35% absolute ethyl alcohol, 20% isopropyl alcohol, 50mM sodium chloride and purified water.

The invention has the beneficial effects that: the invention discovers 2 new lung cancer methylation gene markers CSF2RB gene and DUSP5 gene, searches and optimizes primers and probes of the CSF2RB gene and DUSP5 gene methylation detection, can detect CSF2RB gene and DUSP5 gene methylation fragments with extremely low concentration in a plasma sample, improves the sensitivity of lung cancer patients in diagnosis, and has high specificity.

Drawings

FIG. 1 shows the results of lung cancer sample measurement.

FIG. 2 shows the results of the measurement of a normal sample.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The reagents and starting materials used in the following examples were all commercially available unless otherwise specified.

Example 1 discovery of Lung cancer-specific methylated genes CSF2RB and DUSP5

Genomic DNA from 2 normal persons and genomic DNA from cancer tissues and adjacent normal tissues of 6 lung cancer patients were sonicated at 500ng each to construct a genomic DNA fragment of about 300 bp.

Mu.g of 6 XHis-labeled MBD2bt was pre-incubated with 500ng of genomic DNA of Escherichia coli JM110, and then bound to Ni-NTA magnetic beads. Will be treated by ultrasonic500ng each of the genomic DNAs isolated from normal human and lung cancer patients in a binding buffer (10 mM Tris-HCl, 50mM NaCl, 1mM EDTA, 1mM DTT, 3mM MgCl) ₂ 0.1% Triton-X100, 5% glycerol, 25mg/ml BSA) was reacted with the magnetic beads at 4 ℃ for 20 minutes. The beads were then washed 3 times with 500 μ L of binding buffer containing 700mM NaCl, and then methylated DNA bound to MBD2bt was isolated using the QiaQuick PCR purification kit.

Methylated DNA bound to MBD2bt was amplified with the genomic DNA amplification kit and 4. Mu.g of the amplified DNA was Cy 4-labeled with BioPrime Whole genome labeling System I. In order to indirectly compare the degree of methylation between normal humans and lung cancer patients, reference DNAs were constructed, the same amount of genomic DNAs from 6 lung cancer patients were mixed with each other, the genomic DNA mixture was amplified using a genomic DNA amplification kit, and Cy 3-labeling of 4 μ g of the amplified genomic DNAs was performed using a BioPrime whole genome labeling system I (Invitrogen corporation). The reference DNA was mixed with DNA of normal human and lung cancer patients, respectively, and then hybridized with 244K human CpG microarray (Agilent Corp.). After hybridization, the DNA mixture was subjected to a washing process and then scanned with an Agilent scanner. Signal values from the microarray images were calculated by calculating the relative difference in signal intensity between normal human and lung cancer patient samples using the Feature Extraction program v.9.5.3.1 (Agilent).

39870 probes having Cy3 signal values exceeding 115.3 among a total of 24 arrays were screened by a cross-gene error model using the GeneSpring 7.3 program (Agilent). The lung tissue of a normal person and a tissue that seems to be normal and adjacent to the lung tissue were used as the same group, and analysis of variance (ANOVA test) was performed, whereby 3467 probes (p < 0.05) were selected. From these probes, 1026 probes highly methylated in lung cancer tissues were further selected, and 2 biomarker candidate genes (CSF 2RB gene, DUSP5 gene) showing hypermethylation were selected from these probes. Pyrosequencing of the above 2 genes demonstrated high methylation in lung cancer cell lines, indicating that these genes are suitable as biomarkers for lung cancer diagnosis.

Example 2 CSF2RB Gene and DUSP5 Gene methylation detection primer design

The primers and probes for CSF2RB gene, DUSP5 gene and reference gene specifically comprise the following components:

specific primers and probes for CSF2RB gene methylation:

a forward primer: 5'-CCAGCCACATCACCTGCAGGTG-3' SEQ ID NO;

reverse primer: 5'-TGCCTGGTGTGGAGGCTGCCGAGCC-3' SEQ ID NO;

and (3) probe: 5'FAM-TCGGGCCACGTAGGTGCTGCTG-BHQ 1' SEQ ID NO;

specific primers and probes for DUSP5 gene methylation:

a forward primer: 5'-CGTCAACCTCAACTCGGTGGTGC-3' SEQ ID NO;

reverse primer: 5'-TGCTGTGCTCCCGTAATGGCTTGGC-3' SEQ ID NO;

and (3) probe: 5'FAM-CGCACTTGGATGCATGGTAGGCA-BHQ 1' SEQ ID NO;

specific primers and probes for reference gene GAPDH:

a forward primer: 5'-GTTTGGGTGGTGTAGGAGT-3' SEQ ID NO;

reverse primer: 5'-ACTATTCCTACAACCAAAACC-3' SEQ ID NO;

and (3) probe: 5'FAM-TGGAGGTGTTTTTAATGTTT-BHQ 1' SEQ ID NO.

Example 3 detection kit for methylation of CSF2RB Gene and DUSP5 Gene

The kit comprises the primer and the probe in the embodiment 1, and further comprises a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent comprises a nucleic acid lysis adsorption solution, a proteinase K solution, magnetic beads, a washing solution and an eluent.

The nucleic acid lysis adsorption solution comprises the following components: 2M guanidine hydrochloride, 15% TritonX-100, 30mM Tris-HCl, 2% SDS, 3M guanidine isothiocyanate, 15% absolute ethanol, 15% isopropanol, 35% glycerol, purified water.

The washing solution comprises a washing solution A, a washing solution B and a washing solution C, wherein the washing solution A and the washing solution B consist of 5M guanidine hydrochloride, 30mM sodium chloride, 15% absolute ethyl alcohol, 15% isopropanol and purified water; the washing solution C comprises 25% of absolute ethyl alcohol, 25% of isopropanol, 40mM of sodium chloride and purified water.

The conversion solution comprises the following components: 50% ammonium bisulfite, 15% ammonium sulfite, 20% sodium bisulfite, 4M sodium hydroxide, purified water.

The dosage of the primers, the probes and the reaction system of the kit is shown in the table 1-2:

TABLE 1 methylated premix (1 part by weight)

Components	Add volume (μ L)
		CSF2RB Forward primer (10. Mu.M)	0.5
CSF2RB reverse primer (10. Mu.M)	0.5
		DUSP5 Forward primer (10. Mu.M)	0.5
DUSP5 reverse primer (10. Mu.M)	0.5
		CSF2RB Probe (10. Mu.M)	0.2
DUSP5 Probe (10. Mu.M)	0.2
		GAPDH forward primer F (10. Mu.M)	0.3
GAPDH reverse primer R (10. Mu.M)	0.3
		GAPDH probe (10. Mu.M)	0.2
Water (W)	6.8

TABLE 2 25ul reaction system configuration (1 part)

Components	Dosage (mu L)
		2 XDNA polymerase Mix	12.5
Methylated premix	10.5
		DNA template	2

Example 4 detection of methylation of the CSF2RB Gene and DUSP5 Gene

3 histologically confirmed lung cancer samples and 3 normal samples were selected at the clinic stage in a three-tumor hospital.

Plasma separation: collecting 10mL of blood by using a free nucleic acid blood collection tube, putting the blood collection tube into a centrifuge for centrifugation for 12min, transferring the plasma into a 15mL centrifuge tube, centrifuging for 12min again, collecting 3mL of plasma, adding the plasma into a new centrifuge tube, and storing the collected plasma sample at-20 ℃.

Extraction of free DNA: adding 3mL of a plasma sample into a 15mL centrifuge tube, sequentially adding 350 [ mu ] L of a proteinase K solution, 8.25mL of a nucleic acid lysis adsorption solution and 40 [ mu ] L of magnetic beads, carrying out vortex mixing, placing the centrifuge tube in a 56 ℃ for 10min, placing the centrifuge tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste solution, adding 1mL of a washing solution A, mixing uniformly to ensure that the magnetic beads are thoroughly resuspended, transferring the suspended magnetic beads into a 1.5mL centrifuge tube, placing the centrifuge tube in the magnetic rack for magnetic attraction for 2min, sucking away all the waste solution, adding 0.6mL of a washing solution B, mixing uniformly to ensure that the magnetic beads are thoroughly resuspended, placing the centrifuge tube in the magnetic rack for magnetic attraction for 2min, sucking away all the waste solution, removing the residual liquid by using a 50 [ mu ] L gun head, transferring the centrifuge tube onto a nonmagnetic test tube, opening a cover of the centrifuge tube, drying for 2min, adding 50 [ mu ] L of the magnetic beads, placing the centrifuge tube in the magnetic rack for magnetic attraction for 2min, incubating, and mixing uniformly, placing the centrifuge tube in the magnetic rack for 2mL eluent at room temperature, and placing the centrifuge tube into a 2.5 ℃ for PCR.

Free DNA sulfite conversion: adding 100 mu L of sulfite solution into a 0.2mL PCR tube of 50 mu L DNA, covering the centrifugal tube, performing vortex mixing, then performing short-time centrifugation, placing the centrifugal tube in a common PCR instrument for reaction, setting the reaction conditions to be 95 ℃,5min, 60 ℃,10min, transferring the DNA solution after the reaction is finished into a new 1.5mL centrifugal tube, adding 600 mu L of binding solution and 2 mu L of magnetic beads, performing vortex mixing, standing at room temperature for 5min, placing the centrifugal tube in a magnetic frame for magnetic attraction for 2min, sucking all the waste solution, adding 500 mu L of washing solution B, performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended, placing the centrifugal tube in a magnetic frame for magnetic attraction for 2min, sucking all the waste solution, adding 500 mu L of desulfonation solution, performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended, standing at room temperature for 15min, placing the centrifugal tube in the magnetic frame for magnetic attraction for 2min, sucking all the waste solution, adding 500 mu L of washing liquid B, carrying out vortex mixing to ensure thorough resuspension of magnetic beads, placing a centrifugal tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 mu L of washing liquid B, carrying out vortex mixing to ensure thorough resuspension of magnetic beads, placing the centrifugal tube in the magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 250 mu L of washing liquid C, carrying out vortex mixing to ensure thorough resuspension of magnetic beads, placing the centrifugal tube in the magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, removing the residual liquid as much as possible by using a 50 mu L gun head, transferring the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, drying at room temperature for 2min, adding 50 mu L of eluent, covering the tube cover, carrying out vortex mixing to resuspension of magnetic beads, incubating the centrifugal tube at 56 ℃ for 4min, placing the centrifugal tube in the magnetic frame for magnetic attraction for 2min, and transferring all the eluent to a new 1.5mL PCR tube for later use.

Detecting a target gene by fluorescent quantitative PCR: taking out the PCR reaction solution from the kit, thawing at room temperature, fully thawing, slightly oscillating and uniformly mixing, and performing instantaneous low-time low-speed centrifugation for later use; adding DNA of a sample to be detected into the PCR reaction tubes prepared with the reagents respectively, adding 25 mu L of PCR reaction solution into each tube, covering the tube cover tightly, and centrifuging at a low speed instantaneously.

Set up the PCR reaction program as table 3:

TABLE 3

The signal acquisition channels are FAM, VIC and Cy5.

Results are judged as in table 4:

TABLE 4

The detection results of all the 3 normal samples are negative, the detection results of all the 3 lung cancer samples are positive, the detection results of the lung cancer samples are shown in figure 1, and the detection results of the normal samples are shown in figure 2, so that the primer provided by the invention is proved to be excellent in lung cancer detection specificity.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A lung cancer methylation gene marker, wherein the gene marker is CSF2RB gene and/or DUSP5 gene.

2. A detection kit for CSF2RB gene and/or DUSP5 gene methylation, which is characterized by comprising primers and probes for CSF2RB gene and DUSP5 gene; the sequences of the primer and the probe aiming at the CSF2RB gene are shown in a sequence table SEQ ID NO: 1. SEQ ID NO:2 and SEQ ID NO:3 is shown in the specification; the sequences of the primer and the probe aiming at the DUSP5 gene are shown in a sequence table SEQ ID NO: 4. SEQ ID NO:5 and SEQ ID NO: and 6, respectively.

3. The CSF2RB gene and/or DUSP5 gene methylation detection kit of claim 2, further comprising primers and probes for the internal reference gene GAPDH; the sequences of the primer and the probe aiming at the reference gene GAPDH are shown in a sequence table SEQ ID NO: 7. SEQ ID NO:8 and SEQ ID NO: shown at 9.

4. The CSF2RB gene and/or DUSP5 gene methylation detection kit of claim 2, further comprising nucleic acid extraction reagents comprising nucleic acid lysis sorbent solutions, proteinase K solutions, magnetic beads, wash solutions, eluents.

5. The CSF2RB gene and/or DUSP5 gene methylation detection kit according to claim 4, wherein the nucleic acid lysis sorbent solution comprises: 2M guanidine hydrochloride, 15% Triton X-100, 30mM Tris-HCl, 2% SDS, 3M guanidine isothiocyanate, 15% absolute ethanol, 15% isopropanol, 35% glycerol, purified water.

6. The kit for detecting methylation of the CSF2RB gene and/or DUSP5 gene according to claim 4, wherein said proteinase K solution comprises: 30mg/ml proteinase K, 50mM Tris-HCl, 40mM potassium chloride, 50mM sodium chloride, 20mM calcium chloride, 15% Triton X-100, 40% glycerol, purified water.

7. The kit for detecting methylation of the CSF2RB gene and/or DUSP5 gene of claim 4, wherein said kit further comprises a sulfite conversion reagent; the sulfite conversion reagent comprises conversion solution, binding solution, desulfonation solution, magnetic beads, washing solution and eluent.

8. The CSF2RB gene and/or DUSP5 gene methylation detection kit according to claim 7, wherein said transformation fluid consists of: 50% ammonium bisulfite, 15% ammonium sulfite, 20% sodium bisulfite, 4M sodium hydroxide, purified water.

9. The CSF2RB gene and/or DUSP5 gene methylation detection kit according to claim 7, wherein said binding solution consists of: 15% TritonX-100, 30mM Tris-HCl, 5M guanidinium isothiocyanate, 3M sodium chloride, 15% absolute ethanol, 15% isopropanol, 40% glycerol, purified water.

10. The kit for detecting methylation of the CSF2RB gene and/or the DUSP5 gene according to claim 7, wherein said desulfonation solution comprises the following components: 1M sodium hydroxide, 50mM Tris-HCl, 35% absolute ethyl alcohol, 20% isopropyl alcohol, 50mM sodium chloride and purified water.