CN107164508A - Gene marker for detecting liver cancer and application thereof - Google Patents

Abstract

The invention relates to a gene marker for detecting liver cancer and its application. The present invention also relates to a method for detecting liver cancer using the gene marker.

Description

Gene markers for detecting liver cancer and uses thereof

technical field

The present invention relates to the field of clinical molecular diagnosis of liver cancer. Specifically, the present invention relates to a method and a kit for detecting the presence of liver cancer by detecting the content of 5-hydroxymethylcytosine in liver cancer gene markers through high-throughput sequencing.

Background technique

Liver cancer is one of the most common malignancies worldwide. According to the statistics of the World Health Organization in 2008, there are 748,300 new cases and 695,900 deaths in the world every year, of which more than 50% occur in China. Among primary liver cancers, 70%-85% are hepatocellular carcinoma (HCC). At present, the 5-year survival rate of liver cancer is only 3%-5%, because most patients are already in the middle and advanced stages when they see a doctor, and the best time for treatment has been lost. Therefore, early detection, early diagnosis and early treatment are the key to improving the quality of life and prolonging the survival time of patients.

At present, the detection of HCC is mainly through imaging, tissue biopsy, serological detection and so on. However, imaging is easily affected by operator experience, depends on equipment, and is expensive. Especially in the case of limited medical resources, its accuracy is difficult to guarantee, and it is difficult to be widely and routinely applied. Tissue biopsy is currently the gold standard for clinically diagnosing liver cancer. However, tissue biopsy has great limitations, such as the difficulty of surgical sampling, or the inconvenient puncture of some cancer sites, and the puncture itself will also bring certain clinical risks. Repeated puncture Screening will bring great pain to patients. The most widely used serological test is the detection of alpha-fetoprotein (AFP), but the sensitivity and specificity of AFP to early liver cancer are not high, for example, in some patients with non-liver cancer chronic liver disease, such as many chronic hepatitis and liver cirrhosis In patients, serum AFP was also elevated.

In the early screening of liver cancer, the most difficult thing is the screening of small liver cancer. Small liver cancer is also called subclinical liver cancer or early liver cancer. There are no obvious symptoms and signs of liver cancer clinically. It generally refers to the maximum diameter of a single cancer nodule in hepatocellular carcinoma not exceeding 3 cm or the sum of the diameters of two cancer nodules not exceeding 3 cm. of liver cancer. The standard for small liver cancer in my country is: the largest diameter of a single cancerous nodule should not exceed 3 cm; the number of multiple cancerous nodules should not exceed two, and the sum of their largest diameters should be less than 3 cm. The surgical resection rate of small liver cancer is as high as 93.6%, the prognosis is better, and the survival rate is higher. Therefore, early screening of small liver cancer has important clinical significance. At present, the screening of small liver cancer mainly adopts methods such as ultrasonography, imaging diagnosis and detection of serum alpha-fetoprotein. However, as mentioned above, the accuracy and specificity of these traditional methods for the diagnosis of small HCC are not high.

Therefore, finding new liver cancer markers, especially markers for early warning monitoring and early diagnosis, is of great significance for improving the diagnosis rate of early liver cancer, realizing early intervention and treatment, and reducing the mortality rate of liver cancer.

Contents of the invention

The inventors performed high-throughput sequencing on normal samples and liver cancer samples, and analyzed the content of 5-hydroxymethylcytosine (5-hmC) in each gene, unexpectedly found a number of very informative Genetic markers that can be used to detect liver cancer.

Therefore, a first aspect of the present invention relates to genetic markers for the detection of liver cancer, comprising one or more genes selected from the group consisting of: FAT atypical cadherin 1 (FAT1), estrogen-related receptor gamma (ESRRG) , γ-aminobutyric acid class A receptor β3 subunit (GABRB3), TNF receptor superfamily member 11b (TNFRSF11B), receptor-interacting serine/threonine kinase 4 (RIPK4), rearranged L-myc fusion protein (RLF), solute carrier family 13 member 5 (SLC13A5), cytochrome P450 oxidoreductase (POR), and DeltexE3 ubiquitin ligase (DTX1). Preferably, the gene markers include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine genes selected from the following: FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR, and DTX1. More preferably, the gene markers include FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR and DTX1.

The present invention also relates to the use of the above gene markers in detecting liver cancer.

A second aspect of the present invention relates to a method for detecting liver cancer, comprising the steps of:

(a) Determination of the 5-hmC content of the gene markers of the present invention in normal samples and test samples;

(b) using the 5-hmC content of the gene marker in the normal sample as a reference, standardizing the 5-hmC content of the corresponding gene marker in the subject sample;

(c) mathematically correlating the normalized 5-hmC content of said genetic marker and obtaining a score; and

(d) Obtaining a test result according to the score, and a score P greater than 0.5 indicates that the subject sample has liver cancer.

In one embodiment, the sample is free DNA fragments in the subject or normal human body fluid, or complete genomic DNA derived from organelles, cells and tissues. Among them, the body fluids are blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic juice, etc.

In one embodiment, the 5-hmC content of the gene markers described in the present invention can be determined by any method known to those skilled in the art, for example including but not limited to, glucosylation method, restriction endonuclease method, chemical labeling method, precipitation method combined with high-throughput sequencing methods, single-molecule real-time sequencing (SMRT), oxidative bisulfite sequencing (OxBS-Seq), etc. The principle of the glucosylation method is to use T4 bacteriophage β-glucose transferase (β-GT) to transfer glucose to the hydroxyl position in the presence of the glucose donor substrate uridine diphosphate glucose (UDP-Glu), thereby Generates β-glucosyl-5-hydroxymethylcytosine (5-ghmC). At the same time, isotope-labeled substrates can be used for quantification. Based on the glucosylation method, the restriction endonuclease method and chemical labeling method were further developed. The principle of the restriction endonuclease method is that the glucosylation reaction changes the enzymatic cutting characteristics of some restriction endonucleases. Methylation-dependent restriction enzymes MspI and HpaII recognize the same sequence (CCGG), but their sensitivity to methylation status is different: MspI recognizes and cleaves 5-methylcytosine (5-mC) and 5-hmC, but cannot cleave 5-ghmC; HpaII cleaves only completely unmodified sites, and any modification on cytosine (5-mC, 5-hmC, 5-ghmC) hinders cleavage. If the CpG site contains 5-hmC, the band can be detected after glycosylation and enzymatic hydrolysis, but there is no band in the unglycosylated control reaction; at the same time, qPCR can be used for quantitative analysis. In addition, other restriction endonucleases also hinder 5-ghmC digestion and can be applied to 5-hmC detection (such as: GmrSD, MspJI, PvuRtslI, TaqI, etc.). The principle of the chemical labeling method is: chemically modify the glucose on the substrate of the enzyme reaction into UDP-6-N3-glucose, transfer 6-N3-glucose to the hydroxymethyl position, and generate N3-5ghmC. Subsequently, by adding a molecule of biotin to each 5-hmC by click chemistry, combined with next-generation high-throughput DNA sequencing technology or single-molecule sequencing technology, the distribution of 5-hmC in genomic DNA can be analyzed. The precipitation method is to modify 5-hmC in a special way and then specifically capture it from genomic DNA, and perform sequencing analysis. Oxidative bisulfite sequencing is the first method to quantitatively sequence 5-hmC with single-base resolution. First, 5-hmC is oxidized with KRuO4 to generate 5-formylcytosine (5fC), and then regenerated Bisulfite sequencing. During this process, 5-hmC is first oxidized to 5fC and then deaminated to form U. Usually, multiple detection methods are used simultaneously for quantitative detection of 5-hmC.

In one embodiment of the present invention, the 5-hmC content of the gene marker of the present invention is determined by using a chemical labeling method combined with high-throughput sequencing. In this specific embodiment, the method for determining the 5-hmC content of the gene marker of the present invention comprises the following steps: fragmenting the DNA of samples from liver cancer patients and normal persons; repairing the ends of the fragmented DNA and End-filling; Ligate the end-filled DNA with the sequencing adapter to obtain the ligation product; label the 5-hydroxymethylcytosine in the ligation product by labeling reaction; enrich the DNA containing 5-hydroxymethylcytosine label fragments to obtain enriched products; perform PCR amplification on the enriched products to obtain a sequencing library; perform high-throughput sequencing on the sequencing library to obtain sequencing results; determine the content of 5-hydroxymethylcytosine in the gene according to the sequencing results. Among them, the labeling reaction includes: i) using glycosyltransferase to covalently link the sugar with the modification group to the hydroxymethyl group of 5-hydroxymethylcytosine, and ii) directly or indirectly linking the sugar with biotin Click chemistry substrate reacts with 5-hydroxymethylcytosine with a modifying group. Wherein, step i) and step ii) can be carried out sequentially, and can also be carried out simultaneously in one reaction. This labeling method reduces the sample size required for sequencing, and the biotin label on 5-hydroxymethylcytosine makes it show a higher kinetic signal in sequencing, improving the accuracy of nucleotide identification. In this embodiment, the glycosyltransferases include, but are not limited to: T4 bacteriophage β-glucosyltransferase (β-GT), T4 bacteriophage α-glucosyltransferase (α-GT) and the same Or derivatives, analogs, or recombinases with similar activities; the sugars with modification groups include but are not limited to: sugars with azide modification (such as 6-N3-glucose) or other chemical modifications (such as carbonyl, mercapto, hydroxyl, carboxyl, carbon-carbon double bond, carbon-carbon triple bond, disulfide bond, amine group, amide group, diene, etc.), among which sugars with azide modification are preferred The chemical groups used to indirectly link biotin and click chemistry substrates include but are not limited to: carbonyl, sulfhydryl, hydroxyl, carboxyl, carbon-carbon double bonds, carbon-carbon triple bonds, disulfide bonds, amine groups, Amide, Diene. In this embodiment, the DNA fragments containing the 5-hmC marker are preferably enriched by means of a solid phase material. Specifically, the 5-hydroxymethylcytosine-labeled DNA fragments can be bound to the solid phase material by solid-phase affinity reaction or other specific binding reactions, and then unbound DNA fragments can be removed by multiple washings. Solid phase materials include but are not limited to silicon wafers or other chips with surface modifications, such as artificial polymer beads (preferably 1nm-100um in diameter), magnetic beads (preferably 1nm-100um in diameter), agarose beads, etc. (preferably 1nm-100um in diameter). The washing solution used in the solid-phase enrichment is a buffer well known to those skilled in the art, including but not limited to: containing Tris-HCl, MOPS, HEPES (pH=6.0-10.0, concentration between 1mM and 1M), NaCl (O -2M) or surfactant such as Tween20 (0.01%-5%) buffer. In this embodiment, PCR amplification is preferably performed directly on the solid phase to prepare the sequencing library. If necessary, after performing PCR amplification on the solid phase, the amplified product can be recovered and then subjected to a second round of PCR amplification to prepare a sequencing library. The second round of PCR amplification can be performed by conventional methods known to those skilled in the art. Optionally, one or more purification steps may be further included in the process of preparing the sequencing library. Any purification kit known to those skilled in the art or commercially available can be used in the present invention. Purification methods include, but are not limited to: gel electrophoresis, gel-cutting recovery, silica gel membrane spin column method, magnetic bead method, ethanol or isopropanol precipitation method or a combination thereof. Optionally, the sequencing library is quality checked prior to high throughput sequencing. For example, fragment size analysis of libraries and absolute quantification of library concentrations using qPCR methods. Sequencing libraries that pass quality checks can be used for high-throughput sequencing. Then a certain number (1-96) of libraries containing different barcodes were mixed at the same concentration and sequenced on the machine according to the standard on-machine method of the next-generation sequencer to obtain the sequencing results. Various next-generation sequencing platforms and their associated reagents known in the art can be used in the present invention.

In one embodiment of the present invention, it is preferred to compare the sequencing results with the standard human genome reference sequence, and select the sequences that are compared to the gene markers of the present invention, that is, select the alignment site and gene features (such as group protein modification site, transcription factor binding site, gene exon intron region, gene promoter, etc.) to represent the modification level of 5-hmC on the gene, so as to determine the 5-hmC The content on the gene marker. It is preferable to remove low-quality sequencing sites from the sequencing results before performing the comparison. The factors to measure the quality of the sequencing sites include but are not limited to: base quality, reads quality, GC content, repeated sequences, and the number of Overrepresented sequences. Various comparison software and analysis methods involved in this step are known in the art.

In one embodiment of the present invention, determining the 5-hmC content of a gene marker refers to determining the 5-hmC content on the full length of the gene marker or measuring the 5-hmC content of a certain fragment on the gene marker or its combination.

According to the present invention, after measuring the 5-hmC content of each gene marker, using the 5-hmC content of the gene marker in the normal sample as a reference, the 5-hmC content of the corresponding gene marker in the subject sample standardization. For example, if the 5-hmC content of the same gene marker in the normal sample and the subject sample are X and Y respectively, then the normalized 5-hmC content of the gene marker in the subject sample is Y/X.

According to the present invention, after the data is standardized, the standardized 5-hmC content of each gene marker is mathematically correlated to obtain a score, so as to obtain the detection result according to the score. As used herein, "mathematical correlation" refers to any computational method or machine learning method that correlates the 5-hmC content of a gene marker from a biological sample with a diagnosis of liver cancer. Those of ordinary skill in the art understand that different calculation methods or tools can be selected for providing the mathematical association of the present invention, such as elastic network regularization, decision trees, generalized linear models, logistic regression, highest score pairs, neural networks, linear and Quadratic Discriminant Analysis (LQA and QDA), Naive Bayes, Random Forests, and Support Vector Machines.

In one embodiment of the present invention, the specific steps of mathematically correlating the standardized 5-hmC content of each gene marker and obtaining a score are as follows: multiply the normalized 5-hmC content of each gene marker by a weighting coefficient to obtain the gene The predictive factor t of the marker; the predictive factor t of each gene marker is added to obtain the total predictive factor T; the total predictive factor T is subjected to Logistic transformation to obtain the score P; if P>0.5, the subject sample has Liver cancer; if P≤0.5, the subject's sample is normal. The weighting coefficients described herein refer to the factors that may affect the 5-hmC content (such as the subject's region, age, gender, lower than, smoking history, drinking history, family history, etc.) Coefficients obtained by various advanced statistical analysis methods known to personnel.

The third aspect of the present invention also relates to a kit for detecting liver cancer using the above-mentioned gene markers, which includes reagents and instructions for determining the 5-hmC content of the above-mentioned gene markers. Reagents for determining the 5-hmC content of gene markers are known to those skilled in the art, such as T4 bacteriophage β-glucotransferase and isotope labeling (for glucosylation), restriction enzymes (for restriction endonuclease method), glycosyltransferase and biotin (for chemical labeling method), PCR and sequencing reagents, etc.

Compared with the prior art, the method for detecting liver cancer in the present invention is based on the 5-hmC content on the gene marker, so a wider range of DNA sample sources can be used. Therefore, the method for detecting liver cancer in the present invention has the following advantages: (1) safe and non-invasive, even asymptomatic people have high acceptance of the test; (2) DNA has a wide range of sources, and there is no detection blind spot in imaging (3) High accuracy, high sensitivity and specificity for early liver cancer, suitable for early screening of liver cancer; (4) Convenient operation, good user experience, and easy dynamic monitoring of liver cancer recurrence and metastasis. The gene marker of the present invention can be combined with other clinical indicators to provide more accurate judgment for liver cancer screening, diagnosis, treatment and prognosis.

Description of drawings

Figure 1: The results of using the liver cancer gene markers of the present invention to distinguish liver cancer samples from healthy controls.

Figure 2: The result of using the liver cancer gene markers of the present invention to distinguish small liver cancer samples from healthy controls.

detailed description

The present invention will be described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can better understand the present invention and implement it. It should be noted that those skilled in the art should understand that the drawings and the embodiments of the present invention are only for the purpose of illustration, and shall not constitute any limitation to the present invention. In the case of no contradiction, the embodiments in the present application and the features in the embodiments can be combined with each other.

Example 1. Screening of Liver Cancer Gene Markers

(1) Extract plasma DNA:

10ng of plasma DNA was extracted from samples from 20 liver cancer patients and 20 normal people. This step can be carried out using any methods and reagents suitable for extracting plasma DNA well known to those skilled in the art.

(2) End-fill the plasma DNA, overhang A and connect with the sequencing adapter:

Prepare a reaction mixture containing 50uL plasma DNA, 7uL End Repair&A-Tailing Buffer and 3uL End Repair&A-Tailing Enzyme mix according to the Kapa Hyper Perp Kit instructions (total volume is 60uL), incubate at 20°C for 30 minutes, then incubate at 65°C for 30 minutes minute. Prepare the following ligation reaction mixture in a 1.5mL low adsorption EP tube: 5uL Nuclease free water, 30uL Ligation Buffer and 10uL DNA Ligase. Add 5uL of sequencing adapters to 45uL ligation reaction mixture, mix, heat at 20°C for 20 minutes, and then keep at 4°C. The reaction product was purified using AmpureXP beads, and eluted with 20 uL of Tris-HCl (10 mM, pH=8.0) and EDTA (0.1 mM) buffer to obtain the final DNA ligation sample.

(3) Labeling 5-hydroxymethylcytosine:

Prepare a labeling reaction mixture with a total volume of 26uL: azide-modified uridine diphosphate glucose (i.e., UDP-N3-Glu, with a final concentration of 50uM), β-GT (with a final concentration of 1uM), Mg ²⁺ (final concentration 25 mM), HEPES (pH=8.0, final concentration 50 mM) and 20 uL DNA from the above step. The mixture was incubated at 37°C for 1 hour. The mixture was taken out and purified with AmpureXPbeads to obtain purified 20uL DNA.

Then, 1 uL of biotin-linked diphenylcyclooctyne (DBCO-Biotin) was added to the above purified 20uL DNA, reacted at 37°C for 2 hours, and then purified with AmpureXP beads to obtain a purified labeled product.

(4) Solid-phase enrichment of DNA fragments containing labeled 5-hydroxymethylcytosine:

First, prepare magnetic beads as follows: remove 0.5uL C1 streptadvin beads (lifetechnology) and add 100uL buffer (5mM Tris, pH=7.5, 1M NaCl, 0.02% Tween20), vortex for 30 seconds, then wash with 100uL (5mM Tris, pH=7.5, 1M NaCl, 0.02% Tween20) wash the magnetic beads 3 times, finally add 25uL binding buffer (10mM Tris, pH=7.5, 2M NaCl, 0.04% Tween20 or other surfactants), and mix uniform.

Then, add the purified labeled product obtained in the above steps to the magnetic bead mixture, and mix in a rotary mixer for 15 minutes to fully combine.

Finally, the magnetic beads were washed 3 times with 100uL of washing solution (5mM Tris, pH=7.5, 1M NaCl, 0.02% Tween20), centrifuged to remove the supernatant, and 23.75uL of nuclease-free water was added.

(5) PCR amplification:

Add 25uL of 2X PCR master mix and 1.25uL of PCR primers to the final system of the above steps (the total volume is 50uL), and perform amplification according to the temperature and conditions of the following PCR reaction cycles:

The amplified product was purified with AmpureXP beads to obtain the final sequencing library.

(6) Perform high-throughput sequencing after quality inspection of the sequencing library:

The concentration of the obtained sequencing library was determined by qPCR, and the size and content of DNA fragments in the library were determined with Agilent2100. The sequencing library that passed the quality inspection was mixed at the same concentration and sequenced with Illumina Hiseq 4000.

(7) Determine the 5-hmC content and weighting coefficient of each gene marker

The obtained sequencing results were subjected to preliminary quality control evaluation, and after removing low-quality sequencing sites, the reads that met the sequencing quality standard were compared with the human standard genome reference sequence using the Bowtie2 tool. Then use featureCounts and HtSeq-Count tools to count the number of reads to determine the 5-hmC content of each gene marker. At the same time, using the results of high-throughput sequencing, the factors that may affect the content of 5-hmC were used as covariates, and the weighted coefficients of each gene marker were obtained through logistic regression and elastic network regularization. The results are shown in Table 1.

Table 1: Average normalized 5-hmC content and weighting coefficients of liver cancer gene markers of the present invention

As mentioned above, the average normalized 5-hmC content refers to the ratio of the average 5-hmC content of the gene marker in liver cancer samples to the average 5-hmC content of the same gene marker in normal samples. As can be seen from Table 1, there is a significant difference in the 5-hmC content of the liver cancer gene markers of the present invention in normal samples and liver cancer samples, and except for RLF, the 5-hmC content of the other gene markers is relatively normal. A significant increase.

Example 2. Effectiveness of Liver Cancer Gene Markers

This example verifies the effectiveness of the liver cancer gene markers of the present invention for detecting liver cancer.

Measure the 5-hmC content of 9 liver cancer gene markers of the present invention in the first batch of 96 samples (50 routine liver cancers and 46 healthy controls) according to the method for embodiment 1, and determine the weighting coefficient of each genetic marker .

The normalized 5-hmC content of each gene marker is multiplied by the corresponding weighting coefficient to obtain the predictor t of the gene marker, and then the predictor t of each gene marker is added to obtain the total predictor T, and then The total predictor T was Logistically transformed according to the following formula to obtain the score P:

If P>0.5, the subject sample has liver cancer; if P≤0.5, the subject sample is normal.

Figure 1 shows the results of differentiating the batch of samples according to the method of the present invention. As shown in Figure 1, the method of the present invention can achieve a sensitivity of 90% and a specificity of 91%.

In addition, the nine liver cancer gene markers of the present invention are used to screen for small liver cancer. As shown in Figure 2, in the samples of 42 small liver cancer patients and 42 healthy controls, the sensitivity and specificity of screening small liver cancer using the liver cancer gene markers of the present invention are about 83% and 83%.

Claims (10)

1. the gene marker for detecting liver cancer, including it is one or more selected from following gene：FAT atypia cadherins 1 (FAT1), estrogen-related receptor γ (ESRRG), the subunits (GABRB3) of gamma aminobutyric acid A receptoroids β 3, TNF receptor superfamilies Member 11b (TNFRSF11B), acceptor interaction serine/threonine kinase 4 (RIPK4), the L-myc fusion proteins reset (RLF), the member 5 (SLC13A5) of sapiens's Solute Carrier family 13, cytochrome P450 reductase (POR) and Deltex E3 ubiquitin Ligase (DTX1).

2. the gene marker described in claim 1, including FAT1, ESRRG, GABRB3, TNFRSF11B, RIPK4, RLF, SLC13A5, POR and DTX1.

3. purposes of the gene marker described in claim 1 or 2 in the method for detecting liver cancer.

4. a kind of method for detecting liver cancer, comprises the following steps：

(a) 5-hydroxymethyl cytosine of the gene marker in normal specimens and Samples subjects described in claim 1 or 2 is determined The content of (5-hmC)；

(b) with the 5-hmC contents of gene marker described in normal specimens as reference, by corresponding gene in Samples subjects The 5-hmC content standards of mark；

(c) the 5-hmC contents to the normalised gene marker in step (b) carry out mathematical, and are scored P；With

(d) testing result is obtained according to the scoring P, scoring P shows that the Samples subjects suffer from liver cancer more than 0.5.

5. the method described in claim 4, wherein step (a) are to determine the 5- in the gene marker total length or its fragment HmC content.

6. the method described in claim 4, wherein the sample is the DNA pieces dissociated in normal person or subject's body fluid Section, or the complete genome group DNA in organelle, cell and tissue.

7. the method described in claim 6, wherein the body fluid be blood, urine, sweat, sputum, excrement, cerebrospinal fluid, ascites, Hydrothorax, bile or pancreas liquid.

8. the reagent of the 5-hmC contents for determining the gene marker described in claim 1 or 2 is being prepared for detecting liver cancer Kit in purposes.

9. a kind of kit for being used to detect liver cancer, including：

(a) it is used for the reagent for determining the 5-hmC contents of the gene marker described in claim 1 or 2；With

(b) specification.

10. the kit described in claim 9, wherein the 5-hmC contents refer to the gene marker total length or its fragment On 5-hmC content.