DNA methylation biomarker combination for prostate cancer detection and application of DNA methylation biomarker combination

Abstract

The invention discloses a DNA methylation biomarker combination for prostate cancer detection and application of the DNA methylation biomarker combination, and belongs to the technical field of biological medicine. The DNA methylation biomarker combination comprises a combination of methylation sites indicated by CG in the SEQ ID NO: 1 sequence of the USP44 gene and the SEQ ID NO: 2 sequence of the HIF3A gene. It is reported for the first time that the methylation levels of the USP44 gene and the HIF3A gene are detected at the same time to conduct auxiliary diagnosis on the prostatic cancer, a sample urine detection result shows that the sensitivity is 97.14%, the specificity is 97.78%, and the non-invasive and reliable prostatic cancer detection method is provided and has good application prospects in early screening of the prostatic cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a DNA methylation biomarker combination for prostate cancer detection and an application thereof. Background Art

[0002] Prostate cancer is one of the most common cancers in male patients. Although prostate cancer usually has a slow course, it is the third leading cause of cancer death in men. According to data from the World Health Organization, there will be 115,000 new prostate cancer patients in China in 2020 and 134,000 in 2022, showing an upward trend. The domestic prostate cancer mortality rate is 4.4 / 100,000, which is higher in urban areas than in rural areas. The incidence rate increases with age, with a peak age of over 65 years old. 98% of prostate cancers are adenocarcinomas, most of which are multifocal and asymptomatic in the early stage. After the disease develops, progressive urinary difficulties and even incontinence may occur, and nerve compression may cause pain. In the late stage, it may invade the bladder, seminal vesicles, and vascular nerve bundles, and bone metastasis is prone to occur. According to international statistics, the 5-year survival rate of patients with clinically localized prostate cancer after receiving standardized treatment is close to 100%, while the 5-year survival rate of patients with metastatic prostate cancer is only 30%. Because prostate cancer is insidious and progresses slowly, screening for prostate cancer in high-risk populations, identifying early prostate cancer patients and giving standardized treatment are important means to improve the prognosis of prostate cancer patients in my country.

[0003] At present, the clinical diagnosis of prostate cancer mainly relies on rectal examination, puncture biopsy, serum prostate-specific antigen (PSA) test, transrectal prostate ultrasound and pelvic MRI. If the PSA level in the serum is higher than 4ng / mL, there is a 25% chance of having prostate cancer, and if it is higher than 10ng / mL, there is a 50% chance. Since the PSA level in the blood of patients with benign lesions will also increase, a tissue biopsy is usually required for confirmation after the PSA test. Rectal examination is extremely important in the early diagnosis of prostate cancer, with an accuracy rate of 50%-70%, while rectal ultrasound and pelvic MRI can not only directly create images of prostate tissue, but also assist in puncture biopsy to target abnormal areas.

[0004] Existing studies have shown that the methylation frequency of CpG sites of related genes in prostate tumor tissue cells is significantly different from that in healthy or benign hyperplasia tissue cells, and can be used as a biological target for auxiliary diagnosis of prostate. However, existing DNA methylation biomarkers for prostate cancer detection have problems such as low sensitivity and poor specificity. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA methylation biomarker combination for prostate cancer detection and its application, so as to solve the problems of low sensitivity and poor specificity of existing DNA methylation biomarkers for prostate cancer detection.

[0006] In a first aspect, the present invention provides a DNA methylation biomarker combination for prostate cancer detection, comprising a combination of methylation sites indicated by CG in the SEQ ID NO: 1 sequence of the USP44 gene and the SEQ ID NO: 2 sequence of the HIF3A gene.

[0007] In the present invention, the inventors have found that when the above-mentioned DNA methylation biomarker combination is used for joint methylation detection and used for auxiliary diagnosis of prostate cancer, it has significantly improved sensitivity and specificity.

[0008] In a second aspect, the present invention provides use of the above-mentioned DNA methylation biomarker combination in the preparation of a product for prostate cancer detection.

[0009] In a third aspect, the present invention provides a kit for detecting prostate cancer, comprising a reagent for detecting the methylation level of the above-mentioned DNA methylation biomarker combination.

[0010] In some embodiments, the kit further comprises primers and probes for the USP44 gene, and primers and probes for the HIF3A gene.

[0011] In some preferred embodiments, the two ends of the probe are connected to a fluorescent group and a quenching group, respectively.

[0012] In some more preferred embodiments, the fluorescent group includes FAM, VIC, and the quenching group includes MGB.

[0013] In some embodiments, the nucleotide sequence of the primers for the USP44 gene is shown as SEQ ID NO:3-4, and the nucleotide sequence of the probe is shown as SEQ ID NO:5; the nucleotide sequence of the primers for the HIF3A gene is shown as SEQ ID NO:6-7, and the nucleotide sequence of the probe is shown as SEQ ID NO:8.

[0014] In some embodiments, the kit further comprises primers and probes for internal reference genes, and the internal reference genes comprise the ACTB gene.

[0015] In some preferred embodiments, the two ends of the probe are connected to a fluorescent group and a quenching group, respectively.

[0016] In some more preferred embodiments, the fluorescent group comprises CY5 and the quenching group comprises MGB.

[0017] In some embodiments, the nucleotide sequences of the primers for the internal reference gene are shown in SEQ ID NOs:9-10, and the nucleotide sequence of the probe is shown in SEQ ID NO:11.

[0018] In some embodiments, the kit further comprises at least one of a nucleic acid extraction reagent, a methylation conversion reagent, and a PCR reaction reagent.

[0019] In some embodiments, the methylation conversion reagent comprises a bisulfite conversion reagent.

[0020] In the present invention, during the methylation treatment, bisulfite can convert all unmethylated cytosines into uracil, while the methylated cytosines remain unchanged. Specifically, after the treatment, the unmethylated C in the target gene sequence is converted to U, and in the subsequent fluorescence quantitative PCR reaction, the primer probe specifically captures and binds to the methylated C site to emit fluorescence, while the unmethylated target gene cannot bind to the primer probe and does not emit fluorescence.

[0021] In some embodiments, the sample tested by the kit includes a urine sample.

[0022] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention reports for the first time the simultaneous detection of the methylation levels of the USP44 gene and the HIF3A gene for auxiliary diagnosis of prostate cancer, and the sample urine test results show that the sensitivity is 97.14% and the specificity is 97.78%. The present invention provides a non-invasive and reliable prostate cancer detection method, which has good application prospects in the early screening of prostate cancer. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0025] Example 1

[0026] This embodiment provides a kit for prostate cancer detection, which includes primers and probe sequences for detecting the methylation level of DNA methylation biomarkers.

[0027] Specifically, the DNA methylation biomarkers for prostate cancer detection include SEQ ID NO: 1 sequence of USP44 gene and SEQ ID NO: 2 sequence of HIF3A gene. The specific sequences are shown in Table 1 below, wherein CG in different sequences indicates methylation sites, which are indicated by underlines.

[0028] Table 1 Sequences of methylation sites of the two markers

[0029]

[0030] For the methylation site sequences of the above-mentioned different target genes, primers and probe sequences for detecting the methylation levels of the above-mentioned sequences were designed, as shown in Table 2 below.

[0031] Table 2 Primer and probe sequences

[0032]

[0033]

[0034] The kit also includes at least one of a nucleic acid extraction reagent, a methylation conversion reagent, and a PCR reaction reagent.

[0035] Example 2

[0036] This embodiment provides a method for detecting prostate cancer using the kit in Embodiment 1, which specifically comprises the following steps:

[0037] 1. Extraction of urine samples

[0038] The human DNA in urine samples was extracted using a kit (Nucleic Acid Extraction Kit (Magnetic Bead Method) of Youzhiyou Medical Technology Co., Ltd., E-Han Medical Equipment No. 20220684). The standard operating procedure includes the following steps:

[0039] 1) Lysis: Take an appropriate amount of 1.5 mL centrifuge tubes, add 400 μL of lysis solution, 200 μL of the sample to be tested, 10 μL of magnetic bead suspension and 20 μL of proteinase K to each tube, shake and mix for 10 seconds, warm at 65°C for 10 minutes, centrifuge briefly, place the centrifuge tube on a magnetic rack for about 2 minutes, and discard the supernatant;

[0040] 2) Add 600 μL of rinse solution 1, shake and mix for 10 seconds, centrifuge instantly, adsorb on a magnetic rack for 2 minutes, and discard the supernatant;

[0041] 3) Add 600 μL of rinse solution 2, shake and mix for 10 seconds, centrifuge instantly, adsorb on a magnetic rack for 2 minutes, and discard the supernatant;

[0042] 4) Add 600 μL of rinse solution 2 again, shake and mix for 10 seconds, centrifuge instantly, adsorb on a magnetic rack for 2 minutes, discard the supernatant, and dry for 5 minutes;

[0043] 5) Elution: Add 30-40 μL of elution buffer, shake and mix for 30 seconds to break up the magnetic beads, incubate at 60°C for 5 minutes, and then vortex at high speed for 30 seconds;

[0044] 6) Instantaneously centrifuge and collect the liquid in the tube cap and drop it into the tube, transfer it to the magnetic rack until the magnetic beads are completely adsorbed, and then carefully transfer the liquid to a new centrifuge tube to obtain the nucleic acid solution.

[0045] 2. Bisulfite conversion

[0046] The sample DNA obtained in the above steps was methylated. DNA methylation was performed using a commercial kit (EZDNA Methylation-Lightning TM Kit (ZYMO), catalog number: D5030. ) was processed, including the following steps:

[0047] 1) Add the reaction solution and DNA sample to a 200 μL PCR tube according to the ratio in Table 3;

[0048] Table 3 Reaction system

[0049]

[0050] 2) Mix the reaction solution at room temperature;

[0051] 3) The reaction is carried out on a common PCR instrument, which takes about 1.15 hours; (If the maximum allowable volume of the PCR instrument cannot reach 140 μL, it can be set to the maximum reaction volume, such as 100 μL). The reaction program is shown in Table 4 below:

[0052] Table 4 Reaction procedure

[0053] step time Incubation temperature transsexual 8min 98℃ Incubation 60min 54℃ <20h 4℃

[0054] 4) Place the PCR tube into the instrument to start the reaction. Be sure to use a PCR instrument with a heated cover.

[0055] 5) After transformation, centrifuge briefly;

[0056] 6) Prepare the corresponding number of spin columns and collection tubes and assemble them, and add 600 μL of M-Binding Buffer to the spin columns;

[0057] 7) Transfer the mixture in step (6) to a centrifuge column and mix by slowly pipetting;

[0058] 8) Centrifuge at the maximum speed of the centrifuge for 1 minute, remove the waste liquid and put the centrifuge column back into the collection tube;

[0059] 9) Add 100 μL of M-Wash Buffer to each tube and continue centrifugation at maximum speed for 1 minute to remove the waste liquid;

[0060] 10) Add 200 μL of L-Desulphonation Buffer and incubate at room temperature for 15 minutes; (If there is white precipitate in Buffer BD, avoid transferring it to the mixed solution; seal Buffer BD immediately after taking it to prevent oxidation);

[0061] 11) Centrifuge at maximum speed for 1 minute, remove the waste liquid and put the centrifuge column back into the collection tube;

[0062] 12) Add 200 μL of M-Wash Buffer, centrifuge at maximum speed for 1 minute, and remove the waste liquid;

[0063] 13) Repeat step 12;

[0064] 14) Transfer the column to a new 1.5 mL centrifuge tube and centrifuge at maximum speed for 3 minutes to remove the waste liquid;

[0065] 15) Place the opened spin column in a clean 1.5 mL centrifuge tube and incubate at room temperature for 5 minutes;

[0066] 16) Add 20 μL of M-Elution Buffer to the centrifuge column and place it in the center of the filter membrane. Centrifuge at 15,000 g or 12,000 rpm for 1 minute. To increase the yield of purified DNA, add the liquid after centrifugation to the column and centrifuge again to increase the recovery rate.

[0067] The DNA nucleic acid template is obtained through the above steps. The product can be directly used in downstream experiments. If it needs to be stored, it can be placed in a -20°C refrigerator.

[0068] 3. Fluorescence quantitative PCR detection

[0069] The DNA converted by bisulfite in the above step was subjected to fluorescent quantitative PCR (qPCR), and the fluorescent group FAM and the quenching group MGB were connected to the two ends of the nucleotide sequence of the probe for the USP44 gene; the fluorescent group VIC and the quenching group MGB were connected to the two ends of the nucleotide sequence of the probe for the HIF3A gene; the fluorescent group CY5 and the quenching group MGB were connected to the two ends of the nucleotide sequence of the probe for the ACTB gene; wherein the reaction system and reaction procedure of qPCR are shown in Tables 5-6 below:

[0070] Table 5 Reaction system

[0071] 2*PCR buffer 12.5μL USP44-F (10 μM) 0.5μL USP44-R (10 μM) 0.5μL USP44-P (5 μM) 0.5μL HIF3A-F (10 μM) 0.5μL HIF3A-R (10 μM) 0.5μL HIF3A-P (5 μM) 0.5μL ACTB-F (10 μM) 0.5μL ACTB-R (10μM) 0.5μL ACTB-P (5μM) 0.5μL Hs Taq 0.2μL ROX 0.4μL DNase / RNase-Free Distilled water 2.4μL DNA template 5μL Total volume 25μL

[0072] Table 6 Reaction procedure

[0073]

[0074] 4. Interpretation of results

[0075] After the negative control, positive control and internal control are qualified, the sample test results can be judged according to the following Table 7-8.

[0076] Table 7 Gene detection results

[0077]

[0078] Note: △Ct value = target gene Ct value - internal standard gene Ct value

[0079] Table 8 Sample test results

[0080]

[0081] Example 3

[0082] This example uses the method in Example 2 to test actual samples. Specifically, urine DNA samples from 35 prostate cancer patients (numbered 1-35), 35 negative cases (36-70) and 10 patients with urinary system inflammation (71-80) are tested. Among them, the samples of prostate cancer patients and urinary system inflammation patients are from a hospital in Wuhan or Chongqing, and the normal samples are from volunteer urine samples. The test results are shown in Table 9 below:

[0083] Table 9 Results of Fluorescence Quantitative PCR Tests of Clinical Samples

[0084]

[0085]

[0086] According to the test results in Table 9, the sensitivity and specificity were calculated, and the specific results are shown in Table 10 below:

[0087] Table 10 Sensitivity and specificity test results

[0088]

[0089]

[0090] It can be seen from the data in Table 10 that simultaneous detection of the methylation levels of USP44 gene and HIF3A gene for auxiliary diagnosis of bladder cancer has strong specificity and high sensitivity.

[0091] In summary, the present invention provides a non-invasive and reliable method for detecting prostate cancer, which has good application prospects in the early screening of prostate cancer.

[0092] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0093] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A DNA methylation biomarker combination for prostate cancer detection, characterized in that: It includes a combination of methylation sites indicated by CG in the sequence of SEQ ID NO: 1 of the USP44 gene and the sequence of SEQ ID NO: 2 of the HIF3A gene.

2. Use of the DNA methylation biomarker combination as claimed in claim 1 in the preparation of a product for prostate cancer detection.

3. A kit for detecting prostate cancer, characterized in that: Comprising a reagent for detecting the methylation level of the DNA methylation biomarker combination according to claim 1.

4. The kit according to claim 3, characterized in that The kit also includes primers and probes targeting USP44 gene, and primers and probes targeting HIF3A gene.

5. The kit according to claim 4, characterized in that The nucleotide sequences of the primers for the USP44 gene are shown in SEQ ID NOs: 3-4, and the nucleotide sequence of the probe is shown in SEQ ID NO: 5; the nucleotide sequences of the primers for the HIF3A gene are shown in SEQ ID NOs: 6-7, and the nucleotide sequence of the probe is shown in SEQ ID NO:

8.

6. The kit according to claim 3, characterized in that The kit also includes primers and probes for internal reference genes, and the internal reference genes include ACTB genes.

7. The kit according to claim 6, characterized in that The nucleotide sequences of the primers for the internal reference gene are shown in SEQ ID NOs:9-10, and the nucleotide sequence of the probe is shown in SEQ ID NO:

11.

8. The kit according to claim 3, characterized in that The kit also includes at least one of a nucleic acid extraction reagent, a methylation conversion reagent, and a PCR reaction reagent.

9. The kit according to claim 8, characterized in that The methylation conversion reagent includes a bisulfite conversion reagent.

10. The kit according to claim 3, characterized in that The samples tested by the kit include urine samples.

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