Kit for quantitative detection of FOXK1 gene promoter region methylation and application thereof in identification of hysteromyoma and hysterosarcoma

Abstract

The invention discloses a kit for quantitatively detecting methylation of an FOXK1 gene promoter region and application of the kit to identification of hysteromyoma and hysterosarcoma, the sequence of the FOXK1 gene promoter region is chr7: 4682295-4685295, the reference genome version is GRCh37 / hg19, and the FOXK1 gene promoter region has a nucleotide sequence shown in SEQ ID NO.1. The FOXK1 gene promoter region has a nucleotide sequence shown in SEQ ID NO.2. The FOXK1 gene promoter region has a nucleotide sequence shown in SEQ ID NO.2. The kit comprises a pair of primers FOXK1-Me-F and FOXK1-Me-R for detecting the methylation degree of the FOXK1 gene and a probe FOXK1-Me-Probe, a pair of primers FOXK1-unMe-F and FOXK1-unMe-R for detecting the non-methylation degree of the FOXK1 gene and a probe FOXK1-unMe-Probe, and a pair of primers B2M-F and B2M-R for a reference gene B2M and a probe B2M-Probe. By detecting a specific gene methylation marker of cfDNA in peripheral blood and particularly utilizing an MS-qPCR technology, the invention provides a non-invasive or minimally invasive early differential diagnosis method for uterine sarcoma, which is high in sensitivity, relatively simple and convenient to operate and controllable in cost. The method is helpful for distinguishing benign and malignant lesions before an operation, provides an important basis for clinical decision making, avoids unnecessary operation risks, and improves prognosis of a patient.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, and in particular relates to a kit for quantitative detection of methylation in the promoter region of the FOXK1 gene and its application in differentiating uterine fibroids from uterine sarcomas. Background Technology

[0002] Uterine sarcoma is a rare and poorly prognostic malignant tumor of the female reproductive tract, with an incidence of 0.36 / 100,000. It is most common in women aged 45-55 and presents with atypical clinical manifestations. Uterine sarcoma has the highest malignancy among gynecological tumors, with an extremely poor prognosis. The median survival is about 10 months, and the 5-year survival rate is only 15%-25%. Uterine sarcoma is often discovered incidentally during myomectomy. In current clinical practice, accurately differentiating between benign uterine fibroids and malignant uterine sarcomas preoperatively presents a significant challenge.

[0003] Liquid biopsy based on peripheral blood cell DNA (cfDNA) offers a new approach and method for the early diagnosis of tumors. Currently, there is still a lack of reliable differential diagnosis methods based on cfDNA methylation detection that specifically target uterine sarcomas and effectively differentiate them from benign uterine fibroids. Therefore, achieving a non-invasive or minimally invasive early differential diagnosis method for uterine sarcomas by detecting specific gene methylation in peripheral blood cfDNA combined with imaging examinations has significant clinical implications. Summary of the Invention

[0004] The purpose of this invention is to provide a kit for quantitative detection of methylation in the promoter region of the FOXK1 gene and its application in differentiating uterine fibroids from uterine sarcomas. This kit helps to differentiate between benign and malignant lesions preoperatively, provides important evidence for clinical decision-making, avoids unnecessary surgical risks, and improves patient prognosis.

[0005] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a kit for quantitatively detecting methylation of the FOXK1 gene promoter region, wherein the sequence of the FOXK1 gene promoter region is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19. The kit comprises... A pair of primers FOXK1-Me-F and FOXK1-Me-R for detecting the degree of FOXK1 gene methylation, and a probe FOXK1-Me-Probe, wherein the sequence of FOXK1-Me-F is shown in SEQ ID NO.1, the sequence of FOXK1-Me-R is shown in SEQ ID NO.2, and the sequence of FOXK1-Me-Probe is shown in SEQ ID NO.3; A pair of primers FOXK1-unMe-F and FOXK1-unMe-R for detecting the degree of unmethylation of the FOXK1 gene, and a probe FOXK1-unMe-Probe, wherein the sequence of FOXK1-unMe-F is shown in SEQ ID NO.4, the sequence of FOXK1-unMe-R is shown in SEQ ID NO.5, and the sequence of FOXK1-unMe-Probe is shown in SEQ ID NO.6; A pair of primers B2M-F and B2M-R for the internal reference gene B2M, and a probe B2M-Probe, wherein the sequence of B2M-F is shown in SEQ ID NO.7, the sequence of B2M-R is shown in SEQ ID NO.8, and the sequence of B2M-Probe is shown in SEQ ID NO.9.

[0006] In the above technical solution, the 5' end of the FOXK1-Me-Probe is equipped with a fluorescent group FAM, and the 3' end is equipped with a quenching group BHQ1; The FOXK1-unMe-Probe has a fluorescent group HEX at its 5' end and a quenching group BHQ1 at its 3' end; The B2M-Probe has a fluorescent group HEX at its 5' end and a quenching group BHQ1 at its 3' end.

[0007] In the above technical solution, when using the kit for quantitative detection of methylation, the qPCR reaction program is as follows: 95℃ for 5 min for one cycle, 95℃ for 15 s for 45 cycles, and 60℃ for 30 s for 45 cycles.

[0008] In the above technical solutions, when using the kit for quantitative methylation detection, the sample source is peripheral blood from patients diagnosed with uterine fibroids by imaging examination.

[0009] In the above technical solution, when using the kit for quantitative methylation detection, each sample is subjected to three biological replicates, and the lowest Ct value among the three replicates is taken. Samples with Ct values ​​between 22 and 28 for the internal reference gene B2M are considered as quality control samples. The quality control samples are then subjected to further statistical analysis to calculate the methylation level Q of the FOXK1 gene.

[0010] Samples with a Q < 30% were identified as uterine sarcomas, while samples with a Q ≥ 30 were identified as uterine fibroids.

[0011] Secondly, the present invention provides the application of the above-mentioned kit in the preparation of products for differentiating uterine fibroids from uterine sarcomas.

[0012] Thirdly, the present invention provides the application of FOXK1 gene promoter region methylation as a biomarker in the preparation of reagents, kits or devices for differentiating uterine fibroids from uterine sarcomas, wherein the sequence of the FOXK1 gene promoter region is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19.

[0013] Fourthly, this invention provides a method for detecting methylation in the promoter region of the FOXK1 gene, wherein the sequence of the promoter region of the FOXK1 gene is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19. The method includes the following steps: S1, cfDNA was extracted from peripheral blood using a cfDNA extraction kit; S2, bisulfite conversion was performed using a sulfidation kit, followed by purification; S3, Collect the purified cDNA for Taqan probe-based methylation qPCR detection; wherein the primers and probes used for methylation qPCR detection include A pair of primers FOXK1-Me-F and FOXK1-Me-R for detecting the degree of FOXK1 gene methylation, and a probe FOXK1-Me-Probe, wherein the sequence of FOXK1-Me-F is shown in SEQ ID NO.1, the sequence of FOXK1-Me-R is shown in SEQ ID NO.2, and the sequence of FOXK1-Me-Probe is shown in SEQ ID NO.3; A pair of primers FOXK1-unMe-F and FOXK1-unMe-R for detecting the degree of unmethylation of the FOXK1 gene, and a probe FOXK1-unMe-Probe, wherein the sequence of FOXK1-unMe-F is shown in SEQ ID NO.4, the sequence of FOXK1-unMe-R is shown in SEQ ID NO.5, and the sequence of FOXK1-unMe-Probe is shown in SEQ ID NO.6; A pair of primers B2M-F and B2M-R for the internal reference gene B2M, and a probe B2M-Probe, wherein the sequence of B2M-F is shown in SEQ ID NO.7, the sequence of B2M-R is shown in SEQ ID NO.8, and the sequence of B2M-Probe is shown in SEQ ID NO.9.

[0014] In the above technical solution, in S3, the qPCR reaction program is as follows: 95℃ for 5 min for one cycle, 95℃ for 15 s for 45 cycles, and 60℃ for 30 s for 45 cycles.

[0015] Fifthly, the present invention provides the application of the above method in the preparation of products for differentiating uterine fibroids from uterine sarcomas.

[0016] The beneficial effects of this invention are as follows: By detecting specific gene methylation markers of cfDNA in peripheral blood, particularly using MS-qPCR technology, this invention provides a non-invasive or minimally invasive, highly sensitive, relatively simple, and cost-effective method for the early differential diagnosis of uterine sarcoma. This method helps to differentiate between benign and malignant lesions preoperatively, providing important evidence for clinical decision-making, avoiding unnecessary surgical risks, and improving patient prognosis. Attached Figure Description

[0017] Figure 1 This is a comparison chart of the methylation levels of the FOXK1 gene between uterine sarcoma and uterine fibroids, showing a significant statistical difference between the two. Detailed Implementation

[0018] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can 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, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0019] This invention provides a method for early differential diagnosis of uterine sarcoma based on cell-free DNA (cfDNA) methylation detection. The core of this method is to identify the benign or malignant nature of the subject's uterine tumor by detecting the degree of methylation in the FOXK1 gene promoter region in cfDNA extracted from the subject's peripheral blood plasma.

[0020] 1. Methylation detection: cfDNA was extracted from peripheral blood using a cfDNA extraction kit; bisulfite conversion was performed using a sulfidation kit, followed by purification; the purified cDNA was collected for methylation detection using the Taqan probe method by qPCR. The primers and probes for FOXK1 gene methylation detection are shown in Table 1 below.

[0021] Table 1: Primers and probes for quantitative qPCR detection of FOXK1 and B2M gene methylation.

[0022] In this context, FOXK1 and B2M represent gene names; "gene name-Me-F", "gene name-Me-R", and "gene name-Me-Probe" represent the upstream primer, downstream primer, and probe used to detect the methylation level of the gene; "gene name-unMe-F", "gene name-unMe-R", and "gene name-unMe-Probe" represent the upstream primer, downstream primer, and probe used to detect the demethylation level of the gene; and "gene name-F", "gene name-R", and "gene name-Probe" represent the upstream primer, downstream primer, and probe used to detect the demethylation level of the gene.

[0023] Table 2: Methylation qPCR reaction system

[0024] Table 3: Methylation qPCR Reaction Conditions

[0025] The above-mentioned kit was used for quantitative methylation detection to differentiate between uterine fibroids and uterine sarcomas. The procedure was as follows: each sample was tested in triplicate, and the lowest Ct value from the three replicates was used. Samples with a Ct value between 22 and 28 for the internal reference gene B2M were considered quality control samples. These quality control samples were then used for further statistical analysis. The methylation level Q of the FOXK1 gene was calculated. A Q < 30% identified the sample as uterine sarcoma, and a Q ≥ 30 identified the sample as uterine fibroids.

[0026]

[0027] Example 1 1. Biomarkers for the differential diagnosis of uterine sarcoma Based on the results of RRBS sequencing of 18 pairs of uterine sarcoma tissues and 18 pairs of uterine fibroid tissues, the differences in methylation levels between the two tissues were compared and analyzed to screen for differential methylation markers in uterine sarcoma. The methylation of the FOXK1 gene showed significant differences between uterine sarcoma and uterine fibroid tissues (Table 4). The sequence of the FOXK1 gene promoter region is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19.

[0028] Table 4: Differences in FOXK1 gene methylation between uterine sarcomas and uterine fibroids in the early RRBS sequencing of this invention

[0029] 2. Methylation detection methods cfDNA was extracted from peripheral blood using the Anlong Gene Free cfDNA Extraction Kit [Nucleic Acid Extraction Kit (AL-CF50)]. The extracted cfDNA was then converted to bisulfite using a DNA bisulfite conversion kit (centrifuge column type) (DP215-02). After conversion, the DNA was used for quantitative qPCR detection of methylation using probe-based qPCR premix (EM701-01). The relevant primers and probes are shown in Table 5, the detection system in Table 6, and the reaction conditions in Table 7.

[0030] Table 5: Primers and probes for quantitative qPCR detection of FOXK1 and B2M gene methylation.

[0031] Table 6: Methylation qPCR Detection System

[0032] Table 7: Methylation qPCR Reaction Conditions

[0033] Specific applications of methylation detection in uterine sarcomas: The study included 15 patients with uterine sarcoma (numbered US001-US015) and 25 patients with uterine fibroids (numbered LM001-LM025). Each participant underwent the experimental testing described in Part II above. Each sample was tested in triplicate, and the lowest Ct value among the three replicates was used. Samples with Ct values ​​of 22-28 for the internal reference gene B2M were considered quality control samples, and these quality control samples were then subjected to further statistical analysis. The results showed that the degree of FOXK1 methylation was significantly lower in the uterine fibroid group compared to the uterine sarcoma group.

[0034] Table 8: Experimental Data

[0035] Example 2 Accuracy Experiment Peripheral blood samples from 5 cases of uterine sarcoma and 15 cases of uterine fibroids were selected for FOXK1 gene methylation detection. The following experiments were performed on each sample according to Example 1.

[0036] Genomic DNA extraction was performed using the Tiangen Genomic DNA Extraction Kit [Tiangen Biochemical Blood Genomic DNA Extraction Kit (Catalog No.: DP348)]. Other sulfurization and methylation amplification detection methods were performed as described in Example 1.

[0037] Table 9: Sample Accuracy Test

[0038] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A kit for quantitative detection of methylation in the promoter region of the FOXK1 gene, characterized in that: The sequence of the FOXK1 gene promoter region is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19. The kit includes... A pair of primers FOXK1-Me-F and FOXK1-Me-R for detecting the degree of FOXK1 gene methylation, and a probe FOXK1-Me-Probe, wherein the sequence of FOXK1-Me-F is shown in SEQ ID NO.1, the sequence of FOXK1-Me-R is shown in SEQ ID NO.2, and the sequence of FOXK1-Me-Probe is shown in SEQ ID NO.3; A pair of primers FOXK1-unMe-F and FOXK1-unMe-R for detecting the degree of unmethylation of the FOXK1 gene, and a probe FOXK1-unMe-Probe, wherein the sequence of FOXK1-unMe-F is shown in SEQ ID NO.4, the sequence of FOXK1-unMe-R is shown in SEQ ID NO.5, and the sequence of FOXK1-unMe-Probe is shown in SEQ ID NO.6; A pair of primers B2M-F and B2M-R for the internal reference gene B2M, and a probe B2M-Probe, wherein the sequence of B2M-F is shown in SEQ ID NO.7, the sequence of B2M-R is shown in SEQ ID NO.8, and the sequence of B2M-Probe is shown in SEQ ID NO.

9.

2. The kit according to claim 1, characterized in that: The FOXK1-Me-Probe has a fluorescent group FAM at its 5' end and a quenching group BHQ1 at its 3' end; The FOXK1-unMe-Probe has a fluorescent group HEX at its 5' end and a quenching group BHQ1 at its 3' end; The B2M-Probe has a fluorescent group HEX at its 5' end and a quenching group BHQ1 at its 3' end.

3. The kit according to claim 1, characterized in that: When using the kit for quantitative methylation detection, the qPCR reaction program is as follows: 95℃ for 5 min for one cycle, 95℃ for 15 s for 45 cycles, and 60℃ for 30 s for 45 cycles.

4. The kit according to claim 1, characterized in that: When using the kit to perform quantitative methylation detection, the sample source is peripheral blood from patients diagnosed with uterine fibroids by imaging examination.

5. The kit according to claim 1, characterized in that: When performing quantitative methylation detection using the kit, each sample was subjected to three biological replicates. The lowest Ct value among the three replicates was taken. Samples with Ct values ​​between 22 and 28 for the internal reference gene B2M were considered quality control samples. These quality control samples were then subjected to further statistical analysis to calculate the methylation level Q of the FOXK1 gene. Samples with a Q < 30% were identified as uterine sarcomas, while samples with a Q ≥ 30 were identified as uterine fibroids.

6. The use of the kit according to any one of claims 1-5 in the preparation of a product for differentiating uterine fibroids from uterine sarcomas.

7. The application of FOXK1 gene promoter region methylation as a biomarker in the preparation of reagents, kits, or devices for differentiating uterine fibroids from uterine sarcomas, characterized in that: The sequence of the promoter region of the FOXK1 gene is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19.

8. A method for detecting methylation in the promoter region of the FOXK1 gene, characterized in that: The sequence of the FOXK1 gene promoter region is chr7:4682295-4685295, and the reference genome version is GRCh37 / hg19. The method includes the following steps: S1, cfDNA was extracted from peripheral blood using a cfDNA extraction kit; S2, bisulfite conversion was performed using a sulfidation kit, followed by purification; S3, Collect the purified cDNA for Taqan probe-based methylation qPCR detection; wherein the primers and probes used for methylation qPCR detection include A pair of primers FOXK1-Me-F and FOXK1-Me-R for detecting the degree of FOXK1 gene methylation, and a probe FOXK1-Me-Probe, wherein the sequence of FOXK1-Me-F is shown in SEQ ID NO.1, the sequence of FOXK1-Me-R is shown in SEQ ID NO.2, and the sequence of FOXK1-Me-Probe is shown in SEQ ID NO.3; A pair of primers FOXK1-unMe-F and FOXK1-unMe-R for detecting the degree of unmethylation of the FOXK1 gene, and a probe FOXK1-unMe-Probe, wherein the sequence of FOXK1-unMe-F is shown in SEQ ID NO.4, the sequence of FOXK1-unMe-R is shown in SEQ ID NO.5, and the sequence of FOXK1-unMe-Probe is shown in SEQ ID NO.6; A pair of primers B2M-F and B2M-R for the internal reference gene B2M, and a probe B2M-Probe, wherein the sequence of B2M-F is shown in SEQ ID NO.7, the sequence of B2M-R is shown in SEQ ID NO.8, and the sequence of B2M-Probe is shown in SEQ ID NO.

9.

9. The method according to claim 8, characterized in that: In S3, the qPCR reaction program is as follows: 95℃ for 5 min for one cycle, 95℃ for 15 s for 45 cycles, and 60℃ for 30 s for 45 cycles.

10. The use of the method according to any one of claims 8-9 in the preparation of a product for differentiating uterine fibroids from uterine sarcomas.

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