Detection method and application of miR-34a gene promoter region DNA methylation

Abstract

The invention relates to the technical field of molecular biological detection, in particular to a detection method for DNA methylation of a miR-34a gene promoter region and application. According to the method, the bisulfite reaction system is treated by two tubes, and the strategy of adjusting the reaction time according to the DNA dosage is combined, so that the conversion efficiency of the unmethylated cytosine is improved to 90% or above, and is obviously superior to that of the existing single-tube reaction.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a method and application for detecting DNA methylation in the promoter region of the miR-34a gene. Background Technology

[0002] DNA methylation, a core regulatory mechanism in epigenetics, participates in cell proliferation, differentiation, and disease development by influencing gene transcription activity. Abnormal methylation of tumor suppressor gene promoter regions leads to gene silencing, a key driver of cancer development. miR-34a, a crucial microRNA molecule in the p53 transcriptional network regulatory pathway, plays a vital role in biological processes such as cell cycle arrest, apoptosis induction, senescence regulation, and stem cell differentiation. Studies have shown that miR-34a is significantly downregulated in various malignant tumors (such as lung cancer, colorectal cancer, liver cancer, and breast cancer), and its expression loss is closely related to tumor progression, metastasis, and treatment resistance. Promoter region DNA methylation has been confirmed as one of the main mechanisms of miR-34a gene epigenetic silencing; therefore, detecting the methylation status at this site is of great significance for early tumor diagnosis, prognostic assessment, and the selection of therapeutic targets.

[0003] When cells suffer DNA damage or other stress stimuli, p53 is activated and induces miR-34a expression, which in turn inhibits cell proliferation and promotes apoptosis by targeting and suppressing multiple oncogenes. In malignant tumors, abnormally high methylation often occurs in the CpG islands of the miR-34a gene promoter region, leading to the suppression of its expression. This epigenetic silencing disrupts the p53-miR-34a negative feedback loop, enhancing the survival and proliferation of tumor cells. Multiple clinical studies have confirmed that the miR-34a promoter methylation status is significantly correlated with tumor stage, prognosis, and treatment response. For example, in colorectal cancer, the frequency of miR-34a methylation increases with tumor progression and is positively correlated with lymph node metastasis and poor prognosis; in non-small cell lung cancer, its methylation status can serve as a biomarker for predicting EGFR-TKI treatment response.

[0004] Among existing DNA methylation detection methods, methylation-specific PCR is widely used due to its simplicity and low cost. However, it has the following technical drawbacks: First, during the bisulfite conversion process, the large volume of a single tube reaction can easily lead to uneven system composition and insufficient conversion efficiency of unmethylated cytosine, affecting detection accuracy. Second, primer design for the miR-34a gene targets multiple non-critical regions instead of focusing on functionally defined CpG islands, resulting in low detection specificity. Third, insufficient rinsing steps can lead to residual bisulfite and impurities inhibiting subsequent PCR reactions and increasing non-specific amplification interference.

[0005] Therefore, there is an urgent need to develop a methylation detection method that targets the key regions of the miR-34a gene promoter, has high transformation efficiency, and high specificity. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low transformation efficiency, insufficient specificity, and large non-specific interference in existing miR-34a gene methylation detection methods, and to provide a standardized and reliable method for detecting miR-34a gene promoter region DNA methylation. To achieve the above objective, this invention adopts the following technical solution: The first aspect of this invention provides a method for detecting DNA methylation in the promoter region of the miR-34a gene, comprising the following steps: S1. Sample processing: Extract genomic DNA from the sample to be tested; S2. Bisulfite conversion: Genomic DNA is mixed with bisulfite conversion reagent, and the total reaction system is divided into two reaction tubes for conversion. S3. Purification: The product after the conversion reaction is purified to remove residual bisulfite and impurities; S4. Methylation-specific PCR amplification: Using the DNA purified in S3 as a template, PCR amplification was performed using methylation-specific primer pairs and non-methylation-specific primer pairs designed for the CpG islands in the promoter region of the miR-34a gene. S5. Detection and analysis: The PCR amplification products of S4 were detected by electrophoresis, and the methylation status of the miR-34a gene promoter region was determined based on the electrophoresis results.

[0007] In some preferred embodiments, in S2, the total reaction system of the bisulfite conversion reaction is divided into two reaction tubes, with a total reaction system volume of 120 μL, which is divided equally into two reaction tubes, with each tube having a reaction volume of 60 μL.

[0008] In some preferred embodiments, in S2, the reaction time of the transformation reaction is determined according to the initial amount of genomic DNA used: when the amount of DNA used is ≤500ng, the reaction time is 25-35 minutes; when the amount of DNA used is >500ng, the reaction time is 55-65 minutes.

[0009] In some preferred embodiments, in S3, the purification process employs an adsorption column method, and sequentially uses rinsing solution PW, rinsing solution DB, and then rinsing again with rinsing solution PW.

[0010] In some preferred embodiments, in S3, the specific steps of the purification process include: rinsing twice with rinsing solution PW, and then treating with rinsing solution DB at room temperature for 15 minutes.

[0011] In some preferred embodiments, in S3, the sequences of the methylation-specific primer pair are as follows: the forward primer is shown in SEQ ID No:1, and the reverse primer is shown in SEQ ID No:2; the sequences of the non-methylation-specific primer pair are as follows: the forward primer is shown in SEQ ID No:3, and the reverse primer is shown in SEQ ID No:4.

[0012] In some preferred embodiments, in S4, the CpG island is located at 1272-1380 bp in the miR-34a gene promoter region and has a length of 109 bp.

[0013] In some preferred embodiments, in S4, the methylation-specific primer pairs and the non-methylation-specific primer pairs are designed for the CpG islands, with the expected amplification product length being 173 bp.

[0014] In some preferred embodiments, in S5, the electrophoretic detection is agarose gel electrophoresis.

[0015] In some preferred embodiments, in S5, the electrophoresis detection is agarose gel electrophoresis, performed at 120V for 30 minutes; the judgment criteria are as follows: when only the methylation-specific primer pair amplifies a 173bp product band, it is judged as methylation positive; when only the non-methylation-specific primer pair amplifies a 173bp product band, it is judged as non-methylation positive; when both amplify a 173bp product band, it is judged as partial methylation.

[0016] In some preferred embodiments, the reaction procedure for the methylation-specific PCR amplification includes: pre-denaturation at 95°C for 5 minutes; followed by 35 cycles, each cycle including denaturation at 94°C for 20 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 20 seconds; and a final extension at 72°C for 5 minutes.

[0017] The second aspect of this invention provides an application of a method for detecting DNA methylation in the promoter region of the miR-34a gene, which can be used for early cancer diagnosis and tumor prognosis assessment.

[0018] The technical solution adopted in this invention can achieve the following beneficial effects: 1. High conversion efficiency: By treating the bisulfite reaction system in two tubes and adjusting the reaction time according to the amount of DNA used, the conversion efficiency of unmethylated cytosine is increased to over 90%, which is significantly better than the existing single-tube reaction (conversion efficiency is usually 70-80%). 2. High detection specificity: Primers designed focusing on the functional CpG island of the miR-34a gene promoter 1272-1380bp, combined with "two PW + one DB" washing steps, reduce non-specific amplification rate by more than 50% and achieve a detection accuracy of more than 95%. 3. Simple and reproducible operation: All experimental steps are based on conventional molecular biology instruments (such as PCR instruments and horizontal electrophoresis instruments), the reagents are all commercial products, the operating parameters are well-defined (such as centrifugation at 12000 rpm for 1 min and annealing at 60℃ for 30 sec), and can be stably replicated in different laboratories; 4. Significant clinical value: It can be directly used for early cancer diagnosis (such as detecting miR-34a methylation status in peripheral blood) and tumor prognosis assessment, providing a reliable molecular marker detection method for clinical diagnosis and treatment. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, forming part of the present invention. The illustrative embodiments of the present invention and their descriptions explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 : Prediction results of CpG islands in the miR-34a gene promoter region. Horizontal axis: miR-34a gene promoter sequence position (1-2200bp); Vertical axis: CpG island analysis parameters (GC content percentage, Obs / Exp value); Annotated region: CpG islands are located at 1272-1380bp, with a size of 109bp, GC content > 50.0%, and Obs / Exp > 0.60, meeting the CpG island identification criteria.

[0020] Figure 2 Electrophoresis results of PCR products for miR-34a gene methylation detection. Lane M: DL2000 DNA Marker, band sizes are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; Lane 1: miR-34aM1 primer set (methylation specific), arrow indicates a 173bp specific band (methylation positive); Lane 2: miR-34aU1 primer set (non-methylation specific), no band; Result interpretation: The promoter region of the miR-34a gene in this sample is methylated positive. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0022] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0023] Example 1 This embodiment provides a method for detecting DNA methylation in the promoter region of the miR-34a gene, including the following steps: S1. Sample processing: Extract genomic DNA from the sample to be tested; (1) Genomic DNA was extracted from human esophageal cancer cells KYSE150 using a DNA extraction kit. The purity and concentration of DNA were detected by a spectrophotometer: the A260 / A280 ratio was ensured to be 1.93 and the DNA concentration was adjusted to 100 ng / μL.

[0024] (2) Primer design is shown in Table 1: Table 1

[0025] S2. Bisulfite conversion: (1) Preparation of reaction system: Prepare two reaction systems (60 μL each) in sterile centrifuge tubes according to the proportions in Table 2. Table 2

[0026] S3. Purification: The product after the conversion reaction is purified to remove residual bisulfite and impurities; (1) The procedure for bisulfite DNA methylation is shown in Table 3; Table 3

[0027] (2) Adsorption and rinsing: After the reaction, the two reaction solutions were mixed and transferred to a 1.5 mL centrifuge tube. 600 μL of binding solution PB (5 times the volume) was added, and the mixture was inverted 10 times. The mixture was added to the adsorption column CB1 in two portions (300 μL each time, with the adsorption column placed in the matching collection tube). The mixture was incubated at room temperature for 2 min, centrifuged at 12000 rpm at room temperature for 1 min, and the waste liquid in the collection tube was discarded. The adsorption column CB1 was then returned to the collection tube. 600 μL of rinsing solution PW was added to the adsorption column CB1, and the mixture was centrifuged at 12000 rpm at room temperature for 1 min. The waste liquid was discarded, and the adsorption column was... Return the column to the collection tube; add 600 μL of wash buffer DB to the adsorption column CB1, let it stand at room temperature for 15 min, centrifuge at 12000 rpm at room temperature for 1 min, discard the waste liquid, and return the adsorption column to the collection tube; repeat by adding 600 μL of wash buffer PW, centrifuge at 12000 rpm at room temperature for 1 min, discard the waste liquid; return the adsorption column CB1 to the collection tube, centrifuge at 12000 rpm at room temperature for 1 min to remove residual wash buffer, and then place the adsorption column at room temperature for 5 min until the surface of the adsorption membrane is no longer shiny (ensure it is completely dry to avoid residual wash buffer affecting subsequent PCR).

[0028] (3) Elution and recovery: Transfer the adsorption column CB1 to a clean 1.5 mL centrifuge tube, add 20 μL of elution buffer EB to the middle of the adsorption membrane, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, collect the elution buffer (i.e. the purified methylated transformed DNA) in the centrifuge tube, and store at -20℃ for later use.

[0029] S4. Methylation-specific PCR amplification: PCR reaction system preparation: Prepare 20 μL of reaction system in PCR tubes according to the proportions in Table 4; Table 4

[0030] (1) PCR amplification program: Pre-denaturation: 95℃, 5min (to completely unwind the DNA template and activate DNA polymerase); Cyclic reaction (35 times): 94℃ denaturation for 20sec (DNA double strand unwinding) → 60℃ annealing for 30sec (primer specifically binds to template) → 72℃ extension for 20sec (DNA polymerase synthesizes daughter strand); Final extension: 72℃, 5min (to ensure complete synthesis of daughter strand); Incubation: 4℃, store (to prevent product degradation).

[0031] (2) Detection of PCR products Prepare a 1.5% agarose gel (containing 0.5 μg / mL nucleic acid dye), heat to dissolve, pour into a gel bath, insert a comb, and let stand at room temperature for 30 min to allow the gel to solidify. Place the solidified gel into a horizontal electrophoresis tank, add 1×TAE electrophoresis buffer (2 mm above the gel surface), and carefully remove the comb; add 5 μL of 6×Loading Buffer to each PCR product tube, mix well, and then add 10 μL to the gel loading wells. Simultaneously, add 5 μL of LDL2000 DNA Marker (TAKARAD502A) to adjacent loading wells; set the electrophoresis parameters: voltage 120V, time 30 min, and start electrophoresis. After electrophoresis, the gel was placed in a UV analyzer and observed and photographed under 254nm UV light: if a specific 173bp band appeared at the corresponding position in the miR-34aM1 group and no band appeared in the miR-34aU1 group, the miR-34a gene promoter region was determined to be methylated positive; if a specific 173bp band appeared in the miR-34aU1 group and no band appeared in the miR-34aM1 group, it was determined to be unmethylated positive; if both groups showed a 173bp band, it was determined to be partially methylated; the blank control group showed no bands, proving that there was no cross-contamination in the experiment and the results were valid.

[0032] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A method for detecting DNA methylation in the promoter region of the miR-34a gene, characterized in that, Includes the following steps: S1. Sample processing: Extract genomic DNA from the sample to be tested; S2. Bisulfite conversion: Genomic DNA is mixed with bisulfite conversion reagent, and the total reaction system is divided into two reaction tubes for conversion. S3. Purification: The product after the conversion reaction is purified to remove residual bisulfite and impurities; S4. Methylation-specific PCR amplification: Using the DNA purified in S3 as a template, PCR amplification was performed using methylation-specific primer pairs and non-methylation-specific primer pairs designed for the CpG islands in the promoter region of the miR-34a gene. S5. Detection and analysis: The PCR amplification products of S4 were detected by electrophoresis, and the methylation status of the miR-34a gene promoter region was determined based on the electrophoresis results.

2. The detection method according to claim 1, characterized in that, The overall reaction system for the bisulfite conversion reaction was carried out in two separate reaction tubes.

3. The detection method according to claim 1 or 2, characterized in that, In S2, the reaction time of the transformation reaction is determined according to the initial amount of genomic DNA used: when the amount of DNA used is ≤500ng, the reaction time is 25-35 minutes; when the amount of DNA used is >500ng, the reaction time is 55-65 minutes.

4. The detection method according to claim 1, characterized in that, In S3, the purification process uses an adsorption column method, and washes are performed sequentially with wash solution PW, wash solution DB, and then wash again with wash solution PW.

5. The detection method according to claim 4, characterized in that, In S3, the sequences of the methylation-specific primer pair are as follows: the forward primer is shown in SEQ ID No:1, and the reverse primer is shown in SEQ ID No:2; the sequences of the non-methylation-specific primer pair are as follows: the forward primer is shown in SEQ ID No:3, and the reverse primer is shown in SEQ ID No:

4.

6. The detection method according to claim 1, characterized in that, In S4, the CpG island is located at 1272-1380 bp in the miR-34a gene promoter region and has a length of 109 bp.

7. The detection method according to claim 6, characterized in that, In S4, the methylation-specific primer pair and the non-methylation-specific primer pair are designed for the CpG island, and the expected amplification product length is 173 bp.

8. The detection method according to claim 1, characterized in that, In S5, the electrophoresis detection is agarose gel electrophoresis, performed at 120V for 30 minutes. The judgment criteria are as follows: when only the methylation-specific primer pair amplifies a 173bp product band, it is judged as methylation positive; when only the non-methylation-specific primer pair amplifies a 173bp product band, it is judged as non-methylation positive; when both amplify a 173bp product band, it is judged as partial methylation.

9. The detection method according to claim 1, characterized in that, The reaction procedure for the methylation-specific PCR amplification includes: pre-denaturation at 95°C for 5 minutes; followed by 35 cycles, each cycle consisting of denaturation at 94°C for 20 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 20 seconds; and a final extension at 72°C for 5 minutes.

10. An application of a method for detecting DNA methylation in the promoter region of the miR-34a gene, characterized in that, It is used for early cancer diagnosis and tumor prognosis assessment.

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