CN113355420A - JAK3 promoter methylation detection primer composition, application and detection method - Google Patents
JAK3 promoter methylation detection primer composition, application and detection method Download PDFInfo
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Abstract
The invention discloses a JAK3 promoter methylation detection primer composition, application and a detection method, wherein the methylation level of the JAK3 promoter cg04557677 is detected by designing detection primer compositions cg04557677-F, cg04557677-Rbio and cg04557677-S and utilizing a pyrosequencing technology. The detection method for the methylation level of the JAK3 promoter has high sensitivity and accuracy, and has important significance for pathological stage and early detection, prognosis and risk assessment of kidney cancer.
Description
Technical Field
The invention belongs to the technical field of molecular biological detection, and particularly relates to a JAK3 promoter methylation detection primer composition, application and a detection method.
Background
The kidney cancer is a malignant tumor originated from the parenchyma of the kidney, the high-incidence age of the kidney cancer is 50-70 years old, the kidney cancer accounts for 2-3% of adult malignant tumors, the second place of the incidence rate of the malignant tumor of the urinary system in China is just second to the bladder cancer, and the incidence rate of the cancer gradually rises in recent years. According to the research, the 5-year survival rates of patients with different risks after renal cancer surgery are different, the low-risk patients are ranked in the leaders at a rate of 90%, while the high-risk patients are only 42%, and 62% of the middle-risk patients are not optimistic. Due to the influence of various factors, the survival rate of the kidney cancer patients in China is lower than that of the data. The etiology of kidney cancer is not clear at present, and the kidney cancer may be related to factors such as obesity, smoking, hypertension treatment, heredity and the like, wherein the hereditary kidney cancer accounts for 2-4% of the total number of the kidney cancer. The pathogenesis of the kidney cancer is more secret, patients at early stage often have no obvious symptoms, but when the patients have triple signs of the kidney cancer (hematuria, pain and tumor), the kidney cancer is suggested to be developed to the late stage, and the optimal treatment period is missed, so the early diagnosis and the early treatment of the kidney cancer are particularly important.
Renal cancer has no obvious symptoms in the early stage, and is difficult to find due to the hidden position. The diagnosis of kidney cancer is mainly based on imaging examinations including abdominal Ultrasound (US), Magnetic Resonance Imaging (MRI) and X-ray Computed Tomography (CT), in addition to clinical symptoms based on the "triple sign of kidney cancer". The ultrasonic examination is currently carried out by using multi-purpose color ultrasound, can identify the kidney parenchymal tumor and the kidney cystic mass, but is difficult to distinguish from other kidney parenchymal lesions even if the ultrasound contrast is carried out; CT is the most common and important means for diagnosing kidney cancer at present, theoretically, the position, the range and the form of a substantial kidney tumor can be clearly displayed, a kidney with early canceration can be found, the strengthened tumor is generally strengthened, and the transparent cell cancer is similar to liver cancer and shows a strengthening mode of 'fast forward and fast out'; MRI can clearly display the tumor morphology, invasion range and adhesion degree from various angles, mainly has a clear stage of kidney cancer, and is also helpful for diagnosing whether cancer embolus is combined or not.
The protein encoded by the JAK3 gene is a receptor tyrosine kinase, a member of the Janus kinase family, which includes JAK1, JAK2, JAK3 and TYK 2. JAK3 is expressed primarily in immune cells and signals through tyrosine phosphorylation after being activated by interleukins. JAK/STAT is a very important signal pathway through which many cytokines such as IFN, IL-2, etc. and growth factors such as EGF, CSF, etc. induce proliferation, differentiation and apoptosis of cells. Therefore, the search for a DNA marker in the JAK3 gene has an important role in the diagnosis and prognosis evaluation of renal cancer.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a JAK3 promoter methylation detection primer composition, an application and a detection method, wherein the detection primer composition is designed, and the level of methylation of the cg04557677 of a JAK3 promoter is detected by a pyrosequencing technology, so that the method has high sensitivity and accuracy and has important significance for pathological staging and early detection, prognosis and risk assessment of kidney cancer.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the first aspect of the invention provides a JAK3 promoter methylation detection primer composition, which comprises an upstream primer cg04557677-F, a downstream primer cg04557677-Rbio and a sequencing primer cg04557677-S, wherein the nucleotide sequence of cg04557677-F is shown as SEQ ID NO.1, the nucleotide sequence of g04557677-Rbio is shown as SEQ ID NO.2, and the nucleotide sequence of cg04557677-S is shown as SEQ ID NO. 3.
In a preferred embodiment, the methylation site of the JAK3 promoter is cg04557677, and cg04557677 is located 340bp upstream of the transcription start site of the gene promoter.
The second aspect of the invention provides an application of a JAK3 promoter methylation detection primer composition in preparation of a JAK3 promoter methylation detection tool.
In a preferred embodiment, the detection means includes, but is not limited to, as a separate detection reagent, a detection kit.
The invention also provides application of the JAK3 promoter methylation detection primer composition in preparation of a renal cancer detection tool.
In a preferred embodiment, the methylation site of the JAK3 promoter in the above application is cg04557677, and the cg04557677 is located 340bp upstream of the transcription start site of the gene promoter.
In a third aspect, the invention provides a kit for detecting methylation of the JAK3 promoter, the kit comprising the primer composition described above.
The fourth aspect of the invention provides a method for detecting the methylation of the JAK3 promoter by using the primer composition, which comprises the following steps;
s1, extracting DNA of a sample to be detected;
s2, transforming and purifying the sample DNA extracted in the step S1;
s3, carrying out PCR amplification on the DNA obtained in the step S2 by using an upstream primer cg04557677-F and a downstream primer cg 04557677-Rbio;
s4, pyrosequencing the amplified PCR product obtained in step S3 by using the sequencing primer cg 04557677-S.
In a preferred embodiment, the concentration of DNA in the sample in step S2 is greater than 50 ng/. mu.L, and the total amount of the sample is greater than 500 ng.
In a preferred embodiment, the reaction system for amplification in step S3 is as follows:
adding water to 25 μ L, and reacting at 98 deg.C for 10s, 55 deg.C for 30s, and 72 deg.C for 30s for 40 cycles; 72 ℃ for 1 min; and keeping at 4 ℃.
In the invention, the accession number of the JAK3(Janus kinase 3) gene in genebank is NC-000019.10, the JAK3 gene is located on chromosome 19, and the total length of the genome sequence is 23290bp (17824782-.
The invention has the following beneficial effects:
(1) the invention discovers that the JAK3 promoter methylation site can be used as a novel kidney cancer DNA marker, and the detection primer of JAK3 promoter methylation is designed, so that the detection primer is used for detecting kidney cancer, and the invention has important values in early detection, diagnosis and prognosis of kidney cancer.
(2) The methylation level of the JAK3 promoter can be used as the standard of the grading and staging of the kidney cancer, has simple and convenient operation, strong feasibility and high accuracy, has clinical diagnosis and guidance significance, and has medium and large application in preparing tools for detecting the kidney cancer.
(3) The methylation detection method disclosed by the invention is wide in application range, DNA methylation exists in various tumors widely when the mutation site on the JAK3 gene is detected, and the DNA methylation modification is stable and not easy to degrade, so that the preservation and the transportation are convenient.
Drawings
FIG. 1 is a graph of the methylation level at the cg04557677 site as a function of the expression level in renal cancer and as a function of JAK3 expression and patient survival time;
FIG. 2 is a graph of methylation level at the cg04557677 site as a function of staging of renal cancer patients;
FIG. 3 shows the results of the methylation level and survival time of cg04557677 site from renal cancer patients applied for by tissue specimen bank of tumor hospital of Zhongshan university.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the description herein, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
First, methylation site of JAK3 gene
First, according to methylation chip data in a gene TCGA database, compared with normal tissues, the methylation site cg04557677 on JAK3 gene is abnormally hypomethylated in kidney cancer, and meanwhile, the methylation degree of the site is found to be negatively related to the expression of JAK3, and the higher the methylation degree of cg04557677 is, the longer the survival time is, and the result is shown in FIG. 1, wherein FIG. 1-a shows that the hypomethylation of cg04557677 in kidney cancer is shown, FIG. 1-b shows that the methylation degree of cg04557677 is negatively related to the expression of JAK3, and FIG. 1-c shows that the higher the methylation degree of cg04557677 of a patient is, the longer the survival time is shown.
Based on the analysis of the TCGA database renal clear cell carcinoma data, cg04557677 exhibited hypomethylation levels in patients with high grade, in patients with high T grade, in patients with high N grade, and in patients with distant metastases, the results of the analysis are shown in FIG. 2, where FIG. 2-a shows that cg04557677 exhibited hypomethylation in patients with high grade, FIG. 2-b shows that cg04557677 exhibited hypomethylation in patients with high T grade, FIG. 2-c shows that cg04557677 exhibited hypomethylation in patients with high grade, FIG. 2-d shows that cg04557677 exhibited hypomethylation in patients with high N grade, and FIG. 2-e shows that cg04557677 exhibited hypomethylation in patients with distant metastases.
Method for detecting methylation of JAK3 promoter
1. Primers cg04557677-F, cg04557677-Rbio and cg04557677-S were designed, and were synthesized by the company biologies (sangon biotech), with specific primer sequences as follows:
cg04557677-F(SEQ ID NO:1)：AGATGGGTAAATTGAGGTAATAAGG；
cg04557677-Rbio(SEQ ID NO:2)：CTCAACCCACTCTAAACCCACTTATTAAAT；
cg04557677-S(SEQ ID NO:3)：ATTGAGGTAATAAGGGT。
2. DNA samples of tumor tissues and normal tissues (or body fluids of patients, such as blood, urine, etc.) were extracted according to a commercial genome extraction kit (Tiangen Biochemical technology (Beijing) Ltd., product No. DP 304).
3. Taking 0.5-1 μ g DNA sample, using Methylation transformation kit EZ DNA Methylation-GoldTMKit (ZYMO, D5006) was transformed and purified.
3.1 transformation of samples
3.1.1 Using a NanoDrop ND-2000 spectrophotometer, the sample concentration was determined to be greater than 50 ng/. mu.L and the total amount of sample was greater than 500 ng.
3.1.2 reagent preparation:
reagent 1: add 900. mu. L H2O, 300. mu. L M-Dilution Buffer, 50. mu. L M-resolving Buffer to a CT Conversion Reagent tube and shake for 10min at room temperature.
Reagent 2: to M-Wash Buffer was added 96mL of 100% ethanol.
3.1.3 reagents for bisulfite conversion reaction were prepared in a 200. mu.L PCR tube and mixed well according to Table 1 below.
TABLE 1
| Reagent | Volume (μ L) |
| |
20 |
| |
130 |
| Total volume | 150 |
3.1.4 reaction sequence: denaturation at 98 deg.C for 8 min; incubating at 64 deg.C for 2.5 h; and keeping at 4 ℃.
3.2 purification of the samples
3.2.1 to Zymo-SpinTM600. mu. L M-Binding Buffer was added to the IC purification column.
3.2.2 mu.L of the transformed sample was added to a purification column containing M-Binding Buffer and mixed by inversion.
3.2.3 centrifuge at greater than 10,000g for 30s and discard the waste stream.
3.2.4 Add 100. mu. L M-Wash Buffer to the column, centrifuge for 30s, discard the solution.
3.2.5 mu. L M-depletion Buffer was added to the column, and the column was left at room temperature for 15-20min, centrifuged for 30s, and discarded.
3.2.6 adding 200 mu L M-Wash Buffer into the purification column, centrifuging for 30s, repeatedly adding 200 mu L M-Wash Buffer, centrifuging for 30s, and discarding the solution.
3.2.7 Place the column in a new 1.5mL centrifuge tube, add 10. mu. L M-Elution Buffer, and centrifuge for 30s to elute the DNA.
4. PCR amplification
4.1 PCR amplification was carried out using the transformed DNA sample as a template according to the reaction system shown in Table 2 below.
TABLE 2
| Reagent | Volume of |
| 10×EpiTap PCR Buffer | 2.5μL |
| 25mM MgCl2 | 2.5μL |
| dNTP Mixture(2.5mM each) | 3μL |
| EpiTap HS(5U/μL) | 0.125μL |
| cg04557677-F(10μM) | 1μL |
| cg04557677-S(10μM) | 1μL |
| Transformed DNA template | 2μL |
| Adding water to | 25μL |
Among them, EpiTap HS, MgCl2, 10 × EpiTap PCR Buffer, dNTP mix were purchased from TaKaRa, cat # R110A.
3.2 the reaction sequence is: 10s at 98 ℃, 30s at 55 ℃, 30s at 72 ℃ and 40 cycles; 72 ℃ for 1 min; hold at 4 ℃.
4. 10. mu.L of the PCR product was subjected to sequencing analysis using a Pyromark Q48 real-time quantitative pyrophosphate sequencer.
Third, methylation detection of clinical samples
The methylation levels of 72 renal cancer tumor tissues and 12 normal tissue samples from the tumor hospital of Zhongshan university were examined using the method described in example 2, and the results are shown in Table 3 below.
TABLE 3
The methylation level referred to in the present application means that the methylation level of normal tissue is higher than that of renal cancer tumor tissue in the sample overall, and as can be seen from table 3, the methylation level of cg04557677 site of 12 normal tissues is about 50% or so, and the methylation level of cg04557677 site of 84 renal cancer tumor tissues is lower than 50%, although the individual data is the same as the methylation level of normal tissue, the methylation level of JAK3 promoter in renal cancer tumor tissue is obviously reduced in the whole data, which indicates that the methylation level of the promoter of JAK3 gene is closely related to the occurrence of renal cancer.
Fourth, tissue specimen detection
The methylation level of the tissue specimen of a renal cancer patient is detected by the method described in example 2, and the detection result is shown in the following figure 3, wherein the figure 3 shows that the methylation level of cg04557677 in the tissue specimen of the renal cancer patient is obviously lower than that of normal tissues, and the hypermethylated patient has longer survival time.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and is not intended to limit the practice of the invention to these embodiments. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
SEQUENCE LISTING
<110> Hunan Lingkang medical science and technology Co., Ltd
<120> JAK3 promoter methylation detection primer composition, application and detection method
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
agatgggtaa attgaggtaa taagg 25
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
ctcaacccac tctaaaccca cttattaaat 30
<210> 3
<211> 17
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
attgaggtaa taagggt 17
Claims (10)
1. The primer composition for detecting the methylation of the JAK3 promoter comprises an upstream primer cg04557677-F, a downstream primer cg04557677-Rbio and a sequencing primer cg04557677-S, wherein the nucleotide sequence of cg04557677-F is shown as SEQ ID NO.1, the nucleotide sequence of cg04557677-Rbio is shown as SEQ ID NO.2, and the nucleotide sequence of cg04557677-S is shown as SEQ ID NO. 3.
2. The primer composition for detecting methylation of the JAK3 promoter according to claim 1, wherein the methylation site of the JAK3 promoter is cg04557677, and the cg04557677 is located 340bp upstream of the transcription start site of the gene promoter.
3. Use of the JAK3 promoter methylation detection primer composition of claim 1 in the preparation of a JAK3 promoter methylation detection tool.
4. The use according to claim 3, wherein said detection means include, but are not limited to, as a separate detection reagent, detection kit.
5. Use of the JAK3 promoter methylation detection primer composition of claim 1 in the preparation of a renal cancer detection tool.
6. The use of any one of claims 3 to 5, wherein the methylation site of the JAK3 promoter is cg04557677, and the cg04557677 is located 340bp upstream of the transcription start site of the gene promoter.
7. A kit for detecting the methylation of the JAK3 promoter, comprising the primer composition of claim 1.
8. A method for detecting the methylation of the JAK3 promoter, which comprises the steps of using the primer composition according to claim 1;
s1, extracting DNA of a sample to be detected;
s2, transforming and purifying the sample DNA extracted in the step S1;
s3, carrying out PCR amplification on the DNA obtained in the step S2 by using an upstream primer cg04557677-F and a downstream primer cg 04557677-Rbio;
s4, pyrosequencing the amplified PCR product obtained in step S3 by using the sequencing primer cg 04557677-S.
9. The method of claim 8, wherein the concentration of DNA in the sample is greater than 50 ng/. mu.L and the total amount of DNA in the sample is greater than 500ng in step S2.
10. The method for detecting the methylation of the JAK3 promoter according to claim 8, wherein the reaction system amplified in step S3 is as follows:
adding water to 25 μ L, and reacting at 98 deg.C for 10s, 55 deg.C for 30s, and 72 deg.C for 30s for 40 cycles; 72 ℃ for 1 min; and keeping at 4 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170175197A1 (en) * | 2014-01-29 | 2017-06-22 | Caris Mpi, Inc. | Molecular profiling of immune modulators |
| US20190264286A1 (en) * | 2016-06-08 | 2019-08-29 | University Of Iowa Research Foundation | Compositions and methods for detecting predisposition to cardiovascular disease |
| CN110785490A (en) * | 2017-04-19 | 2020-02-11 | 鹍远基因公司 | Compositions and methods for detecting genomic variations and DNA methylation status |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170175197A1 (en) * | 2014-01-29 | 2017-06-22 | Caris Mpi, Inc. | Molecular profiling of immune modulators |
| US20190264286A1 (en) * | 2016-06-08 | 2019-08-29 | University Of Iowa Research Foundation | Compositions and methods for detecting predisposition to cardiovascular disease |
| CN110785490A (en) * | 2017-04-19 | 2020-02-11 | 鹍远基因公司 | Compositions and methods for detecting genomic variations and DNA methylation status |
Non-Patent Citations (1)
| Title |
|---|
| FEIGUO LIANG等: "JAK3 is a potential biomarker and associated with immune infiltration in kidney renal clear cell carcinoma", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
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