CN113416783A - Detection primer composition for NLRP3 promoter methylation, application and detection method - Google Patents
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Abstract
The invention discloses a primer composition for detecting methylation of NLRP3 promoter, application and a detection method, wherein the level of methylation of NLRP3 promoter cg21919599 is detected by a pyrosequencing technology through designing a primer composition for detecting upstream primer cg21919599-F, downstream primer cg21919599-Rbio and sequencing primer cg 21919599-S. The method for detecting the methylation level of the NLRP3 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 primer composition for detecting NLRP3 promoter methylation, an application and a detection method.
Background
Renal cancer is one of the common malignant tumors threatening human health. The incidence of kidney cancer in China is always in a remarkable rising trend in recent years, the annual growth rate of the incidence of kidney cancer is as high as 5 percent, and the kidney cancer becomes one of the tumors with the fastest growth rate and mortality rate in China. Early renal cancer is limited in the kidney envelope, and the 5-year survival time is up to more than 90%; however, when the tumor spreads outside the envelope or metastasizes far away, the treatment of the tumor is quite difficult and the prognosis is poor, and the 5-year survival time is only about 15%. More importantly, because early renal cancer often has no clinical symptoms and lacks of molecular markers for early diagnosis of renal cancer clinically, nearly 30% of patients have early diagnosis, namely late stage. Therefore, there is a great need to find simple and easy molecular markers in clinic to realize early diagnosis of renal cancer, thereby improving the overall survival time of renal cancer patients.
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.
NLRP3 inflammasome, an important component of innate immunity, plays an important role in the immune response and disease development in the body. Due to their ability to be activated by various types of pathogens or danger signals, NLRP3 inflammasome plays a key role in a variety of disease processes, including the initially identified familial periodic autoinflammatory response, to type II diabetes, alzheimer's disease, and atherosclerosis, among others. Therefore, as the core of inflammatory response, NLRP3 inflammasome may provide a new target for the treatment of various inflammatory diseases.
The NLRP3 receptor protein contains PYD, NACHT and LRR three structural domains, in the case of inflammatory corpuscle assembly, the PYD structural domain and the PYD structural domain of ASC combine to form a same type PYD-PYD interaction (PPI), while the CARD structural domain of ASC can combine with the CARD structural domain of effector protein to form a similar CARD-CARD interaction, so that the ASC plays a bridge role here to connect the receptor protein and the effector protein. Caspase-1 is finally activated by the above interaction, inducing its self-cleavage and activation when the receptor protein is stimulated by an agonist. Activated caspase-1 can cleave and promote the maturation of interleukins and other cytokines. The release of the interleukins plays an important role in the inflammatory response of the cells. In addition, caspase-1 maturation also plays a role in rendering cells non-mortal for cell death. The agonists of NLPR3 are quite broad, and there are roughly several of them: cytosolic stress of calcium ions, Reactive Oxygen Species (ROS) stress, cytosolic stress of hydrogen protons, potassium ion efflux, bacterial mRNA, oxidative mitochondrial DNA stress, lysosomal instability, and the like. Also because of this, the specific mechanism of action and signaling axis of agonist-receptor proteins has not been fully understood. Recent studies suggest that the disintegration of the reverse network structure of golgi may play a crucial role in signal transmission.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a primer composition for detecting NLRP3 promoter methylation, an application and a detection method, wherein the primer composition is designed, and the level of NLRP3 promoter cg21919599 methylation is detected by a pyrosequencing technology, so that the method has high sensitivity and accuracy and has important significance for pathological stage 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 invention provides a primer composition for detecting methylation of NLRP3 promoter, which comprises upstream primer cg21919599-F, downstream primer cg21919599-Rbio and sequencing primer cg21919599-S, wherein the nucleotide sequence of cg21919599-F is shown as SEQ ID NO.1, the nucleotide sequence of cg21919599-Rbio is shown as SEQ ID NO.2, and the nucleotide sequence of cg21919599-S is shown as SEQ ID NO. 3.
In a preferred embodiment, the methylation site of the NLRP3 promoter is cg21919599, and the cg21919599 is located 2789bp upstream of the transcription start site of the gene promoter.
The second aspect of the invention provides an application of a primer composition for detecting methylation of NLRP3 promoter in preparation of a tool for detecting methylation of NLRP3 promoter.
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 primer composition for detecting NLRP3 promoter methylation in preparation of a detection tool for detecting kidney cancer.
In a preferred embodiment, the methylation site of the NLRP3 promoter in the application is cg21919599, and the cg21919599 is 2789bp upstream of the transcription starting site of the gene promoter.
In a third aspect, the invention provides a tool for detecting methylation of NLRP3 promoter, which comprises the primer composition.
The fourth aspect of the invention provides a method for detecting methylation of NLRP3 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 cg21919599-F and a downstream primer cg 21919599-Rbio;
s4, pyrosequencing the PCR product amplified in step S3 by using the sequencing primer cg 21919599-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 ℃.
The accession number of the NLRP3(NLR family pyridine domain linking 3) gene in genebank is NC-000001.11, the gene is positioned on chromosome 1, and the total length of the genome sequence is 32661bp (247416163-247448823).
The invention has the following beneficial effects:
(1) the invention discovers that the NLRP3 promoter methylation site can be used as a new kidney cancer DNA marker, and the detection primer for NLRP3 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 NLRP3 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, and compared with the detection of mutation sites on the NLRP3 gene, the methylation of DNA is widely existed in various tumors, and the DNA methylation modification is stable, is not easy to degrade, and is convenient to store and transport.
Drawings
FIG. 1 is a graph of methylation level at the cg21919599 site as a function of expression level in renal cancer and expression of NLRP3 and patient survival time;
FIG. 2 is a graph showing the relationship between the methylation level at the cg21919599 site and the staging grade of renal cancer patients;
FIG. 3 shows the results of the methylation level and survival time of cg21919599 site of renal cancer patients applied from tissue specimen bank of tumor hospital at 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.
Methylation site of NLRP3 gene
First, based on methylation chip data in gene TCGA database, it was found that methylation site cg21919599 on NLRP3 gene is abnormally hypomethylated in kidney cancer compared with normal tissue, and at the same time, the methylation degree of this site was found to be negatively correlated with the expression of NLRP3 and the higher the methylation degree of cg21919599, the longer the survival time, and the results are shown in FIG. 1, wherein FIG. 1-a shows that cg21919599 is hypomethylated in kidney cancer, FIG. 1-b shows that cg21919599 is negatively correlated with the expression of NLRP3, and FIG. 1-c shows that the higher the methylation degree of cg21919599 of patient is, the longer the survival time is.
Based on the analysis of renal clear cell carcinoma data in the TCGA database, cg21919599 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 metastasis, and the results of the analysis are shown in fig. 2, in which fig. 2-a shows that cg21919599 exhibited hypomethylation in patients with high grade, fig. 2-b shows that cg21919599 exhibited hypomethylation in patients with high T grade, fig. 2-c shows that cg21919599 exhibited hypomethylation in patients with high grade, fig. 2-d shows that cg21919599 exhibited hypomethylation in patients with high N grade, and fig. 2-e shows that cg21919599 exhibited hypomethylation in patients with distant metastasis.
Method for detecting methylation of NLRP3 promoter
1. Primers cg21919599-F, cg21919599-Rbio and cg21919599-S were designed, which were synthesized by the company biologies (Sangon Biotech) and have the following specific sequences:
cg21919599-F(SEQ ID NO:1)：GTTGTAGAGGGTAGTGTTAGAGGTAA；
cg21919599-Rbio(SEQ ID NO:2)：AACAAAAAAAATATAATCTCCACTTACAT；
cg21919599-S(SEQ ID NO:3)：AGGTGAATTTTAGTATGGA。
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
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 69 renal cancer tumor tissues and 8 normal tissue samples from the Zhongshan university tumor hospital were measured by 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 tissues is higher than that of renal cancer tumor tissues in the sample overall, and as can be seen from table 3, the methylation level of cg21919599 site of 8 normal tissues is above 40%, and the methylation level of cg21919599 site of 69 renal cancer tumor tissues is below 40%, although the individual data is similar to the methylation level of normal tissues, the methylation level of NLRP3 promoter in renal cancer tumor tissues is obviously reduced from the whole data, which indicates that the promoter methylation level of NLRP3 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 cg21919599 in the tissue specimen of the renal cancer patient is obviously lower than that of normal tissues, and the survival time of hypermethylated patients is longer.
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> detection primer composition for NLRP3 promoter methylation, application and detection method
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gttgtagagg gtagtgttag aggtaa 26
<210> 2
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
aacaaaaaaa atataatctc cacttacat 29
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
aggtgaattt tagtatgga 19
Claims (10)
1. The primer composition for detecting methylation of the NLRP3 promoter is characterized by comprising an upstream primer cg21919599-F, a downstream primer cg21919599-Rbio and a sequencing primer cg21919599-S, wherein the nucleotide sequence of cg21919599-F is shown as SEQ ID NO.1, the nucleotide sequence of cg21919599-Rbio is shown as SEQ ID NO.2, and the nucleotide sequence of cg21919599-S is shown as SEQ ID NO. 3.
2. The primer composition for detecting methylation of the NLRP3 promoter according to claim 1, wherein the methylation site of the NLRP3 promoter is cg21919599, and the cg21919599 is located 2789bp upstream of the transcription start site of the gene promoter.
3. The application of the primer composition for detecting the methylation of the NLRP3 promoter in the preparation of a tool for detecting the methylation of the NLRP3 promoter according to claim 1.
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. The application of the primer composition for detecting the methylation of the NLRP3 promoter according to claim 1 in the preparation of a tool for detecting kidney cancer.
6. The use of any one of claims 3 to 5, wherein the NLRP3 promoter methylation site is cg21919599, and the cg21919599 is located 2789bp upstream of the transcription start site of the gene promoter.
7. A tool for detecting methylation of NLRP3 promoter, wherein the tool comprises the primer composition of claim 1.
8. A method for detecting methylation of NLRP3 promoter by using the primer composition according to claim 1, which comprises the steps of;
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 cg21919599-F and a downstream primer cg 21919599-Rbio;
s4, pyrosequencing the PCR product amplified in step S3 by using the sequencing primer cg 21919599-S.
9. The method for detecting methylation of the NLRP3 promoter according to claim 8, wherein the concentration of DNA in the sample is more than 50ng/μ L and the total amount of DNA in the sample is more than 500ng in step S2.
10. The method for detecting methylation of NLRP3 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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| Title |
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| MINGYI JU等: "Pan-cancer analysis of NLRP3 inflammasome with potential implications in prognosis and immunotherapy in human cancer", 《BRIEFINGS IN BIOINFORMATICS》 * |
| QIAN LONG等: "Prognostic, clinicopathological, and immune correlation of NLRP3 promotermethylation in kidney renal clear cell carcinoma", 《CLIN. TRANSL. MED.》 * |
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