CN113981099B - Detection method and application of methylation sites of human AURKB and TM4SF20 genes - Google Patents
Detection method and application of methylation sites of human AURKB and TM4SF20 genes Download PDFInfo
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Abstract
The invention discloses a detection method and application of methylation sites of human AURKB and TM4SF20 genes, and designs unique primers thereof, comprising a PCR amplification primer and a methylation sequencing primer, wherein the sequence of a PCR upstream primer of the AURKB gene site cg11009596 is as follows: the sequence of the downstream primer of biotin-5'-GAGTGGGTAGATGATTAGGTAGATTAGA-3' is as follows: 5'-CAACACACTAACCCCAATCTAAA-3', the sequence of the methylation sequencing primer is: 5'-CCCTACCTCCTTCCAA-3'; the PCR upstream primer sequence of the TM4SF20 gene locus cg20683151 is as follows: 5'-TTAATTGAGTTAGGGGTGATTATGA-3', the downstream primer sequences are: the sequence of the biotin-5'-AAACCAACAAACTAAATCCATTACA-3' methylation sequencing primer is as follows: 5'-GTTAGGGGTGATTATGAT-3'. According to the invention, specific sites of AURKB genes and TM4SF20 genes are screened in primary hepatocellular carcinoma patients, PCR primers and pyrosequencing primers aiming at the two sites are designed, and methylation levels of the specific sites can be rapidly and accurately quantitatively detected by a pyrosequencing technology.
Description
Technical Field
The invention relates to the technical field of liver cancer treatment, in particular to a detection method and application of methylation sites of human AURKB and TM4SF20 genes.
Background
Liver cancer is a malignant tumor with third mortality rate ranking in the global scope, and is also a second cancer 'killer' in our country. At present, the treatment of liver cancer mainly comprises surgery, radiotherapy and chemotherapy, and other treatment methods such as immunotherapy, traditional Chinese medicine and the like are combined; however, the pathogenesis of liver cancer is not clear and an effective treatment target is lacking, so that the treatment effect of patients in middle and later stages is poor and the survival rate is low. Research shows that liver cancer is an epigenetic regulated process. DNA methylation is one of the hot spots of current epigenetic research, and refers to the process of transferring methyl groups to certain bases of DNA by taking S-adenosylmethionine as a methyl donor under the mediation of DNA methyltransferase (DNAmethyltransferase, DNMT). DNA methylation abnormalities are considered to be a significant cause of tumor development. Research on the methylation change of the specific locus of the liver cancer related gene is helpful for understanding the pathogenesis of the liver cancer and provides an effective action target for preventing and treating the liver cancer.
The AURKB gene is located in region 3 of chromosome 17, short arm 1, and encodes a member of the aurora kinase subfamily of serine/threonine kinases. Genes encoding the other two members of this subfamily are located on chromosomes 19 and 20. These kinases are involved in the regulation of chromosomal arrangement and segregation during mitosis and meiosis by binding to microtubules. A pseudogene of this gene is located on chromosome 8. High expression of AURKB gene was reported by ArihiroAihara to be associated with recurrence of liver cancer. However, the relation between methylation of DNA specific sites of AURKB gene and liver cancer is not reported at home and abroad.
The TM4SF20 gene is located in the long arm 3 region 6 of human chromosome 2 and encodes a protein that is a member of the four transmembrane L6 superfamily. Members of this family play a role in various cellular processes, including cell proliferation, motility, and adhesion, through interactions with integrins. In human brain tissue, the gene is expressed at high levels in the parietal, occipital, hippocampus, pontine, white matter, corpus callosum and cerebellum. Knocking out the homologous genes in mice resulted in a neurobehavioral phenotype suggesting enhanced motor coordination. Deletion mutations in human genes are associated with specific language disorder-5. However, the gene has no research report related to cancer at present, and the methylation abnormality of the promoter region is not clearly related to liver cancer.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the problem that the relation between methylation of DNA specific sites of AURKB genes and liver cancer is not proposed in the prior art, and provides a detection method and application of methylation sites of human AURKB and TM4SF20 genes.
2. Technical proposal
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the methylation site of the human AURKB gene related to liver cancer is screened by a methylation chip to screen the difference between methylation spectrums in primary liver cell cancer and corresponding paracancerous tissues, and the methylation level of one site cg11009596 of the AURKB gene in DNA of the primary liver cell cancer is obviously lower than that of the paracancerous tissues, the difference has significance, and the nucleic acid sequence of the specific site is:
TGGGCGCTGGTCTCACCGCCCCCGCCCTGCTATCGTCCCTACCTCCTTCCAGCCCTGCGGCGTGCGCGCAGGCCAGCCCAACGGACCCTCTGATCTACCTGATCATCTGCCCACTCCCGGCG；
the base underlined is the exact position of the methylation site 215 bases from the transcription initiation site.
Preferably, the pyrosequencing method quantitatively detects the specific methylation site cg11009596 of the human AURKB gene, and the primers for the specific methylation site include a PCR primer and a methylation sequencing primer, wherein the sequence of the PCR upstream primer is as follows: the sequence of the downstream primer of biotin-5'-GAGTGGGTAGATGATTAGGTAGATTAGA-3' is as follows: 5'-CAACACACTAACCCCAATCTAAA-3', the sequence of the methylation sequencing primer is: 5'-CCCTACCTCCTTCCAA-3'.
The invention also provides a methylation site of the human TM4SF20 gene, a methylation chip screens the difference between methylation spectrums in primary hepatocellular carcinoma and corresponding paracancerous tissues, and the methylation level of one site cg20683151 of the TM4SF20 gene in DNA of the primary hepatocellular carcinoma is obviously lower than that of the paracancerous tissues, the difference is obvious, and the nucleic acid sequence of the specific site is:
GAACTACTCCTAACAGCAGTAGAACCAGCAGGCTGAATCCATTGCAGGATGTCCATCCTTCGCAGCAGGTCATGGTCACCCCTGGCTCAGAAACGTTGTCAAAGTGGCTATGCTTGCATGGT；
this site is 11 bases from the transcription initiation site.
The invention also provides a pyrosequencing method for quantitatively detecting the specific methylation site cg20683151 of the human TM4SF20 gene, wherein the specific methylation site primers comprise PCR primers and methylation sequencing primers, and the PCR upstream primer sequence is as follows: 5'-TTAATTGAGTTAGGGGTGATTATGA-3'; the downstream primer sequences were: biotin-5'-AAACCAACAAACTAAATCCATTACA-3'; the sequence of the methylation sequencing primer is as follows: 5'-GTTAGGGGTGATTATGAT-3'.
Preferably, the method for detecting methylation sites of human AURKB and TM4SF20 genes comprises the following steps:
step 1: extracting genome DNA of primary hepatocellular carcinoma tissue and paracancestral tissue respectively, performing bisulfite conversion on the genome DNA of all samples respectively, and converting unmethylated cytosine into uracil, wherein methylated cytosine is unchanged;
step 2: designing PCR primers and sequencing primers aiming at the upstream and downstream sequences of sites (cg 11009596 and cg 20683151) by taking the human genome DNA converted by bisulfite as a template;
step 3: pyrosequencing was performed using the PCR primers and sequencing primers using the bisulfite converted human genomic DNA as a template, and the methylation level of the (cg 11009596, cg 20683151) sites was analyzed.
Preferably, the methylation degree of the TM4SF20 gene cg20683151 is detected by using pyrosequencing, and the primary hepatocellular carcinoma is significantly different from the corresponding paracancerous tissue; the methylation degree of the site in the primary liver cancer is obviously reduced, and the change of methylation quantitative information of the site provides support for understanding pathogenesis of the liver cancer and finding out tumor prevention and treatment targets with potential clinical significance.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) In the invention, specific DNA methylation sites of genes related to primary hepatocellular carcinoma crowd are found, a pyrosequencing PCR primer and a sequencing primer are designed aiming at the specific sites, and methylation of the specific sites can be rapidly and accurately quantitatively detected through pyrosequencing.
In the invention, the methylation level of the cg11009596 locus in the primary hepatocellular carcinoma group is detected to be obviously lower than that of the cancer tissue from the same sample source, the methylation level of the cg20683151 locus is detected to be obviously lower than that of the cancer tissue from the same sample source, and the methylation level is consistent with the screening result of a methylation chip. The cg11009596 and cg20683151 sites are shown to be specific sites in the AURKB and TM4SF20 genes, respectively.
Drawings
FIG. 1 is an agarose gel electrophoresis of an AURKB gene PCR amplification product.
FIG. 2 is a graph showing results of pyrosequencing of AURKB gene, in which in-frame columnar portions show methylation sites and methylation percentages; 1 is cancer tissue, 2 is paracancerous tissue.
FIG. 3 is an agarose gel electrophoresis chart of a PCR amplification product of the TM4SF20 gene.
FIG. 4 is a graph showing the results of the pyrophosphate sequencing of the TM4SF20 gene, wherein the in-frame columnar portions reveal the methylation sites and the methylation percentages; 1 is cancer tissue, 2 is paracancerous tissue.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments.
Example 1:
referring to fig. 1-4, methylation of liver cancer related human AURKB gene, screening the methylation chip for differences between methylation spectra in primary hepatocellular carcinoma and corresponding paracancerous tissue, and finding that in DNA of primary hepatocellular carcinoma, methylation level of cg11009596 at one site of AURKB gene is significantly lower than that of paracancerous tissue, the differences are significant, and the nucleic acid sequence of the specific site is:
TGGGCGCTGGTCTCACCGCCCCCGCCCTGCTATCGTCCCTACCTCCTTCCAGCCCTGCGGCGTGCGCGCAGGCCAGCCCAACGGACCCTCTGATCTACCTGATCATCTGCCCACTCCCGGCG；
the base underlined is the exact position of the methylation site 215 bases from the transcription initiation site.
In the invention, a pyrosequencing method is used for quantitatively detecting a specific methylation site cg11009596 of a human AURKB gene, and primers of the specific methylation site comprise a PCR primer and a methylation sequencing primer, wherein the sequence of the PCR upstream primer is as follows: the sequence of the downstream primer of biotin-5'-GAGTGGGTAGATGATTAGGTAGATTAGA-3' is as follows: 5'-CAACACACTAACCCCAATCTAAA-3', the sequence of the methylation sequencing primer is: 5'-CCCTACCTCCTTCCAA-3'.
In the invention, the methylation site of the human TM4SF20 gene is screened by a methylation chip to screen the difference between methylation spectrums in primary hepatocellular carcinoma and corresponding paracancerous tissues, and the methylation level of one site cg20683151 of the TM4SF20 gene in DNA of the primary hepatocellular carcinoma is obviously lower than that of the paracancerous tissues, the difference is remarkable, and the nucleic acid sequence of the specific site is: GAACTACTCCTAACAGCAGTAGAACCAGCAGGCTGAATCCATTGCAGGATGTCCATCCTTCGCAGCAGGTCATGGTCACCCCTGGCTCAGAAACGTTGTCAAAGTGGCTATGCTTGCATGGT；
This site is 11 bases from the transcription initiation site.
The pyrosequencing method quantitatively detects the specific methylation site cg20683151 of the human TM4SF20 gene, wherein the primers of the specific methylation site comprise PCR primers and methylation sequencing primers, and the sequence of the PCR upstream primer is as follows: 5'-TTAATTGAGTTAGGGGTGATTATGA-3'; the downstream primer sequences were: biotin-5'-AAACCAACAAACTAAATCCATTACA-3'; the sequence of the methylation sequencing primer is as follows: 5'-GTTAGGGGTGATTATGAT-3'.
The detection method of the methylation sites of the human AURKB and TM4SF20 genes comprises the following steps:
step 1: extracting genome DNA of primary hepatocellular carcinoma tissue and paracancestral tissue respectively, performing bisulfite conversion on the genome DNA of all samples respectively, and converting unmethylated cytosine into uracil, wherein methylated cytosine is unchanged;
step 2: designing PCR primers and sequencing primers aiming at the upstream and downstream sequences of sites (cg 11009596 and cg 20683151) by taking the human genome DNA converted by bisulfite as a template;
step 3: pyrosequencing was performed using the PCR primers and sequencing primers using the bisulfite converted human genomic DNA as a template, and the methylation level of the (cg 11009596, cg 20683151) sites was analyzed.
In the invention, the methylation degree of the TM4SF20 gene cg20683151 is detected by applying pyrophosphoric acid sequencing to the application of methylation sites of the human AURKB and the TM4SF20 gene, and the primary hepatocellular carcinoma has a significant difference from corresponding paracancerous tissues; the methylation degree of the site in the primary liver cancer is obviously reduced, and the change of methylation quantitative information of the site provides support for understanding pathogenesis of the liver cancer and finding out tumor prevention and treatment targets with potential clinical significance.
Specific site analysis
In the present invention, the difference between methylation spectra in primary hepatocellular carcinoma and corresponding paracancerous tissue was screened using an InfiniumHumanMethylation27BeadChip methylation chip, and it was found that in genomic DNA of primary hepatocellular carcinoma, one site of the AURKB gene promoter region (which site has a number (cg 11009596) in the methylation chip described above) had a significantly lower methylation level than that of the paracancerous group, and one site of the TM4SF20 gene promoter region (cg 20683151) had a significantly lower methylation level than that of the paracancerous group.
AURKB gene specific site (SEQ ID NO. 1): TGGGCGCTGGTCTCACCGCCCCCGCCCTGCTATCGTCCCTACCTCCTTCCAGCCCTGCGG [ CG ] TGCGCGCAGGCCAGCCCAACGGACCCTCTGATCTACCTGATCATCTGCCCACTCCCGGCG
The sequence of the specific site of the TM4SF20 gene is (SEQ ID No. 5): GAACTACTCCTAACAGCAGTAGAACCAGCAGGCTGAATCCATTGCAGGATGTCCATCCTT [ CG ] CAGCAGGTCATGGTCACCCCTGGCTCAGAAACGTTGTCAAAGTGGCTATGCTTGCATGGT
To detect the accuracy of the screening results, relevant specific primers were designed and the specificity of the specific sites was verified by pyrosequencing.
In the present invention, the human genomic DNA extraction comprises the steps of:
s1, collecting cancer tissues and paracancerous tissues of 42 primary hepatocellular carcinoma patients, extracting genome DNA of each sample, and performing the operation according to the specification of Germany QIAGENQIAamp DNAminikit (Cat. No. 51304).
S2, performing agarose gel electrophoresis analysis on the obtained genomic DNA sample.
S3, bisulfite conversion
The bisulfite conversion was performed using the conversion kit EpiTectFastDNABisulfite kit (Cat. No. 59824) from QIAGEN, germany, and the procedure was followed according to the kit instructions.
S4, designing a primer, namely designing a pyrosequencing PCR primer and a sequencing primer by using genome DNA converted by bisulfite as a template and using PyroMark assaydesign2.0 software of QIAGEN company, and synthesizing the primer by Shanghai biological engineering Co.
In the present invention, pyrosequencing comprises the steps of:
s1.PCR amplification: the genome DNA converted by the bisulfite is used as a template, and the PCR reaction is carried out under the following conditions:
reaction conditions: 95 ℃ for 15min;94℃30sec,60℃1min,72℃1min (45 cycles); and at 72℃for 10min.
S2, performing 2% agarose gel electrophoresis on the PCR product after the reaction is finished, and verifying the specificity and accuracy of the product, as shown in figures 1 and 3.
S3, pyrosequencing: on the QIAGENPyroMarkQ48ID platform, pyrosequencing was performed, with specific steps according to the instrument instructions.
In the present invention, the methylation results of the DNA methylation detection result show that: the methylation level of the cg11009596 site cancer-associated group is significantly higher than that of the cancer group, the average methylation level of the site cancer-associated group is 16.00+ -6.12, and the average methylation level of the cancer group is 2.06+ -0.60 (p < 0.01); the methylation level of the cg20683151 site cancer-associated group was significantly higher than that of the cancer group, with an average methylation level of 91.84.+ -. 1.79 and an average methylation level of 58.53.+ -. 23.35 (p < 0.01). The sequencing charts are shown in figures 2 and 4 respectively.
In the invention, the research result shows that in primary hepatocellular carcinoma, the methylation level of the cg11009596 locus of the AURKB gene is obviously lower than that of a paracancerous group, and the methylation level of the cg20683151 locus of the TM4SF20 gene is obviously higher than that of the paracancerous group. Therefore, the pyrosequencing PCR primer and the sequencing primer designed aiming at the cg11009596 and cg20683151 loci can be used for quantitatively detecting the methylation level of two loci in primary hepatocellular carcinoma.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (2)
1. The use of a reagent for detecting the methylation level of a methylation site in a human AURKB gene in the preparation of a kit for assisting in diagnosing liver cancer, wherein the methylation site is cg11009596, and the nucleic acid sequence of the methylation site is: TGGGCGCTGGTCTCACCGCCC CCGCCCTGCTATCGTCCCTACCTCCTTCCAGCCC TGCGGCGTGCGCGCAGGCCAGCC CAACGGACCCTCTGATCTACCTGATCATCTGCCCAC TCCCGGCG; the base at the underline is the exact position of the methylation site, which is 215 bases from the transcription initiation site; the liver cancer is primary liver cancer.
2. Use of a reagent for the preparation of a kit for detecting the methylation level of a methylation site in a human AURKB gene according to claim 1, wherein the reagent comprises: designing a PCR primer and a sequencing primer aiming at an upstream and downstream sequence of a site cg11009596, wherein the sequence of the PCR upstream primer is as follows: the sequence of the downstream primer of biotin-5'-GAGTGGGTAGATGATTAGGTAGATT AGA-3' is as follows: 5'-CAACACACTAACCCCAATCTAAA-3', the sequence of the methylation sequencing primer is: 5'-CCCTACCTCCTTCCAA-3'.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103276095A (en) * | 2013-06-07 | 2013-09-04 | 浙江省医学科学院 | Primer for quantitatively detecting methylation levels of specific sites of human DMBT1 gene through pyrosequencing method |
| CN106480019A (en) * | 2015-09-01 | 2017-03-08 | 杨小丽 | The detection in people NUDT2 and PCDH8 gene methylation site and application |
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| CN103276095A (en) * | 2013-06-07 | 2013-09-04 | 浙江省医学科学院 | Primer for quantitatively detecting methylation levels of specific sites of human DMBT1 gene through pyrosequencing method |
| CN106480019A (en) * | 2015-09-01 | 2017-03-08 | 杨小丽 | The detection in people NUDT2 and PCDH8 gene methylation site and application |
Non-Patent Citations (2)
| Title |
|---|
| AURKB as a Promising Prognostic Biomarker in Hepatocellular Carcinoma;Xiao Jingchuan and Zhang Yingai;Evolutionary Bioinformatics;第17卷;1-10 * |
| Landscape of cancer diagnostic biomarkers from specifically expressed genes;Yao Lv等;Briefings in Bioinformatics;第21卷(第6期);2175-2184 * |
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