CN115786501A - Enhancer functional site related to colorectal cancer early screening and auxiliary diagnosis and application thereof - Google Patents
Enhancer functional site related to colorectal cancer early screening and auxiliary diagnosis and application thereof Download PDFInfo
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Abstract
The invention relates to an enhancer functional site marker related to colorectal cancer early screening and auxiliary diagnosis and application thereof, wherein the marker is rs4810856. Large-scale population data and biological functional experiments prove that the rs4810856 locus has enhancer function regulation activity, and an individual carrying rs4810856[ C ] genotype is more easily combined with a transcription factor ZEB1, so that the chromatin interaction of the region and a promoter of a target gene is influenced, the expression levels of the target genes PREX1, CSE1L and STAU1 are promoted, the cancer cell proliferation is promoted, and the risk of individual colorectal cancer is increased. By skillful primer and probe design aiming at rs4810856 and depending on fluorescent quantitative PCR, the rs4810856 dangerous site detection can be carried out on normal people, so that high risk people of colorectal cancer are identified, and diagnosis of patients with colorectal cancer is assisted.
Description
Technical Field
The invention relates to the fields of genetic engineering and tumor medicine, in particular to an enhancer functional site related to colorectal cancer early screening and application thereof.
Background
Colorectal cancer is one of common digestive tract malignant tumors and seriously threatens human life health. The incidence rate of the colorectal cancer is the third place of malignant tumors in China, and with the development of the national economic level and the change of the life style and dietary structure of residents, the incidence rate and the mortality rate of colorectal cancer in China tend to rise year by year, and the colorectal cancer becomes a major public health problem to be solved urgently in China and even all over the world. Currently, the most effective screening method for colorectal cancer is fecal occult blood test (F I T) combined with colonoscopy. However, this method also has problems such as low detection rate and low patient acceptance. Therefore, if early screening and early diagnosis can be more accurately carried out on high-risk individuals with colorectal cancer, the acceptance of patients can be higher, and a great amount of medical expenses can be saved for the country.
In recent years, an individual screening scheme based on molecular genetics becomes the key point for screening high-risk group of colorectal cancer. Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation, a class of molecular markers that have been shown to be associated with risk of tumorigenesis, reflecting individual genetic background differences. To date, GWAS has identified over 100 genetic susceptibility SNPs for colorectal cancer, and of the susceptibility SNPs discovered by GWAS, more than 90% of non-coding regions located in the genome can influence colorectal cancer risk by participating in gene expression regulation. While GWAS is rapidly developed, with the support of genome high-throughput detection technology, researchers find that non-coding regions contain many Cis-Regulatory elements (CRE), such as promoters, enhancers, insulators, etc., which can play a great role in gene expression regulation, thereby affecting tumorigenesis and susceptibility. With the rise of epigenomics and three-dimensional genomics, researchers are becoming more and more aware of the characteristics and mechanisms of action of cress. For example, CREs are often enriched in Chromatin opening regions as found by the Chromatin Transposase accessibility sequencing (ATAC-seq) technique, and naked DNA makes it easier to bind to Transcription Factor (TF), thereby participating in gene expression regulation. Nucleosomes near cress are often found to have specific histone modifications, including H3K27ac, H3K27me3 and H3K4me1, by Chromatin Immunoprecipitation sequencing (ChIP-sequencing) techniques. These specific epigenetic markers can help predict the activity status of cress and differentiate between different cress. In addition, with the continuous maturation of High-throughput chromatin three-dimensional Conformation Capture technology (High-Through Chromosome formation Capture, high-C). Researchers have found that Chromatin can spatially close to enhancers and promoters by three-dimensional folding to form a loop (Chromatin-Looping), thereby activating gene transcription.
However, how to efficiently integrate these multiomic data to find CREs and determine their target genes in genome-wide scale has always been a great challenge. Recently, in order to optimize the prediction of CREs and their target genes, researchers developed an Activity-by-Contact Model (ABC Model), and identified the Activity enhancer element in the genome-wide range and determined the target gene regulated by the Activity-by-Contact Model by integrating ATAC-seq and H3K27ac ChIP-seq signal peaks representing the Activity state (Activity) of the regulatory element and Hi-C data representing the chromatin Contact frequency (Contact). The ABC model is based on a simple biochemical concept that the quantitative effect of a remote candidate regulatory element on target gene expression should depend on its Activity as an enhancer (Activity), weighted by its chromatin Contact frequency with the target gene promoter (Contact); and the relative contribution of a candidate regulatory element to the expression of the target gene is determined by dividing the quantitative impact of the element by the total quantitative impact of all candidate regulatory elements in the region of the target gene. The ABC score corresponding to each enhancer-target gene is finally obtained by the model so as to evaluate the strength of the enhancer on the gene expression regulation effect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an enhancer function SNP locus marker for early screening and auxiliary diagnosis of colorectal cancer and application thereof.
The technical scheme for solving the technical problems is as follows:
an enhancer function site marker related to early colorectal cancer screening and auxiliary diagnosis, wherein the marker is rs4810856.
The specific amplification primer of the enhancer functional site marker related to colorectal cancer early screening and auxiliary diagnosis is provided with the sequence of rs4810856 as shown in SEQ ID NO:1 and SEQ ID NO:2.
the specific probe of the enhancer function site marker related to colorectal cancer early screening and auxiliary diagnosis is characterized in that the probe sequence of rs4810856 is SEQ ID NO:3 and SEQ ID NO:4.
the application of the detection reagent of the enhancer functional site marker related to colorectal cancer early screening and auxiliary diagnosis in the preparation of a colorectal cancer auxiliary diagnosis kit.
An early colorectal cancer screening and auxiliary diagnosis kit is used for detecting rs4810856 in peripheral blood DNA.
The colorectal cancer early screening and auxiliary diagnosis kit comprises a specific amplification primer of the SNP marker and a specific probe of the SNP marker.
The invention has the beneficial effects that: provides a technical method for screening high risk group of colorectal cancer from molecular biology and gene diagnosis level. The method is based on the fact that an ABC model integrates multiple groups of chemical technologies in the previous period, and the rs4810856 locus is related to colorectal cancer susceptibility of Chinese population. Through carrying out ingenious primer and probe design to rs4810856 site, can rely on fluorescence quantitative PCR, can carry out rs4810856 dangerous site's detection to normal crowd to the high risk crowd of discerning the colorectal cancer, supplementary colorectal cancer early screening and diagnosis, this technical method design benefit, simple and easy feasible, the result is accurate reliable, can promote in hospitals at all levels, to the sick risk that assesses the colorectal cancer provides help, help carrying out colorectal cancer screening and early intervention to this type of crowd clinically.
Drawings
FIG. 1 is a flow chart of ABC spectrometry for identifying susceptibility locus rs4810856 of colorectal cancer;
FIG. 2 is a schematic diagram of the population screening and potential regulation effect of the enhancer site rs 4810856;
FIG. 3 is a graph of the AUC of colorectal cancer risk prediction model based on SNP locus rs 4810856;
FIG. 4 is a diagram showing the experimental results of rs4810856 dual luciferase reporter gene;
FIG. 5 is a schematic diagram showing regulation of expression levels of target genes PREX1, CSE1L and STAU1 by rs4810856 genotype;
fig. 6 schematic diagram of CRISPR/Cas9 construction of single nucleotide mutations of rs4810856 in SW480 and HCT116 cells;
FIG. 7 schematic representation of the effect of the rs4810856 site on the proliferative capacity of colorectal cancer cells;
FIG. 8 is a schematic diagram showing the effect of overexpression of the target genes PREX1, CSE1L and STAU1 on the proliferative capacity of colorectal cancer cell lines;
FIG. 9 is a diagram illustrating the effect of overexpression of PREX1, CSE1L and STAU1 genes on subcutaneous neoplasia in nude mice.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The technical scheme for solving the technical problems is as follows:
an enhancer function site marker related to early colorectal cancer screening and auxiliary diagnosis, wherein the marker is rs4810856.
The sequence of the specific amplification primer of rs4810856 is SEQ ID NO:1 and SEQ ID NO:2.
the specific probe of the enhancer function site marker related to colorectal cancer early screening and auxiliary diagnosis has the probe sequence of SEQ ID NO:3 and SEQ ID NO:4.
the application of the detection reagent of the enhancer functional site marker related to colorectal cancer early screening and auxiliary diagnosis in the preparation of colorectal cancer auxiliary diagnosis kits.
A kit for early screening and auxiliary diagnosis of colorectal cancer is used for detecting rs4810856 in peripheral blood DNA.
The kit comprises a specific amplification primer of the SNP marker and a specific probe of the SNP marker.
Specifically, the technical solution of the present invention to solve the problem includes: (1) establishing a unified specimen library and a database: standard procedures (SOPs) were used to collect blood samples from subjects meeting the standards and the system collected complete demographic and clinical data. (2) genotype detection: colorectal cancer cases and healthy controls are selected, an ABC model is combined with multiple groups of mathematics such as ATAC-seq, CHIP-seq, RNA-se and Hi-C, the screened positive association markers are more accurately found out through the SNP markers rs4810856 (3) related to colorectal cancer incidence, and verification is carried out on independent adult group samples to judge the association stability. (4) development of colorectal cancer auxiliary diagnosis kit: an early screening and auxiliary diagnosis kit is developed according to genetic markers with significant difference in genotype distribution frequency in colorectal cancer cases and healthy controls.
Specifically, the experimental method of the research of the invention mainly comprises the following steps:
first, we collected 10 fresh colorectal cancer tissue samples surgically excised from the affiliated college of science and technology university in Huazhong. 10 samples were subjected to ATAC-seq, H3K27ac ChIP-seq and RNA-seq experiments, respectively. An ABC model system is used for integrating ATAC-seq, H3K27acChIP-seq, RNA-seq and Hi-C omics data, hi-C experimental data are obtained by downloading from an ENCODE database, the influence of activity of an enhancer and chromatin interaction with a target gene promoter on gene expression is comprehensively considered, then an enhancer element and a target gene regulated by the enhancer element are identified in a whole genome range, and an enhancer-target gene mapping map is drawn. 26,877 significant SNPs sites of the ABC enhancer are identified by taking the ABC score of more than or equal to 0.02 as a significance threshold, and the specific flow is shown in figure 1.
Next, we get fromThree colorectal cancer GWAS data sets of European population are downloaded from dbGaP database, and after the data are merged, 17,789 colorectal cancer cases and 19,951 control samples are contained. The basic demographic characteristics are shown in table 1. Based on the unconditional Logistic regression model, and after correcting for age and gender confounders, we calculated the association of the identified ABC enhancer SNPs with colorectal cancer susceptibility and plotted a manhattan plot therefrom, with the results shown in panel a in figure 2. During the calculation, we used P value < 0.05 as the significance threshold, and identified 4,847 ABC enhancer SNPs associated with colorectal cancer risk. Among them, we found that rs4810856 is located in the enhancer region: the chr20:48682097-48683408 has the strongest gene expression regulation effect and can simultaneously regulate three target genes: PREX1, CSE1L and STAU1, the results are shown in Panel B of FIG. 2. The results of the case-control study showed that individuals carrying the rs4810856 risk genotype had an increased risk of colorectal cancer of 4% compared to normal individuals in the european population samples (OR =1.04, 95% ci -5 )。
TABLE 1 basic data of colorectal cancer patients and normal controls of European and Chinese populations used in the study
TABLE 2 European population sample rs4810856 colorectal cancer Risk Association analysis results
Meanwhile, 6,024 colorectal cancer patients with complete medical record data and 10,022 normal controls without tumor history are collected from the two areas of Beijing Wuhan in China, and the people are subjected to genotyping. Patients are confirmed by histopathology without age restriction; normal controls had no history of tumor disease and no signs of tumor upon physical examination. Meanwhile, information such as sex, age, smoking and drinking of the study object is collected. Each subject had informed consent to participate in the study and donated 2ml of peripheral venous blood for isolation to prepare genomic DNA for lymphocytes. The basic demographic characteristics are shown in table 1.
We used the two recruited population samples for two stage population verification, calculating the association of SNP locus rs4810856 and colorectal cancer susceptibility separately for the two stage population samples by applying the unconditional logistic regression model, and correcting gender, age, smoking and drinking. The results of two-stage case contrast analysis of Chinese population show that rs 4810856C is carried]Individuals at risk genotype with increased risk of colorectal cancer by 30% and 14%, respectively (OR =1.30, 95% ci -4 ；OR＝1.14，95％CI:1.05-1.22，P＝3.76×10 -4 ). To enhance the test efficacy of the case-control study, samples from both stages were pooled and tested for association of rs4810856 with colorectal cancer susceptibility. The results of the Chinese population case contrast study are consistent with the results of colorectal cancer samples of European population, and rs4810856 is carried]The risk of an individual of the at-risk genotype for colorectal cancer is increased by 16% (OR =1.16, 95% ci -7 ) The detailed results are shown in Table 3.
TABLE 3 Chinese population sample rs4810856 colorectal cancer risk correlation analysis results
* And (4) calculating by adopting a Logistic regression model, and correcting the sex, the age, the smoking and the drinking conditions.
The association analysis result of the large sample population at the early stage shows that the site is a colorectal cancer risk site, so that a colorectal cancer risk prediction model is established by using the risk SNP site. A formula is constructed, and three genotypes of SNP and sex, age, smoking and drinking conditions are comprehensively considered. Wherein, for SNP genotyping, wild homozygous type = "1", heterozygous type = "2", and mutant homozygous type = "3"; for gender, "1" for men and "0" for women; for age, greater than or equal to 60 years is "1" and less than 60 years is "0". In the analysis, the multi-factor logistic regression coefficient beta is taken as a weight, and the formula based on the rs4810856 typing risk score is obtained as follows:
chinese population risk score = (0.1064 × gender score) + (-0.0121 × age score) + (0.2015 × smoking score) + (-0.1129 × drinking score) + (-0.0648 × rs8100241 type score).
European population risk score = (-0.1889 x score for gender) + (-0.0296 x score for age) + (-0.0437 x rs8100241 score for type)
By plotting the AUC curve, the area under the model curve is 0.591 and 0.561 in european population and chinese population, respectively, and the results are shown in fig. 3.
In order to functionally analyze the colorectal cancer risk association of the rs4810856 marker, the action mechanism of the SNP locus influencing the occurrence and development of colorectal cancer is explored.
First, we verified the functional regulation function of the enhancer in the region of rs4810856 by reporter gene experiments. DNA fragments containing different alleles of rs4810856 were constructed in a reporter plasmid vector in a forward or reverse direction, and co-transfected with an internal control plasmid pRL-SV40 into two human colorectal cancer cell lines of SW480 and HCT116, respectively, followed by detecting dual-luciferase gene expression and comparing relative luciferase activity differences among different groups. We find that the reporter gene activity of the recombinant plasmid containing rs4810856[ C ] allele is obviously higher than that of pGL3-Promoter no-load group no matter the insertion direction of the DNA fragment, and the DNA fragment of the target site has the activity of an enhancer. The reporter activity of the rs4810856[ T ] genotype group is significantly reduced compared to the rs4810856[ C ] genotype group. The results of the above experiments show that the region where rs4810856 is located has enhancer activity, and the transcriptional activation effect of the enhancer at the site rs4810856[ C ] is stronger than that of rs4810856[ T ], and the results are shown in FIG. 4. The eQTL analysis result shows that the rs4810856 locus with enhancer activity is closely related to the expression levels of target genes PREX1, CSE1L and STAU1 in the CRC tissues of a human sample, and the result is shown in FIG. 5.
In order to deeply explore the single base gene editing of rs4810856 and the influence of target genes on colorectal cancer cell proliferation, cell proliferation experiments, clone formation experiments, subcutaneous tumor formation of nude mice in vivo and other experiments are respectively carried out. In a cell proliferation experiment, an rs4810856 mononucleotide mutant cell line in SW480 and HCT116 cells is constructed by using CRISPR/Cas9 as an experimental model, the influence of an rs4810856 site on a colorectal cancer cell malignant phenotype is researched, and the construction process is shown in figure 6. The cell proliferation capacity of the rs4810856[ C ] carrying cell line was significantly greater compared to the rs4810856[ T ] cell line, and the results are shown in FIG. 7. Then, we used the stable transgenic cell lines with PREX1, CSE1L and STAU1 respectively over-expressed and jointly over-expressed as experimental models to examine the influence of the target genes on the proliferation capacity of the colorectal cancer cells. Experiments show that compared with blank control group cells, the clone forming capability of the cell line respectively over-expressed by the target gene is obviously enhanced, and the clone forming capability of the target gene and the over-expressed cell line is strongest, and the results are shown in FIG. 8. In an in-vivo nude mouse subcutaneous tumor formation experiment, a stable cell line is injected into the subcutaneous part of a nude mouse, the subcutaneous tumor volume of the nude mouse between different groups is measured periodically, and a subcutaneous tumor formation growth curve is drawn. We found that nude mice injected with PREX1, CSE1L and STAU1 over-expressing cell lines, respectively, had significantly increased subcutaneous tumor volume compared to nude mice injected with the blank control cell line, while nude mice injected with the target gene in combination with the over-expressing cell line had the greatest subcutaneous tumor volume, and the results are shown in fig. 9. Cell phenotype and in vivo nude mouse subcutaneous tumorigenesis experimental results show that PREX1, CSE1L and STAU1 can play a certain synergistic effect to promote the proliferation of colorectal cancer cells.
The results, combined with the large-scale population analysis, bioinformatics analysis, and rigorous experimental design described above, show that the rs4810856 risk site located in the 20q13.13 chromatin enhancer region significantly increases the risk of colorectal cancer in individuals carrying [ C ] allelic variation relative to individuals carrying rs4810856[ T ]. And the action mechanism is that the binding capacity of rs4810856 and a transcription factor ZEB1 is influenced, so that the interaction between the region where the gene is located and chromatin of a promoter of a target gene is inhibited, the expression level of the target genes PREX1, CSE1L and STAU1 is further reduced, the activation of an AKT signal pathway is inhibited, and the incidence risk of colorectal cancer is reduced. Therefore, the rs4810856 risk site located in a non-coding region has a close relationship with the colorectal cancer disease risk, and is expected to be clinically applied to assist the early diagnosis of colorectal cancer and discover the high risk group of colorectal cancer at an early stage.
The experimental method comprises the following steps:
1. peripheral blood DNA extraction:
we extract DNA by the conventional phenol-chloroform method, which comprises the following steps:
1) About 3ml of the anticoagulated blood was centrifuged at 5,000 Xg for 15min at room temperature, and the upper layer was discarded, leaving about 0.3ml of blood cells. 0.5ml of freshly prepared extraction buffer with a final concentration of 20. Mu.g/ml RNase was added, mixed well and incubated at 37 ℃ for 1h.
2) Proteinase K was added to a final concentration of 100. Mu.g/ml, mixed well and incubated overnight at 37 ℃.
3) 0.7ml of Tris buffer equilibrated phenol (pH = 7.0) was added to each tube, mixed well and centrifuged at 8,000 Xg for 15min at room temperature.
4) Transferring the upper layer liquid into another 1.5ml centrifuge tube, adding 0.7ml of phenol-chloroform (1); centrifuge at 8,000 Xg for 15min at room temperature.
5) The supernatant was transferred to another clean 1.5ml centrifuge tube, 10% by volume of 10M ammonium acetate solution was added, 2 volumes of pre-cooled absolute ethanol were added, and the mixture was allowed to stand at-20 ℃ for 2h to precipitate DNA.
6) Washing the precipitated DNA with 75% ethanol, centrifuging at 12,000 Xg for 15min, and discarding the upper liquid; the column was washed with 75% ethanol, centrifuged at 12,000 Xg for 15min, and the supernatant was discarded.
7) The tube is inverted on absorbent paper, after ethanol is completely volatilized, a proper TE buffer solution is added into each tube, and the tube is placed at 4 ℃ for a week and then stored at-20 ℃ for later use.
2. Genotyping
The typing platform used was TaqMan genotyping technology (ABI 7900HT Real Time PCR system, applied Biosystems), and 5. Mu.l of PCR reaction system is shown in Table 4:
TABLE 4 TaqMan genotyping System preparation
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, followed by 45 cycles of 95 ℃ for 15sec and 60 ℃ for 1min, and cooling to 4 ℃.
The primers and probes used in the reaction were as follows:
rs4810856 primer:
a forward primer: caatacctctcttattagtac (SEQ ID NO: 1)
Reverse primer: caggggagggtctgtttaata (SEQ ID NO: 2)
rs4810856 probe:
a forward probe: fam-acctacatcttc-mgb (SEQ ID NO: 3)
Reverse probe: vic-acctacacctttc-mgb (SEQ ID NO: 4)
3. Cell proliferation assay
1) Cell counting
Before CCK-8 proliferation experiments and clone formation experiments are carried out, cell counting is needed to ensure that cells with proper quantity are inoculated in different groups of pore plates. This experiment uses the blood count board to carry out the cell count, and this count board comprises two counting cells that the size is the same, and every counting cell comprises nine 1mm square again, and every square can hold 0.1mm3 liquid volume. The specific counting steps are as follows:
(1) digesting the cell pore plate after being cultured for a period of time to obtain cell suspension, and fully and uniformly mixing the cell suspension as much as possible to scatter cells.
(2) And (5) disinfecting the counting plate and then covering a cover glass.
(3) Aspirate 10mL of cell suspension and drip slowly into the counting plate along the slide edge to ensure no air bubbles are generated between the coverslip and the counting plate).
(4) Placing for 1min, observing a counting plate under a microscope, and counting squares at four corners according to the following principle: a. for cells pressed against the square border, only the upper and left cells are counted, b. If the cells clump, one cell is counted.
(5) Cell suspension concentration = total number of cells/4 × 104 cells/mL in the four-cornered squares.
2) CCK-8 cell proliferation assay
The research adopts CCK-8 kit (Dojindo, japan) to detect the proliferation capacity of colorectal cancer cells.
(1) The cell suspension was aspirated at 100mL and diluted to a cell count of about 2,000, and the cells were seeded in a 96-well plate.
(2) Cell activity assays were performed at four time points, 24h, 48h, 72h and 96 h. 10mL of CCK-8 reagent was added to each well, shaken up and down and left and right to mix gently, and returned to the cell incubator for further incubation for 1.5h.
(3) Absorbance measurements were performed using a microplate reader, the wavelength was set at 450nm, and cell proliferation curves for different experimental groups were plotted based on absorbance values at four time points to compare the differences.
3) Clone formation assay
(1) After 36h of transfection of the cells, the cells were counted and diluted to the appropriate concentration. 2mL of the cell suspension was added to each well of the 6-well plate so that the cell amount was about 1,000, and the culture was continued for about 2 weeks.
(2) During this period, the cell morphology was observed every 2 to 3 days and the medium was replaced with fresh one. If cell clones are observed under a microscope, fixed staining can be performed.
(3) The 6-well plate was removed from the incubator, the waste was aspirated and washed 2 times by slow addition of PBS. After each well of PBS was aspirated, 2mL of methanol solution was added, and the mixture was left at room temperature for 30min.
(4) After methanol was removed by suction, 0.25% crystal violet solution was added and left to stand in the dark for 30min for dyeing.
(5) After dyeing is finished, absorbing waste liquid in the hole, cleaning the hole plate by using double distilled water until the visual field is clear, and taking a picture for storage.
In conclusion, we systematically integrated multiple sets of mathematical data including ATAC-seq, CHIP-seq, RNA-seq and Hi-C by using ABC model, thereby constructing the colorectal cancer enhancer-target gene mapping map. Furthermore, the method combines the technical methods of case control research, biological experiments and the like of large samples of European population and Chinese population. It was found that rs4810856, located in the 20q13.13 region, had the strongest gene expression regulatory effect, and that individuals carrying the [ C ] allele had a significantly increased risk of developing colorectal cancer compared to rs4810856[ T ] individuals. Mechanistically, we found that rs4810856, located in the 20q13.13 region of chromatin, can simultaneously regulate three target genes: PREX1, CSE1L and STAU1. And the binding capacity of rs4810856[ C ] and a transcription factor ZEB1 is stronger than that of rs4810856[ T ] carriers, so that the chromatin interaction of the target gene promoter in the region where the rs4810856[ C ] is located is influenced, the expression levels of the target genes PREX1, CSE1L and STAU1 are further promoted, and the AKT signal pathway activation is influenced, so that the colorectal cancer morbidity risk is promoted. Therefore, the rs4810856 variation positioned in a non-coding region has close relation with the colorectal cancer disease risk, and is expected to be clinically applied to assist the early diagnosis of colorectal cancer and discover colorectal cancer patients at an early stage.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. An enhancer functional site related to early colorectal cancer screening and auxiliary diagnosis, wherein the functional site is rs4810856.
2. The specific amplification primer of enhancer functional site related to colorectal cancer early screening and aided diagnosis according to claim 1, wherein the sequence of the specific amplification primer of rs4810856 is SEQ ID NO:1 and SEQ ID NO:2.
3. the specific probe of enhancer functional site related to colorectal cancer early screening and auxiliary diagnosis according to claim 1, wherein the probe sequence of rs4810856 is SEQ ID NO:3 and SEQ ID NO:4.
4. use of the detection reagent for enhancer functional site related to colorectal cancer early screening and auxiliary diagnosis according to claim 1 in the preparation of colorectal cancer auxiliary diagnosis kit.
5. An early colorectal cancer screening and auxiliary diagnosis kit is characterized by being used for detecting rs4810856 in peripheral blood DNA.
6. The kit for early screening and aided diagnosis of colorectal cancer according to claim 5, comprising an amplification primer specific to the enhancer functional site according to claim 2 and a probe specific to the enhancer functional site according to claim 3.
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Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005304497A (en) * | 2004-03-25 | 2005-11-04 | Joji Inasawa | Method for detecting cancer using specified cancer-related gene and method for inhibiting the cancer |
| WO2006138275A2 (en) * | 2005-06-13 | 2006-12-28 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| US20080015160A1 (en) * | 2005-11-04 | 2008-01-17 | Johji Inazawa | Method for detecting cancer and a method for suppressing cancer |
| WO2009055011A1 (en) * | 2007-10-26 | 2009-04-30 | Ludwig Institute For Cancer Research Ltd. | Enhancer signatures in the prognosis and diagnosis of cancers and other disorders |
| US20100267028A1 (en) * | 2008-12-24 | 2010-10-21 | Boris Pasche | Tgfbr1 HAPLOINSUFFICIENCY MODIFIES RISK FOR CANCER |
| CN102084000A (en) * | 2008-02-01 | 2011-06-01 | 总医院有限公司 | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| CA3012783A1 (en) * | 2016-01-28 | 2017-08-03 | The University Of Melbourne | Methods for assessing risk of developing colorectal cancer |
| CN107254531A (en) * | 2017-06-28 | 2017-10-17 | 南京医科大学 | The genetic biomarkers thing of early onset colorectal cancer auxiliary diagnosis and its application |
| CN108220394A (en) * | 2018-01-05 | 2018-06-29 | 清华大学 | Identification method, system and its application of gene regulation sex chromatin interaction |
| WO2018146033A1 (en) * | 2017-02-07 | 2018-08-16 | F. Hoffmann-La Roche Ag | Non-invasive test to predict recurrence of colorectal cancer |
| CN110283908A (en) * | 2019-06-04 | 2019-09-27 | 华中科技大学 | A kind of colorectal cancer auxiliary diagnosis SNP marker and its application |
| CN110592213A (en) * | 2019-09-02 | 2019-12-20 | 深圳市新合生物医疗科技有限公司 | Gene panel for prediction of neoantigen load and detection of genomic mutations |
| WO2020257353A1 (en) * | 2019-06-17 | 2020-12-24 | The Board Of Trustees Of The Leland Stanford Junior University | Diagnostics and treatments based upon molecular characterization of colorectal cancer |
| KR20210066635A (en) * | 2019-11-28 | 2021-06-07 | 의료법인 성광의료재단 | Compositions, kits and methods for diagnosing cancer resistant to cancer therapeutics |
| WO2022135552A1 (en) * | 2020-12-25 | 2022-06-30 | 上海善准生物科技有限公司 | Colorectal cancer molecular typing and survival risk factor gene cluster, diagnostic product, and application |
-
2022
- 2022-07-02 CN CN202210779427.0A patent/CN115786501B/en active Active
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005304497A (en) * | 2004-03-25 | 2005-11-04 | Joji Inasawa | Method for detecting cancer using specified cancer-related gene and method for inhibiting the cancer |
| WO2006138275A2 (en) * | 2005-06-13 | 2006-12-28 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| US20080015160A1 (en) * | 2005-11-04 | 2008-01-17 | Johji Inazawa | Method for detecting cancer and a method for suppressing cancer |
| WO2009055011A1 (en) * | 2007-10-26 | 2009-04-30 | Ludwig Institute For Cancer Research Ltd. | Enhancer signatures in the prognosis and diagnosis of cancers and other disorders |
| CN102084000A (en) * | 2008-02-01 | 2011-06-01 | 总医院有限公司 | Use of microvesicles in diagnosis, prognosis and treatment of medical diseases and conditions |
| US20100267028A1 (en) * | 2008-12-24 | 2010-10-21 | Boris Pasche | Tgfbr1 HAPLOINSUFFICIENCY MODIFIES RISK FOR CANCER |
| CA3012783A1 (en) * | 2016-01-28 | 2017-08-03 | The University Of Melbourne | Methods for assessing risk of developing colorectal cancer |
| WO2018146033A1 (en) * | 2017-02-07 | 2018-08-16 | F. Hoffmann-La Roche Ag | Non-invasive test to predict recurrence of colorectal cancer |
| CN107254531A (en) * | 2017-06-28 | 2017-10-17 | 南京医科大学 | The genetic biomarkers thing of early onset colorectal cancer auxiliary diagnosis and its application |
| CN108220394A (en) * | 2018-01-05 | 2018-06-29 | 清华大学 | Identification method, system and its application of gene regulation sex chromatin interaction |
| CN110283908A (en) * | 2019-06-04 | 2019-09-27 | 华中科技大学 | A kind of colorectal cancer auxiliary diagnosis SNP marker and its application |
| WO2020257353A1 (en) * | 2019-06-17 | 2020-12-24 | The Board Of Trustees Of The Leland Stanford Junior University | Diagnostics and treatments based upon molecular characterization of colorectal cancer |
| CN110592213A (en) * | 2019-09-02 | 2019-12-20 | 深圳市新合生物医疗科技有限公司 | Gene panel for prediction of neoantigen load and detection of genomic mutations |
| KR20210066635A (en) * | 2019-11-28 | 2021-06-07 | 의료법인 성광의료재단 | Compositions, kits and methods for diagnosing cancer resistant to cancer therapeutics |
| WO2022135552A1 (en) * | 2020-12-25 | 2022-06-30 | 上海善准生物科技有限公司 | Colorectal cancer molecular typing and survival risk factor gene cluster, diagnostic product, and application |
Non-Patent Citations (5)
| Title |
|---|
| ENSEMBL GENOME BROWSER: ""rs4810856 (SNP) - Flanking sequenc-，Homo_sapiens-Ensembl genome browser 106", pages 1 - 2, Retrieved from the Internet <URL:apr2022.archive.ensembl.org/Homo_sapiens/Variation/Sequence?db=core;r=20:48681644-48682644;v=rs4810856;vdb=variation;vf=177261167> * |
| JINHUA WANG: "P-REX1 amplification promotes progression of cutaneous melanoma via the PAK1/P38/MMP-2 pathway", 《CANCER LETTERS》, pages 66 - 75 * |
| 朱颖: "肠内营养剂对结直肠癌患者术后体征及并发症的影响", 《结直肠肛门外科》, pages 429 - 434 * |
| 李迎君;刘冰;景方圆;丁烨;何青芳;钟要红;范春红;: "UCA1表达水平及其启动子区域rs7255437单核苷酸多态性与结直肠癌的关系", 预防医学, no. 09, pages 884 - 888 * |
| 缪小平: "结直肠癌遗传流行病与药物基因组学研究概述", 《药物流行病学杂志》, vol. 23, no. 6, pages 371 - 375 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117106912A (en) * | 2023-08-21 | 2023-11-24 | 武汉大学 | Application of detection reagent of enhancer RNA functional site rs3094296 in preparation of colorectal cancer auxiliary diagnosis kit |
| CN117106912B (en) * | 2023-08-21 | 2024-02-02 | 武汉大学 | Enhancer RNA functional site related to colorectal cancer high-risk group identification and early screening and application thereof |
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