CN112553344B - Biomarker related to colorectal cancer and application thereof - Google Patents
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Abstract
The invention provides a biomarker related to colorectal cancer and application thereof, wherein intestinal microorganism, namely, Micromonas sp (Parvimonas spp.) is taken as a marker 1), BMP3 gene is methylated as a marker 2), and the content of human DNA is taken as a marker 3). The invention also provides application of the marker as a detection target in preparing a detection reagent or a kit, application in assessing colorectal cancer risk and a corresponding colorectal cancer risk assessment model. The marker has the potential of being used as a colorectal cancer marker, and the method and the model for evaluating the colorectal cancer of the patient according to the marker have high sensitivity and good specificity, have the function of non-invasive auxiliary diagnosis of the colorectal cancer, and have good application prospect and practical significance.
Description
Technical Field
The invention relates to the technical field of biotechnology, in particular to a colorectal cancer related biomarker and application thereof.
Background
Colorectal cancer (CRC), also known as Colorectal cancer, is a collective term for colon and rectal cancers. The incidence and mortality of colorectal cancer in China keep rising, and 2018 Chinese cancer statistical reports show that the incidence and mortality of colorectal cancer in China are respectively at the 3 rd and 5 th positions in all malignant tumors, 37.6 thousands of new cases and 19.1 thousands of death cases, and most cases belong to middle and late stages when diagnosis is confirmed. The onset of colorectal cancer is a multi-factor and multi-step process, which is a process of interaction between internal factors of the body and external factors such as environment, diet, living habits and the like. Epidemiological research results show that social development conditions, lifestyle and dietary patterns are all closely related to the occurrence of colorectal cancer. Studies have shown that a high-fat, high-protein, low-cellulose diet may increase the risk of colorectal cancer, and such a diet structure may slow down intestinal motility, prolong food retention time, and increase the chances of carcinogens coming into contact with the intestine. Fried smoked foods may contain carcinogenic components acting on colorectal cancer, while pickled foods are also associated with colorectal cancer. In the analysis of occupational physical activity it was found that the occupational category, which is long-term or frequently in place, is at a risk of colorectal cancer of 1.4 times the occupational activity with greater physical activity.
Stages 0, I, II, III and IV are usually divided according to the invasion degree of colorectal cancer, lymph node metastasis conditions around tumors and the like. Clinical data show that the 5-year survival rate of colorectal patients at the 0 and I stages after operation reaches more than 90%, the II stage is 68.4%, the III stage is 39.7%, and the IV stage is only 10.2%, wherein misdiagnosis and missed diagnosis are very serious. Literature data shows that the misdiagnosis rate of colorectal cancer is as high as 60-70%, and about 64% -85% of patients can obtain correct diagnosis after more than 6 months, so that part of patients lose the best cure time. In addition, 96% of colorectal cancers develop from adenomatous polyps, which take about 10-15 years to develop into tumors, so that early detection and removal of polyps can significantly reduce the risk of canceration.
The mainstream colorectal cancer screening techniques include stool examination, endoscopy and imaging examination. The detection cost of guaiac fecal occult blood test (gFOBT) in fecal examination is low, and the method is a traditional method for noninvasive screening of colorectal cancer, but human hemoglobin is subjected to non-specific detection, interference factors are more, and false positive is possibly caused by diet, medicines and the like; an immunochemical fecal occult blood test (FIT) is characterized in that a detection result is standardized by detecting globin in feces, the specificity and the sensitivity are higher than those of gFOBT, but the FIT is easily influenced by a sample environment; fecal DNA detection is a new noninvasive screening method, non-specific interference factors are avoided, the U.S. Food and Drug Administration (FDA) has approved a technology for screening and rectal cancer detection through fecal DNA, the specificity and sensitivity of fecal DNA detection are remarkably improved compared with FIT, and the colorectal tumor can be more accurately detected by combining the fecal DNA detection technology with FIT, but the colorectal cancer cost based on fecal DNA detection is higher. The colonoscopy is used simultaneously in the endoscopy, the accuracy is high, but the endoscopy is invasive inspection, the operation process is complex, the risks of bleeding, perforation, complication and the like exist, and the acceptance degree of a patient is low. The imaging examination of colorectal cancer includes pneumobarium double colon radiography, colon CT imaging and the like, and although the imaging examination method is safe and non-invasive, the imaging examination method is difficult to find for some flat adenomas and early colorectal cancer.
Intestinal microorganisms, which are part of the human immune system, are important participants in the inflammatory and immune responses of the human body, and can reflect the individual health status to a certain extent, and in colorectal cancer patients, intestinal microbial flora is often disordered, specifically, beneficial bacteria are reduced and harmful bacteria are increased, wherein fusobacterium nucleatum is proved to participate in the carcinogenic process. During the onset of colorectal cancer, the intestinal epithelial cells are significantly exfoliated, which is manifested as an increase in the human DNA content in the stool. In addition, the early occurrence of the colorectal cancer is greatly related to the methylation of promoter regions of colorectal cancer related genes, and the methylation level of a bone morphogenetic protein gene (BMP 3) is closely related to the occurrence, development and metastasis of tumors and can be used as a biological characteristic of the early occurrence of the colorectal cancer.
With the development of the field of precise health, a colorectal cancer early screening method with accuracy, non-invasiveness and high cost performance is clinically needed. Meanwhile, the contents of microorganisms and human DNA in the intestinal tract and the methylation of BMP3 gene have great potential as molecular markers, and can be used as molecular markers for noninvasive colorectal cancer detection. Therefore, a colorectal cancer related biomarker and application thereof are provided.
Disclosure of Invention
In view of the above-mentioned shortcomings in the background art, the present invention is directed to provide a biomarker associated with colorectal cancer and use thereof, which can assist in diagnosing colorectal cancer by detecting the level of pseudomonas sp, the methylation level of BMP3 gene promoter and the Human DNA Content (HDC) in stool; the detection method comprises the steps of separating and extracting DNA from a fecal sample, carrying out fixed-slice qPCR determination by using a TaqMan probe method, judging the methylation level or DNA content of the marker in the sample by calculating the difference value of the Ct value of the three markers and the Ct value of an internal reference gene, and further evaluating the risk of colorectal cancer occurrence of a sample individual. The noninvasive detection mode has accuracy and cost performance superior to those of the traditional detection method, and can be used for auxiliary diagnosis of colorectal cancer.
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a biomarker associated with colorectal cancer, the biomarker comprising 3 of:
marker 1) Micromonospora (Parvimonas spp.);
marker 2) BMP3 gene methylation;
marker 3) human DNA content.
Wherein, the marker 1) probe primer combination of the detection reagent of the genus Micromonospora (Parvimonas spp.) is as follows: the sequence of the forward primer is shown as SEQ ID NO.1, the sequence of the reverse primer is shown as SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO. 3.
Wherein, the probe primer combination of the marker 2) BMP3 gene methylation detection reagent is as follows: the sequence of the forward primer is shown as SEQ ID NO.4, the sequence of the reverse primer is shown as SEQ ID NO.5, and the sequence of the probe is shown as SEQ ID NO. 6.
Wherein, the probe primer combination of the marker 3) human DNA content detection reagent is as follows: the sequence of the forward primer is shown as SEQ ID NO.7, the sequence of the reverse primer is shown as SEQ ID NO.8, and the sequence of the probe is shown as SEQ ID NO. 9.
The invention discloses a method for detecting colorectal cancer, which is characterized in that the genus Micromonospora (Parvimonas spp.) is a general name of a plurality of unknown strains in the genus Micromonospora, and the marker 1) Micromonospora (Parvimonas spp.) has obvious difference in two groups of people and has the potential of being used as a colorectal cancer marker by analyzing and comparing the abundance difference of floras in fecal samples of colorectal cancer patients and healthy individuals.
The marker 2) BMP3 gene methylation sequence provided by the invention is a nucleotide sequence with the length of 380bp from 1660 th to 2039 th on a BMP3 gene promoter, and the sequence has 16 known methylation sites, so that the methylation detection sensitivity of the BMP3 gene is high. The BMP3 gene methylation upstream and downstream primers and the probe provided by the invention can effectively amplify the sequences and detect fluorescence signals.
The marker 3) the human DNA content provided by the invention means that in the process of colorectal cancer, the desquamation of intestinal epithelial cells is obviously increased, and the human DNA content is specifically increased, so that the human DNA content can also reflect the health condition of human intestinal tracts, and the marker has the potential of being used as a colorectal cancer marker.
The marker provided by the invention has high sensitivity and good specificity, and the cooperation of the three markers is beneficial to auxiliary diagnosis or evaluation of the risk of colorectal cancer, can be used for early screening of colorectal cancer, and has good application prospect and practical significance.
Further, the amount or level of the marker is provided based on calculation of the gene sequence fragment thereof.
Further, the content or level information of the marker is used for training a colorectal cancer risk assessment model and assessing colorectal cancer risk.
In a second aspect, the present invention provides a reagent for detecting a marker as described in the first aspect.
The reagent may be a combination of the primer and probe with a marker or other reagent for determining the level or amount of the marker.
In a third aspect, the present invention provides a marker according to the first aspect or a reagent according to the second aspect for use, wherein the use comprises preparation of a colorectal cancer auxiliary diagnostic reagent or preparation of a colorectal cancer auxiliary diagnostic kit.
In a fourth aspect, the present invention provides a colorectal cancer risk assessment model.
The colorectal cancer risk assessment model was trained in 357 healthy samples and 354 colorectal cancer patients using a random forest machine learning algorithm with the content of the three markers and the sample grouping information as described in the first aspect. Meanwhile, ten times of cross validation is used, so that errors caused by one-time validation are avoided.
The model has high evaluation performance and can assist in diagnosing the colorectal cancer risk of an individual.
In a fifth aspect, the present invention provides a method for risk assessment of colorectal cancer in an individual, comprising the steps of:
step 1) extracting and purifying DNA fragments from the individual excrement sample, and dividing the DNA fragments into two parts;
step 2) treating a part of the sample DNA fragments obtained in the step 1) by using bisulfite conversion solution for methylation detection of BMP3 gene;
step 3) carrying out quantitative qPCR by using a TaqMan probe method, and detecting the content of a target gene fragment of a marker and the content of an internal reference gene in a sample, wherein the marker 2) BMP gene methylation detection uses the sample DNA fragment obtained by the step 2) after treatment, and the marker 1) Micromonospora and the marker 3) human DNA content detection uses the DNA fragment in the sample obtained by the step 1);
step 4) calculating the level or content of the corresponding marker according to the content of the target gene fragment of the marker in the step 3) and the content of the internal reference gene;
and 5) inputting the level or content of the marker obtained in the step 4) into the colorectal cancer risk assessment model according to the fourth aspect, and assessing the colorectal cancer risk of the individual.
The method for colorectal cancer risk assessment is realized by relying on the colorectal cancer risk assessment model according to the fourth aspect, and by using the background data of 357 healthy samples and 354 colorectal samples, the colorectal cancer risk of the individual to be tested under the background of the data is assessed. And (5) prompting the change of the dietary life style or further carrying out colorectal cancer screening diagnosis in a hospital for the individuals with higher risk.
In the present invention, the method for assessing the risk of colorectal cancer in an individual according to the fifth aspect provides a non-invasive auxiliary detection method for early diagnosis of colorectal cancer.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a marker related to colorectal cancer, which has the potential of being used as a colorectal cancer diagnosis marker, can be used for auxiliary diagnosis and risk assessment of colorectal cancer, has good specificity, high sensitivity and high cost performance, assesses colorectal cancer risk from multiple layers and reduces the possibility of colorectal cancer occurrence;
2. the invention provides a method for assessing the risk of colorectal cancer of an individual, which does not need intestinal tract preparation and can increase the compliance of patients;
3. the invention provides a microorganism marker related to colorectal cancer and a detection reagent of the microorganism marker, which can be used for preparing a colorectal cancer diagnosis reagent or a kit and have good application prospect and practical significance;
4. the model for assessing colorectal cancer risk provided by the invention has the advantages of good model performance and strong assessment capability, and can assist in diagnosing the colorectal cancer risk of an individual.
Drawings
FIG. 1 shows the evaluation results of colorectal cancer risk assessment model.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following embodiments, but the scope of the present invention is not limited by the specific embodiments, and it should be understood that the claims are only directed to the described embodiments, and not to the whole 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.
The data used in the present invention have meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:
"biomarker" refers to a biochemical marker that can mark changes or likely changes in the structure or function of systems, organs, tissues, cells and subcellular systems, and can be used for disease diagnosis, disease staging or evaluation of the safety and efficacy of new drug therapies in target populations. In the present invention, "biomarker" refers to intestinal microbial markers, human DNA content in feces, and individual BMP3 gene methylation.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1, extraction of fecal DNA samples.
(1) Collecting an individual excrement sample, immediately freezing the sample, and placing the sample on ice before an experiment;
(2) weighing 200mg of fixed excrement, adding 800 mu L of excrement DNA extraction buffer solution into a 2mL centrifuge tube, fully shaking and uniformly mixing for 5min, and centrifuging for 1min at 1800 g;
(3) taking 50 mu L of suspension from the suspension, putting the suspension into a 1.5mL centrifuge tube, adding 800 mu L of lysate, uniformly mixing by vortex oscillation, cracking at 70 ℃ for 5min, centrifuging for 5min, and transferring supernatant into a clean 1.5mL centrifuge tube;
(4) adding 20 μ L of the mixed magnetic beads, vortex shaking for 20s, standing at room temperature for 4min, placing on a magnetic frame, standing for 20s, and sucking the supernatant;
(5) adding 500 mu L of washing solution I, carrying out vortex oscillation for 20s, uniformly mixing magnetic beads, placing on a magnetic frame, standing for 20s, and discarding the supernatant;
(6) adding 750 mu L of washing liquid II, carrying out vortex oscillation for 20s, uniformly mixing magnetic beads, placing on a magnetic frame, standing for 20s, and discarding the supernatant;
(7) repeating the step (6) once to remove all liquid as much as possible;
(8) placing on a magnetic frame, uncovering, and drying for 7-8min to remove all liquid as much as possible;
(9) adding 50 mu L of buffer solution or double distilled water, carrying out vortex oscillation for 15s, uniformly mixing magnetic beads, heating at 65 ℃ for 7min (during the period of 10s of vortex oscillation), carrying out vortex oscillation for 15s, placing on a magnetic frame, standing for 2min, and absorbing supernatant into a collecting pipe to obtain the fecal DNA sample.
The obtained sample is divided into two parts, one part is used for quantitative detection of the markers 1) and 3), and the other part is used for quantitative detection of the marker 2) after being treated by the bisulfate.
Example 2, quantitative detection of markers.
Quantitative detection of the markers 1) and 3) adopts a Taqman qPCR method, and quantitative detection of the marker 2) adopts an MSP (methylation Specific PCR) method, wherein the used probes and primers are shown in Table 1.
TABLE 1 probes and primers for markers
The specific steps of the content detection of the marker 1) and the marker 3) are described below by taking the TaqMan Master Mix kit product of Suzhou New sea Biotechnology corporation as an example.
(1) The reaction was carried out according to the qPCR reaction system shown in Table 2 to prepare a PCR reaction solution.
TABLE 2 qPCR reaction System
(2) After the PCR reaction solution is prepared, the mixture is evenly mixed and centrifuged upside down, and is subpackaged into a 96-hole PCR reaction plate, centrifuged for 2min at 2000g, and then placed in a PCR instrument for reaction after being sealed.
(3) The qPCR reaction was performed using a two-step PCR reaction method, and the procedure was set as shown in table 3.
TABLE 3 two-step PCR reaction procedure
(4) And according to the Ct value output by the instrument, the content of the Micromonospora and the human DNA takes 16S rDNA as an internal reference, the content of the target fragment of the microbial marker in the sample is relatively and quantitatively calculated, and the result is the abundance of the microbial marker.
Similarly, marker 2) quantitative determination of BMP3 gene methylation was performed using the MSP method using a sodium bisulfate-modified DNA sample and an internal reference gene corresponding to BMP3 gene fragment as the MSP product.
Example 3, colorectal cancer risk assessment model.
The colorectal cancer risk assessment model is trained and tested by using a random forest algorithm and three marker abundances of 357 healthy samples and 354 colorectal cancer patient samples, and is finally established, and the method comprises the following specific steps:
step 1) stool DNA samples were extracted from the stools of 357 healthy and 354 colorectal cancer samples using the method as described in example 1 and the extracted samples were divided into two parts, one of which was treated with bisulfate;
step 2) the content of the target gene fragment of the three markers and the content of the internal reference gene in all samples were determined using the method as described in example 2, wherein marker 1) and marker 3) used 16S rDNA as internal reference, and marker 2) used MSP product corresponding BMP3 gene fragment as internal reference;
step 3) calculating to obtain the abundance of the corresponding marker according to the content of the target gene fragment of the marker in the step 2) and the content of the internal reference gene;
and 4) training a colorectal cancer risk assessment model by using the marker abundance and grouping information of all samples obtained in the step 3) and a random forest algorithm, and verifying internal data.
The verification of the model uses ten times of cross verification, so that errors caused by one-time verification are avoided.
Referring to fig. 1, the numbers in the blocks in the ith row and jth column indicate how many samples corresponding to the ith row grouping are predicted as the grouping indicated by the jth column. CTR means healthy sample and CRC means colorectal cancer patient sample.
Wherein, the specificity (True negative rate = number of correct negative groups/(number of correct negative groups + number of false positive groups)) reaches 0.86, and the sensitivity (True positive rate = number of correct positive groups/(number of correct positive groups + number of false negative groups)) reaches 0.87, thereby assisting in diagnosing colorectal cancer risk.
Example 4, colorectal cancer risk assessment.
Step 1) extracting a fecal DNA sample from the feces of a sample to be tested using the method as described in example 1 and dividing the extracted sample into two parts, one of which is treated with bisulfate;
step 2) same as example 3, step 2);
step 3) same as example 3, step 3);
and 4) inputting the relative abundance of the three colorectal cancer related markers into a colorectal cancer risk assessment model, wherein the output result of the model is the colorectal cancer risk of the individual.
The evaluation results will be shown in percentage form, as: "healthy 0.862; cancer 0.138 ". The judgment result is divided by 0.5, and the result with the proportion higher than 0.5 is taken as the result of the colorectal cancer risk assessment model.
It should be noted that the step method provided in this embodiment is only used for assessing the colorectal cancer risk of the individual, and has no diagnostic effect, and when the assessment result shows "cancer", it only indicates that the risk of detecting the colorectal cancer of the individual is higher, indicating that the colorectal cancer screening and diagnosis need to be further performed in the hospital.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (3)
1. The application of the machine learning model random forest in preparing products for evaluating colorectal cancer risk is characterized in that input variables of the machine learning model random forest are the abundance of colorectal cancer biomarkers;
the colorectal cancer biomarkers include the following 3:
marker 1) Micromonospora (Parvimonas spp.);
marker 2) BMP3 gene methylation;
marker 3) human DNA content;
wherein, the marker 1) probe primer combination of the detection reagent of the genus Micromonospora (Parvimonas spp.) is as follows: the sequence of the forward primer is shown as SEQ ID NO.1, the sequence of the reverse primer is shown as SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO. 3;
wherein, the probe primer combination of the marker 2) BMP3 gene methylation detection reagent is as follows: the sequence of the forward primer is shown as SEQ ID NO.4, the sequence of the reverse primer is shown as SEQ ID NO.5, and the sequence of the probe is shown as SEQ ID NO. 6;
wherein, the probe primer combination of the marker 3) human DNA content detection reagent is as follows: the sequence of the forward primer is shown as SEQ ID NO.7, the sequence of the reverse primer is shown as SEQ ID NO.8, and the sequence of the probe is shown as SEQ ID NO. 9.
2. The use of claim 1, wherein the determination of the abundance of markers 1) and 3) is qPCR quantitative determination and the determination of the amount of marker 2) is MSP.
3. A construction method of a colorectal cancer risk assessment product based on a machine learning model random forest is characterized by comprising the following steps:
step 1) extracting fecal DNA samples from the feces of 357 healthy samples and 354 colorectal cancer samples, and dividing the extracted samples into two parts, wherein one part is treated with bisulfate;
step 2) detecting the contents of the target gene fragments of the three markers and the contents of internal reference genes in all samples by using the determination method of claim 2, wherein the markers 1) and 3) use 16S rDNA as an internal reference, and the marker 2) uses an MSP product corresponding to BMP3 gene fragment as the internal reference;
step 3) calculating to obtain the abundance of the corresponding marker according to the content of the target gene fragment of the marker in the step 2) and the content of the internal reference gene;
and 4) training a colorectal cancer risk assessment model by using the marker abundance and grouping information of all samples obtained in the step 3) and a random forest algorithm, and verifying internal data.
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