CN111996255B - Diagnostic marker for colorectal malignant polyps and use thereof - Google Patents
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Abstract
The invention provides miRNA markers related to colorectal malignant polyps, a reagent for detecting the miRNA markers and application of the miRNA markers.
Description
Technical Field
The invention belongs to the field of molecular auxiliary diagnosis, and particularly relates to an application of the molecular auxiliary diagnosis in colorectal malignant polyp diagnosis.
Background
Colorectal cancer (CRC) is one of the most common malignant tumors in the world, and the incidence rate of the CRC is 2 nd of the malignant tumors in the digestive system of China, and the CRC is in an increasing trend, so that the CRC seriously jeopardizes the life and health. Morson presented a sequential theory of colorectal adenoma canceration in 1974, believing that the vast majority of colorectal cancers originate from adenomas, and this adenoma-adenocarcinoma transition has now been recognized as the most classical mode of colorectal carcinogenesis. Therefore, the adenoma closely related to the colorectal cancer can be diagnosed and treated early, the incidence rate of the colorectal cancer can be effectively reduced, and the prognosis of the patient can be improved.
Currently, endoscopic resection and surgery are the two main treatment modalities for colorectal adenomas. Compared with surgical operations, the treatment of colorectal adenoma by endoscopic resection has the advantages of small wound, quick recovery, low cost and the like, and the indications of endoscopic treatment are continuously expanded along with the continuous development of endoscopic technologies. Endoscopic resection is safe and effective for small colorectal adenomas and has a low recurrence rate, which has become the treatment of choice, but remains controversial for larger diameter adenomas. In Chinese colorectal cancer diagnosis and treatment Specification (2015 edition), colorectal polyp with the diameter of more than 2.5cm is considered to have high cancer rate and is recommended to be cleaned; the guidelines for colorectal cancer diagnosis and treatment of the Chinese Society of Clinical Oncology (CSCO) (2017 edition) show that the treatment strategy for colorectal polyps with diameters larger than 2.5cm is relatively fuzzy, and surgical treatment is taken as an alternative. The endoscope center of Zhongshan Hospital affiliated to the university of Compound Dan is used as a larger center for domestic colorectal tumor endoscope treatment, and the clinical experience and follow-up data of the center are combined to discover that the recurrence rate and the metastasis rate of colorectal polyps after endoscopic resection are lower even if the colorectal polyps with the diameter of more than 2.5cm, so that the risks and the benefits brought by surgical operations still need to be further researched. In addition, the judgment of lesion canceration and infiltration degree of larger polyps in the initial diagnosis also influences important factors of subsequent treatment schemes. The multipoint biopsy is the main method for pathological diagnosis of the focus for the first time, but the pathological result of endoscopic biopsy and the pathological result after resection have certain difference, and the research reports that the difference rate is as high as 39.8%. In recent years, although the ultrasonic endoscope is gradually applied to preoperative evaluation of rectal polyp endoscopic resection, the accuracy of judging the lesion canceration degree is still not ideal, and the accuracy is greatly influenced by equipment limitation and the experience of an operating doctor. Therefore, the current preoperative diagnosis method cannot reflect the real condition of the focus and can not achieve the definite diagnosis of the nature, invasion and depth of polyp. Based on the clinical treatment decision requirement, a proper colorectal adenoma molecular typing method is established, the canceration degree of the focus is accurately predicted, a doctor is helped to make a reasonable treatment scheme, a part of cases are hopefully changed from the traditional surgical operation into endoscopic treatment, and the medical resources are saved to a certain extent while the patient is benefited.
In recent years, the research of molecular markers characteristic to tumors based on epigenetics provides a new approach for tumor classification detection, and microRNA plays an important role in the detection. Michael et al first proposed by studies that there was a specific miRNA expression profile in colorectal tumor tissues (Michael M Z, et al. Molcancer Res, 2003); other researchers found various differential miRNA expression by measuring miRNA expression levels in CRC tissue and normal tissue paired with CRC tissue. There is no report in the art of mirnas and their mechanisms for early diagnosis and prognostic assessment of colorectal cancer.
Disclosure of Invention
The invention aims to provide a reagent for detecting miRNA and application thereof in colorectal malignant polyp diagnosis.
In a first aspect, the invention provides an isolated RNA from a mammal comprising any one or two, three, four, five or more mirnas or functional fragments thereof selected from the following mirnas or pri-miRNA or pre-miRNA thereof, or variants having at least 70% identity thereto: has-miR-129-5p, has-miR-150-3p, has-miR-193a-3p, has-miR-195-3p, has-miR-214-3p, has-miR-345-5p, has-miR-378a-5p, has-miR-584-5p, has-miR-16-5p, has-miR-21-5p and has-miR-1228-3p.
In one or more embodiments, the RNA comprises a sequence selected from any one or two, three, four, five or more of SEQ ID NOs 1-12 or a functional fragment thereof or a pri-miRNA or pre-miRNA thereof, or a variant thereof having at least 70% identity thereto.
In one or more embodiments, the RNA comprises at least any one of the following groups of mirnas, or functional fragments thereof, or pri-miRNA or pre-miRNA thereof, or variants having at least 70% identity thereto:
(1) has-miR-150 and has-miR-193a;
(2) has-miR-378a, has-miR-150 and has-miR-193a;
(3) has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(4) has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(5) has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(6) has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(7) has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(8) has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(9) has-miR-584, has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a; or
(10) has-miR-129, has-miR-584, has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a.
In a second aspect, the invention provides a primer or probe for detecting RNA as described in the first aspect herein.
In one or more embodiments, the primer comprises the sequence of any one of SEQ ID NO 13-SEQ ID NO 24 or a variant thereof having at least 90% sequence identity thereto.
In one or more embodiments, the primer comprises the sequence of any one of SEQ ID No. 25 to SEQ ID No. 37 or a variant thereof having at least 90% sequence identity thereto.
In a third aspect the invention provides a medium bearing a sequence of an RNA as described in the first aspect herein. The medium is used to align with RNA sequencing data to determine the presence or amount of the RNA.
In one or more embodiments, the sequence of the RNA is selected from the sequences of any one, two, three, four, five or more of the following mirnas: has-miR-129-5p, has-miR-150-3p, has-miR-193a-3p, has-miR-195-3p, has-miR-214-3p, has-miR-345-5p, has-miR-378a-5p, has-miR-584-5p, has-miR-16-5p, has-miR-21-5p and has-miR-1228-3p. Preferably, the sequence of the RNA is selected from the sequences of any one, two, three, four or five of the following mirnas: has-miR-150, has-miR-193a, has-miR-214, has-miR-378a and has-miR-1228.
In one or more embodiments, the sequence of the RNA is selected from any one or more of the sequences set forth in SEQ ID NOS 1-12.
In one or more embodiments, the medium is a card printed with the sequence, such as paper, plastic, metal, glass card.
In one or more embodiments, the medium is a computer readable medium storing the sequence and a computer program which, when executed by a processor, performs the steps of: comparing the RNA sequencing data of the sample with the sequence, thereby obtaining the presence and level of RNA comprising the sequence in the sample.
In a further aspect of the invention there is provided a kit for diagnosing colorectal cancer, identifying the benign or malignant colorectal polyps, or judging/selecting/aiding in the selection of colorectal polyps or a surgical modality for colorectal cancer or a post-operative intervention comprising an agent for detecting the level of RNA as described in the first aspect of the invention and/or a medium as described in the third aspect of the invention and optionally a control sample.
In one or more embodiments, the agent is a primer or probe according to the second aspect of the invention.
In one or more embodiments, the colorectal cancer is a colorectal malignancy or a colorectal malignant polyp.
In one or more embodiments, the kit further comprises a substance selected from one or more of the following: PCR buffer solution, polymerase, dNTP, restriction endonuclease, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance.
In a further aspect the invention provides the use of a reagent and/or medium for the manufacture of a kit for diagnosing colorectal cancer, identifying benign or malignant colorectal polyps, or judging/selecting/aiding in the selection of colorectal polyps or surgical modalities or post-operative interventions for colorectal cancer, said reagent being for use in detecting the level in a sample of RNA selected from any one or two, three, four, five or more of the following mirnas or functional fragments thereof or pri-miRNA or pre-miRNA thereof, or variants having at least 70% identity thereto: has-miR-129-5p, has-miR-150-3p, has-miR-193a-3p, has-miR-195-3p, has-miR-214-3p, has-miR-345-5p, has-miR-378a-5p, has-miR-584-5p, has-miR-16-5p, has-miR-21-5p and has-miR-1228-3p.
In one or more embodiments, the RNA comprises at least any one of the following groups of mirnas, or functional fragments thereof, or pri-miRNA or pre-miRNA thereof, or variants having at least 70% identity thereto:
(1) has-miR-150 and has-miR-193a;
(2) has-miR-378a, has-miR-150 and has-miR-193a;
(3) has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(4) has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(5) has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(6) has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(7) has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(8) has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a;
(9) has-miR-584, has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a; or
(10) has-miR-129, has-miR-584, has-miR-345, has-miR-21, has-miR-195, has-miR-16, has-miR-214, has-miR-1228, has-miR-378a, has-miR-150 and has-miR-193a.
In one or more embodiments, the reagent is a primer or probe according to the second aspect of the invention.
In one or more embodiments, the medium is a medium according to the third aspect of the present invention.
In one or more embodiments, the colorectal cancer is a colorectal malignancy or colorectal malignant polyp.
In one or more embodiments, diagnosing colorectal cancer, identifying colorectal polyps benign and malignant, classifying colorectal cancer subtypes, or judging/selecting/aiding in selecting colorectal polyps or colorectal cancer surgical modalities or post-operative interventions includes: comparing with a control sample, or obtaining a score based on the level of said RNA, diagnosing colorectal cancer, identifying benign or malignant colorectal polyps, or judging/selecting/aiding in the selection of colorectal polyps or a colorectal cancer surgical modality or post-operative intervention, based on the comparison result or score.
In one or more embodiments, the sample is from a mammal, preferably from a human. The sample is from a tissue, cell or body fluid, such as colorectal intestinal tissue, colorectal polyps, adenomas, blood, serum or plasma, preferably colorectal polyps. In one or more embodiments, the sample is a colorectal polyp biopsy, preferably a fine needle biopsy.
In one or more embodiments, the kit further comprises a substance selected from one or more of the following: PCR buffer solution, polymerase, dNTP, restriction endonuclease, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance.
The present invention also provides a method for diagnosing colorectal cancer, identifying benign or malignant colorectal polyps, or judging/selecting/aiding in the selection of colorectal polyps or a colorectal cancer surgical modality or a postoperative intervention, comprising: (1) obtaining the level of an RNA described herein in a sample from a subject; (2) Comparing to a reference sample, or obtaining a score based on the level of said RNA, e.g. by calculation; (3) Diagnosing colorectal cancer, identifying benign and malignant colorectal polyps, or judging/selecting/assisting in selecting colorectal polyps or colorectal cancer operation modes or postoperative intervention measures according to the comparison result or score in the step (2).
In one or more embodiments, the method further comprises, prior to step (1): and (3) extracting, quality testing and/or storing sample RNA.
In one or more embodiments, the RNA level is increased or decreased.
In one or more embodiments, the scoring in step (2) is obtained by constructing a Support Vector Machine (SVM), logistic regression, and/or random forest model calculations.
In one or more embodiments, step (3) comprises: the subject sample has a change in RNA level compared to the control sample, and colorectal cancer is diagnosed when the RNA level meets a threshold.
In one or more embodiments, step (3) comprises: when the score meets a threshold, colorectal cancer is diagnosed.
In one or more embodiments, the sample is from a mammal, preferably from a human. The sample is from a tissue, cell or body fluid, such as colorectal intestinal tissue, colorectal polyps, adenomas, blood, serum or plasma, preferably colorectal polyps. In one or more embodiments, the sample is a colorectal polyp biopsy, preferably a fine needle biopsy.
The present invention also provides an apparatus for diagnosing colorectal cancer, identifying benign or malignant colorectal polyps, or determining/selecting/assisting in the selection of colorectal polyps or a surgical modality for colorectal cancer or a post-operative intervention, the apparatus comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor upon executing the program implements the steps of: (1) obtaining the level of an RNA described herein in a sample from a subject; (2) Comparing to a reference sample, or obtaining a score based on the level of said RNA, e.g. by calculation; (3) Diagnosing colorectal cancer, identifying benign and malignant colorectal polyps, or judging/selecting/assisting in selecting colorectal polyps or colorectal cancer operation modes or postoperative intervention measures according to the comparison result or the score in the step (2).
In one or more embodiments, the method further comprises, prior to step (1): and (3) extracting, quality testing and/or storing sample RNA.
In one or more embodiments, the RNA level is increased or decreased.
In one or more embodiments, the scoring in step (2) is obtained by constructing a Support Vector Machine (SVM), logistic regression, and/or random forest model calculations.
In one or more embodiments, step (3) comprises: the subject sample has a change in RNA level compared to the control sample, and colorectal cancer is diagnosed when the RNA level meets a threshold.
In one or more embodiments, step (3) comprises: when the score meets a threshold, colorectal cancer is diagnosed.
In one or more embodiments, the sample is from a mammal, preferably from a human. The sample is from a tissue, cell or body fluid, such as colorectal intestinal tissue, colorectal polyps, adenomas, blood, serum or plasma, preferably colorectal polyps. In one or more embodiments, the sample is a colorectal polyp biopsy, preferably a fine needle biopsy.
The invention has the advantages that: the inventor searches about molecular markers and mechanisms thereof for early diagnosis and prognosis evaluation of colorectal tumors, performs transcriptome sequencing on normal tissues-adenoma tissues-adenocarcinoma tissues derived from the same case by a second-generation sequencing technology, and obtains a differential expression microRNA group profile. Then, through combining qRT-PCR with related literature reports, miRNA stably expressed in a large number in both normal tissues and tumor tissues are selected to form a prediction model candidate target set. By collecting more than 150 adenoma samples subjected to endoscopic resection in recent years in the center, combining clinical information, pathological results and prognosis of patients, and quantitatively analyzing the expression level of candidate targets in 150 samples by using qRT-PCR, a molecular early warning model based on differential microRNA is established. When the model is applied to judging the adenoma canceration, the AUC =0.865, the sensitivity is 87% and the specificity is 71% are obtained by ROC curve analysis. Therefore, the model has effective classification capacity and high accuracy.
The invention carries out canceration diagnosis based on miRNA of the colorectal adenoma tissue sample, and similar research reports and patent applications are not available at home and abroad, so that a brand-new clinical diagnosis application technology is expected to be formed. According to the colorectal high-risk adenoma molecular early warning model obtained through the research, a clinician can judge an operation mode and postoperative intervention measures according to the basic condition and focus characteristics of a patient, and a more reasonable treatment scheme is formulated, so that the patient is benefited.
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FIG. 1, area under ROC curve (AUC) of the exemplary 5 gene model.
Detailed Description
The invention researches the relation between miRNA expression level and colorectal cancer. Aims to improve the accuracy of colorectal cancer minimally invasive diagnosis by using a miRNA marker group as a colorectal cancer diagnosis marker through a minimally invasive method.
As used herein, "plurality" refers to any integer. Preferably, the "plurality" of the "one or more" can be any integer within the range, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11.
As used herein, "colorectal cancer" includes malignant tumors or malignant polyps occurring anywhere in the colon or rectum, including adenocarcinoma, squamous carcinoma and mucinous carcinoma. Colorectal cancer as referred to herein refers primarily to colorectal malignancies or colorectal malignant polyps.
The "microRNA" or "miRNA" as described herein is a non-coding single-stranded RNA molecule encoded by an endogenous or exogenous gene. The miRNAs exist in various forms, including the most primitive pri-miRNA, a pre-miRNA (microRNA precursor) formed by processing the pri-miRNA once, and a mature miRNA formed by cutting the pre-miRNA with Dicer enzyme. miRNA or functional fragments thereof are involved in regulation and control of post-transcriptional gene expression in animals and plants. As used herein, a "functional fragment" refers to a fragment of miRNA that retains the regulatory function of gene expression, typically 5-25nt, preferably 18-25nt in length.
The inventors have found that colorectal cancer is associated with expression levels of one or more mirnas or functional fragments thereof or pri-miRNA or pre-miRNA thereof, or variants thereof having at least 70% identity thereto, selected from the group consisting of: has-miR-129-5p, has-miR-150-3p, has-miR-193a-3p, has-miR-195-3p, has-miR-214-3p, has-miR-345-5p, has-miR-378a-5p, has-miR-584-5p, has-miR-16-5p, has-miR-21-5p and has-miR-1228-3p. The sequences of the above miRNAs are shown in Table 1 and SEQ ID NOS: 1-12 herein. Preferably, the RNA comprises at least has-miR-150, has-miR-193a, has-miR-214, has-miR-378a, and has-miR-1228 or functional fragments thereof or pri-miRNA or pre-miRNA thereof, or variants having at least 70% identity thereto. Thus, detection of the levels of the above mirnas can diagnose colorectal cancer, identify colorectal polyps benign or malignant, or judge/select/aid in the selection of colorectal polyps or colorectal cancer surgical modalities or post-operative interventions.
Herein, methods for detecting miRNA levels are well known in the art, such as sequencing, RT-PCR, northern blot analysis, microarray analysis, and quantitative Real-Time PCR. Accordingly, the present invention relates to reagents for detecting miRNA levels. Illustratively, the reagent for detecting DNA methylation may be a reagent used in one or more methods selected from the group consisting of: RT-PCR, qPCR, DNA or RNA sequencing, northern, fluorometric methods, high resolution melting curve methods, chip-based miRNA profiling, mass spectrometry (e.g., flight mass spectrometry). Thus, the reagent for detecting miRNA levels may comprise one or more of: PCR buffer solution, polymerase, dNTP, primer, probe, restriction endonuclease, enzyme digestion buffer solution, fluorescent dye, fluorescent quencher, fluorescent reporter, exonuclease, alkaline phosphatase, internal standard and reference substance. RNA sequencing as described herein is the process of DNA sequencing of RNA after reverse transcription, for example miRNA sequencing based on second generation sequencing technologies. Sequencing procedures and reagents required are known in the art, e.g., extraction and purification to obtain sample RNA,3', 5' linker, construction of library, and machine sequencing. Based on the measured miRNA sequence information, the expression level of the miRNA can be obtained, so that the change of the expression level can be analyzed. Alternatively, the presence or expression level of certain mirnas in a sample can be obtained by comparing the determined miRNA sequence information to the certain miRNA sequences.
In detection methods involving amplification of DNA or RNA, the reagents include primers and/or probes. As used herein, a "primer" refers to a nucleic acid molecule having a specific nucleotide sequence that directs the synthesis at the initiation of nucleotide polymerization. The primers are typically two oligonucleotide sequences synthesized by man, one primer complementary to one DNA template strand at one end of the target region and the other primer complementary to the other DNA template strand at the other end of the target region, which functions as the initiation point for nucleotide polymerization. Primers designed artificially in vitro are widely used in Polymerase Chain Reaction (PCR), qPCR, sequencing, probe synthesis, and the like. Typically, primers are designed such that the amplified products are 50-150 bp, 60-140, 70-130, 80-120bp in length. Methods for designing PCR primers for a template are known in the art. Preferably, the product is 50-100bp in length.
As used herein, a "probe" refers generally to a DNA or RNA sequence that hybridizes to a target sequence under stringent conditions. Typically, the probe sequence is labeled with a fluorescent reporter at the 5 'end and a quencher at the 3' end. In discussing primers or probes, the term "recognize" or "hybridize" as used herein refers to hybridization of a primer or probe to a template sequence under stringent or highly stringent conditions as are known in the art, e.g., high stringency conditions can be hybridization and washing of a membrane in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.
The present invention also relates to kits for diagnosing colorectal cancer comprising the probes, primers and other reagents described herein for detecting miRNA expression levels, including but not limited to: PCR buffer, polymerase, dntps, restriction enzymes, digestion buffer, fluorescent dye, fluorescent quencher, fluorescent reporter, exonuclease, alkaline phosphatase, internal standard, control, and optionally instructions for detecting miRNA expression levels.
The invention also relates to a medium comprising the sequence of a miRNA described herein for alignment with RNA sequencing data to determine the presence or amount of said RNA. In particular embodiments, the sequence of the RNA is selected from the sequence of any one, two, three, four, five or more of the following mirnas: has-miR-129-5p, has-miR-150-3p, has-miR-193a-3p, has-miR-195-3p, has-miR-214-3p, has-miR-345-5p, has-miR-378a-5p, has-miR-584-5p, has-miR-16-5p, has-miR-21-5p and has-miR-1228-3p. Preferably, the sequence of the RNA is selected from the sequences of any one, two, three, four or five of the following mirnas: has-miR-150, has-miR-193a, has-miR-214, has-miR-378a and has-miR-1228. The medium may be one that can provide sequence information of the miRNA in any form, such as a card printed with the sequence, e.g., paper, plastic, metal, glass card; a computer readable medium having stored thereon the sequence. The computer-readable medium further stores a computer program which, when executed by the processor, performs the steps of: comparing the RNA sequencing data of the sample to the sequence, thereby obtaining the presence and level of RNA comprising the sequence in the sample.
The term "variant" or "mutant" as used herein refers to a polynucleotide that has a nucleic acid sequence altered by insertion, deletion or substitution of one or more nucleotides compared to a reference sequence, while retaining its ability to hybridize to other nucleic acids. A mutant according to any of the embodiments herein comprises a nucleotide sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence and retains the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn from NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions, or substitutions) in the reference sequence and in the nucleotide sequence, while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to within 1-10, such as 1-8, 1-5, or 1-3. The substitution may be a substitution between purine nucleotides and pyrimidine nucleotides, or a substitution between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, conservative substitutions with nucleotides of similar or analogous properties are not typically made in the art to alter the stability and function of the polynucleotide. Conservative substitutions are, for example, exchanges between purine nucleotides (A and G), exchanges between pyrimidine nucleotides (T or U and C). Thus, substitution of one or more sites with residues from the same in the polynucleotides of the invention will not substantially affect their activity. Specifically, the primer variants of the present invention can achieve stringent hybridization to the target sequence and direct amplification; the probe variants of the invention can achieve stringent hybridization and detection with a target sequence.
The invention also provides a method for diagnosing colorectal cancer, identifying benign and malignant colorectal polyps, or judging/selecting/assisting in selecting colorectal polyps or a colorectal cancer operation mode or postoperative intervention screening, comprising the following steps of: (1) detecting the level of an RNA described herein in a sample from a subject; (2) Comparing to a reference sample, or obtaining a score based on the level of said RNA, e.g. by calculation; (3) Diagnosing colorectal cancer, classifying colorectal cancer subtypes, or judging/selecting/assisting in selecting colorectal polyps or colorectal cancer surgical modes or postoperative interventions according to the comparison results or scores of step (2). Typically, the method further comprises, prior to step (1): and (3) extracting, quality testing and/or storing sample RNA.
The step (3) may include: the subject sample has an increased or decreased expression level of miRNA compared to the control sample. When the expression level meets a certain threshold, colorectal cancer is identified. Alternatively, the step (3) comprises: when the score meets a threshold, colorectal cancer is identified. This involves performing a mathematical analysis of the measured expression levels to obtain a score; and for the detected sample, judging the result to be positive when the score is larger than the threshold value, namely colorectal cancer, and judging the result to be negative, namely benign polyp. Methods of conventional mathematical analysis and processes of determining thresholds are known in the art, and exemplary methods are mathematical models such as logistic regression, support vector machines, and random forest models. For example, for the differential miRNA markers, logistic regression, support vector machine, or random forest models are constructed for two sets of samples, and the accuracy, sensitivity, and specificity of the test results and the area under the predictive value characteristic curve (ROC) (AUC) are used by the models to statistically test the sample prediction scores in the set.
For example, when five miRNAs, namely has-miR-150, has-miR-193a, has-miR-214, has-miR-378a and has-miR-1228 are diagnosed, the expression levels of the miRNAs are subjected to multiple logistic regression analysis, and a five-gene model can be obtained: logit (P) =5.96203-0.79819 has-miR-150-1.32400 has-miR-193a +0.83361 has-miR-214+1.61468 has-miR-378a-1.17075 has-miR-1228. When the threshold of the model is-0.189 to 0.003, the specificity is 70.79%. In particular, when the model threshold is-0.08, sensitivity =87.23%, specificity =70.69%. Colorectal cancer is diagnosed when the score is greater than-0.08, and benign polyps are diagnosed when the score is less than-0.08.
Herein, the sample is from a mammal, preferably a human. The sample may be from any organ (e.g., colon, rectum), tissue (e.g., epithelial tissue, connective tissue, muscle tissue, and neural tissue), cell (e.g., colorectal polyp biopsy), or bodily fluid (e.g., blood, plasma, serum, interstitial fluid, urine). Typically, so long as the sample comprises miRNA. Illustratively, the sample is a colorectal polyp biopsy, preferably a fine needle biopsy.
Furthermore, a computer-readable storage medium storing a computer program is disclosed, the computer program stored on the storage medium being executable by a computer to perform the method for diagnosing colorectal cancer described herein. The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. If implemented in software as a computer program product, the functions may be stored on or transmitted over as one or more instructions or code on a computer-readable medium. Computer-readable media includes both computer storage media and communication media including any medium that facilitates transfer of a computer program from one place to another. A storage media may be any available media that can be accessed by a computer. By way of example, and not limitation, such computer-readable media can comprise RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to carry or store desired program code in the form of instructions or data structures and that can be accessed by a computer. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.
Examples
Example 1, experimental methods and materials
Sample processing
And (3) adding 5-10 times of 10% volume neutral formalin for fixation within 30min after sampling the polyp tissue meeting the grouping requirement, preserving at normal temperature, and completing embedding and FFPE sample within 24 hours.
Sequencing process
And (3) detecting miRNA of the sample by using NGS second-generation sequencing: extracting and purifying to obtain sample RNA, connecting 3 'and 5', constructing a library, and performing machine sequencing.
qRT-PCR
A plurality of 10-micron slices are cut from an FFPE sample, 1-3 slices are selected according to the tissue area size for detection by a qRT-PCR method, and the flow is shown in figure 1. Wherein the reverse transcription into cDNA program is: 60min at 42 ℃; 5min at 85 ℃; the PCR detection program is as follows: activating enzyme at 95 deg.C for 10min; denaturation at 95 ℃ for 15s; extending at 60 ℃ for 60s; the denaturation and extension are repeated for 40 cycles, and the fluorescence collection phase is the extension phase.
Primer and method for producing the same
Reverse transcription stem-loop primers for mirnas are shown below: has-miR-129-5p, SEQ ID NO; has-miR-150-3p SEQ ID NO; has-miR-193a-3p, SEQ ID NO; has-miR-195-3p, SEQ ID NO; has-miR-214-3p, SEQ ID NO; has-miR-345-5p, SEQ ID NO; has-miR-378a-5p, SEQ ID NO; has-miR-584-5p, SEQ ID NO; cel-miR-39-3p, SEQ ID NO; has-miR-16-5p, SEQ ID NO; has-miR-21-5p, SEQ ID NO; has-miR-1228-3p SEQ ID NO.
PCR primers for miRNA are shown below: has-miR-129-5p, SEQ ID NO; has-miR-150-3p, SEQ ID NO; has-miR-193a-3p, SEQ ID NO; has-miR-195-3p, SEQ ID NO; has-miR-214-3p, SEQ ID NO; has-miR-345-5p, SEQ ID NO; has-miR-378a-5p, SEQ ID NO; has-miR-584-5p, SEQ ID NO; cel-miR-39-3p, SEQ ID NO; has-miR-16-5p, SEQ ID NO; has-miR-21-5p, SEQ ID NO; has-miR-1228-3p, SEQ ID NO; general reverse primer, SEQ ID NO:37.
Example 2 discovery set
After NGS second-generation sequencing detection is carried out on a normal mucosa biopsy specimen, an early cancer endoscope biopsy specimen and a colorectal cancer operation specimen of the same patient, the change of the expression level of some miRNAs is found to have consistency. The following mirnas were selected as candidate genes: has-miR183-5p, has-miR195-3p, has-miR378a-5p, has-miR150-3p, has-miR584-5p, has-miR21-5p, has-miR182-5p, has-miR214-3p, has-miR30a-3p, and has-miR129-5p.
To further search for mirnas that are closely related to colorectal cancer among them, qRT-PCR was performed on 187 historical tissue samples, and 7 mirnas that had relatively significant differences between the experimental and control groups were screened out of 10 candidate mirnas: hsa-miR-129-5p (vs. carcinoma: p =0.0757, vs. adenoma: p = 0.0134), hsa-miR-150-3p (vs. carcinoma: p =0.0137, vs. adenoma: p = 0.0008), hsa-miR-195-3p (vs. carcinoma: p =0.0048, vs. adenoma: p = 0.0008), hsa-miR-21-5p (vs. carcinoma: p =0.0165, vs. adenoma: p = 0.0021), hsa-miR-214-3p (vs. carcinoma: p =0.0551, vs. adenoma: p = 0.0026), hsa-miR-378a (vs. carcinoma: p =0.0034, vs. adenoma: p = 0.4), and a-miR-5 p (vs. carcinoma: p = 584: 0100.0100.0004).
Meanwhile, 4 mirnas were selected and included in the study in combination with the reports of the relevant literature: hsa-miR-193a-3p, hsa-miR-345-5p, hsa-miR-16-5p and hsa-miR-1228-3p. miRNA that are not expressed in human cells (Cel-miR-39) were selected as internal references for each experiment.
The sequence information of 11 under-study mirnas and one reference miRNA is shown in table 1:
TABLE 1
| miRNA ID | Login number | Sequence numbering |
| has-miR-129-Sp | MIMATOOOO242 | SEQ ID NO:I |
| has-miR-I50-3p | MIMATOOO46IO | SEQ ID NO:2 |
| has-miR-193a-3p | MIMATOOOO459 | SEQ ID NO:3 |
| has-miR-I95-3p | MIMATOOO4615 | SEQ ID NO:4 |
| has-miR-2I4-3p | MIMATOOOO27I | SEQ ID NO:5 |
| has-miR-345-Sp | MIMATOOOO772 | SEQ ID NO:6 |
| has-miR-378a-Sp | MIMATOOOO73I | SEQ ID NO:7 |
| has-miR-584-Sp | MIMATOOO3249 | SEQ ID NO:8 |
| Cel-miR-39-3p | MIMATOOOOOIO | SEQ ID NO:9 |
| has-miR-I6-5p | MIMATOOOOO69 | SEQ ID NO:10 |
| has-miR-2I-Sp | MIMATOOO4494 | SEQ ID NO:II |
| has-miR-I228-3p | MIMATOOO5583 | SEQ ID NO:12 |
Example 3 training set
3.1 sample grouping criteria:
and (3) inclusion standard:
a) Age 18 years old, male and female;
b) Performing colorectal enteroscopy biopsy on adenomatous polyps with the diameter of more than or equal to 2cm, and performing biopsy, wherein the pathological diagnosis is clear; or the sample is removed by operation, and the pathological diagnosis is definite;
exclusion criteria
a) Populations that do not meet inclusion criteria;
b) The crowd with unclear sample background information;
c) People with a history of tumor diseases.
3.2 grouping criteria:
classifying the sample into cancerous lesion, high-grade neoplastic lesion, low-grade neoplastic lesion and inflammatory disease according to pathological diagnosis of wax stone;
determining the grade of all wax blocks of the focus according to the highest grade of the pathology of the focus;
the cancer and high grade tumor are disease groups (positive), and the low grade and inflammation are control groups (negative).
3.3 number of grouped samples:
all samples were from 107 lesions of 56 patients, with a total of 152 samples (wax block count);
the disease groups are as follows: 94 cases; 58 cases in the control group.
3.4 sample detection:
sample handling and qRT-PCR were performed as described in the commercial Experimental methods.
3.5 data processing and analysis:
processing the original results, uniformly setting Threshold, setting Baseline to Auto, and recording the obtained result as Ct n . The original detection result is subjected to homogenization treatment by using internal reference, dCt n ＝Ct n -Ct Internal reference Using normalized dCt n For subsequent statistical analysis.
3.5.1 Single target, ROC analysis as follows:
TABLE 2
As can be seen, the single target classification performance is not good, and the AUC is less than 0.8
3.5.2 multiple Gene model Logistic Regression (Logistic Regression) analysis.
The P maximum target in the fitted variables was eliminated each time. The results of the different target number fits are summarized in tables 3 and 4.
TABLE 3
TABLE 4
And selecting a model with the maximum AUC, namely a five-gene model, of which all the target differences are obvious, and further verifying and analyzing.
3.5.3 five Gene model verification analysis
TABLE 5 five Gene model coefficients and Standard error
| Variables of | Coefficient of performance | Error of study | Wald | P |
| has-miR-150 | -0.79819 | 0.29678 | 7.2332 | 0.0072 |
| has-miR-193a | -1.32400 | 0.32544 | 16.5509 | ＜0.0001 |
| has-miR-214 | 0.83361 | 0.38050 | 4.7998 | 0.0285 |
| has-miR-378a | 1.61468 | 0.49370 | 10.6967 | 0.0011 |
| has-miR-1228 | -1.17075 | 0.57051 | 4.2111 | 0.0402 |
| A constant value | 5.96203 | 1.53345 | 15.1164 | 0.0001 |
Namely Logit (P) =5.96203-0.79819 has-miR-150-1.32400 has-miR-193a +0.83361 has-miR-214+1.61468 has-miR-378a-1.17075 has-miR-1228.
The ROC curves are shown in fig. 1, and the associated data are shown in the table below. The area under the ROC curve is 0.865, the significance level P is less than 0.0001, and the difference is significant.
TABLE 6
| Area under ROC curve (AUC) | 0.865 |
| Standard error a | 0.0298 |
| 95% confidence interval b | 0.807-0.923 |
| Z statistics | 12.263 |
| Significance level P (area = 0.5) | ＜0.0001 |
3.6 clinical Performance assessment
Clinical case results are shown in the table below. When the predicted model Cut off value was Logit (p) = -0.08, sensitivity =87.23% and specificity =70.69%. When the Cut off value is = -0.189-0.003, the specificity is 70.69 percent
TABLE 7
Example 4 verification set
4.1 sample grouping criteria:
simultaneous training set
4.2 number of grouped samples:
all samples were from 114 lesions of 114 patients, with a total of 114 samples (wax block count);
the disease groups are as follows: 76 cases; control group 38 cases
4.3 sample detection:
simultaneous training set
4.4 data processing and analysis:
processing the original result, uniformly setting Threshold, setting Baseline to Auto, and recording the result as Ct n . The original detection result is subjected to homogenization treatment by using internal reference, dCt n ＝Ct n -Ct Internal reference Using normalized dCt n And calculating Loxit (p). Namely Logit (P) =5.96203-0.79819 has-miR-150-1.32400 has-miR-193a +0.83361 has-miR-214+1.61468 has-miR-378a-1.17075 has-miR-1228. And (5) counting the Logit (p).
4.5 clinical Performance assessment
Clinical case results are shown in the table below. The validation set showed sensitivity =87.23%, specificity =70.69%, and total agreement rate 80.7%, comparable to the training set performance. The description model has excellent classification capability and high accuracy.
TABLE 8
Sequence listing
<110> Zhongshan Hospital affiliated to Fudan university
Junshi Biological Technology (Shanghai) Co., Ltd.
<120> diagnostic marker for colorectal malignant polyps and use thereof
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Claims (16)
1. And (2) primers for detecting RNA, wherein the RNA at least comprises has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p.
2. The primer of claim 1, wherein the RNA comprises at least one of the following miRNA groups:
(1) has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(2) has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(3) has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(4) has-miR-584-5p, has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p; or
(5) has-miR-129-5p, has-miR-584-5p, has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p.
3. The primer of claim 2, wherein the sequence of has-miR-129-5p is shown as SEQ ID NO. 1, the sequence of has-miR-150-3p is shown as SEQ ID NO. 2, the sequence of has-miR-193a-3p is shown as SEQ ID NO. 3, the sequence of has-miR-195-3p is shown as SEQ ID NO. 4, the sequence of has-miR-214-3p is shown as SEQ ID NO. 5, the sequence of has-miR-345-5p is shown as SEQ ID NO. 6, the sequence of has-miR-378a-5p is shown as SEQ ID NO. 7, the sequence of has-miR-584-5p is shown as SEQ ID NO. 8, the sequence of has-miR-16-5p is shown as SEQ ID NO. 10, the sequence of has-miR-21-5p is shown as SEQ ID NO. 11, and the sequence of has-miR-1228-3p is shown as SEQ ID NO. 12.
4. The primer of claim 1, comprising a sequence set forth in any one of SEQ ID NOs 14, 15, 17, 19, 22, and 24.
5. The primer of claim 1, comprising a sequence set forth in any one of SEQ ID NOs 26, 27, 29, 31, 34, and 36.
6. A medium comprising a sequence of RNA, wherein the RNA comprises at least has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p, and has-miR-193a-3p.
7. The medium of claim 6, wherein the RNA comprises at least one miRNA of any one of the following groups:
(1) has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(2) has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(3) has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p;
(4) has-miR-584-5p, has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p; or
(5) has-miR-129-5p, has-miR-584-5p, has-miR-345-5p, has-miR-21-5p, has-miR-195-3p, has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p.
8. The medium of claim 7, wherein the sequence of has-miR-129-5p is shown as SEQ ID NO:1, the sequence of has-miR-150-3p is shown as SEQ ID NO:2, the sequence of has-miR-193a-3p is shown as SEQ ID NO:3, the sequence of has-miR-195-3p is shown as SEQ ID NO:4, the sequence of has-miR-214-3p is shown as SEQ ID NO:5, the sequence of has-miR-345-5p is shown as SEQ ID NO:6, the sequence of has-miR-378a-5p is shown as SEQ ID NO:7, the sequence of has-miR-584-5p is shown as SEQ ID NO:8, the sequence of has-miR-16-5p is shown as SEQ ID NO:10, the sequence of has-miR-21-5p is shown as SEQ ID NO:11, and the sequence of has-miR-1228-3p is shown as SEQ ID NO: 12.
9. The medium according to any of the claims 6-8, wherein the medium is a card printed with the sequence or a computer readable medium storing the sequence and a computer program which when executed by a processor performs the steps of: comparing the RNA sequencing data of the sample with the sequence, thereby obtaining the presence and level of RNA comprising the sequence in the sample.
10. A kit for diagnosing colorectal cancer, or selecting colorectal polyps or a surgical modality of colorectal cancer or a post-operative intervention, comprising the primer of any one of claims 1-5 and/or the medium of any one of claims 6-8 and optionally a control sample.
11. The kit of claim 10, wherein the colorectal cancer is a colorectal malignancy or a colorectal malignant polyp.
12. Use of a primer according to any one of claims 1 to 5 or a medium according to any one of claims 6 to 8 for the preparation of a kit for the diagnosis of colorectal cancer, or for the selection of colorectal polyps or surgical modalities or post-operative interventions for colorectal cancer.
13. The use of claim 12, wherein the colorectal cancer is a colorectal malignancy or a colorectal malignant polyp.
14. An apparatus for diagnosing colorectal cancer, or selecting a colorectal polyp or a surgical modality for colorectal cancer or a post-operative intervention, the apparatus comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program performs the steps of: (1) obtaining the level of RNA in the sample; (2) Comparing to a reference sample, or obtaining a score based on the level of said RNA; (3) Diagnosing colorectal cancer, or selecting colorectal cancer surgery or a colorectal cancer surgery modality or post-surgery intervention according to the comparison or score of step (2), wherein the RNA comprises at least has-miR-16-5p, has-miR-214-3p, has-miR-1228-3p, has-miR-378a-5p, has-miR-150-3p and has-miR-193a-3p.
15. The device of claim 14, wherein the sample is from animal tissue, cells, or bodily fluids.
16. The device of claim 15, wherein the sample is from colorectal bowel tissue or colorectal polyps or adenomas.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101988060A (en) * | 2009-07-30 | 2011-03-23 | 江苏命码生物科技有限公司 | Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof |
| CN102439169A (en) * | 2008-11-13 | 2012-05-02 | 复旦大学 | Compositions and methods for micro-rna expession profiling of colorectal cancer |
| CN102933719A (en) * | 2009-12-24 | 2013-02-13 | 复旦大学 | Compositions and methods for microrna expession profiling in plasma of colorectal cancer |
| WO2015194956A1 (en) * | 2014-06-19 | 2015-12-23 | Stichting Vu-Vumc | Biomarker for colorectal cancer |
| CN106661619A (en) * | 2014-06-13 | 2017-05-10 | 东丽株式会社 | Colorectal cancer detection kit or device, and detection method |
| CN107779504A (en) * | 2016-08-24 | 2018-03-09 | 北京旷博生物技术股份有限公司 | Colorectal cancer microRNA molecule mark and application thereof |
| CN109182507A (en) * | 2018-10-19 | 2019-01-11 | 浙江大学 | For diagnosing blood plasma miRNA molecular marker, kit and its application of colorectal polypus |
-
2020
- 2020-08-24 CN CN202010855030.6A patent/CN111996255B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439169A (en) * | 2008-11-13 | 2012-05-02 | 复旦大学 | Compositions and methods for micro-rna expession profiling of colorectal cancer |
| CN101988060A (en) * | 2009-07-30 | 2011-03-23 | 江苏命码生物科技有限公司 | Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof |
| CN102933719A (en) * | 2009-12-24 | 2013-02-13 | 复旦大学 | Compositions and methods for microrna expession profiling in plasma of colorectal cancer |
| CN106661619A (en) * | 2014-06-13 | 2017-05-10 | 东丽株式会社 | Colorectal cancer detection kit or device, and detection method |
| WO2015194956A1 (en) * | 2014-06-19 | 2015-12-23 | Stichting Vu-Vumc | Biomarker for colorectal cancer |
| CN107779504A (en) * | 2016-08-24 | 2018-03-09 | 北京旷博生物技术股份有限公司 | Colorectal cancer microRNA molecule mark and application thereof |
| CN109182507A (en) * | 2018-10-19 | 2019-01-11 | 浙江大学 | For diagnosing blood plasma miRNA molecular marker, kit and its application of colorectal polypus |
Non-Patent Citations (3)
| Title |
|---|
| "2018 版《中国结直肠癌肝转移诊断和综合治疗指南》解读";秦新裕等;《临床外科杂志》;20190131;第27卷(第1期) * |
| characterization of microrna expression in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620);Wei Yan et al.;《OncoTargets and Therapy》;20181231;第4701页摘要部分 * |
| Serum microRNA signatures and metabolomics have high diagnostic value in colorectal cancer using two novel methods;Hai-Ning Liu et al.;《Cancer Science》;20181231;第109卷;第1188页表格1 * |
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