CN114107514A

CN114107514A - miRNA molecular marker for colorectal cancer diagnosis and kit thereof

Info

Publication number: CN114107514A
Application number: CN202210097760.3A
Authority: CN
Inventors: 常子嵩; 庄晶玲; 杨扬; 李英; 李薇
Original assignee: Laipu Tianjin Biomedical Technology Co ltd; Tianjin Oudelai Biological Medicine Technology Co ltd
Current assignee: Laipu Tianjin Biomedical Technology Co ltd; Tianjin Oudelai Biological Medicine Technology Co ltd
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-03-01

Abstract

The invention provides a miRNA molecular marker for colorectal cancer diagnosis and a kit thereof, wherein the miRNA molecular marker comprises at least one human fecal occult blood diagnosis molecule of miR-144-3p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p, and preferably further comprises at least one of miR-144-5p, miR-92a, miR-135b and miR-421. The invention also provides a kit for detecting the early colorectal cancer of the miRNA molecular marker, preferably 9 miRNAs have expression in colorectal cancer patients which is obviously higher than that of a control group, and the expression difference is more than 2 folds (the CT value in fluorescence quantitative PCR is less than 30), so that the miRNA molecular marker can be used as a biomarker for diagnosing the colorectal cancer. One or more of the 9 miRNAs can be used as a biomarker for colorectal cancer diagnosis, and the sensitivity and the specificity of combined diagnosis are higher than those of the traditional fecal occult blood test and single miRNA diagnosis.

Description

miRNA molecular marker for colorectal cancer diagnosis and kit thereof

Technical Field

The invention belongs to the field of biological detection, relates to a detection method and a kit for colorectal cancer early screening, and relates to identification of 9 miRNA molecular markers for performing multiplex real-time fluorescent quantitative PCR detection.

Background

Tumor markers are synthesized and secreted by tumor cells through gene expression, or are abnormally produced or expressed in response to tumor, and play an important role in early detection, diagnosis and prognosis of tumors. With the development of biotechnology, new tumor markers are continuously discovered, and the screening method of the tumor markers is fundamentally changed.

The clinical application of tumor markers requires three phases: 1. a laboratory research screening stage; 2. in the clinical large sample verification stage, whether the large sample can be used as an index of a screening marker or not is determined, and a diagnostic standard of the large sample is determined; 3. a randomized controlled trial was designed to evaluate the value of using this screening tool in a population.

The tumor markers commonly used at present have 3 functions: 1. a screening function; 2. a diagnostic function; 3. prognosis and monitoring efficacy.

With the development of biotechnology, various novel markers are gradually discovered, and the specificity and sensitivity are continuously improved, representative of the markers are oncogenes, cancer suppressor genes and products thereof, tumor DNA, cytokines and receptors thereof, tumor miRNA, tumor stem cells and the like; the combined diagnosis technology of tumor markers also becomes a focus of attention.

Colorectal cancer (CRC) is a malignant lesion of the intestinal mucosal epithelium under the action of various carcinogenic factors such as environment or heredity, is one of three major malignant tumors in the world, and is also a common digestive tract malignant tumor in China. In recent years, with the improvement of economic level of China, the improvement of living standard of people and the continuous change of eating habits, the incidence rate of colorectal cancer of China is increased year by year, and the incidence rate is 3-5 of malignant tumors. The data of the Chinese colorectal cancer diagnosis and treatment standard (2015 edition) show that the morbidity and mortality of CRC in 2011 of China are 23.03/10 ten thousand and 11.11/10 ten thousand respectively, and urban areas are far higher than rural areas.

Colorectal cancer is hidden, early lesions are located in mucous membrane layers and submucosa, no clinical symptoms or unobvious symptoms exist, and the colorectal cancer is easy to ignore, but with the development and the deterioration of the disease conditions, the conditions of hematochezia, emaciation, abdominal pain and the like can appear, and most of patients with the symptoms are in middle and late stages and lose the early treatment opportunity. Colorectal cancer is a highly preventable cancer. The vast majority of colorectal cancers originate from adenomas, and the process takes 3 to 17 years to develop, which provides an opportunity for early screening. The colorectal cancer is diagnosed after obvious symptoms appear, the 5-year survival rate is only 30%, and if the colorectal cancer is detected at early stage and is cut by an operation, the 5-year survival rate can reach 97%. Therefore, screening of asymptomatic people, finding early cancers and precancerous lesions and performing intervention treatment are the keys for reducing the incidence rate of colorectal cancer and improving the survival rate of patients, and can greatly reduce the social public health medical cost.

The pathological tissue staining-based detection grading method is still the current clinical diagnosis gold standard, and the method is suitable for the tumor grading before and after operation. The early stage screening of colorectal cancer is only used as a secondary screening means to test specimens collected by an enteroscope, and the primary screening method adopts fecal occult blood detection (FOBT). FOBT is also the first screening experiment method for screening early colorectal cancer recommended in the consensus of screening and diagnosis and treatment of early colorectal cancer and precancerous lesions in China. Such non-invasive, rapid and easily portable assays are often used for preliminary testing in medical institutions and for home self-testing. The method has moderate price, convenient operation and small burden on patients, and can provide enough sensitivity detection results for early screening. People with healthy body function have no bleeding phenomenon in intestinal tract under normal conditions, and the FOBT result is negative. There are currently two FOBT detection methods: chemical methods (gFOBT) and immunological methods (iFOBT/FIT).

Micro RNA (MicroRNA, miRNA) is a non-coding small-molecule RNA of about 22 nucleotides in length in eukaryotes, which has been highly conserved evolutionarily. miRNA can cause target mRNA degradation or inhibit translation of the target mRNA through base complementary pairing specific to the target mRNA, so that the transcribed gene is regulated and controlled, and the miRNA is used as a potential oncogene or cancer suppressor gene to participate in the generation and development processes of various tumors. Studies have shown that some mirnas are demonstrated to be aberrantly expressed in certain types of cancer. In addition, such variant expression may play an important role in diagnostic and therapeutic treatment by regulating apoptosis, proliferation, differentiation and development of colorectal tumor cells.

Research has shown that miRNA plays an important role in important biological processes (such as participating in regulating tumor proliferation, migration and apoptosis) in the process of generating and developing colorectal cancer. Research of Iorio, Michael et al shows that the expression of miR-143 and miR-145 in various malignant tumors such as colorectal cancer is related to the occurrence and development of the tumors. The miRNA has a good specific detection effect on molecular indications of colorectal cancer, but the sensitivity needs to be improved, and false negative omission is easy to generate.

The method prompts whether the molecular characteristics and the detection method of miRNA can be combined with the pathological diagnosis principle of FOBT, and the FOBT network for early colorectal cancer diagnosis is constructed by a noninvasive detection method of a new molecular marker of a stool sample, so that the individual difference is reduced, the detection rate is improved, the purpose of early diagnosis is achieved, the illness state of a patient can be controlled, the survival period is prolonged, and the development of cancer is avoided.

Disclosure of Invention

Aiming at the prior art, the invention aims to systematically screen the colorectal cancer miRNA to serve as a specific molecular marker for early diagnosis of colorectal cancer and design an early detection kit.

The invention provides a miRNA molecular marker for diagnosing colorectal cancer, which is a human fecal occult blood diagnostic molecule, and comprises at least one of miR-144-3p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5p, and the expression level of the colorectal cancer group is up-regulated relative to the normal control group miR-144-3p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5 p.

Preferably, the miRNA molecular marker further comprises miR-144-5 p. More preferably, the expression level of miR-144-5p is up-regulated.

Preferably, the miRNA molecular markers comprise at least two of miR-144-3p, miR-451a, miR-486-5p, miR-363-3p, miR-144-5p or miR-20b-5 p.

Preferably, the miRNA molecular marker comprises at least one of miR-144-3p, miR-451a, miR-486-5p, miR-20b-5p, and further comprises at least one of miR-363-3p or miR-144-5 p.

The preferable marker combination mode is that the miRNA molecular marker comprises a combination of four markers of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, and further comprises one or two of miR-363-3p or miR-144-5 p;

the preferable marker combination mode is that the miRNA molecular marker comprises miR-363-3p and miR-144-5p, and also comprises one or more of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5 p;

the preferable marker combination mode is that the miRNA molecular marker comprises a combination of six markers of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5 p.

Any one of the above preferred miRNA molecular markers further comprises at least one of miR-92a, miR-135b and miR-421.

The preferable marker combination mode is that the miRNA molecular marker comprises at least one of six markers of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p and at least one of three markers of miR-92a, miR-135b and miR-421;

the preferable marker combination mode is that the miRNA molecular marker comprises a combination of six markers of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p and at least one of three markers of miR-92a, miR-135b and miR-421;

the preferable marker combination mode is that at least one of four markers of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p is included, at least one of two markers of miR-363-3p or miR-144-5p is also included, and at least one of three markers of miR-92a, miR-135b and miR-421 is also included;

the preferable marker combination mode is that the miRNA molecular marker comprises a combination of four markers of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, further comprises one or two of miR-363-3p or miR-144-5p, and further comprises at least one of three markers of miR-92a, miR-135b and miR-421;

the preferable marker combination mode is that the miRNA molecular marker comprises miR-363-3p and miR-144-5p, and also comprises one or more of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, and also comprises at least one of miR-92a, miR-135b and miR-421;

in a preferred marker combination mode, the miRNA molecular markers comprise at least one of six markers of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p and at least one of three markers of miR-92a, miR-135b and miR-421, and do not comprise other markers except the nine markers;

the preferred marker combination mode is that the miRNA molecular markers comprise a combination of six markers of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p and at least one of three markers of miR-92a, miR-135b and miR-421, and do not comprise other markers except the nine markers;

the preferable marker combination mode is that at least one of four markers of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p is included, at least one of two markers of miR-363-3p or miR-144-5p is also included, at least one of three markers of miR-92a, miR-135b and miR-421 is also included, and other markers except the nine markers are not included;

the preferred marker combination mode is that the miRNA molecular markers comprise a combination of four markers of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, one or two of miR-363-3p or miR-144-5p, at least one of three markers of miR-92a, miR-135b and miR-421 and do not comprise other markers except the nine markers;

the preferable marker combination mode is that the miRNA molecular markers comprise miR-363-3p and miR-144-5p, and also comprise one or more of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, and also comprise at least one of miR-92a, miR-135b and miR-421, and do not comprise other markers except the nine markers.

Any one of the above preferred miRNA molecular markers consists of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421.

Any one of the above is preferably used for constructing a FOBT (Forsythia-based diagnostic protocol) network for diagnosing the rectal cancer by detecting the change of the expression level of the miRNA molecular marker, so as to diagnose the rectal cancer.

Any one of the above is preferably that the detection means for diagnosing the rectal cancer by detecting the expression level change of the miRNA molecular marker is a detection means based on nucleic acid amplification and/or a detection means based on nucleic acid molecule hybridization principle, and preferably includes at least one of PCR, digital PCR, isothermal amplification technology, cross primer amplification technology, fluorescent quantitative PCR technology, high resolution melting curve (HRM), quantitative fluorescent polymerase chain reaction technology of repeated sequence markers, multiple ligation dependent probe amplification, colloidal gold immunochromatographic test strip, lateral chromatography test strip, limit PCR, photo-PCR, Cast PCR and pulse PCR, nested PCR, PCR chip, molecular beacon nucleic acid detection technology, and real-time fluorescent quantitative PCR.

The above techniques are conventional in the prior art, and for further explanation of specific experimental methods, the following are examples of specific operation modes of the experimental methods, but the operation method of the present invention is not limited thereto.

High resolution dissolution curve (HRM) analysis technique: a method of producing a High-resolution metallic analysis for rapid detection of a sample in Mycobacterium tuberculosis [ J ] Journal of clinical microbiology, 2013, 51 (10): 3294-9.

Digital PCR: ye Pen et al, The diagnostic access of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of tissues with a chromatographic carrier A systematic review and meta-analysis [ J ]. Plous one, 2021, 16 (3): e0248775-e0248775.

Isothermal amplification technique: ahuja amine and Somvanshi visual Single, Diagnosis of plant-specific chemicals using loop-mediated isothermal amplification (LAMP): A review [ J ] Crop Protection, 2021, 147

Cross primer amplification technology: application of the cross primer amplification technology in tuberculosis diagnosis [ J ] tuberculosis and journal of pulmonary health, 2018,7(04):284-287.

Fluorescent quantitative PCR technology: xuTing fluorescent quantitative PCR technical application reviews [ J ] livestock and poultry industry, 2010(10) 48-50.

Quantitative fluorescent polymerase chain reaction technology of repeated sequence markers: the use of the QF-PCR technique in combination with karyotyping in prenatal diagnosis of amniotic fluid [ J ]. Hainan medicine 2020,31(20):2617-2619.

Multiplex ligation-dependent probe amplification: wuhui, Xiangxiangji, Zhongchangsheng MLPA technology has clinical value of SMA prenatal diagnosis [ J ] intelligent health, 2020,6(22):76-77.

Colloidal gold immunochromatography test strip: the application of Qinlidede, Nanwenlong, Chenyiping, colloidal gold immunochromatographic test strip in poultry disease detection and the prospect [ J ]. Chinese animal quarantine, 2016,33(08):78-81+85.

Lateral chromatography test paper strip: zeifen, Chenwei, lateral chromatography test paper strip detects aflatoxin M1[ J ] in milk on site, proceedings of Combined Fertilizer industry university (Nature science edition), 2021,44(06):845-850.

Limit PCR, photo PCR, Cast PCR and pulse PCR: yan Wen, Yongg, novel PCR technique [ J ]. university of Lanzhou proceedings (medical edition), 2017,43(01):60-65.

Nested PCR: huang Gong, Xuchao, Li Bao, Xiaoting, Yi Kun, Liu Gong Zheng, Wang Wei Yan, Zhao Gui Hua, Wei Yan Bin, Wang used bin, Zhao Long Lei, Wei Qing Width application study of nested PCR technique in input oval malaria diagnosis [ J ]. J. J. parasite and parasite diseases in China, 2015,33(01):49-51+57.

PCR chip: establishment and preliminary application of PCR chip of genes related to colorectal cancer transfer [ J ] academic newspaper of southern medical university, 2011,31(07): 1169-.

Molecular beacon nucleic acid detection techniques: chen faithfully, Wang Sheng, Sunxian, molecular beacon nucleic acid detection technology research progress [ J ]. Biochemical and biophysical progress, 1998(06):6-10.

Any one of the above is preferably based on the fluorescent quantitative PCR technology, and combined with the miRNA molecular marker to construct the FOBT network for diagnosing the colorectal cancer. And (3) comparing the excrement of the patient and the excrement of a control group by adopting a fluorescent quantitative PCR method, and screening miRNA markers with significant difference in expression. In order to solve the technical problems, the invention adopts the technical scheme that a multiple RT-PCR detection method and a kit of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421 are provided, and the rapid and accurate quantification and diagnosis method of 9 miRNA molecular markers is realized.

The CRC confirmation mode colonoscopy is invasive, complex in examination mode, high in requirement on equipment medical staff, painful for patients and prone to causing complications, and is obviously not practical as a conventional popularization examination mode. Research shows that the excrement contains not only the exfoliated normal colorectal cells but also the exfoliated CRC cells, so that the abnormal expression molecules of the tumor can be detected as the molecular markers for CRC screening by extracting the cell markers in the excrement. FOBT has the characteristics of simplicity, convenience, easy operation of the detection process, high patient acceptance, strong popularity and the like as a conventional screening means of CRC, but has lower diagnosis sensitivity to CRC and higher false positive rate.

The invention finally determines 9 miRNAs as molecular markers for detecting the rectal cancer through a large amount of data analysis, and determines a new method for detecting the rectal cancer by combining with FOBT, a common early indication of the colorectal cancer. The 9 miRNAs selected by the invention are expressed in a small amount in the colorectal epithelium or are not expressed, but exist in a large amount in whole blood. mirnas are readily degraded in a variety of biological media, including blood and feces. Therefore, the present invention seeks to evaluate the use of miRNA markers as novel fecal occult blood markers, thereby serving as molecular markers for early diagnosis of colorectal cancer. The molecular marker provided by the invention and the method for constructing the colorectal cancer diagnosis FOBT network based on the combination of the fluorescent quantitative PCR technology and the miRNA molecular marker overcome the defects of lower diagnosis sensitivity and higher false positive rate of FOBT as a CRC detection means in the prior art.

Any one of the above preferred methods for constructing the FOBT network for diagnosing the rectal cancer based on the combination of the fluorescent quantitative PCR technology and the miRNA molecular marker comprises:

step (1): calculating correlation coefficient tests in excrement of the miRNA molecular marker colorectal cancer patients and normal contrast persons, calculating Pearson correlation coefficients by using SPSS statistical product and service solution software, and evaluating the similarity of the miRNA molecular marker expression;

step (2): detecting the expression conditions of the miRNA molecular markers in excrement of colorectal cancer patients and normal contrast persons by a reverse transcription real-time quantitative RT-PCR technology, screening candidate miRNA molecular markers, using a sample set consisting of the colorectal cancer patients and the normal contrast persons as a training set, analyzing the sensitivity and specificity of the candidate miRNA molecular markers on colorectal cancer diagnosis by a receiver operation characteristic curve, and evaluating the diagnosis efficiency of a candidate miRNA molecular marker diagnosis model by the area under the curve to obtain the selected miRNA molecular markers;

and (3): and classifying and statistically analyzing the data of the selected miRNA molecular markers in the feces of the colorectal cancer patients and the normal contrast persons by using a support vector machine, and finally calculating to obtain a colorectal cancer diagnosis calculation formula based on the selected miRNA molecular markers.

Any one of the above is preferred, the colorectal cancer diagnosis calculation formula is derived from the sum of the selected miRNA molecular marker and its corresponding term, further preferred,

the corresponding item of the selected miRNA molecular marker miR-92a is 0.929X 2^-ΔCT _{（miR-92a）}；

The selected miRNA molecular marker miR-135b has a corresponding item of 0.946X 2^-ΔCT _{（miR-135b）}；

The corresponding item of the selected miRNA molecular marker miR-421 is 0.797 x 2^-ΔCT _{（miR-421）}；

The corresponding item of the selected miRNA molecular marker miR-144-3p is 0.910 multiplied by 2^-ΔCT _{（miR-144-3p）}；

The corresponding item of the selected miRNA molecular marker miR-144-5p is 0.922 x 2^-ΔCT _{（miR-144-5p）}；

The corresponding item of the selected miRNA molecular marker miR-451a is 0.943X 2^-ΔCT _{（miR-451a）}；

The corresponding item of the selected miRNA molecular marker miR-486-5p is 0.748 multiplied by 2^-ΔCT _{（miR-486-5p）}；

The corresponding item of the selected miRNA molecular marker miR-363-3p is 0.771 multiplied by 2^-ΔCT _{（miR-363-3p）}；

The selected miRNA molecular marker miR-20b-5p has a corresponding item of 0.791 x 2^-ΔCT _{（miR-20b-5p）}. In a preferred embodiment of the present invention, preferably, the selected miRNA molecular markers in step (2) are miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421. Further preferably, the calculation formula for colorectal cancer diagnosis based on the selected miRNA molecular markers is as follows: miRNA-based Panel I = 0.929X 2^-ΔCT _{（miR-92a）}+0.946×2^-ΔCT _{（miR-135b）}+0.797×2^-ΔCT _{（miR-421）}+0.910×2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}+0.791×2^-ΔCT _{（miR-20b-5p）}。

In a preferred embodiment of the present invention, preferably, the miRNA molecular markers in step (2) comprise a combination of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p 6 miRNA molecular markers. Further preferably, the calculation formula for colorectal cancer diagnosis based on the selected miRNA molecular markers is as follows: miRNA-based Panel III = 0.910X 2- Δ CT (miR-144-3 p) + 0.922X 2- Δ CT (miR-144-5 p) + 0.943X 2- Δ CT (miR-451 a) + 0.748X 2- Δ CT (miR-486-5 p) + 0.771X 2- Δ CT (miR-363-3 p) + 0.791X 2- Δ CT (miR-20 b-5 p).

Any one of the above preferred, the PCR primer in the RT-PCR technology in step (2) comprises a PCR forward primer consisting of at least one of the following nucleotide sequences: seq ID NO: 1, Seq ID NO: 2, Seq ID NO: 3, Seq ID NO: 4, Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8 or Seq ID NO: 9, or a nucleotide sequence shown in the specification.

Any one of the above preferred, the reverse primer in the RT-PCR technology of step (2) comprises Seq ID NO: 10, and a PCR reverse universal primer consisting of the nucleotide sequence shown in the specification.

Any one of the above-mentioned preferred methods is that the reverse transcription primer in the RT-PCR technology of step (2) comprises a reverse transcription primer consisting of at least one of the following nucleotide sequences: seq ID NO: 11, Seq ID NO: 12, Seq ID NO: 13, Seq ID NO: 14, Seq ID NO: 15, Seq ID NO: 16, Seq ID NO: 17, Seq ID NO: 18 or Seq ID NO: 19.

Any one of the above preferred, the RT-PCR technology in step (2) is labeled by using Taqman probe, and the probe comprises at least one of the following nucleotide sequences: seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 22, Seq ID NO: 23, Seq ID NO: 24, Seq ID NO: 25, Seq ID NO: 26, Seq ID NO: 27 or Seq ID NO: 28.

The invention also provides a colorectal cancer screening kit prepared by using the miRNA molecular marker, which is used for detecting the miRNA molecular marker based on real-time fluorescent quantitative PCR. Preferably, the PCR forward primer comprises at least one nucleotide sequence selected from the group consisting of: seq ID NO: 1, Seq ID NO: 2, Seq ID NO: 3, Seq ID NO: 4, Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8 or Seq ID NO: 9, or a nucleotide sequence shown in the specification.

Preferably in any of the above, the kit comprises Seq ID NO: 10, and a PCR reverse primer consisting of the nucleotide sequence shown in the specification.

Preferably, in any of the above cases, the kit comprises a reverse transcription primer consisting of at least one of the following nucleotide sequences: seq ID NO: 11, Seq ID NO: 12, Seq ID NO: 13, Seq ID NO: 14, Seq ID NO: 15, Seq ID NO: 16, Seq ID NO: 17, Seq ID NO: 18 or Seq ID NO: 19.

Preferably, in any of the above cases, the kit comprises a probe consisting of at least one of the following nucleotide sequences: seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 22, Seq ID NO: 23, Seq ID NO: 24, Seq ID NO: 25, Seq ID NO: 26, Seq ID NO: 27 or Seq ID NO: 28.

The expression level of the candidate tumor marker is detected in feces of the colorectal cancer group and the control group. Results the expression levels of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421 are verified by RT-PCR, so that the early colorectal cancer is detected.

And (3) analyzing the correlation between the miRNA expression level and the colorectal cancer disease in the feces of the colorectal cancer patients and the feces of the control group by using RT-PCR (reverse transcription-polymerase chain reaction), wherein the miRNA expression level is obviously different, and the change trend is consistent with the disease development. The biological function of the miRNA is researched, an early colorectal cancer diagnosis network is established, and the miRNA can be used as a novel marker for colorectal cancer diagnosis and disease dynamic monitoring.

The invention systematically screens miRNA molecular markers specific to colorectal cancer in feces, determines the miRNA molecular markers with statistical significance difference between a colorectal cancer group and a control group, then uses a regression model to analyze the value of the miRNA molecular markers in early detection of colorectal cancer, and based on discovery of miRNA and construction of a diagnosis model, the invention provides a miRNA RT-PCR detection kit for multiple early screening of colorectal cancer.

The invention has the advantages that:

9 miRNA molecular markers with obviously different expressions in feces of a colorectal cancer group and a control group are obtained through RT-PCR experiments, the expression quantity of the 9 miRNA molecular markers in the colorectal cancer group is obviously higher than that of the control group, and early patients with colorectal cancer can be accurately identified, wherein the 9 miRNA molecular markers are miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421. The expression of the 9 miRNA molecular markers in colorectal cancer patients is obviously higher than that of a control group, the expression difference is more than 2 folds (the CT value in fluorescent quantitative PCR is less than 30), and the miRNA molecular markers can be used as biomarkers for colorectal cancer diagnosis. The 9 miRNA molecular markers can be used as biomarkers for colorectal cancer diagnosis, and the sensitivity and specificity of combined diagnosis are higher than those of the traditional fecal occult blood test and single miRNA diagnosis.

Description of the drawings:

the calculation method and software related to the invention are the calculation method and software in the prior art. The receiver operating characteristic curve (ROC curve) and the Support Vector Machine (SVM) refer to specific calculation methods. caret 6.0-86 and R4.0.3 refer to The caret package (Classification and Regression Training) which is a comprehensive tool package created for data Training to solve The Classification and Regression problem, is calculated in The caret package by using The R language, and is a conventional calculation tool.

ROC cites the application and development of the ROC curve in medical diagnostics [ J ]. university of southeast (medical edition), 2003(01): 67-70.

The SVM is introduced from (1) learning work, about statistical learning theory and support vector machine (J), 2000(01) 36-46.

Drawings

Figure 1 graph of similarity results for miRNA molecule marker expression.

FIG. 2 is a graph showing the results of ROC curve analysis of the diagnostic efficacy of 9 miRNA molecular markers on colorectal cancer.

FIG. 3 ROC curves for the SVM classifier in the test set (24 samples).

Figure 49 results of importance of miRNA molecular markers in classifier.

FIG. 59 is a graph of the amplification of miRNA molecular markers.

FIG. 69 miRNA molecular marker standard curves.

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are given to aid the understanding of the present invention and should not be construed as limiting the scope of the invention.

The invention provides a multiple early screening colorectal cancer miRNA RT-PCR detection method, which utilizes the reverse transcription primer sequence, the forward primer, the universal reverse primer and the Taqman probe (5 '-3') of the 9 miRNA molecular markers, the enzyme system, the RT-PCR reaction system, the positive control and the negative control, detects the 9 miRNA molecular markers by using the RT-PCR technology, and diagnoses early colon cancer by combining a diagnosis model.

Example 1

Example 1 provides the procedures for the detection method of the present invention and a method for constructing a diagnostic model

1.1 origin of specimen

50 colorectal cancer patients from the study were enrolled and collected over the same time period as a control group with 50 healthy examiners who reported no obvious abnormalities, no tumor history and matched age and gender with the colorectal cancer patients.

1.2 extraction of Total RNA from feces

Diluting feces by volume of 1: 2 with PBS, fully vortexing, and freeze thawing for 1 time; vortexing the frozen and thawed sample for 3-5 min, and centrifuging at 8000g at 4 ℃ for 10 min; adding 750 mu L of RNAasso Plus into 250 mu L of supernatant, standing for 5min, adding 200 mu L of chloroform, continuously and violently shaking for 15 s, and standing for 2 min; centrifuging at 12000 g at 4 deg.C for 15 min; sucking 400 μ L of supernatant into a new sterile EP tube, adding equal volume of 4 deg.C pre-cooled isopropanol, mixing with vortex, and standing at room temperature for 5 min; centrifuging at 12000 g at 4 deg.C for 10 min, and removing supernatant; adding 1 mL of 75% ethanol diluted by DEPC treated water into the precipitate; centrifuging at 12000 g at 4 deg.C for 10 min, and removing supernatant; the mixture was allowed to stand at room temperature for 5min, and the pellet (i.e., RNA) was dissolved in 30. mu.L of DEPC-treated water and stored at-80 ℃ until use.

1.3 reverse transcription PCR

Taking 1 μ l of total RNA extract in feces as template, according to reverse transcription kit (TaKaRa company), the reverse transcription primer is shown in Table I, and the test system is as follows:

mixing the system, keeping the temperature at 65 ℃ for 5min, rapidly cooling on ice for 2min, and adding the following reagents:

mixing, and performing reverse transcription in a PCR instrument: the cDNA was obtained at 42 ℃ for 45 min and 95 ℃ for 5 min.

1.4 RT-PCR detection

The miRNA to be detected is detected by using the designed specific forward primer, reverse primer and Taqman probe, the forward primer, the reverse primer and the Taqman probe are shown in the table I, and the RT-PCR system comprises the following specific operations:

all the components are added into an eight-connection pipe, and each group of three compound holes are fully and uniformly mixed to avoid bubbles. And (3) computer detection, wherein the setting procedure is as follows: at 95 ℃ for 30 s; 30s at 95 ℃,31 s at 55 ℃ and 40 cycles; 95 ℃ for 60s, 55 ℃ for 30s and 95 ℃ for 30 s. The experiment is repeated three times, the average value is taken, the relative expression quantity of miRNA is analyzed, and the result is shown in table two.

1.5 similarity of miRNA expression

Calculating Correlation coefficients of 9 miRNA molecules in feces of 50 colorectal cancer patients and 50 normal controls, and calculating Pearson Correlation Coefficient (Pearson Correlation Coefficient) by using SPSS (statistical Product and Service solutions) statistical products and Service solution software, wherein the Pearson Correlation Coefficient is used for measuring whether two data sets are on one line or not and measuring linear relation between range variables, and the homogeneity of each miRNA molecule can be predicted. The correlation coefficient of the Pearson value is 0.8-1.0 extremely strong correlation; 0.6-0.8 strong correlation; 0.4-0.6 moderate correlation; 0.2-0.4 weakly correlated; 0.0-0.2 are very weakly or not correlated. In this example, the results of RT-PCR of the rectal cancer group and the normal control group 2^-ΔCTSubstituting the value into SPSS software, and calculating to obtain a Pearson result smaller than 04, showing no linear correlation between colorectal cancer patients and normal controls, significance less than 0.005 (P<0.005). FIG. 1 shows the similarity of miRNA expression, wherein con is a normal control, case is a patient with colorectal cancer, and the RT-PCR results 2 of the rectal cancer group and the normal control group^-ΔCTValues were carried into the SPSS software and figure 1 was generated with the intensity of the data as the ordinate, showing that there was no linear correlation between colorectal cancer patients and normal controls. The SPSS described in this embodiment is applicable to the present invention as long as it is software capable of calculating relevant data processing, and the SPSS version 20.0 is preferably used.

1.6 diagnostic modeling

Analyzing the relative expression quantity of miRNA in serum and excrement by using a data processing method of delta CT (Delta CT), wherein CT is the cycle number required by the reaction reaching a threshold value and the relative expression quantity of each miRNA, and 2 is used^-ΔCTAnd (4) showing.

The calculation is carried out by taking hsa-miR-200b-3p (MIMAT 0000318) as an internal reference, and the miRNA sequence of the miRNA is shown as Seq ID NO: shown at 40. The primers (reverse transcription primer sequence: Seq ID NO: 41, forward primer: Seq ID NO: 42) and probe sequence (Seq ID NO: 43) for internal reference miR-200b-3p are shown in Table I and sequence Listing.

Checking amplification curve, setting 3 multiple holes for each sample, subtracting Ct value of corresponding hsa-miR-200b-3p internal reference from each multiple hole, subtracting delta Ct of control group from delta Ct value of colorectal cancer group and taking opposite number, finally performing power operation of 2 for comparison-delta-Ct, calculating change number of miRNA of each hole colorectal cancer group relative to control group, and taking final relative expression result as 2^-△CtIt is shown that the calculation process is performed in a PCR fluorescence quantitative instrument, which is a conventional basic operation method for those skilled in the art.

Wherein: Δ Ct =Δct^{Colorectal cancer group}-△Ct^{Control group}；

Δ CT ^{Colorectal cancer group}=CT^{Colorectal cancer group} _{Target gene}-CT^{Rectal cancer group} _{Internal reference gene}；

ΔCT^{Control group}=CT^{Control group} _{Target gene}-CT^{Control group} _{Internal reference gene}。

Data analysis was performed using SPSS 20.0 software, data were expressed as mean ± standard deviation (means ± SD), group comparisons were by t-test, and P <0.05 was considered statistically different.

Detecting the expression conditions of 9 miRNA molecules in the feces of 50 colorectal cancer patients and 50 normal controls by a reverse transcription real-time quantitative RT-PCR technology, screening candidate miRNA, and using 100 samples consisting of 50 colorectal cancer patients and 50 normal controls as a training set. Sensitivity and specificity for colorectal cancer diagnosis are analyzed by receiver operating characteristic curve (ROC curve). The diagnostic efficacy of the diagnostic model was assessed by the area under the curve. ROC curve analysis shows that the Area AUC (Area under the curve of the subject who diagnoses colorectal cancer) under the miRNA (AUC of ROC) is respectively miR-486-5p to be 0.748(95% CI: 0.643-0.835); miR-144-3p is 0.910(95% CI: 0.847-0.965); miR-20b-5p is 0.791(95% CI: 0.690-0.883); miR-421 is 0.797(95% CI: 0.709-0.881); miR-451a is 0.943(95% CI: 0.899-0.982); miR-92a is 0.929 (95% CI: 0.881-0.979); miR-363-3p is 0.771 (95% CI: 0.667-0.876); miR-135b is 0.946 (95% CI: 0.898-0.994); miR-144-5p is 0.922 (95% CI:0.872-0.972), and the results are shown in FIG. 2. The invention carries out the determination of the diagnostic efficacy of a large number of cancer and rectal cancer markers, and the 9 miRNA markers finally screened and determined for the invention are displayed in the example, and the 9 miRNA markers have good effects on the sensitivity and specificity of colorectal cancer diagnosis.

ROC curve of 1.7 SVM classifier in test set

The 9 miRNA molecular data were classified in the stools of 50 colorectal cancer patients and 50 normal controls using a Support Vector Machine (SVM), also called a trained linear SVM, or SVMLinear.

The support vector machine method is based on VC dimension theory and structure risk minimization principle of statistical learning theory, and seeks an optimal compromise between model complexity (namely learning precision of specific training samples) and learning capacity (namely capacity of identifying any sample without error) according to limited sample information so as to obtain the best popularization capacity (or generalization capacity).

The diagnostic formula is optimized by training the linear SVM (SVMLinear) based on caret 6.0-86 and R4.0.3.

Model training method (calculation process of SVM optimization diagnosis formula):

samples were randomly divided into training and validation sets (75%/25%)

And searching a parameter C in the training set through ten-fold cross validation to minimize the RMSE of the model, and constructing SVMLinitial by using the set parameter C and all the training sets.

Statistical analysis of the characteristics revealed that 9 miRNA molecules were significantly different between the two types of tissues of 50 colorectal cancer patients and 50 normal controls of feces (P.ltoreq.0.01). 24 samples were taken and the sensitivity, specificity, accuracy of the classification test and the area under the line AUC of the subject operating characteristic curve were verified using the SVM method for 9 features with significant differences to be 1.000, see fig. 3.

The support vector machine method is based on the risk minimum theory and the dimensional theory, and realizes the error-free identification of the sample and the optimal compromise scheme between the classification precision of the training sample, so that the method has better classification effect and popularization capability. The core content of the support vector machine is that the original low-dimensional samples are transformed into a high-dimensional space in a mapping mode, and a hyperplane is established in the high-dimensional space. Two hyperplanes parallel to the hyperplane are established on two sides of the hyperplane, and an additional segmentation hyperplane is established to maximize the distance between the hyperplane parallel to the two sides. In general, the larger the distance between two parallel hyperplanes, the better the corresponding classification effect, and the smaller the error.

Since svmLinear has no calculation method for the built-in feature import, the feature import set by the invention is replaced by the area under the ROC curve corresponding to each feature (namely the area under the ROC curve corresponding to each miRNA in the invention). feature import refers to the coefficients of miRNA in the following formulaAnd (4) selecting. 9 miRNA molecules, and establishing miRNA-based Panel I through the results of ROC curves of SVM classifiers in a test set (24 samples) and the ROC curve analysis of 9 miRNAs, wherein the formula is as follows: miRNA-based Panel I = 0.929X 2^-ΔCT _{（miR-92a）}+0.946×2^-ΔCT _{（miR-135b）}+0.797×2^-ΔCT _{（miR-421）}+0.910×2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}+0.791×2^-ΔCT _{（miR-20b-5p）}As shown in fig. 4, the importance of 9 mirnas in the classifier is shown for the coefficient of each miRNA in the formula, and the larger the data coefficient in the formula is, the larger the proportion is, and the RT-PCR test result of each miRNA in the training set (2)^-ΔCTValues, i.e. the test results in table two) into the diagnostic model formula, the range of colorectal cancer patients is 15.88-24.49; the range of normal control is 9.17-15.51.

Substituting into the RT-PCR test result of the normal control in the verification set, wherein the value of the normal control is 13.28, and the diagnosis model shows correct display when the value is in the range of the normal control; using the colorectal cancer patients RT-PCR test results, 19.24 was in the colorectal cancer patients range and the diagnostic model showed correct.

In practical application, 2 is determined according to the fecal occult blood sample of the detected person^-ΔCTSubstituting the miRNA-based Panel I formula, and according to the interval of the result, if the result falls within the range of 15.88-24.49, indicating that the colorectal cancer is possibly ill, and if the result falls within the range of 9.17-15.51, indicating that the colorectal cancer is normal.

Example 2

Example 2 provides a kit for early screening colorectal cancer based on real-time fluorescent quantitative PCR detection of miRNA molecular markers. Early screening of colorectal cancer is performed by using the 9 miRNA molecular markers provided in example 1 and a formula miRNA-based Panel I obtained based on the 9 miRNA molecular markers.

2.1 extraction of miRNA

The specific operation method is 1.2.

2.2 RT-PCR amplification of miRNA

Using the miRNA extracted in 2.1 as a template, adding an enzyme system, an RT-PCR reaction system, a forward and reverse primer pair set and a fluorescent probe set, and preparing an amplification reaction system; setting reaction conditions to perform RT-PCR reaction to obtain an amplification curve, and performing result analysis on the amplification curve, as shown in Table six. Preferably, three replicate wells are simultaneously amplified for each sample, the RNA template of the sample in the amplification reaction system (1) is replaced by a negative standard, and RNase free H is added₂And O, adding an enzyme system, an RT-PCR reaction system, a forward and reverse primer pair set and a fluorescent probe set to prepare an amplification reaction system as a negative control hole.

As a preferred scheme of the miRNA RT-PCR detection kit for the multiple early screening of the colorectal cancer, the kit comprises the following components: the primer design preferably comprises the following steps: obtaining 9 miRNA (miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421) related gene sequences in TargetScan (http:// www.targetscan.org/vert _72 /); according to the primer design principle, respectively designing a specific primer and a fluorescent probe aiming at the specific target fragment, as shown in the table I;

all probes of the fluorescent probe set contain a 5 'end fluorescent reporter group and a 3' end fluorescent quenching group, the 5 'end fluorescent reporter group of the first probe in the fluorescent probe set is FAM, and the 3' end fluorescent quenching group is MGB; in the fluorescent probe set, a fluorescent reporter group at the 5 'end of a second probe is CY3, and a fluorescent quencher group at the 3' end is MGB; in the fluorescent probe set, a 5 'end fluorescent reporter group of an internal reference quality control probe is ROX, a 3' end fluorescent quenching group is MGB, and the fluorescent reporter group can be selected from one of FAM, Cy5, VIC and ROX; the fluorescence quenching group is preferably one of BHQ1 and MGB; the fluorescent reporter groups are preferably FAM, Cy5, VIC and ROX, and are respectively marked with different probes to distinguish the fluorescent signals of different miRNAs. In example 2, 9 markers of the present invention were aligned with a plurality of fluorescent reporter systems to obtain reporter combinations that can detect the 9 markers simultaneously and accurately with little interference, and one of the preferred fluorescent signal combinations is shown in table three.

As a preferred scheme of the fluorescent quantitative RT-PCR detection kit for early screening of multiple colorectal cancers, the kit comprises the following components: the primer probes comprise reverse transcription primer sequences of 9 miRNAs and U6 (human miRNA internal reference), forward primers, universal reverse primers and Taqman probes (5 '-3'), and the first is a primer and a probe sequence for early screening 9 miRNA specificity RT-PCR identification of colorectal cancer:

the invention provides the following technical scheme: a multiple early screening colorectal cancer miRNA RT-PCR detection kit comprises RT-PCR reaction liquid, enzyme reaction liquid, a primer probe mixture, sterile nuclease-free water, a positive control and a negative control;

the RT-PCR reaction solution comprises dNTP, Mg2+ and PCR buffer solution; the enzyme reaction solution comprises reverse transcriptase and Taq enzyme; the primer probe mixture comprises specific primers and probes of 9 miRNAs;

preferably, the kit further comprises a positive standard: the 9 standard products comprise 9 miRNA sequence fragments (RNA fragments shown as SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36 and 37); internal reference quality control products: a miRNA solution containing a reference gene U6 sequence fragment; negative standard substance: RNase free H₂O; RT-Taq mix reagents.

Preferably, the positive standard plasmid contains 9 kinds of miRNA standard, and the preparation method comprises the following steps: the concentration is 1X 10²copies/mL; and (3) standard substance 2: the concentration is 1X 10³copies/mL; and (3) standard substance: the concentration is 1X 10⁴copies/mL; and (4) standard substance: the concentration is 1X 10⁵copies/mL; and (5) standard substance: the concentration is 1X 10⁶copies/mL; a standard curve was constructed. Fig. 5 shows the amplification profile of 9 mirnas. Figure 6 shows standard curves for 9 mirnas, where the standard curve for each miRNA is:

miR-144-3p：Y=-3.373X+17.069，R²=0.999；

miR-144-5p：Y=-3.401X+15.72，R²=0.999；

miR-486-5p：Y=-3.356X+15.662，R²=0.999；

miR-451a：Y=-3.693X+19.036，R²=0.996；

miR-363-3p：Y=-4.416X+20.389，R²=0.992；

miR-20b-5p：Y=-3.486X+29.607，R²=1.000；

miR-92a：Y=-3.355X+26.978，R²=1.000；

miR-135b：Y=-3.305X+29.576，R²=0.998；

miR-421：Y=-3.777X+30.779，R²=0.998；

x is the relative concentration of 9 miRNA sequence fragments, and is artificially set to be the concentration of gradient dilution by taking 10 times as a gradient; y is Ct value.

Example 3

Example 3 similar to example 2 except that the miR-144-3p reverse transcription primer sequences are as described in Seq ID NO: 38, the reverse transcription primer sequence of miR-144-5p is shown as Seq ID NO: 39, as shown in table five. See Table five on the end of text, alternative primers.

Example 4

Example 4 is similar to examples 1 to 3, except that the content of two miRNAs, namely miR-144-3p and miR-144-5p is detected, and the miR-144-3p and miR-144-5p are used as markers for detecting colorectal cancer.

The colorectal cancer diagnosis calculation formula based on the selected miRNA molecular marker is as follows: miRNA-based Panel II = 0.910X 2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}

The range of colorectal cancer patients is 2.00-2.71; the range of normal control is 3.65-5.08. The invention systematically screens colorectal cancer specific tumor markers respectively, and then constructs a colorectal cancer miRNA-based Panel II diagnosis model for noninvasive detection.

Example 5

Example 5 is similar to examples 1 to 3, except that the content of 6 miRNAs, miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5p is measured, and miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5p is used as a marker for detecting colorectal cancer.

The colorectal cancer diagnosis calculation formula based on the selected miRNA molecular marker is as follows: miRNA-based Panel III =0.910 × 2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}+0.791×2^-ΔCT _{（miR-20b-5p）}

The range of colorectal cancer patients is 11.02-15.25; the range of normal control is 7.25-9.88.

Example 6

Example 6 is similar to examples 1 to 3, except that the miRNA content of miR-144-5p and miR-363-3p is detected, and miR-144-5p and miR-363-3p are used as the markers for detecting colorectal cancer.

The colorectal cancer diagnosis calculation formula based on the selected miRNA molecular marker is as follows: miRNA-based Panel IV =0.922 × 2^-ΔCT _{（miR-144-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}

The range of colorectal cancer patients is 1.73-2.23; the range of normal control is 2.95-4.87.

Preferably, in example 6, besides miR-144-5p and miR-363-3p, one or more of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p can be selected and used as a marker for detecting colorectal cancer together with miR-144-5p and miR-363-3 p.

Example 7

Example 7 is similar to examples 1 to 3, except that miRNA content of miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p is detected, and miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p are used as markers for detecting colorectal cancer.

The colorectal cancer diagnosis calculation formula based on the selected miRNA molecular marker is as follows: miRNA-based Panel v =0.910 × 2^-ΔCT _{（miR-144-3p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.791×2^-ΔCT _{（miR-20b-5p）}

The range of colorectal cancer patients is 5.52-7.64; the range of normal control is 8.07-10.37.

Preferably, in example 7, besides miR-144-3p, miR-451a, miR-486-5p and miR-20b-5p, miR-144-5p and/or miR-363-3p can be selected, and can be used as a marker for detecting colorectal cancer together with miR-144-3p, miR-451a, miR-486-5p and miR-20b-5 p.

Example 8

And detecting the content of the three miRNAs miR-92a, miR-144-3p and miR-363-3 p.

The colorectal cancer diagnosis calculation formula based on the selected miRNA molecular marker is as follows: miRNA-based Panel VI = 0.929X 2- Δ CT (miR-92 a) + 0.910X 2- Δ CT (miR-144-3 p) + 0.771X 2- Δ CT (miR-363-3 p).

The range of the colorectal cancer patients is 2.59-4.51; the range of normal control is 5.63-9.23.

The invention systematically screens colorectal cancer specific tumor markers respectively, and then constructs a colorectal cancer miRNA-based Panel series diagnosis model for noninvasive detection.

TABLE-primer and Probe sequences

TABLE II RT-q PCR detection of miRNA molecule expression in colorectal cancer group and control group

Group carrying epitrifluorophore

TABLE IV RT-PCR reaction procedure

TABLE five primer alternatives

Sequence listing

<110> Tianjin Oudelai biomedical science and technology, Inc

Lapu (Tianjin) biomedical science and technology Co., Ltd

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Claims

1. A miRNA molecular marker for diagnosing colorectal cancer, wherein the miRNA molecular marker is a human fecal occult blood diagnostic molecule, and comprises at least one of miR-144-3p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5p, and the expression level of the colorectal cancer group is up-regulated relative to a normal control group miR-144-3p, miR-451a, miR-486-5p, miR-363-3p or miR-20b-5 p.

2. The miRNA molecular marker of claim 1, wherein the FOBT network for diagnosing the colorectal cancer is constructed by detecting the change of the expression level of the miRNA molecular marker, and the means for diagnosing the rectal cancer by detecting the change of the expression level of the miRNA molecular marker is nucleic acid amplification-based detection means and/or nucleic acid molecule hybridization principle-based detection means.

3. The miRNA molecular marker of claim 2, wherein the FOBT network for diagnosing the colorectal cancer is constructed by detecting the change of the expression amount of the miRNA molecular marker based on the fluorescent quantitative PCR technology and the miRNA molecular marker in combination, and is constructed by the following method:

step (2): detecting the expression conditions of the miRNA molecular markers in excrement of colorectal cancer patients and normal contrast persons by a reverse transcription real-time quantitative RT-PCR technology, screening candidate miRNA, using a sample set consisting of the colorectal cancer patients and the normal contrast persons as a training set, analyzing the sensitivity and specificity of the candidate miRNA on colorectal cancer diagnosis by a receiver operation characteristic curve, and evaluating the diagnosis efficiency of the candidate miRNA diagnosis model by the area under the curve to obtain the selected miRNA molecular markers;

4. The miRNA molecular marker of any one of claims 1 to 3, comprising a combination of miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p and miR-20b-5p 6 human fecal occult blood miRNA molecular markers.

5. The miRNA molecular marker of claim 4, wherein the selected miRNA molecular markers are miR-144-3p, miR-144-5p, miR-451a, miR-363-3p, miR-486-5p and miR-20b-5p, and the colorectal cancer diagnosis calculation formula based on the selected miRNA molecular markers is as follows: miRNA-based Panel III =0.910 × 2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}+0.791×2^-ΔCT _{（miR-20b-5p）}。

6. The miRNA molecular marker of any one of claims 1 to 3, wherein the miRNA molecular marker is used in combination with a non-fecal occult blood diagnostic molecule comprising at least one of miR-92a, miR-135b and miR-421.

7. The miRNA molecular marker of claim 6, wherein the selected miRNA molecular markers are miR-144-3p, miR-144-5p, miR-451a, miR-486-5p, miR-363-3p, miR-20b-5p, miR-92a, miR-135b and miR-421, and the colorectal cancer diagnosis calculation formula based on the selected miRNA molecular markers is as follows: miRNA-based Panel I = 0.929X 2^-ΔCT _{（miR-92a）}+0.946×2^-ΔCT _{（miR-135b）}+0.797×2^-ΔCT _{（miR-421）}+0.910×2^-ΔCT _{（miR-144-3p）}+0.922×2^-ΔCT _{（miR-144-5p）}+0.943×2^-ΔCT _{（miR-451a）}+0.748×2^-ΔCT _{（miR-486-5p）}+0.771×2^-ΔCT _{（miR-363-3p）}+0.791×2^-ΔCT _{（miR-20b-5p）}。

8. A colorectal cancer screening kit prepared using the miRNA molecular marker of any one of claims 1 to 7, wherein the miRNA molecular marker is detected based on real-time fluorescent quantitative PCR.

9. The kit of claim 8, comprising a PCR forward primer consisting of at least one nucleotide sequence of: seq ID NO: 1, Seq ID NO: 2, Seq ID NO: 3, Seq ID NO: 4, Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8 or Seq ID NO: 9, or a nucleotide sequence shown in the specification.

10. The kit of claim 8, comprising a reverse transcription primer consisting of at least one nucleotide sequence selected from the group consisting of: seq ID NO: 11, Seq ID NO: 12, Seq ID NO: 13, Seq ID NO: 14, Seq ID NO: 15, Seq ID NO: 16, Seq ID NO: 17, Seq ID NO: 18 or Seq ID NO: 19; a probe comprising at least one of the following nucleotide sequences: seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 22, Seq ID NO: 23, Seq ID NO: 24, Seq ID NO: 25, Seq ID NO: 26, Seq ID NO: 27 or Seq ID NO: 28.