CN101942502B - Pancreatic cancer marker, and detection method, kit and biochip thereof - Google Patents
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Abstract
The invention provides a pancreatic cancer marker, and a detection method, a kit and a biochip thereof. The pancreatic cancer marker provided by the invention contains 36 kinds of micro ribonucleic acids which stably exist in the serum/plasma of a person receiving the test and can be detected. The invention also provides a kit and a biochip of tools or elements for detecting the pancreatic cancer marker. The combination, the method, the kit and the biochip provided by the invention can be used for auxiliary diagnosis and differential diagnosis of pancreatic cancers, predication of occurrence and recrudescence of disease complications, evaluation of the curative effect, screening of active ingredients of medicaments, evaluation of pesticide effectiveness and the like, and has the advantages of wide detection range, high sensitivity, low detection cost, readily available raw materials, easy storage of samples and the like; and the method is widely applied to work related to the general survey of pancreatic cancers, improves the specificity and sensitivity which are low in the single marker due to individual difference, obviously increases clinical detection rate of pancreatic cancers and becomes an effective method for early diagnosis of pancreatic cancers.
Description
Technical field
The invention belongs to biological technical field, relate to separation, the qualitative and quantitative analysis of miRNA molecule in human serum/blood plasma, also relate to the various clinical indication of carcinoma of the pancreas simultaneously.Specifically, the present invention is a kind of method that detects miRNA in Patients with Pancreatic Cancer serum/plasma, by the variation of miRNA in Patients with Pancreatic Cancer serum/plasma, diagnosis of pancreatic cancer and chronic pancreatitis in vitro, judgement carcinoma of the pancreas pathogenic process, the generation of prediction carcinoma of the pancreas complication and the probability of carcinoma of the pancreas recurrence and the prognosis of carcinoma of the pancreas, and analyze drug effect and curative effect.
Background technology
Carcinoma of the pancreas is the tumour of a kind of mortality ratio high (～99.9%, after making a definite diagnosis).U.S.'s sickness rate: estimate new cases 32,180 examples, account for 2% of all new cancers for 2005; U.S.'s mortality ratio: estimate death 31,800 examples in 2005 1 year, account for respectively the 4th and the 5th of all cancer associated deaths of men and women reason, account for the 5%-6% of all cancer mortality reasons.European Union's sickness rate: estimate new cases 55100 examples for 2002, European Union's mortality ratio: estimate death 59300 examples in 2002 1 year, difference is not obvious in different sexes and race, and as a rule patient's prognosis is poor.Global carcinoma of the pancreas morbidity in 2002 and dead statistics are as shown in table 1, and wherein number of the infected refers to 2002 and find to suffer from that the number of carcinoma of the pancreas, death toll refer to until within 2002, be diagnosed as carcinoma of the pancreas and in the number of death in 2002.
Table 12002 year global carcinoma of the pancreas morbidity and dead statistics
Therefore, find pancreatic cancer marker and it is carried out to the extremely urgent and important precondition that accurate detection has become the early diagnosis and therapy of carcinoma of the pancreas.Although increasing disease markers has been found and has been applied to the monitoring of generaI investigation, diagnosis and the curative effect of clinical disease, their clinical application effect also exists obvious deficiency.For example, tumor marker alpha-fetoprotein, serum lactic dehydrogenase, carcinomebryonic antigen etc. have been widely used in clinical, but these disease markers also can not meet the needs to early diagnosis of cancer far away, its major cause has two aspects: sensitivity and the specificity of (1) above-mentioned disease markers are relatively low, the index that their detected result can't be made a definite diagnosis as disease; (2) early diagnostic rate of disease should present positive correlation with the effect for the treatment of, and above-mentioned any disease markers is also difficult to meet this requirement of disease early diagnosis.Take cancer as example, owing to existing, Tumor Differentiation classification specificity is excessively strong, the whole susceptibility of tumour is lower, censorship sample is difficult to repeatedly take, sample is preserved the defects such as requirement condition height, simultaneously expensive, therefore under existence conditions, be difficult to the existing tumor marker of wide popularization and application.And some traditional medicine means exist its intrinsic defect as histocyte detects, the position of drawing materials is improper, histocyte sample material deficiency or people will cause mistaken diagnosis for lacking experience etc.Although other technology for example iconography has been widely used in the Checking and diagnosis of disease, it still exists significant limitation on disease degree qualitative.Therefore be at present necessary very much to find and can make up above-mentioned defect novel, sensitive of existing marker and apply disease detection marker easily.
MiRNA, English microRNA by name, is the non-coding strand small ribonucleic acid molecules that a class is about 19 to 23 Nucleotide.They are high conservative on evolving, and with many normal physiological activity of animal, as closely related in biont growth, tissue differentiation, natural death of cerebral cells and energy metabolism etc., also exist closely and contact with the generation of numerous disease and development simultaneously.Nearest research finds that the expression level of several miRNAs in lymphocytic leukemia and Burkitt lymphoma all has downward (Lawrie CH in various degree, Gal S, Dunlop HM et al.Detection of elevated levels of tumor-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.Br J Haematol 2008; 141:672-675); While analyzing the miRNA expression of comparing in people's lung cancer, breast cancer tissue, with respect to healthy tissues, there is variation (Garofalo M in the expression level that finds that there is some tissue specificity miRNAs, Quintavalle C, Di Leva G etal.MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer.Oncogene 2008).Also there are some researches prove that miRNA has affected generation and the development of the cardiovascular disordeies such as myocardial hypertrophy, heart failure, atherosclerosis, and there is close association (Tryndyak VP with metabolic diseases such as type ii diabetes, Ross SA, Beland FA, Pogribny IP.Down-regulationof the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet.Mol Carcinog.2008 Oct21).Between these experimental result prompting miRNA expression and specific variations and Occurrence and development of disease, exist positive connection.
Owing to playing vital role beyond imagination in the expression regulation of miRNA after genetic transcription, therefore there is cognation below with disease in it: first, the variation of miRNA may be the cause of disease, this is because the supressor of disease and the promotion factor may be all the target sites of miRNA, when disorderly expression has first occurred miRNA itself, such as the original disease that suppresses promotes the miRNA expression amount of the factor to reduce, or the miRNA expression amount that suppresses disease supressor has raise, its net result all can cause the variation of downstream series of genes expression and the integral body disorder of some path, and then the generation that induces an illness, secondly, the variation of miRNA may be also the result of disease, this is because when disease (as cancer) occurs, can cause the violent amplification of the loss of chromosome segment, the sudden change of gene or chromosome segment, if miRNA is just in time positioned at this varied sections, its expression amount will occur to change extremely significantly so.Therefore, miRNA molecule can be used as the disease markers that a class is new completely in theory, and its specific variations must produce development with disease and be associated.MiRNA can also, as potential drug target, by the miRNA that suppresses to raise in lysis or the miRNA of crossing down-regulated expression, will likely greatly be alleviated generation and the development of disease simultaneously.
The domestic current existing correlative study of miRNA as disease markers of usining, as Chinese patent application CN100999765A and CN101298630A, they all choose account for the 4th of Cancer Mortality colorectal carcinoma as research object, find after deliberation, during colon benign polypus develops into malignant tumour, some miRNA molecules all exist specific variations, and by measuring the specific variations of miRNA, have set up a kind of more responsive, more accurate method of making a definite diagnosis in early days colorectal carcinoma accordingly.Yet because being not easy to make the widespread use clinically of this method, drawing materials of tissue sample be restricted.
Summary of the invention
For overcoming this defect, the inventor will study sight and invest more easily acquisition, the blood that even just can collect in routine physical examination.Because blood can be circulated to whole body institute in a organized way, and to cell delivery nutrition and remove refuse, so blood can reflect the physiological and pathological situation of whole body, and its detected result has directive significance to HUMAN HEALTH.In known serum/plasma, exist multiple protein, as total protein, albumin, sphaeroprotein etc., multiple lipid, as HDL cholesterol, triglyceride etc., multiple saccharic, pigment, ionogen and inorganic salt, plurality of enzymes, as amylase, alkaline phosphatase, acid phosphatase, Choline lipase, zymohexase etc., also collected the multi-signal molecule from body tissue's organ simultaneously, as cytokine, hormone etc.At present, the diagnosis of disease is only confined to the above-mentioned biochemical indicator in serum/plasma, there is no the report of serum/plasma miRNA.In people's traditional concept, think and there is no ribonucleic acid molecule in serum/plasma, even if having also, can by rnase, be degraded to small molecule segment very soon and can't detect.But, because miRNA molecule is that 19 to 23 nucleotide units form, thering is structural singularity and relative stability, they are very likely present in serum/plasma.The inventor's early-stage Study is verified, in serum/plasma, stably have miRNA, and each disease there is its specific variation collection of illustrative plates (Chen et al:Characterization of microRNAs in serum:a novel class of biomarkers for diagnosis of cancer and other diseases.Cell Res.2008 Oct; 18 (10): 9977).
For finding carcinoma of the pancreas certification mark thing and it accurately being detected, the inventor, based on existing achievement in research, has carried out the research of the following aspects:
(1) specific variations of serum/plasma miRNA in research carcinoma of the pancreas pathogenic process;
(2) by the biochip for detection of serum/plasma miRNA and sequencing technologies, measure the variation of pancreatopathy cancer serum/blood plasma miRNA;
(3) by expressing under carcinoma of the pancreas, chronic pancreatitis and normal physiological state of the screening class serum/plasma miRNA molecular application that difference degree is large in serum/plasma miRNA detection technique, Application and preparation is in biochip and the diagnostic kit in the fields such as diagnosis of pancreatic cancer.
By the above-mentioned research to the dependency of serum/plasma miRNA and carcinoma of the pancreas, applicant has proposed to using the specific miRNA of stable existence in serum/plasma as carcinoma of the pancreas certification mark thing, set up the method for the specific miRNA of stable existence in a kind of vitro detection serum/plasma, by detecting the specific variations of specific miRNA, carry out the early diagnosis of carcinoma of the pancreas, the differential diagnosis of chronic pancreatitis, disease is identified and course of disease monitoring, recurrence and prognosis, the prediction that complication occurs, can further carry out drug effect judgement simultaneously, medicine guide, individualized treatment, effective components of Chinese medicinal screening, plant the researchs such as heap sort.
Therefore, the object of this invention is to provide a kind of pancreatic cancer marker.
Another object of the present invention is to provide a kind of probe combinations for detection of pancreatic cancer marker.
Another object of the present invention is to provide the purposes of above-mentioned pancreatic cancer marker, comprises the corresponding test kit of preparation and biochip.
Another object of the present invention is to provide the method that detects above-mentioned pancreatic cancer marker.
The object of the invention is to realize by the following technical solutions.
On the one hand, first the present invention provides a kind of carcinoma of the pancreas certification mark thing, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA (Mature microRNA), for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 kind: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, mi R-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.Wherein, miR-320 comprises for example miR-320a, miR-320b.
Preferably, described marker comprise following in human serum/blood plasma one or more in the ripe body of stable existence and detectable miRNA, for example 2,3,4,5,6 or 7 kind: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
The present invention also provides a kind of pancreatic cancer marker, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 kind: miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185 and miR-191.
Preferably, described marker comprise following in human serum/blood plasma two or more in the ripe body of stable existence and detectable miRNA, for example 3,4,5,6 or 7 kind: miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191.
Above-mentioned serum/plasma can derive from human body live body, tissue, organ and/or corpse.
On the other hand; the invention provides a kind of detection method of above-mentioned marker, described detection method is selected from one or more in inverse transcription polymerase chain reaction method (RT-PCR), real time fluorescent quantitative poly chain reaction method (Real-time PCR), Northern blot hybridization method (Northern blotting), rnase protection analysis method (RNase protection assay), Solexa sequencing technologies (Solexa sequencing technology) and biochip method.
Preferably, described detection method is RT-PCR method, the RT-PCR method for example comprising the following steps: 1) extract experimenter's the total RNA of serum/plasma, by RNA reverse transcription reaction, obtain cDNA sample; Or the serum/plasma sample of collecting experimenter, the serum/plasma of usining is carried out reverse transcription reaction as damping fluid and is prepared cDNA sample;
2) with miRNA design primer, carry out PCR reaction;
3) carry out the agarose gel electrophoresis of PCR product;
4) after EB dyeing under ultraviolet lamp observations;
Or preferably, described detection method is Real-time PCR method, the Real-time PCR method for example comprising the following steps:
1) extract experimenter's the total RNA of serum/plasma, by RNA reverse transcription reaction, obtain cDNA sample; Or the serum/plasma sample of collecting experimenter, the serum/plasma of usining is carried out reverse transcription reaction as damping fluid and is prepared cDNA sample;
2) with miRNA, design primer;
3) add fluorescent probe to carry out PCR reaction;
4) detect and compare serum/plasma sample with respect to the variation of the amount of miRNA in normal serum/blood plasma.
Specifically, the method for above-mentioned 36 kinds of miRNAs in detection provided by the invention experimenter serum/plasma, can further evaluate the state of human body carcinoma of the pancreas.In described human body serum/plasma, the method for stable existence and detectable 36 kinds of miRNAs comprises: inverse transcription polymerase chain reaction method (RT-PCR), real time fluorescent quantitative poly chain reaction method (Real-time PCR), Northern blot hybridization method (Northern blotting), rnase protection analysis method (RNase protection assay), one or more in Solexa sequencing technologies (Solexa sequencing technology) and biochip method.
Described RT-PCR method comprises the following steps: (1) collects serum/plasma sample, particularly, uses for example total RNA of serum/plasma of Trizol reagent extraction human body, by RNA reverse transcription reaction, obtains cDNA sample; Or the serum/plasma sample of collecting experimenter, the serum/plasma of usining is carried out reverse transcription reaction as damping fluid and is prepared cDNA sample; (2) with miRNA design primer, carry out PCR reaction; (3) carry out the agarose gel electrophoresis of PCR product; (4) after EB dyeing under ultraviolet lamp observations taking pictures.
Described Real-time PCR method comprises the following steps: (1) collects serum/plasma sample, particularly, uses for example Trizol reagent to extract experimenter's the total RNA of serum/plasma, by RNA reverse transcription reaction, obtains cDNA sample; Or the serum/plasma sample of collecting experimenter, the serum/plasma of usining is carried out reverse transcription reaction as damping fluid and is prepared cDNA sample; (2) with miRNA, design primer; (3) add fluorescent probe for example EVA GREEN carry out PCR reaction; (4) analyzing and processing data comparative result, particularly, detect and compare serum/plasma sample with respect to the variation of the amount of miRNA in normal serum/blood plasma.
Described Northern blotting method comprises the following steps: (1) collects serum/plasma sample; (2) by Trizol reagent, extract the total RNA of serum/plasma; (3) carry out sex change PAGE electrophoresis and film shift experiment; (4) prepare isotopic labeling miRNA probe; (5) carry out film hybridization; (6) isotropic substance signal detection, as phosphorus screen scanning detecting result.
Described RNase protection assay method comprises the steps: that (1) carry out the synthetic of antisense RNA probes, isotopic labeling and purifying; (2) collect serum/plasma sample and extract RNA; (3) RNA after extracting is dissolved in hybridization buffer and adds antisense RNA probes to carry out hybridization; (4) add RNase Digestive system to react; (5) carry out electrophoresis and radioautograph; (6) analytical results.
Described Solexa sequencing technology method comprises the steps: (1) collection serum/plasma sample; (2) by Trizol reagent, extract the total RNA of serum/plasma; (3) carry out PAGE electrophoresis and reclaim 17-27nt RNA molecule; (4) adaptor prime enzyme is associated in to 3 of small RNA molecular ' with 5 ' end; (5) carrying out RT-PCR reacts rear and checks order; (6) data analysis and processing.
Described biochip method comprises the steps: (1) by the ripe body storehouse dot matrix of whole more than 500 miRNAs and prepares biochip; (2) collect serum/plasma sample; (3) extract the total RNA of serum/plasma; (4) by the separated next separated miRNA of post; (5) utilize T4RNA ligase enzyme to carry out miRNA fluorescent mark; (6) carry out hybridization with biochip; (7) Data Detection and analysis.
The present invention is by above-mentioned RT-PCR, Real-time PCR, Northern blotting, RNase protection assay, variation tendency and the variable quantity of the methods analysts such as Solexa sequencing technology and biochip serum/plasma miRNA in carcinoma of the pancreas occurs, and the dependency of they and carcinoma of the pancreas.Wherein, first detect and analyze miR-27a, miR-27b, miR-29a, miR-29c, miR-30a, miR-30d, miR-33a, miR-92a, miR-100, miR-101, miR-103, miR-125b, miR-130b, miR-140-3p, miR-148a, miR-192, miR-199a, miR-199a-3p, miR-222, miR-210, miR-215, miR-223, miR-320, miR-361-5p, miR-378, miR-411, miR-483-5p, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-99, miR-122, miR-185, the variation of miR-191 in carcinoma of the pancreas, preparation serum/plasma miRNA biochip is measured the variation of serum/plasma miRNA in various disease, miRNA in various disease serum/plasma is carried out to Solexa sequencing analysis simultaneously.
The serum/plasma of using in aforesaid method derives from experimenter's live body, tissue, organ and/or corpse.
The present invention also provides a kind of prediction, diagnosis, discriminating and/or evaluates the method for carcinoma of the pancreas, and the method comprises the above-mentioned marker of detection, and preferably, the method comprises that the above-mentioned detection method of employing detects above-mentioned marker.
The invention provides above-mentioned pancreatic cancer marker in preparation prediction, diagnosis, discriminating and/or evaluate the reagent of carcinoma of the pancreas or the purposes in instrument.
The present invention also provides a kind of miRNA probe combinations for detection of pancreatic cancer marker, also predict, diagnose and/or evaluate the micro ribonucleic acid probe combinations of carcinoma of the pancreas, described probe combinations comprises one or more in the probe shown in following nucleotide sequence, for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind; Preferably, described probe combinations comprises two or more in the probe shown in following nucleotide sequence, for example 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 or 36 kind:
The present invention also provides a kind of miRNA probe combinations for detection of pancreatic cancer marker, also predict, diagnose and/or evaluate the micro ribonucleic acid probe combinations of carcinoma of the pancreas, described probe combinations comprises one or more in the probe shown in following nucleotide sequence, for example 2,3,4,5,6 or 7 kind:
| miRNA | Corresponding probe sequence | Sequence numbering |
| miR-20a | CTACCTGCACTATAAGCACTTTA | SEQ ID NO.28 |
| miR-21 | TCAACATCAGTCTGATAAGCTA | SEQ ID NO.29 |
| miR-24 | CTGTTCCTGCTGAACTGAGCCA | SEQ ID NO.30 |
| miR-25 | TCAGACCGAGACAAGTGCAATG | SEQ ID NO.31 |
| miR-99 | CACAAGATCGGATCTACGGGTT | SEQ ID NO.33 |
| miR-185 | GAACTGCCTTTCTCTCCA | SEQ ID NO.35 |
| miR-191 | AGCTGCTTTTGGGATTCCGTTG | SEQ ID NO.36 |
The invention provides a kind of test kit for detection of pancreatic cancer marker, also predict, diagnose, differentiate and/or evaluate the test kit of carcinoma of the pancreas, this test kit comprises the instrument that detects above-mentioned marker.Preferably, wherein said instrument comprises the above-mentioned miRNA probe combinations for detection of pancreatic cancer marker; More preferably, described instrument also comprises polysaccharase, deoxyribonucleotide.The miRNA primer of the specific variations relevant to carcinoma of the pancreas screening or its corresponding probe sequence are collected in PCR test kit (RT-PCR or Real-time PCR) and can be prepared diagnosis of pancreatic cancer test kit.
The present invention also provides a kind of biochip for detection of pancreatic cancer marker, also predicts, diagnoses, differentiates and/or evaluate the biochip of carcinoma of the pancreas, and this biochip comprises the element that detects above-mentioned marker.Preferably, wherein said element comprises the above-mentioned miRNA probe combinations for detection of pancreatic cancer marker.Using the reverse complementary sequence of the miRNA of the specific variations relevant to carcinoma of the pancreas screening as probe points at chip, just made specially the serum/plasma miRNA detection of biological chip for carcinoma of the pancreas.
Particularly, in above-mentioned any containing in above a kind of combination to 36 kinds of miRNA markers, method, test kit or biochip, described evaluation experimenter's carcinoma of the pancreas state gives the state of the carcinoma of the pancreas after determinand for measuring experimenter, specifically for the activity that prevents and/or treats carcinoma of the pancreas of screening determinand (medicine that is used for the treatment of carcinoma of the pancreas); The disease that described evaluation experimenter's carcinoma of the pancreas state is diagnosis and/or differential diagnosis experimenter; Described evaluation experimenter's carcinoma of the pancreas state is for evaluating the validity that experimenter's disease is treated; Described evaluation experimenter's carcinoma of the pancreas state predicts for carcinoma of the pancreas is occurred experimenter to, and described generation carcinoma of the pancreas is specially the generation of carcinoma of the pancreas complication and/or the recurrence of carcinoma of the pancreas.The Hazard Factor of carcinoma of the pancreas comprise chronic pancreatitis etc., and pathology evidence has also been found the progressive process of normal pancreatic tissue-hyperplasia-carcinoma of the pancreas.Pancreatitis patient more early occurs as hereditary pancreatitis or tropical pancreatitis, the danger of canceration is also higher, and the time length being also exposed under chronic inflammatory state is topmost Hazard Factor.Have carcinoma of the pancreas and the chronic pancreatitis of chronic pancreatitis background to have certain False Rate, therefore, it is particularly important that differential diagnosis carcinoma of the pancreas in vitro and chronic pancreatitis seem.
At present disease is carried out to traditional biological chemistry and the Protocols in Molecular Biology of clinical diagnosis also more loaded down with trivial details and coarse.What development in recent years was got up likely has gene chip and protein (antibody) chip technology etc. for the new technique of medical diagnosis on disease.The measured mRNA level of gene chip changes the change that can not reflect real protein level completely.Because the biological activity of protein and post transcriptional modificaiton are as glycosylation, phosphorylation etc. are closely related.And for numerous disease detects, biochip technology cannot detect marker molecules in body fluid and blood.Protein (antibody) chip technology and proteomic techniques also have its limitation.In human body, particularly in serum/plasma, contain ten hundreds of proteins and peptides segments, their concentration distribution are wide, clearly the albumen of report seldom, quantification just still less.At the huge Proteomics of this quantity, look for the protein that has close association with specified disease, and understand its effect in lesion tissue and remain an extremely large order, and lacking perfect antibody resource will be a bottleneck problem of restriction antibody chip technical development.Serum/plasma miRNA detection technique, biochip based on serum/plasma miRNA and diagnostic kit are combined as a whole the peculiar property of serum/plasma miRNA and conventional molecular Biological Detection technology dexterously, they analyze the composition of miRNA in pancreatopathy cancer serum/blood plasma rapidly high-throughput, and clinical applicability is extremely strong.Because the physiological status variation of organ-tissue can cause the change that serum/plasma miRNA forms, so serum/plasma miRNA can be used as " disease fingerprint ", realizes the early diagnosis of carcinoma of the pancreas.
In sum, tool of the present invention has the following advantages:
(1) using the specific serum/plasma miRNA filtering out as novel pancreatic cancer marker, have that the pedigree of detecting is wide, highly sensitive, testing cost is low, the advantages such as convenience, sample (deposit serum/plasma-20 ℃) easy to store of drawing materials, the method can be widely used in the related works such as general investigation of desease, becomes the effective means of early diagnosis disease.
(2) serum/plasma miRNA is as new disease markers, by low specificity and the muting sensitivity improving the single individual difference that marker was difficult to overcome and bring, significantly improves the clinical recall rate of disease and realizes the early stage diagnosis and treatment of disease.
(3) advantage of serum/plasma miRNA detection technique is, its detection be a series of disease-related marker, thereby can overcome the difference (being age, sex, race, diet and environment etc.) between individual patient, and this subject matter that single disease markers cannot be gone beyond just.
In a word, the present invention can further be applied to make a definite diagnosis in early days carcinoma of the pancreas, this new serum/plasma pancreatic cancer marker not only provides basic substance for people fully understand the mechanism of carcinoma of the pancreas on molecular level, has also accelerated clinical disease diagnosis and has learned and therapeutic progress.Superiority based on serum/plasma miRNA, believe in the near future, to seriously disease, will become the part of routine physical examination as the serum/plasma miRNA diagnostic techniques of cancer, and the relevant gene therapy of miRNA also can apply widely, conquer these diseases and be no longer a dream.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiments of the invention in detail, wherein:
Fig. 1 shows the RT-PCR result of the part miRNA that in normal human serum, direct-detection arrives;
Fig. 2 shows and extracts in normal human serum RNA and detect the wherein RT-PCR result of miRNA;
In Fig. 1 and Fig. 2, U6 is that molecular weight is the snRNA of 100bp, internal reference molecule as miRNA experiment, remaining 12 code name represents respectively the specific miRNA miR-181a of hemocyte (181a), miR-181b (181b), miR-223 (223), miR-142-3p (142-3p), miR-142-5p (142-5p), miR-150 (150), miRNA miR-1 (1) from cardiac muscle and skeletal muscle, miR-133a (133a), miR-206 (206), miRNA miR-9 (9) from cerebral tissue, miR-124a (124a), and from the miRNA miR-122a (122a) of liver.
Fig. 3 shows respectively the miRNA RT-PCR result of the partially stabilized expression that in mouse, rat, tire ox, calf and horse serum, direct-detection arrives;
Fig. 4 A to 4D shows that 7 species specificity serum/plasma miRNAs are in the result schematic diagram of normal population, chronic pancreatitis and Pancreas cancer patients cluster analysis;
Fig. 5 A to 5C shows that 7 kinds of miRNA detect susceptibility and the specificity schematic diagram of carcinoma of the pancreas;
Fig. 6 shows that 7 kinds of miRNA detect the result figure of the accuracy rate of carcinoma of the pancreas.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
embodiment 1the RT-PCR of miRNA experiment in serum/plasma
Use the various miRNAs of stable existence in RT-PCR scientific discovery reference and animal serum/blood plasma, and its expression amount is quite abundant.Concrete steps are:
(1) collect mouse, rat, normal people and some patient's serum/plasma;
(2) prepare cDNA sample.This operation has two schemes, a kind of scheme is for directly to carry out reverse transcription reaction by 10 μ l serum/plasma, another kind of for using Trizol reagent (Invitrogen company) first to extract the total RNA of serum/plasma (10ml serum/plasma is the RNA of energy enrichment approximately 10 μ g left and right conventionally), then by RNA reverse transcription reaction, obtain cDNA.The reaction system of reverse transcription comprises 4 μ l 5 * AMVbuffer, 2 μ l 10mM each dNTP mixture (Takara company), 0.5 μ l RNase Inhibitor (Takara company), 2 μ l AMV (Takara company) and 1.5 μ l gene specific reverse primer miscellanys.Reactions steps is 16 ℃ hatches 15 minutes, and 42 ℃ are reacted 1 hour, hatch 5 minutes for 85 ℃;
(3) PCR and electrophoresis observation.CDNA is diluted by 1/50, get the cDNA after 1 μ l dilution, add 0.3 μ l Taq enzyme (Takara company), 0.2 μ l 10 μ M forward primers, the general reverse primer of 0.2 μ l 10 μ M, 1.2 μ l 25mM MgCl2,1.6 μ l 2.5mM each dNTP mixture (Takara company), 2 μ l 10 * PCR buffer, 13.5 μ lH2O, 20 μ l systems are carried out PCR.The reaction conditions of PCR is: within 95 ℃, 5 minutes, carry out 1 circulation → 95 ℃, 15 seconds, within 60 ℃, 1 minute, carry out 40 circulations.PCR product is got 10 μ l and is carried out 3% agarose gel electrophoresis, after EB dyeing, under ultraviolet lamp, observes.
Specific experiment the results are shown in Figure 1.Fig. 1 be the serum of taking from normal people be research object, serum is directly carried out to the experimental result of RT-PCR.Select the ripe body of whole more than 500 miRNAs of people to carry out PCR reaction, Fig. 1 is 12 kinds of miRNAs wherein.They are respectively the specific miRNA miR-181a of hemocyte, miR-181b, miR-223, miR-142-3p, miR-142-5p, miR-150, miRNA miR-1, miR-133a, miR-206 from cardiac muscle and skeletal muscle, from miRNA miR-9, the miR-124a of cerebral tissue, and from the miRNA miR-122a of liver.Above-mentioned four kinds of tissue-derived miRNAs can detect in blood as can be seen from the results, be not that the ripe body of whole more than 500 miRNAs has high abundance to express in serum/plasma, some miRNA is very micro-, even can not normally detect.
In order further verifying, first to extract the RNA in normal human serum by these miRNAs of stable existence in serum/plasma, then to select the ripe body of whole more than 500 miRNAs of people to carry out PCR experiment, result as shown in Figure 2.The result of Fig. 2 and the result of Fig. 1 are very identical, and PCR product is single, show that these two kinds of experimental techniques can detect expression and the abundance of human serum/blood plasma miRNA, prove and in human serum/blood plasma, stably have Various Tissues source miRNA.In addition, use the same method and detected expression and the abundance of more than 500 miRNA in mouse, rat, tire ox, calf and horse serum, the miRNA of same discovery different tissue sources has stably express in mouse, rat, tire ox, calf and horse serum, and result as shown in Figure 3.
embodiment 2the real-time PCR of miRNA experiment in serum/plasma
In order to study the special variation of serum/plasma miRNA in carcinoma of the pancreas lysis, carried out the quantitative PCR experiment of serum/plasma miRNA.Quantitative PCR experiment principle and experimental procedure are the same with RT-PCR, and unique not being both added fluorescence dye EVA GREEN in PCR.What instrument used is ABI Prism 7300 quantitative real time PCR Instruments, and reaction conditions is within 95 ℃, 5 minutes, to carry out 1 circulation → 95 ℃, 15 seconds, within 60 ℃, 1 minute, carries out 40 circulations.Data processing method is Δ Δ CT method, and CT is made as the cycle number of reacting while reaching thresholding, and each miRNA can be used equation 2 with respect to the expression amount of standard internal reference
-Δ CTrepresent, wherein Δ CT=CT sample-CT internal reference.Patients serum/plasma sample and normal human serum/plasma sample are directly carried out to reverse transcription reaction, by quantitative PCR, react the wherein amount of contained miRNA.
Choose aplastic anemia, mammary cancer, osteosarcoma, central nervous system lymphoma, diabetic serum sample, the ripe body of whole more than 500 miRNAs of employment simultaneously carries out PCR experiment.Above-mentioned hemocyte specificity miR-181a, miR-181b, miR-223, miR-142-3p, miR-142-5p, the miR-150 mentioning, miRNA miR-1, the miR-133a of cardiac muscle and skeletal muscle, miR-206, from miRNA miR-9, the miR-124a of cerebral tissue, and in normal people and patients serum, carry out the experimental result of quantitative PCR from the miRNA miR-122a of liver.The amount of aplastic anemia, mammary cancer, osteosarcoma, central nervous system lymphoma, diabetic miRNA in blood serum has respectively upper mediation to lower with respect to the ratio of normal people's amount, and same tissue-derived miRNA intensity of variation in various disease is different, show that serum/plasma miRNA has specific variations in various disease, they can be used as the marker of the medical diagnosis on disease that a class is new.
embodiment 3serum/plasma miRNA chip for diagnosis of pancreatic cancer
Chip operation flow process is:
(1) extract total RNA in serum/plasma, denaturing formaldehyde gel electrophoresis detects the quality of total RNA;
(2) separation of miRNA: get the total separated miRNA of Ambion ' s miRNAIsolation Kit (Cat#.1560) for RNA of 50-100 μ g;
(3) fluorescent mark of miRNA sample: utilize T4 RNA ligase enzyme marking method to carry out fluorescent mark, and then by dehydrated alcohol precipitation, after drying up for chip hybridization;
(4) hybridization and cleaning: RNA is dissolved in to (15% methane amide in 16 μ L hybridization solutions; 0.2%SDS; 3 * SSC; 50 * Denhardt ' s solution), in 42 ℃ of hybridization, spend the night.After hybridization finishes, first 42 ℃ of left and right, contain 0.2%SDS, wash 4 minutes in the liquid of 2 * SSC, then in 0.2 * SSC liquid, room temperature is washed 4 minutes, and slide can be used for scanning after drying;
(5) chip scanning: chip scans with LuxScan 10K/A two channels laser scanner;
(6) data are extracted and are analyzed: adopt LuxScan3.0 image analysis software to analyze chip image, picture signal is converted into numerary signal, finally with SAM, analyze and select difference expression gene.
By the large class serum/plasma miRNA probe of the differential expression degree under carcinoma of the pancreas and normal physiological state of quantitative PCR technique and biochip technology double verification, for the preparation of biochip, method is the same.This chip is compared with traditional die, and manufacture craft and operating process do not have significant improvement, but this chip has been simplified probe library, will greatly reduce cost of manufacture and the production time of chip thus, is easy to preparation.Also specific aim and the practicality of chip have been increased simultaneously.This chip is dropped into practice, only need patient's serum/plasma and just can find in early days disease without any need for other tissue, help to instruct and diagnose and treat.
embodiment 4minuteness ribonucleic acid reagent kit for diagnosis of pancreatic cancer and prediction
Diagnosis, the generation of disease complication and the prediction of recurrence for carcinoma of the pancreas, therapeutic evaluation, and the screening of active constituents of medicine is, the manufacture craft of the minuteness ribonucleic acid reagent kit of evaluating drug effect and operating process are based on quantitative and semi-quantitative round pcr and biochip technology.
First by method or the PCR method of order-checking, determine the miRNA that has an above copy in normal serum/blood plasma.Then by quantitative PCR technique and biochip technology, screen expression amount and the large class serum/plasma miRNA of difference degree under carcinoma of the pancreas and normal physiological state, whether the index of carcinoma of the pancreas and diagnosis lesion degree occurs as prediction.The quantity of the serum/plasma miRNA of the every kind of disease of correspondence finally filtering out is 36 kinds, and this is that make on the basis in chip probe storehouse optimized simplified.This test kit comprises the reagent such as a collection of serum/plasma miRNA primer, Taq enzyme, dNTP.
In the present embodiment, the comfortable hospital of all detection sample standard deviations is diagnosed as Pancreas cancer patients, Patients With Chronic Pancreatitis and equity age and other normal people of homogeny (contrast).
First, adopt the method for Solexa order-checking to determine the miRNA that has an above copy in normal serum/blood plasma, by detecting the variation of miRNA in serum/plasma, filter out with normal people's (control group) and compare, 63 kinds of miRNA that change in Pancreas cancer patients serum sample, wherein 44 miRNA raise, and 19 miRNA lower, and concrete outcome is as shown in table 2.
The differential expression sequencing result of miRNA in table 2 Pancreas cancer patients serum sample and control group serum sample
By quantitative PCR technique and biochip technology, filter out expression amount and the large class serum/plasma miRNA of difference degree under disease and normal physiological state, reference table 2, choose wherein mean change multiple surpass 5 and copy number be greater than 10, and in conjunction with laboratory early-stage Study result, selected 36 miRNA and detected index, whether the index of carcinoma of the pancreas and diagnosis lesion degree occurs as prediction, concrete outcome is in Table 3.
36 kinds of miRNA that table 3 is selected
From table 3 in 36 of up-regulated kinds of miRNA, according to the choice criteria of mean change multiple >2 and p value <0.01, further optimize the molecular marked compound that 7 kinds of miRNA detect as carcinoma of the pancreas, be specially miR-20a, miR-21, miR-24, miR-25, miR-99, miR-185 and miR-191, concrete outcome is in Table 4.
7 kinds of miRNA that table 4 is selected
By above-mentioned 7 kinds of miRNA are carried out to cluster analysis, again show that their expression between carcinoma of the pancreas, chronic pancreatitis and normal control serum sample there are differences.Above-mentioned 7 kinds in serum/plasma miRNA as the specificity fingerprint of carcinoma of the pancreas, in normal population and Pancreas cancer patients, change specific analytical results and see Fig. 4 A-D.As known in the figure, can combine clear and definite differentiating pancreatic cancer sample and normal sample according to these 7 kinds of miRNA, and can differentiating pancreatic cancer sample and chronic pancreatitis sample.7 kinds of miRNA combine clear and definite differentiating pancreatic cancer sample and contrast (comprising normal people and chronic pancreatitis) sample.
The concrete data processing of cluster analysis is as follows: for training set (Fig. 4 A is 25 routine Pancreas cancer patients and 25 contrasts), checking collection (Fig. 4 B is 95 routine Pancreas cancer patients and 81 contrasts), (Fig. 4 C is 95 routine Pancreas cancer patients and 82 routine Patients With Chronic Pancreatitis to high risk factor group; Fig. 4 D be 95 routine Pancreas cancer patients, 81 contrast and 82 routine Patients With Chronic Pancreatitis), respectively the absolute expression values of serum miRNA in carcinoma of the pancreas sample is converted to the multiple ratio of contrasting with normal sample, and by its normalization method, cluster and drafting pattern 4A-D (adopting cluster 3.0 softwares mappings to form), the analytical results that in these 7 kinds of serum/plasma, miRNA changes as the specificity fingerprint of carcinoma of the pancreas.Fig. 4 A-D is described in detail as follows.
In Fig. 4 A, right-hand mark word is 7 detected miRNA, and top mark word is respectively detection individual of sample, and normal represents normal people (n=25), concentrates on figure right side; T represents Patients with Pancreatic Cancer (n=25), concentrates on figure left side.This figure has confirmed normal people and Pancreas cancer patients to be distinguished by the detection of 7 miRNA expression levels.
In Fig. 4 B, right-hand mark word is 7 detected miRNA, and top mark word is respectively detection individual of sample, and normal(represents normal people (n=81): concentrate on figure right side; T represents Pancreas cancer patients (n=95), concentrates on figure left side.The further enlarged sample of this figure detects, and has verified by the detection of 7 miRNA expression levels and normal people and Pancreas cancer patients can have been distinguished.
The right-hand mark word of Fig. 4 C is 7 detected miRNA, and top mark word is respectively detection individual of sample, and ch pan(represents Patients With Chronic Pancreatitis (n=82): concentrate on figure right side; T represents Pancreas cancer patients (n=120), concentrates on figure left side.The further enlarged sample of this figure detects, and has verified by the detection of 7 miRNA expression levels and Patients With Chronic Pancreatitis and Pancreas cancer patients can have been distinguished.
Fig. 4 D is depicted as Fig. 4 B and Fig. 4 C samples set originally, this figure right side mark word is 7 detected miRNA, upside mark word is respectively detection individual of sample, nor represents that normal people (n=81) and ch pan(represent Patients With Chronic Pancreatitis (n=82), and normal people and chronic pancreatitis sample concentrate on figure left field; T represents Patients with Pancreatic Cancer (n=120), and carcinoma of the pancreas sample concentrates on figure right side area.Can find out, 7 miRNA can separate carcinoma of the pancreas sample and contrast (comprising normal people and chronic pancreatitis sample) sample area.
Fig. 4 A, 4B, 4C and 4D are carried out to the analysis of risk marking, concrete outcome is in Table 5.In table 5, what the first row of form represented is the risk score mark of assessed sample; The second to eight row is illustrated respectively in training set, checking collection and the concentrated Pancreas cancer patients number of high risk factor under certain risk score mark, Patients With Chronic Pancreatitis number or normal people's number; Adopt statistical analysis software (SAS) to carry out statistical study, setting risk score numerical value is 6, if Sample Risk scoring >=6 is divided into Pancreas cancer patients, if Sample Risk scoring <6 is divided into normal people.
Concrete statistical method is as follows: except controlling each the step variable in whole process, further all data standards are turned to zero-mean and a standard deviation before data clusters.In order to minimize impact assisted layered cluster and the risk score of missing values, adopt K nearest-neighbor method K-Nearest Neighbors (KNN, a kind of method of laying the blame on based on missing data (a method-based missing data imputation)) to estimate the missing values in 19 to 20 intervals.For example, if the miRNA in sample A has a missing values, can in sample A, find same other K miRNA expressing, then find to comprise to other miRNA in case A and express the most similar sample.Can estimate missing values by the weighted mean among sample A from K immediate miRNA.In weighted mean, the weighted value of each miRNA is calculated with the similarity of expressing in itself and miRNA.Set K here and equal 9, use 9 neighbours' miRNA to estimate.In addition the estimation result being drawn by K nearest-neighbor method KNN, is very little for the impact of current result of study.All markers call rate and are all greater than 97.6%, and there is no sample disappearance more than two and plural marker.
At this, used in cluster3.0 with the hierarchical cluster of association mode completely.In order to carry out risk score, be made as t by 95% of the interval upper limit of each miRNA numeric reference in control group, as the threshold value that miRNA expression level corresponding to each sample encoded.The risk score of each miRNA is designated as to S, with calculating equation expression, is:
Wherein, i represents i sample,
represent j miRNA.Consider the weighted of each miRNA assessment pancreatic cancer risk, according to having set up the function of a risk score to each patient to the linear combination of the expression level of miRNA.According to the related data of K miRNA, the risk score function of i sample is:
In the equation above, sij is for miRNA in sample i
risk score.Ws is the weight of the risk score of miRNA j..In order to determine sign and Ws, the model-fitting of 10 single argument logistic regressions is applied to indicate to the various diseases situation of risk score.By the regression coefficient in each risk score, as representing the weight of each miRNA in risk score function, the sign in regression coefficient has determined the sign in risk assessment function.Then, frequency of utilization table and ROC curve are evaluated the diagnosis effect in sample colony.
Table 5 patient and the risk score that contrasts (normal people)
In table, * positive prediction rate, the negative prediction rate of * *
MiRNA detects carcinoma of the pancreas susceptibility and Fig. 5 A-C is shown in by specificity schematic diagram, if the total area (detecting sample totally counts) is one, can find out that area under curve (being confidence level) is corresponding to the training set (Fig. 5 A) of Fig. 4 A, corresponding to the checking collection (Fig. 5 B) of Fig. 4 B and corresponding to Fig. 4 C, the high risk factor collection of 4D (comprises carcinoma of the pancreas, normal people and chronic pancreatitis sample, Fig. 5 C) reach respectively 0.995,0.987 and 0.993.
Figure 6 shows that above-mentioned 7 kinds of miRNA detect the result figure of carcinoma of the pancreas accuracy rate, wherein X-coordinate is detected miRNA kind, ordinate zou is area under curve, and representative adopts 7 kinds of miRNA to detect the accuracy rate (establishing the total area (detecting total sample number) is 1) of carcinoma of the pancreas.Can find out area under curve (being accuracy rate) > 0.98.
Claims (5)
1. a pancreatic cancer marker, it is characterized in that, described marker is included in human serum/blood plasma stable existence and the ripe body miR-185 of detectable miRNA, and described marker is also included in one or more in stable existence in human serum/blood plasma and the ripe body miR-25 of detectable miRNA, miR-99, miR-20a, miR-21, miR-24 and miR-191.
2. the miRNA probe combinations for detection of pancreatic cancer marker, it is characterized in that, described combination comprises the probe shown in SEQ ID NO.35, and described combination also comprises one or more in the probe shown in SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.33 and SEQ ID NO.36:
3. for detection of a test kit for pancreatic cancer marker, it is characterized in that, described test kit comprises probe combinations claimed in claim 2.
4. the test kit for detection of pancreatic cancer marker according to claim 3, is characterized in that, described test kit also comprises polysaccharase and/or deoxyribonucleotide.
5. probe combinations according to claim 2 is in the reagent for the preparation of detection carcinoma of the pancreas or the purposes in instrument.
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