CN101921760A - A serum/plasma miRNA marker associated with breast cancer and its application - Google Patents

Abstract

Translated fromChinese

本发明属于基因工程及肿瘤学领域，公开了一种与乳腺癌相关的血清/血浆miRNA标志物及其应用。该标志物为miR-16、miR-25、miR-222和miR-324-3p的组合。该标志物及其引物可用于制备诊断试剂盒，用于乳腺癌的辅助早期诊断。

The invention belongs to the fields of genetic engineering and oncology, and discloses a serum/plasma miRNA marker related to breast cancer and an application thereof. The marker is a combination of miR-16, miR-25, miR-222 and miR-324-3p. The marker and its primers can be used to prepare a diagnostic kit for auxiliary early diagnosis of breast cancer.

Description

A kind of serum mark and the application thereof relevant with mammary cancer

Invention field

The invention belongs to genetically engineered and oncology, relate to a kind of serum mark and the application thereof relevant with mammary cancer.

Background technology

Mammary cancer is the modal malignant tumour of women.According to estimates, whole world New Development mammary cancer case in 2002 reaches 1,150,000, accounts for 23% of the whole malignant tumours of women morbidity, estimates will reach 140-150 ten thousand in 2010.In China, the sickness rate of mammary cancer is compared low with western countries, but recent two decades is next, and it presents phenomenal growth trend, and average annual growth rate reaches 3-4%, is higher than world's average increment level of 0.5% far away.Though the prognosis of mammary cancer is better,, remain the first cause of women because of cancer mortality because its sickness rate is higher.In China Beijing, Shanghai, city breast cancer incidences such as Tianjin have been in first, second of women's malignant tumour morbidity.2000 to 2005 this in the period of 5, China women with breast cancer patient has increased by 38.5%, annual women because of mammary cancer death reaches 1.3 ten thousand people.Mammary cancer has become the primary malignant tumour that threatens China's women's health, is the great public health problem that needs to be resolved hurrily.

Though mammary cancer does not have special preventive measures at present, but as if energy early discovery, early diagnosis, early treatment, the prognosis situation is generally better.So, in current and quite a while in the future, strive for that early diagnosis is still an elementary tactics of mammary cancer control.The mode of early diagnosis mammary cancer mainly contains the mammary gland self check at present, the photography of breast molybdenum target X line, mammary gland colored multispectral rein in ultrasonic examination, breast duct endoscopy, breast duct lavation, CT and mr etc. are if having lump or the abscess that can't distinguish can carry out the cytology examination by centesis.Although the diagnostic level of mammary cancer constantly rises in recent years, but it is real comparatively ripe or have the breast cancer diagnosis means of better application prospect still few, some breast carcinoma of early stage patient is because lump is less, or soak into light, be expansive growth, show as smooth, movable, clear border, be difficult for distinguishing with innocent tumour.In addition, owing to relate to aspect problems such as Social Culture ethics, some young woman ignore or the generation resisting psychology easily for breast examination, not only can not guarantee to diagnose accuracy to cancer patients's diagnosis, for the patient with breast cancer who is in commitment, especially the diagnosis capability of young patient is more limited.Therefore, we need more clearly and effectively biomarker of discovery badly, and mammary cancer is made clear and definite early diagnosis, and this will help the patient with breast cancer to make early treatment, improve survival rate.

MicroRNAs (being miRNAs) is a focus of oncomolecularbiology research field in recent years, and its maturity state is the little single stranded RNA molecule that a class is about 19-23 Nucleotide, has high conservative in the evolution.It extensively is present in the eukaryote, is one group of not short sequence RNA of coded protein, itself does not have open reading frame (ORF).The major function of MiRNA is to regulate organism intrinsic and body growth, growth, the relevant expression of gene of disease generating process.Since participating in lin-4 that regulation and control nematode sequential grows and being found with let-7, miRNA was selected in the annual ten big technological breakthroughs of Science magazine respectively twice at 2002 and 2003.Prediction miRNAs can regulate and control 5300 Human genomes, promptly 30% of all genes at least in 2005.Along with going deep into of research, increasing miRNAs constantly is found.MiRNA under the spot light lamp has progressively broken away from covering of DNA radiance, becomes " leading role " from " supporting role ", and the middle cardiac status of DNA has been proposed new challenge.In recent years, the relation of miRNA and tumour has become the focus and emphasis of research, has been found that miRNA passes through the expression and the lung cancer of negative regulator gene, mammary cancer, cancer of the stomach, the morbidity height correlation of mammary cancer etc.

Research has confirmed to exist in the blood serum miRNAs of hundreds of kind, and these microRNAs s stable in properties, content enrich, are easy to detection by quantitative, and have significant disease specific.Existing proven technique comprises the technology of qualitative and quantitative miRNA molecule showing that utilizing serum miRNAs will more effective as the method for molecular biosciences mark than traditional differential protein molecule marking method, for biomarker has been opened up the frontier.

Yet, also be not used at present the report of the comparatively stable biomarker of breast cancer diagnosis, if can filter out the serum s of mammary cancer unconventionality expression as biomarker, and develop corresponding diagnostic kit, will be once strong promotion to the diagnosis present situation of China's mammary cancer.

Summary of the invention

Primary and foremost purpose of the present invention is at above-mentioned technical problem, proposes a kind of serum mark relevant with mammary cancer.

Second purpose of the present invention provides the primer of above-mentioned serum mark.

The 3rd purpose of the present invention provides above-mentioned serum mark and the application of primer in preparation mammary cancer auxiliary diagnostic box thereof.

The 4th purpose of the present invention provides the test kit of auxiliary breast cancer diagnosis.

The contriver by separate and the research patient with breast cancer and with the healthy women control serum/blood plasma of its age-matched in miRNAs, seek one group with the high specific of mammary cancer height correlation and the miRNAs of susceptibility, and develop the breast cancer diagnosis test kit that to be convenient to clinical application, for the examination and the diagnosis of mammary cancer provides the data support, provide the data support for finding new small molecule medicine with potential therapeutic value.

The objective of the invention is to realize by following technical proposal:

A kind of serum mark relevant with mammary cancer, this mark is the combination of miR-16, miR-25, miR-222 and miR-324-3p.

The primer of described serum mark, these primers are:

The primer of miR-16 is SEQ ID No.19 and SEQ ID No.20; The primer of miR-25 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-222 is SEQ ID No.17 and SEQ ID No.18; The primer of miR-324-3p is SEQ ID No.13 and SEQ ID No.14.

The application of described serum mark in preparation mammary cancer auxiliary diagnostic box.

The application of the primer of described serum mark in preparation breast cancer diagnosis test kit.

A kind of mammary cancer auxiliary diagnostic box, this test kit is used for detecting blood serum miR-16, miR-25, miR-222 and miR-324-3p.

Described diagnostic kit, this test kit contains the primer of miR-16 in the serum, miR-25 and miR-324-3p.

Described diagnostic kit, the primer of the serum mark that this test kit contains is:

The primer of miR-16 is SEQ ID No.19 and SEQ ID No.20; The primer of miR-25 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-222 is SEQ ID No.17 and SEQ ID No.18; The primer of miR-324-3p is SEQ ID No.13 and SEQ ID No.14.

Described diagnostic kit can also comprise PCR reaction enzyme and reagent commonly used, as reversed transcriptive enzyme, and damping fluid, dNTPs, MgCl2, DEPC water and Taq enzyme etc.; Can also contain standard substance and/or reference substance.

Specifically, the technical scheme that the present invention deals with problems comprises: (1) sets up the sample storehouse and the database of unified standard: (SOP) gathers standard compliant blood sample with Standard operation procedure SOP, demography data and clinical data that systematic collection is complete.(2) serum differential expression spectrum analysis: select the mammary cancer case, with the healthy women contrast of mammary cancer case age-matched, detect its serum express spectra and content, analyze the general character and the characteristic of serum between the contrast of mammary cancer case and healthy women, screening differential expression miRNAs carries out further large sample multistage checking.(3) the blood serum differential expression miRNAs that has screened is carried out quantitative analysis in the large sample crowd, determine the development of relevant serum s (4) serum examination of mammary cancer morbidity and diagnostic kit: according to the special serum exploitation miRNAs diagnostic kit of mammary cancer case and healthy women contrast.

The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), (these data can be used for judging progression of disease for the demography data that systematic collection is complete, clinical data etc., and control disease by stages, factor such as patient age is for the influence of morbidity), and adopted RT-PCR, Real-time PCR method, Solexa sequencing technologies, TaqMan Low DensityArray (TLDA) chip detection etc.

Yan Jiu experimental technique mainly comprises following components specifically:

1. the selection of research sample

(1) the mammary cancer case of clarifying a diagnosis through pathology

(2) perform the operation and chemicotherapy chemicotherapy before the nothing operation before the blood sampling without crossing

(3) healthy women with the case age-matched contrasts

This research adopts 196 routine standard compliant samples to study altogether.

2.Trizol (Invitrogen, Carlsbad CA) extract the total RNA of blood serum with miRNeasy Mini Kit (QIAGEN company), according to a conventional method operation to reagent.Usually can obtain～5 μ g RNA/50ml serum or blood plasma.

3.Solexa order-checking

(1) total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule

(2) will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end

(3) carry out checking order after the RT-PCR reaction

(4) data analysis and processing

(4.TLDA Applied Biosystems company) chip detection

(1) total RNA obtains the cDNA sample by reverse transcription reaction

(2) the cDNA sample reaction of increasing in advance

(3) pre-expansion volume increase thing carries out the TLDA chip detection, obtains the express spectra of miRNA

(4) data analysis and processing

5.Real-time RT-PCR (qRT-PCR) method

(1) gets experimenter's the total RNA of blood serum, obtain the cDNA sample by the RNA reverse transcription reaction;

(2) design primer;

(3) add fluorescent probe or dyestuff and carry out the PCR reaction;

(4) variation of the amount of miRNA in detection and comparison mammary cancer case and the healthy women control serum/plasma sample.

6. diagnostic reagent box preparation method

Solexa order-checking and TLDA chip detecting method comprehensively determine in the contrast of mammary cancer case and healthy women copy difference and differential expression are arranged, and the miRNA of direction unanimity, by qRT-PCR technology screening expression amount and one group of big serum of difference degree in mammary cancer case and healthy women contrast, as the index of auxiliary breast cancer diagnosis.The serum relevant with the mammary cancer morbidity that filters out at last formed diagnostic kit (miR-16, miR-25, miR-222 and miR-324-3p).Diagnostic kit can comprise the primer of these blood serum miRNAs combination, probe, and reagent such as Taq enzyme, dNTP.

7. statistical analysis technique

The difference that demographic characteristics, types of organization and the average expression level of miRNA distribute is compared in utilization x2 check (being used for classified variable) or student t check (being used for the continuous variable) between the research object group.

It is remarkable related that our the result of study discovery of integrated use Solexa order-checking and TLDA chip in exploratory sample population (48 routine mammary cancer cases and the contrast of 48 routine healthy womens) has 10 kinds of miRNAs and mammary cancer incidence to have.The different expression levels that individual miRNAs detects are with 2^{-Δ Ct}Expression, wherein Δ Ct=C_{The T sample}-C_{The T confidential reference items}, we add cel-mir-39 when each sample extraction RNA calculates relative expression quantity as reference.The miRNAs that statistically-significant difference is arranged is further verified in other 50 routine mammary cancer cases and 50 example contrasts, and then observe the degree of stability of this result of study.

The comprehensive indication that constitutes for further these four kinds of miRNAs of research is used for the effect of breast cancer diagnosis, and we have made up a mathematical formula, takes all factors into consideration positive and negative related situation and relation intensity that every kind of miRNA and mammary cancer are fallen ill.Specifically, at first one-sided 95% reference range with exploratory population health women control group miR-16, miR-25, miR-222 and miR-324-3p expression amount is a standard, expression level with these 4 kinds of miRNA is chosen as 0 fen and 1 minute respectively, we are weight with exploratory sample contrast crowd's regression coefficient more then, take all factors into consideration the expression of every kind of miRNA and determine a dangerous score value for each patient.The method of calculation of dangerous score value are as follows: dangerous score value=(scoring of 4.023 * miR-16)+(scoring of 3.646 * miR-25)+(scoring of 2.347 * miR-222)+(scoring of 3.219 * miR-324-3p), the danger of acquisition divide value coefficient and boundary value to be applied directly in checking sample population and the total 196 routine sample population.

Statistical analysis all by special statistical analysis software finish (SAS, v.9.1.3).The horizontal P value of significance,statistical is made as 0.05, and all statistical test are two-tailed test.

Below be further instruction of the present invention:

In above-mentioned 48 routine qualified mammary cancer cases and 48 routine healthy women contrasts, two groups of ages are by individual accurately coupling.We obtain correlated results as exploratory sample through Solexa order-checking test and TLDA chip detection with these two groups of crowds.

According to the Solexa sequence measurement, the inventor detects the miRNA that there are differences expression (2 groups copy number multiple difference is greater than 4) in the serum of " mammary cancer case " group and " healthy women contrast " group and comprises: hsa-let-7b, hsa-miR-1, hsa-miR-103, hsa-miR-107, hsa-miR-10a, hsa-miR-10b, hsa-miR-1246, hsa-miR-1294, hsa-miR-1301, hsa-miR-1307, hsa-miR-151-3p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-206, hsa-miR-22, hsa-miR-222, hsa-miR-223, hsa-miR-25, hsa-miR-30a, hsa-miR-320b, hsa-miR-320c, hsa-miR-324-3p, hsa-miR-330-3p, hsa-miR-339-3p, hsa-miR-342-5p, hsa-miR-361-3p, hsa-miR-375, hsa-miR-409-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-425, hsa-miR-432, hsa-miR-433, hsa-miR-451, hsa-miR-483-5p, hsa-miR-486-5p, hsa-miR-501-3p, hsa-miR-584, hsa-miR-629, hsa-miR-720, hsa-miR-744, hsa-miR-874, hsa-miR-92a, hsa-miR-92b, hsa-miR-99b.

According to the TLDA chip detection, the inventor detects the miRNA that there are differences expression (Δ Δ CT＞2) in the serum of " mammary cancer case " group and " healthy women contrast " group and comprises: has-miR-155, hsa-let-7a*, hsa-let-7b, hsa-let-7e, hsa-let-7g*, hsa-let-7g, hsa-miR-101, hsa-miR-105, hsa-miR-106a, hsa-miR-106b, hsa-miR-10b*, hsa-miR-10b, hsa-miR-122*, hsa-miR-125a-3p, hsa-miR-126*, hsa-miR-126, hsa-miR-130a, hsa-miR-132, hsa-miR-133a, hsa-miR-134, hsa-miR-135a*, hsa-miR-136*, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-1, hsa-miR-144*, hsa-miR-145*, h sa-miR-146a, hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-148b, hsa-miR-149, hsa-miR-150, hsa-miR-151-3p, hsa-miR-15a*, hsa-miR-16-1*, hsa-miR-16, hsa-miR-17*, hsa-miR-17, hsa-miR-182, hsa-miR-183*, hsa-miR-183, hsa-miR-185, hsa-miR-186, hsa-miR-18a*, hsa-miR-18b, hsa-miR-191, hsa-miR-193a-5p, hsa-miR-193b*, hsa-miR-195, hsa-miR-196b, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-19a, hsa-miR-19b, hsa-miR-200c, hsa-miR-205, hsa-miR-20a*, hsa-miR-20a, hsa-miR-20b, hsa-miR-21*, hsa-miR-210, hsa-miR-21, hsa-miR-214, hsa-miR-22*, hsa-miR-222, hsa-miR-223*, hsa-miR-223, hsa-miR-23a, hsa-miR-24, hsa-miR-25, hsa-miR-26a-1*, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27b, hsa-miR-29a, hsa-miR-29b-2*, hsa-miR-29b, hsa-miR-29c*, hsa-miR-29c, hsa-miR-301b, h sa-miR-30a, hsa-miR-30d, hsa-miR-30e*, hsa-miR-30e, hsa-miR-320, hsa-miR-32, hsa-miR-324-3p, hsa-miR-326, hsa-miR-330-3p, hsa-miR-335, hsa-miR-337-3p, hsa-miR-338-3p, hsa-miR-339-3p, hsa-miR-33a*, hsa-miR-340, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, hsa-miR-34a*, hsa-miR-34a, hsa-miR-361-3p, hsa-miR-362-3p, hsa-miR-374a*, hsa-miR-376a, hsa-miR-376c, hsa-miR-378*, hsa-miR-378, hsa-miR-382, hsa-miR-410, hsa-miR-422a, hsa-miR-424, hsa-miR-425*, hsa-miR-425, hsa-miR-429, hsa-miR-432, hsa-miR-449a, hsa-miR-449b, hsa-miR-451, hsa-miR-452, hsa-miR-454*, hsa-miR-454, hsa-miR-455-5p, hsa-miR-486-3p, hsa-miR-486-5p, hsa-miR-493*, hsa-miR-493, hsa-miR-494, hsa-miR-495, hsa-miR-497, hsa-miR-500*, hsa-miR-500, hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-505*, hsa-miR-512-3p, hsa-miR-515-3p, hsa-miR-516a-3p, hsa-miR-517a, hsa-miR-518a-3p, hsa-miR-519b-3p, hsa-miR-521, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-541*, hsa-miR-545*, hsa-miR-545, hsa-miR-548d-5p, hsa-miR-551a, hsa-miR-565, hsa-miR-569, hsa-miR-573, hsa-miR-575, hsa-miR-576-3p, hsa-miR-579, hsa-miR-580, hsa-miR-589*, hsa-miR-589, hsa-miR-590-5p, hsa-miR-597, hsa-miR-598, hsa-miR-601, hsa-miR-605, hsa-miR-616*, hsa-miR-622, hsa-miR-625, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629*, hsa-miR-636, h sa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-642, hsa-miR-645, hsa-miR-652, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-7, hsa-miR-744*, hsa-miR-768-3p, hsa-miR-770-5p, hsa-miR-801, hsa-miR-875-5p, hsa-miR-877, hsa-miR-886-3p, hsa-miR-888, hsa-miR-889, hsa-miR-93*, hsa-miR-933, hsa-miR-93, hsa-miR-941, hsa-miR-942, hsa-miR-9, hsa-miR-944, hsa-miR-99a*.

According to the result that above-mentioned two kinds of methods detect, select the miRNAs that satisfies following condition further to verify with the qRT-PCR method: 1) in the Solexa order-checking these miRNAs at least the copy number in one group of research object (" mammary cancer case " group or " healthy women contrast " are organized) greater than 50 and in the TLDA chip CT value of two groups of research objects all be not more than 35 with the raising detection efficiency; 2) Solexa order-checking reaches 4 times two groups multiple difference, and in the TLDA chip Δ Δ CT greater than 3, two kinds of method gained direction unanimity as a result; 3) Solexa order-checking reaches 8 times two groups multiple difference, and in the TLDA chip Δ Δ CT greater than 2, two kinds of method gained direction unanimity as a result.

The miRNAs that satisfies above-mentioned condition comprises: let-7b, miR-222, miR-25, miR-324-3p, miR-339-3p, miR-451, miR-486, miR-151-3p, miR-30a.In view of miR-16 is used as confidential reference items in some reports, we will include in the subsequent analysis by miR-16.Probe method and dye method qRT-PCR result all find have the expression of 4 kinds of miRNAs (miR-16, miR-25, miR-222, miR-324-3p) in " mammary cancer case " group and " healthy women contrast " group to have significant difference in 48 routine mammary cancer cases and 48 routine healthy women contrasts.

Logistic Regression Analysis result shows that the expression level of these 4 kinds of miRNAs all exists remarkable related with the morbidity of mammary cancer: 4 kinds of miRNAs are high expression level in case all.

According to The above results, with these 4 miRNAss relevant with mammary cancer morbidity other 50 routine mammary cancer cases and with 50 routine healthy womens contrasts of its age-matched in further verify.We find that blood serum high expression level miR-16, miR-25, miR-222, miR-324-3p all are associated with the mammary cancer morbidity, and the result of dye method is consistent with probe method.

The combination of further analyzing these 4 kinds of miRNA is used for the effect of breast cancer diagnosis, find the AUC[Area Under the ROC Curve of its combination to mammary cancer morbidity diagnosis, ROC curve (Receiver Operating CharacteristicCurve, experimenter's performance curve) is area down] compare increase with single miRNA.

According to above-mentioned experimental result, the inventor has prepared a kind of test kit that can be used for breast cancer diagnosis, comprises primer and other detection reagent of measuring stable existence in experimenter's blood serum and detectable ripe miR-16, miR-25, miR-222 and miR-324-3p.

Particularly, the combination of these 4 kinds of miRNAs, perhaps the dependent diagnostic test kit of the combination of primers of these 4 kinds of miRNAs formation helps the diagnosis of mammary cancer, for the clinician quick and precisely grasps the disease of patient state and the degree that is in a bad way, in time take the scheme of preventing and treating of more personalized to provide support, thereby improve patient with breast cancer's survival rate to greatest extent.

Beneficial effect of the present invention:

Blood serum miRNA provided by the invention (microRNAs/miRNAs) mark is as the superiority of the mark of breast cancer diagnosis:

(1) serum s is a kind of new bio mark, be different from the traditional biological mark, not only stable, Wicresoft, be easy to detect, and quantitatively accurately, the susceptibility and the specificity of medical diagnosis on disease will be improved greatly, the successful exploitation of such microRNA biomarker helps the auxiliary diagnosis of mammary cancer, for the development of other diseases biomarker is offered reference.

(2) serum s test kit be a kind of system, comprehensively the diagnosis and the monitoring reagent box, the auxiliary diagnosis that can be used for the patient with breast cancer, help to reflect patient with breast cancer's morbid state, for the clinician quick and precisely grasps conditions of patients, in time takes the scheme of preventing and treating of more personalized to provide support.

(3) adopt tight design and appraisement system, inventor's initial stage adopts Solexa genome sequencing technology that serum s is directly checked order, adopt the TLDA chip detection to obtain the serum s express spectra of the special and unconventionality expression of disease simultaneously, and the method for using qRT-PCR carried out the multistage checking in large sample, adopted two kinds of diverse ways (fluorescent probe method and dye method) to verify; The application of serum s biomarker and diagnostic kit is quickened and has been guaranteed in the application of above method and strategy, also provides reference on method and the strategy for the development of other diseases biomarker.

The present invention is by the influence factor of control age etc. to disease progression, and the research serum is set forth the influence of the miRNAs of unconventionality expression for the mammary cancer progress in the application prospect of mammary cancer auxiliary diagnosis, discloses its examination and diagnostic value.Therefore, the present invention has obtained relevant serum s expression database of mammary cancer morbidity and specificity marker thing; Development and application by serum s biomarker and diagnostic kit, can make that the diagnosis of mammary cancer is more convenient and easy, for the clinician quick and precisely grasps conditions of patients, for the clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug targets with potential therapeutic value offers help.

Description of drawings

Fig. 1. show the expression case line chart of 4 kinds of miRNA of case group and control group.

The expression case line chart that shows mammary cancer case group and healthy women control group.

DS: exploratory sample, IS: checking property sample, Com: merge exploratory sample and checking property sample.

Fig. 2. show the ROC curve of exploratory sample population case group and control group.

Show that mammary cancer case group is the ROC curve of reference to the healthy women control group.

Fig. 3. show the ROC curve of checking property sample population case group and control group.

Show that mammary cancer case group is the ROC curve of reference to the healthy women control group.

Embodiment

The collection of embodiment 1 sample and the arrangement of sample data

The contriver has collected a large amount of patient with breast cancers and the peripheral blood sample of contrast from Jiangsu Prov. Tumour Hospital and Changzhou Wujin District so far in beginning in 2006, and (sample that is used to study is to collect the same period, sampling, packing, preservation condition homogeneous), by the arrangement to the sample data, the contriver has therefrom selected 196 examples to meet the laboratory sample of the sample of following standard as Solexa order-checking, TLDA chip detection and follow-up a series of qRT-PCR checkings:

1, New Development mammary cancer case

2, perform the operation and chemicotherapy chemicotherapy before the nothing operation before the blood sampling without crossing

3, the healthy women with the case age-matched contrasts

And system acquisition situations such as the demography data of these samples, clinical data.

The Solexa of miRNA order-checking experiment in embodiment 2 blood serum

In above-mentioned qualified 48 routine patient with breast cancers and 48 routine healthy women contrasts, two groups of age-matched.These two groups of crowds are obtained correlated results through Solexa order-checking test.Concrete steps are:

1, gets " mammary cancer case " group and " healthy women contrast " group patient's serum 50ml respectively, add isopyknic Trizol reagent;

2, be separated: room temperature is placed 15min, and the volume ratio of pressing 0.2ml chloroform/1ml Trizol reagent then adds chloroform, concussion 15s, room temperature 15min, 12,000g, 4 ℃, centrifugal 15min;

3, water is transferred to the centrifuge tube of new 50ml, 3 step phenol/chloroform are removed the Deproteinization phase;

4, RNA precipitation: water is transferred in the new centrifuge tube, pressed 0.5ml Virahol/1ml Trizol reagent volume and add Virahol, preserve 60min, 12,000g, 4 ℃, centrifugal 60min for-20 ℃;

5, with the resuspended precipitation of 1ml Trizol reagent, suspension is transferred in the centrifuge tube of new 1.5ml;

6, repeated for 2,4 steps (the centrifugal 15min that changes into of the 4th step);

7, RNA washing: remove supernatant, add 75% ethanol, 12,000g, 4 ℃ of centrifugal 5min;

8, measure concentration: can obtain usually～5 μ g RNA/50ml serum;

9, total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule;

10, will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end; 11, carry out RT-PCR reaction back and checking order;

12, data analysis and processing: the miRNA of the serum differential expression that utilization Solexa sequence measurement is found in " mammary cancer case " group and " healthy women contrast " group is enumerated out hereinbefore.

The TLDA chip detection of miRNA in embodiment 3 blood serum

Above-mentioned 48 routine patient with breast cancers and the 48 routine healthy women contrasts of carrying out the Solexa order-checking are obtained correlated results through the TLDA chip detection.Concrete steps are:

1, gets " mammary cancer case " group and " healthy women contrast " group patient's serum 600 μ l respectively, add the Trizol reagent of 3 times of volumes;

2, be separated: room temperature is placed 15min, and adding final concentration is 10^-4The cel-39 (TAKARA) of pmol/ μ l adds and the isopyknic chloroform of blood plasma then as confidential reference items, concussion 50s, room temperature 15min, 14,000rpm, 4 ℃, centrifugal 15min;

3, RNA precipitation: water is transferred to the centrifuge tube of new 15ml, add the dehydrated alcohol of 1.5 times of water volumes, fully mixing;

4, with QIAGEN miRNeasy kit enrichment RNA: draw 700 μ l samples to centrifugal post at every turn, 14, the centrifugal 15s of 000rpm discards filtrate in the collection tube, is repeated to sample standard deviation and crosses post; Add 700 μ l washing lotions 1,14, the centrifugal 15s of 000rpm abandons filtrate in the collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 15s of 000rpm abandons filtrate in the collection tube; Add 500 μ l washing lotions 2,14 again, the centrifugal 2min of 000rpm abandons filtrate in the collection tube; Centrifugal post is put back in the empty collection tube, 14, the centrifugal 2min of 000rpm is with dry centrifuge tube; Centrifuge tube is put into new 1.5ml pipe, add 50 μ l nuclease free water, 10, the centrifugal 1min of 000rpm; Liquid in the pipe is refunded in the centrifugal post, 14, the centrifugal 1min of 000rpm abandons centrifugal post;

5, measure concentration: can obtain usually～1250ng RNA/600 μ l serum;

6, obtain cDNA with the supporting reverse transcription test kit of TLDA chip by the RNA reverse transcription reaction.The reaction system of reverse transcription comprises 0.8 μ l reverse transcriptase primer (10 *), 0.2 μ l 100mM dNTPs mixture, 1.5 μ l reversed transcriptive enzymes (50U/ μ L), 0.8 μ l, 10 * reverse transcription damping fluid, 0.9 μ l 25mM magnesium chloride, 0.1 μ l RNA inhibitor and 0.2 μ l nuclease free water.The total RNA that adds 3 μ l (1-350ng).Reactions steps is 16 ℃ hatched 2 minutes, and 42 ℃ were reacted 1 minute, and 50 ℃ were reacted 1 second, and above-mentioned 3 steps hatched 5 minutes for 85 ℃ through 40 circulating reactions again;

10, the amount that the specific miRNA of chip is increased in advance and expresses required CNDA to increase.The reaction system of pre-amplification comprises: 12.5 μ l increase in advance Master Mix (2 *), 2.5 μ l increase in advance primer (10 *), 7.5 μ l nuclease free water, 2.5 μ l CDNA.Reactions steps is: 95 ℃ 10 minutes → 55 ℃ 2 minutes → 72 ℃ 2 minutes → 95 ℃ 15 seconds, 60 ℃ 4 minutes, 12 the circulation → 99.9 ℃ 10 minutes; Reaction finishes the back and adds 75 μ l 0.1X TE dilution;

11, the pre-expansion of getting after 9 μ l dilute is increased production thing, adds 450 μ l genetic expression Master Mix, and 441 μ l nuclease free water fully behind the mixing, add 100 μ l/ holes on the TLDA chip; The centrifugal 1min of 1000rpm, centrifugal 2 times.What the experiment of TLDA chip was used is ABI Prism 7900 quantitative real time PCR Instruments.Select the specific program of 384-well TaqMan Low DensityArray to react;

12, data analysis and processing: carry out data processing, C with RQ-Manger software_TThreshold value is made as 0.2, Δ C_T=C_TSample-C_TU6(U6 is the endogenous U6 in the TLDA chip, in order to the switchboard differences), the expression amount ratio of two groups of sample serum miRNAs can be used equation Δ Δ C_TExpression, Δ Δ C_T=Δ C_{The T case}-Δ C_{The T contrast}-Δ C_Tcel-39, Δ C wherein_Tcel-39=C_Tcel-39Case-C_{The Tcel-39 contrast}, (difference when cel-39 extracts in order to control RNA).The miRNA of serum differential expression is enumerated out hereinbefore in " mammary cancer case " group that utilization TLDA chip detection is found and " healthy women contrast " group.

The qRT-PCR of miRNA experiment in embodiment 4 blood serum

According to above-mentioned Solexa and TLDA result, select the miRNAs satisfy following condition further to verify with the qRT-PCR method: 1) in the Solexa order-checking these miRNAs at least the copy number in one group of research object (" mammary cancer case " group or " healthy women contrast " are organized) greater than 50 and in the TLDA chip CT value of two groups of research objects all be not more than 35 with the raising detection efficiency; 2) Solexa order-checking reaches 4 times two groups multiple difference, and in the TLDA chip Δ Δ CT greater than 3, two kinds of method gained direction unanimity as a result; 3) Solexa order-checking reaches 8 times two groups multiple difference, and in the TLDA chip Δ Δ CT greater than 2, two kinds of method gained direction unanimity as a result.Primer (seeing Table 2) to 10 miRNAs design reverse transcriptions such as selected let-7b, miR-16, miR-222, miR-25, miR-324-3p, miR-339-3p, miR-451, miR-486, miR-151-3p, miR-30a and qRT-PCR.The single individuality of serum of " mammary cancer case " group and " healthy women contrast " group is carried out the qRT-PCR detection of miRNA, see Table 1.In whole research process, all implement strict Quality Control.Each sample continuous detecting three times.All detect and all to adopt blind method, promptly finish to avoid bias not knowing under the situation of sample background.Carrying out qRT-PCR with dye method and two kinds of methods of probe method respectively detects.

(1) preparation RNA sample: a) get 100 μ l serum; B) the Trizol room temperature that adds 3 times of volumes is placed 15min, and adding final concentration is 10^-4The cel-39 (TAKARA) of pmol/ μ l adds and the isopyknic chloroform of blood plasma then as confidential reference items, concussion 50s, room temperature 15min, 14,000rpm, 4 ℃, centrifugal 15min; C) water is transferred to the centrifuge tube of new 15ml, added the dehydrated alcohol of 1.5 times of water volumes, fully mixing; D) with the miRNeasy kit enrichment RNA of QIAGEN company: draw 700 μ l samples to centrifugal post at every turn, 14, the centrifugal 15s of 000rpm discards filtrate in the collection tube, is repeated to sample standard deviation and crosses post; Add 700 μ l washing lotions 1,14, the centrifugal 15s of 000rpm abandons filtrate in the collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 15s of 000rpm abandons filtrate in the collection tube; Add 500 μ l washing lotions 2,14 again, the centrifugal 2min of 000rpm abandons filtrate in the collection tube; Centrifugal post is put back in the empty collection tube, 14, the centrifugal 2min of 000rpm is with dry centrifuge tube; Centrifuge tube is put into new 1.5ml pipe, add 50 μ l nuclease free water, 10, the centrifugal 1min of 000rpm; Liquid in the pipe is refunded in the centrifugal post, 14, the centrifugal 1min of 000rpm abandons centrifugal post, with the liquid in the pipe as the RNA sample;

(2) probe method: use the ABI test kit.

A) obtain cDNA by the RNA reverse transcription reaction.The reverse transcription reaction system of probe method comprises one or more mixture of 0.15 μ l 100mMdNTPs mixture, 1 μ l reversed transcriptive enzyme (50U/ μ L), 1.5 μ l 10X reverse transcription damping fluids, 0.19 μ l RNA inhibitor and 3 μ l, 5 * reverse transcriptase primer.The total RNA that adds 9.16 μ l.Reactions steps is 16 ℃ hatched 30 minutes, and 42 ℃ were reacted 30 minutes, and hatched 5 minutes for 85 ℃;

B) q-PCR: cDNA is added the dilution of 5 μ l water, get the cDNA after 1 μ l dilutes, add 0.25 μ l20 * MicroRNA detection probes, 2.5 μ l, 2 * genetic expression Master Mix, 1.25 μ l distilled waters, 5 μ l systems are carried out q-PCR.What instrument used is ABI Prism 7900 quantitative real time PCR Instruments, and the reaction conditions of PCR is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 45 circulations in 60 ℃, 1 minute.

(3) dye method:

A) obtain cDNA by the RNA reverse transcription reaction.The reaction system of the reverse transcription of dye method comprises one or more mixture of 4 μ l, 5 * AMV damping fluid, 2 μ l 10mM dNTP mixtures (Takara company), 0.5 μ l RNase inhibitor (Takara company), 2 μ l AMV (Takara company) and 1.5 μ l miRNA specific reverse primers.Reactions steps is 16 ℃ hatched 15 minutes, and 42 ℃ were reacted 1 hour, and hatched 5 minutes for 85 ℃;

B) q-PCR: cDNA is pressed 1/5 volume dilution, get the cDNA after 0.5 μ l dilutes, add 0.15 μ l Taq enzyme (Takara company), 0.5 μ l 20 * EVA GREEN, 0.1 μ l 10 μ M forward primers are a kind of, 0.1 the general reverse primer of μ l 10 μ M (URP:TGGTGTCGTGGAGTCG, SEQ ID No.23), 0.6 μ l 25mM MgCl₂, 0.8 μ l 2.5mM dNTP mixture (Takara company), 1 μ l, 10 * PCR damping fluid, 6.75 μ l distilled waters, 10 μ l systems are carried out q-PCR.What instrument used is ABI Prism 7900 quantitative real time PCR Instruments, and the reaction conditions of PCR is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 45 circulations in 60 ℃, 1 minute.

(4) data processing and analysis

The expression amount ratio of two groups of sample serum miRNAs can be used equation 2^{-Δ Ct}Expression, wherein Δ Ct=C_{The T sample}-C_{The T confidential reference items}, we add cel-39 when each sample extraction RNA calculates relative expression quantity (cel-39:SEQ IDNo.21 and SEQ ID No.22) as reference.

Probe method and dye method qRT-PCR result all find in 96 routine samples, there be the expression of 4 kinds of miRNAs (miR-16, miR-25, miR-222 and miR-324-3p) between two groups to have significant difference, probe method the results are shown in Table 1, table 3, Fig. 1, and the dye method result is because of no longer listing with probe method is similar.

The further research of miRNA qRT-PCR experiment in embodiment 5 serum

According to The above results, these the 4 kinds miRNAss relevant with the mammary cancer morbidity are further detected in the healthy women contrast of other 50 routine patient with breast cancers and 50 routine age-matched.We find that miR-16, miR-25, miR-222 and miR-324-3p all are significantly higher than control group (table 4, Fig. 1) in mammary cancer case group expression of serum, and probe method is consistent equally with the dye method result.

Embodiment 6 utilizes the risk level methods of marking further to analyze the diagnosis of the combination of 4 kinds of miRNA to the mammary cancer morbidity

According to above-mentioned Real-time PCR result, the inventor passes through the analysis to the miRNAs expression level of 2 groups of plasma samples (" mammary cancer case group " and " healthy women control group "), with exploratory population health women control group miR-16, miR-25, one-sided 95% reference range of miR-222 and miR-324-3p expression amount is a standard, these 4 kinds of miRNA are marked, expression amount is 0 minute less than the scoring of the 95th percentile, expression amount is 1 minute more than or equal to the scoring of the 95th percentile, with the regression coefficient is weight, further try to achieve dangerous score value, median (median=2.783) with dangerous score value is a dividing value, draw ROC and come the susceptibility and the specificity of evaluation prediction, and then assess the judgement of these 4 kinds of miRNAs high expression levels the mammary cancer morbidity.Conjoint Analysis to 4 marks finds that in exploratory sample population, these 4 kinds of miRNAs open healthy women control group and mammary cancer carninomatosis example component with 91.7% AUC, and the sensitivity of best stagnation point is 91.7%, specific degree: 91.7%; In the checking sample population, these 4 kinds of miRNAs open healthy women control group and mammary cancer carninomatosis example component with 93.0% AUC, and the sensitivity of best stagnation point is 92.0%, specific degree: 94.0% (Fig. 2,3).Using dividing value false determination ratio in exploratory sample population of dangerous score value is 8.3% (8/96), and false determination ratio is 8% (8/100) (table 5) in checking property sample population.

Therefore, the inventor has proved that employing miR-16, miR-25, miR-222 and miR-324-3p can distinguish healthy women contrast and patient with breast cancer well.

Embodiment 7 is used for the making of the miRNA test kit of mammary cancer auxiliary diagnosis

The making of miRNA test kit and operating process are based on solexa order-checking, TLDA chip detection, technology such as RT-PCR and real-time PCR.Test kit comprises that (primer that comprises following primer: miR-16 is SEQ ID No.19 and SEQ ID No.20 to the serum primer; The primer of miR-25 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-222 is SEQ ID No.17 and SEQ ID No.18; The primer of miR-324-3p is SEQ ID No.13 and SEQ IDNo.14), required enzyme commonly used and/or the reagent of corresponding PCR reaction can also be arranged, as: reversed transcriptive enzyme, damping fluid, dNTPs, MgCl2, remove nuclease water, fluorescence dye or probe, the Taq enzyme, general reverse primer (URP:TGGTGTCGTGGAGTCG, SEQ ID NO:23) etc., can select for use according to the experimental technique of concrete employing, these enzymes commonly used and/or reagent are well known to those skilled in the art, and standard substance and contrast (as nematode mir-39 sample of quantitative markization etc.) and normal reference value (miR-16:5.84*10 can also be arranged in addition^-2, mir-25:2.26*10^-3, miR-222:2.75*10^-3, miR-324-3p:3.60*10^-4).The value of this test kit is only to need blood serum and does not need other tissue sample, detect the variation tendency of miRNA by the fluorescence of simplifying most or probe method, again by this trend auxiliary diagnosis mammary cancer, not only stable, easy to detect, and quantitatively accurately, improve the susceptibility and the specificity of medical diagnosis on disease greatly, therefore with this test kit input practice, can help to instruct the clinical diagnosis of accurately making.

The single factor logistic regression analysis result (95% reference value with the control group expression values is a dividing value) of the exploratory sample population of table 3

The single factor logistic regression analysis result (95% reference value with exploratory sample control group expression values is a dividing value) of table 4 checking property sample population

The misjudgement ratio of the dividing value gained of the exploratory sample population ROC curve of table 5 utilization

Claims (8)

1. a serum mark relevant with mammary cancer is characterized in that this mark is the combination of miR-16, miR-25, miR-222 and miR-324-3p.

2. the primer of the described serum mark of claim 1 is characterized in that this primer is:

The primer of miR-16 is SEQ ID No.19 and SEQ ID No.20; The primer of miR-25 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-222 is SEQ ID No.17 and SEQ ID No.18; The primer of miR-324-3p is SEQ ID No.13 and SEQ ID No.14.

3. the application of the described serum mark of claim 1 in preparation mammary cancer auxiliary diagnostic box.

4. the application of the primer of the described serum mark of claim 2 in preparation mammary cancer auxiliary diagnostic box.

5. a mammary cancer auxiliary diagnostic box is characterized in that this test kit is used for detecting blood serum miR-16, miR-25, miR-222 and miR-324-3p.

6. diagnostic kit according to claim 5 is characterized in that this test kit contains the primer of miR-16 in the serum, miR-25, miR-222 and miR-324-3p.

7. diagnostic kit according to claim 5 is characterized in that this test kit contains the primer of the described serum mark of claim 2.

8. according to claim 6 or 7 described diagnostic kits, it is characterized in that this test kit can also comprise PCR reaction enzyme and reagent commonly used.

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