Blood plasma microRNAs is used to prepare patients with lung adenocarcinoma in sieving and diagnosis female group
The purposes of diagnostic reagent
Technical field
The invention belongs to clinical diagnosis fields, are related to adenocarcinoma of lung sieving and diagnosis reagent, and in particular to blood plasma microRNAs is used
In the purposes for the diagnostic reagent for preparing sieving and diagnosis patients with lung adenocarcinoma.
Background technique
Lung cancer is the most common malignant tumour in the whole world, and the death rate occupies first of malignant tumour.China's lung cancer morbidity rate present by
Year ascendant trend, average annual growth rate nearly 2%.There are many organization type, cancerous lung tissues according to the difference of Pathologic Characteristics for lung cancer
Type is different, and remedy measures are also different.Classified according to 2004 editions WHO, common cancerous lung tissue histological typing is divided into non-small thin
Born of the same parents' cancer (NSCLC) and small cell carcinoma (SCLC).Non-small cell carcinoma is divided into squamous cell carcinoma (SCC), gland cancer (AC) and maxicell again
Cancer (LCC).Different lung cancer clinical therapeutic schemes are different, and outcome is also different.Therefore, in order to improve therapeutic effect, lung cancer
Treatment mode of the therapeutic strategy from traditional based on by stages is changed into histological type and gene mutation as guidance
Individuation, accurately multimodality therapy mode.Individualized treatment improves treatment and the outcome of lung cancer.
Gland cancer is in rising trend in the whole world and China's disease incidence as the most common cancerous lung tissue type.Epidemic disease
It learns result of study and shows that the morbidity of non-small cell lung cancer has apparent gender differences, especially gland cancer.Gland cancer accounts for primary lung cancer
50%, it is the major histological type of non-smoking patient.It is at present for table lung cancer research is most deep, clinical application is most
The small molecule tyrosine kinase inhibitors of skin growth factor Receptor EGFR, and Iressa, Gefitinib and Tarceva are its masters
The representative wanted.With going deep into this micromolecular target therapeutic agent research, facing for its curative effect and patients with lung cancer is gradually found
Bed feature is related, and wherein asian ancestry, women, gland cancer, non-smoking are main feature (Yang Xin outstanding person etc., the salt of its clinical " advantage crowd "
The clinical observation on the therapeutic effect of sour Conmana first-line treatment advanced pulmonary adenocarcinoma, lung cancer in China magazine in July, 2013;Ma Zhiyong etc., easily
Auspicious husky treatment male, non-smoking or the advanced pulmonary adenocarcinoma patient's effectiveness study slightly smoked, medical forum's magazine 2010 11
Month).
These results allow researcher to obtain a supposition: disease incidence of the gland cancer in male and female, pathogenesis and facing
Bed therapeutic effect has differences.In order to adapt to personalized treatment, it is necessary to a point genders to carry out for the research of gland cancer.
Zhongshan Hospital Attached to Fudan Univ doctor Huang Wei is in thesis " tumor marker in three kinds of subtypes cancerous lung tissues
The microRNAs marker for pulmonary cancer diagnosis is had studied in detail in the preliminary screening of the discovery of object, verifying and its target gene ".
Firstly, author is using laser capture microdissection technology from 44 normal lung tissues, 36 gland cancer, 30 squamous carcinomas and 16 small thin
Obtain pure epithelial cell and tumour cell in born of the same parents' cancer, carry out full-length genome microRNAs expression analysis, by variance analysis and
Clustering finds that the microRNAs of 16 differential expressions can distinguish three kinds of lung cancer subtypes (adenocarcinoma of lung, lung squamous cancer and small
Cell lung cancer), and finally determine that 7 therein are further verified as candidate markers;Then, author is in three kinds of pathology
7 candidate's microRNAs tumor markers are verified in hypotype cancerous lung tissue, establish the regression model of diagnosing subtypes,
And analyze the prognostic value of 7 candidate microRNAs, as a result, it has been found that: single candidate microRNA identification normal lung tissue and
The AUC value of adenocarcinoma of lung is undesirable, establishes model discovery hsa-miR-375 and hsa-miR-34a by Logistic regression analysis
Two available best AUC 0.910 that cooperate, sensibility 80%, specificity 97%, susceptibility is lower.That is,
By the research of author, the joint of either single microRNA or multiple microRNAs are unable to efficient diagnosis and distinguish just
Normal lung tissue and adenocarcinoma of lung, accuracy and susceptibility are not fully up to expectations.Moreover, this method is a kind of based on marker in tissue
Method needs to take the lung tissue of patient for detecting, is still a kind of intervention diagnosis method, it has not been convenient to.
It is applicant's understanding that above-mentioned doctoral thesis, which fails to filter out efficient diagnosis, distinguishes normal lung tissue and adenocarcinoma of lung
The key reason of microRNAs is to ignore the gender differences of adenocarcinoma of lung, and sample classification mistake can not obtain ideal knot naturally
Fruit.
Summary of the invention
The purpose of the present invention is to provide the diagnostic reagents that blood plasma microRNAs is used to prepare sieving and diagnosis patients with lung adenocarcinoma
Purposes;Specifically, one group of blood plasma is provided according to gender differences to improve accuracy, susceptibility and the specificity of screening
MicroRNAs marker is used to prepare the diagnostic reagent of patients with lung adenocarcinoma in sieving and diagnosis male population, provides another set blood
Slurry microRNAs marker is used to prepare the diagnostic reagent of patients with lung adenocarcinoma in sieving and diagnosis female group.
Above-mentioned purpose is achieved by the following technical solution:
The technical solution of patients with lung adenocarcinoma is as follows in sieving and diagnosis male population:
One group of blood plasma microRNAs marker for patients with lung adenocarcinoma in sieving and diagnosis male population, by as follows
MicroRNAs composition: miR-224-3p, miR-146a-5p, miR-564, miR-615-5p, miR-601;MiR-224-3p's
Nucleotide sequence is as shown in SEQ ID NO.1;The nucleotide sequence of miR-146a-5p is as shown in SEQ ID NO.2;miR-564
Nucleotide sequence as shown in SEQ ID NO.3;The nucleotide sequence of miR-615-5p is as shown in SEQ ID NO.4;miR-601
Nucleotide sequence as shown in SEQ ID NO.5.
The combination of the microRNAs primer, probe of patients with lung adenocarcinoma, microRNAs in one group of sieving and diagnosis male population
Primer includes primer after primer and quantitative PCR before reverse transcription primer, quantitative PCR: the reverse transcription primer sequence of miR-224-3p is such as
Shown in SEQ ID NO.9, primer sequence is as shown in SEQ ID NO.17 before quantitative PCR, primer sequence such as SEQ ID after quantitative PCR
Shown in NO.25;Primer sequence is such as shown in SEQ ID NO.10, before quantitative PCR for the reverse transcription primer sequence of miR-146a-5p
Shown in SEQ ID NO.18, primer sequence is as shown in SEQ ID NO.25 after quantitative PCR;The reverse transcription primer sequence of miR-564
As shown in SEQ ID NO.11, primer sequence is as shown in SEQ ID NO.19 before quantitative PCR, primer sequence such as SEQ after quantitative PCR
Shown in ID NO.25;Primer sequence is such as shown in SEQ ID NO.12, before quantitative PCR for the reverse transcription primer sequence of miR-615-5p
Shown in SEQ ID NO.20, primer sequence is as shown in SEQ ID NO.25 after quantitative PCR;The reverse transcription primer sequence of miR-601
As shown in SEQ ID NO.13, primer sequence is as shown in SEQ ID NO.21 before quantitative PCR, primer sequence such as SEQ after quantitative PCR
Shown in ID NO.25;The nucleotide sequence of each microRNAs probe is as shown in SEQ ID NO.26.
A kind of diagnostic reagent for patients with lung adenocarcinoma in sieving and diagnosis male population, containing above-mentioned microRNAs primer,
The combination of probe.
A kind of diagnostic kit for patients with lung adenocarcinoma in sieving and diagnosis male population, draws containing above-mentioned microRNAs
The combination of object, probe.
Further, the diagnostic kit also contains the common enzyme of PCR reaction and reagent, such as reverse transcriptase, buffering
Liquid, dNTPs, MgCl2, DEPC water and Taq enzyme etc. and standard items and/or reference substance.
The technical solution of patients with lung adenocarcinoma is as follows in sieving and diagnosis female group:
One group of blood plasma microRNAs marker for patients with lung adenocarcinoma in sieving and diagnosis female group, by as follows
MicroRNAs composition: miR-224-3p, miR-146a-5p, miR-648, miR-523-5p, miR-758-5p;miR-224-3p
Nucleotide sequence as shown in SEQ ID NO.1;The nucleotide sequence of miR-146a-5p is as shown in SEQ ID NO.2;miR-
648 nucleotide sequence is as shown in SEQ ID NO.6;The nucleotide sequence of miR-523-5p is as shown in SEQ ID NO.7;miR-
The nucleotide sequence of 758-5p is as shown in SEQ ID NO.8.
The combination of the microRNAs primer, probe of patients with lung adenocarcinoma, microRNAs in one group of sieving and diagnosis female group
Primer includes primer after primer and quantitative PCR before reverse transcription primer, quantitative PCR: the reverse transcription primer sequence of miR-224-3p is such as
Shown in SEQ ID NO.9, primer sequence is as shown in SEQ ID NO.17 before quantitative PCR, primer sequence such as SEQ ID after quantitative PCR
Shown in NO.25;Primer sequence is such as shown in SEQ ID NO.10, before quantitative PCR for the reverse transcription primer sequence of miR-146a-5p
Shown in SEQ ID NO.18, primer sequence is as shown in SEQ ID NO.25 after quantitative PCR;The reverse transcription primer sequence of miR-648
As shown in SEQ ID NO.14, primer sequence is as shown in SEQ ID NO.22 before quantitative PCR, primer sequence such as SEQ after quantitative PCR
Shown in ID NO.25;Primer sequence is such as shown in SEQ ID NO.15, before quantitative PCR for the reverse transcription primer sequence of miR-523-5p
Shown in SEQ ID NO.23, primer sequence is as shown in SEQ ID NO.25 after quantitative PCR;The reverse transcription primer sequence of miR-758-5p
Column are as shown in SEQ ID NO.16, and primer sequence is such as shown in SEQ ID NO.24, after quantitative PCR for primer sequence before quantitative PCR
Shown in SEQ ID NO.25;The nucleotide sequence of each microRNAs probe is as shown in SEQ ID NO.26.
A kind of diagnostic reagent for patients with lung adenocarcinoma in sieving and diagnosis female group, containing above-mentioned microRNAs primer,
The combination of probe.
A kind of diagnostic kit for patients with lung adenocarcinoma in sieving and diagnosis female group, draws containing above-mentioned microRNAs
The combination of object, probe.
Further, the diagnostic kit also contains the common enzyme of PCR reaction and reagent, such as reverse transcriptase, buffering
Liquid, dNTPs, MgCl2, DEPC water and Taq enzyme etc. and standard items and/or reference substance.
Beneficial effects of the present invention:
One group of blood plasma microRNAs marker provided by the invention can accurately be used for lung gland in sieving and diagnosis male population
Cancer patient, high sensitivity, high specificity, realize male population in patients with lung adenocarcinoma without intervention diagnosis;It is provided by the invention another
One group of blood plasma microRNAs marker can accurately be used for patients with lung adenocarcinoma in sieving and diagnosis female group, high sensitivity, specifically
Property it is strong, realize female group in patients with lung adenocarcinoma without intervention diagnosis.
Detailed description of the invention
Fig. 1 is 5 microRNAs (miR-224-3p, miR-146a-5p, miR-564, miR-615-5p, miR-601)
The ROC curve figure of Combining diagnosis differentiation male's case group and men's health control group;
Fig. 2 is 5 microRNAs (miR-224-3p, miR-146a-5p, miR-648, miR-523-5p, miR-758-
5p) Combining diagnosis distinguishes the ROC curve figure of women case group and women's health control group;
Fig. 3 is that verifying is concentrated with 5 microRNAs (miR-224-3p, miR-146a-5p, miR-564, miR-615-
5p, miR-601) Combining diagnosis distinguish male's case group and men's health control group accuracy figure;
Fig. 4 is that verifying is concentrated with 5 microRNAs (miR-224-3p, miR-146a-5p, miR-648, miR-523-
5p, miR-758-5p) Combining diagnosis distinguish women case group and women's health control group accuracy figure.
Specific embodiment
Technical solution of the present invention and technical effect is discussed in detail with attached drawing combined with specific embodiments below.
Patients with lung adenocarcinoma and healthy volunteer from September, 2014 to during in September, 2015 in Nanjing General Hospital, Nanjing Military Area Command, PLA
It is collected with Nanjing drum tower hospital, blood is taken to agree to through patient and passes through Nanjing General Hospital, Nanjing Military Area Command, PLA and Nanjing drum tower hospital ethics committee
Member can examine.Male's case group is made of 96 Primary Pulmonary Adenocarcinoma male patients, and women case group is by 82 primary pulmonary glands
Cancer female patient composition, take do not performed the operation before blood, chemotherapy, radiotherapy or endocrine therapy.Men's health control group and female
Sex-health control group is made of 58 men's health volunteers and 54 women's health volunteers respectively.The sample age is in 25-
Between 60 years old, age equal nothing between male's case group and men's health control group and women case group and women's health control group
Statistical difference (P value is respectively 0.072 and 0.068).All patients and healthy volunteer pass through histopathology confirmation.It will be male
Property case group, women case group, men's health control group, women's health control group be divided in half into test set and verifying collection at random.
Test set is used for the discovery of blood plasma difference microRNAs, and is used preliminarily for evaluation difference microRNAs marker screening male
In property group in patients with lung adenocarcinoma and screening female group patients with lung adenocarcinoma diagnostic;Verifying collection is for further verifying
The diagnostic accuracy of microRNAs marker.Sample packet situation is as follows:
Embodiment 1: blood plasma difference microRNA discovery and quantitative confirmation based on microRNA chip
One, experimental material
1, instrument reagent
Supercentrifuge, high speed freezing centrifuge, ultraviolet specrophotometer are purchased from Eppendorf company, Germany;
1000 spectrophotometer of NanoDrop is purchased from the silent winged generation that Thermo Scientific company of U.S.'s match;Agilent
2100Bioanalyzer System is purchased from U.S. Agilent company;Common PCR reaction instrument and real-time fluorescence PCR instrument are purchased from beauty
PE company, state;DYCP-31B type electrophoresis apparatus is purchased from 61 Biotechnology Co., Ltd of Beijing;BIO-RAD gel imaging analysis is purchased from
U.S. Bole;Ultra low temperature freezer is purchased from Haier Group.mirvanaTMparisTMKit is purchased from U.S. Ambion company;Agilent
RNA 6000Pico Kit is purchased from U.S. Agilent company;Trizol total RNA extraction reagent is public purchased from U.S. Invitrogen
Department;TaqMan MicroRNAReverse Transcription Kit,TaqMan Universal PCR Master Mix,
CDNA synthetic agent box is purchased from the silent winged generation that Thermo Scientific company of U.S.'s match;Primer, probe student on commission's work biology work
The synthesis of journey (Shanghai) limited liability company.Non- special emphasis instrument and reagent are the conventional use of instrument of those skilled in the art
Device and reagent, and be easily obtained.
2, laboratory sample
Extract test set patients with lung adenocarcinoma and healthy volunteer peripheral blood 2mL in the morning on an empty stomach, is put into EDTA pipe, gently mixes
It is even, anticoagulant substances in blood and pipe are come into full contact with, blood cell breakage is prevented.EDTA pipe is put into 4 DEG C of refrigerators, is divided in 2 hours
From blood plasma.Firstly, 820g revolving speed is centrifuged 10 minutes, upper phase is carefully sucked out, avoids drawing middle white cellular layer.It will be upper
Layer liquid phase is transferred to the 1.5mL centrifuge tube being pre-chilled in advance, and 16000g revolving speed continues centrifugation 10 minutes, so as to further separated plasma and
Haemocyte.The blood plasma obtained after second step centrifugation is transferred to -80 DEG C of refrigerator long-term preservations with the packing of 500 μ L volumes.
Other materials includes that conventional reagent, conventional instrument etc. are the material that those skilled in the art know and are easily obtained
Material.The preparation of related reagent is operated according to product description, or is by conventional method well known to those skilled in the art preparation
It can.
Two, experimental method
1, the extraction of blood plasma total serum IgE
mirvanaTMparisTMKit is for extracting blood plasma total serum IgE, 1000 spectrophotometric determination total serum IgE of NanoDrop
Concentration.Agilent RNA 6000Pico Kit is passed through by Agilent 2100Bioanalyzer to the evaluation of RNA mass
System is completed.Method is referring to respective specification.With RNA complete exponential (RIN) measure total serum IgE quality, the value from 1 to
10 react the quality of sample respectively.RIN >=7-10 indicates high-quality, and RIN >=5 indicates medium quality, and RIN < 5 indicates of poor quality.
In this experiment, the qualified standard using RIN > 5 as extracted total RNA.
2, full-length genome microRNA is analyzed
The detection of microRNA chip and interpretation of result are completed by Shanghai Biochip Co., Ltd.MicroRNA chip packet
Containing 723 people microRNAs and 76 Human virus's microRNAs probes, microRNA data come from Sanger v.10.1 data
Library.Each chip includes eight sample application sites.Total serum IgE (100ng) is marked by Cy3, and chip passes through XDR Scan
(PMT100, PMT5) scans the signal on chip.Label and hybrid process are according to Agilent microRNA chip system explanation
Book operation.Chip image information is converted to intensity value by software, and after eliminating background noisy signal, signal strength indication is directly defeated
Enter to software and is analyzed.In the detection process to sample, it is found that hsa-miR-1228 stablizes expression in blood plasma, therefore select
The original signal that hybridization obtains is standardized as internal reference, obtains the log value with 2 for the truth of a matter by hsa-miR-1228.If
One sample repeats the numerical value of detection on chip, and the coefficient of variation is greater than 15% or positive signal value is less than 5%, it is believed that should
Sample quality is unqualified, will be excluded in next step test.The microRNA that can detecte is defined as being more than 50%
In sample, chip can detect positive signal.
3, real-time fluorescence quantitative PCR
Referring in particular to kit TaqMan MicroRNAReverse Transcription Kit, TaqMan
The specification of MicroRNAAssay, TaqMan Universal PCR Master Mix, each sample applied sample amount are 100ng,
Reaction step is as follows:
100ng RNA sample is added in each RT reaction tube, and Nuclease-free water supplies volume;It is added each
It is slightly centrifuged mixing after reacted constituent, executes following procedure in PCR reaction instrument:
16 DEG C of 30min → 42 DEG C 30min → 85 DEG C 5min → 4 DEG C save.
It is carried out on real-time fluorescence quantitative PCR instrument referring to TaqMan MicroRNAAssay specification, system and condition are such as
Under:
Each reacted constituent is added and is slightly centrifuged after mixing well, each 10 μ L of hole, each sample does three multiple holes.Glimmering
Following procedure is executed on Fluorescent Quantitative PCR instrument:
40 circulations in total.Fluorescence data acquisition, absorbing wavelength 490nm are carried out at 60 DEG C, release wavelength is 530nm.
Cp value is calculated by SDS software by secondary derivatization method.
4, data statistic analysis
The most stable of hsa-miR-1228 using in chip test result is compareed as internal reference, utilizes Agilent Feature
Extraction software carries out data quantitative analysis.The image information of chip passes through Scanner Control Rev.7.0 software
Be converted to density value.Signal is introduced directly into GeneSpring GX10 software after background is eliminated and is standardized.To repeat to test
The Average normalized data comparative studies analysis obtained.Benjamini-Hochberg check and correction non-paired t test (p≤
0.01).Clustering is carried out using hierarchical clustering algorithm software, filters out male's adenocarcinoma of lung
Compareed with men's health, during female pulmonary adenocarcinoma is compareed with women's health with the blood plasma microRNAs of significant difference.
Real-time fluorescence PCR data are standardized using internal reference identical with chip, and non-paired t test determines group difference.P <
0.05 is set as statistical difference.F is examined and T check analysis is expressed by the microRNAs that fluorescence real-time quantitative PCR detects
Significant difference between value is analyzed by SPSS software and is realized, it is bilateral that p < 0.05, which thinks statistically significant,.
Test method without specific conditions, usually according to normal condition, such as described in textbook and experiment guide
Condition be or according to the normal condition proposed by manufacturer well known within the skill of those ordinarily skilled or be easy to know.
Three, experimental result
The discovery of microRNA chip test result, compared with men's health control group, there are a large amount of up-regulations in male's case group
Or the microRNAs of expression is lowered, part microRNAs differential expression is clearly;Compared with women's health control group, women
There are a large amount of up-regulations or lowers the microRNAs of expression in case group, and part microRNAs differential expression is clearly.
Since microRNA chip is indirectly that there are still spies in the analysis process to the research of microRNA expression characteristic
The disadvantages of anisotropic and susceptibility is not high.Therefore, acquired results have certain false positive, the expression for needing to assist other more accurate
Research method is verified, this experiment is identified using fluorescence real-time quantitative RT-PCR.
As a result 13 blood plasma difference microRNA obtain the confirmation of fluorescence real-time quantitative RT-PCR, expression is as follows:
In upper table, the variation of oblique line representation transformation is unobvious.After obtaining above-mentioned blood plasma difference microRNA, carry out real in next step
It tests, single blood plasma difference microRNA is evaluated using Receiver operating curve (ROC curve) and its joint is used for diagnostic region
Divide the diagnostic that male's adenocarcinoma of lung is compareed with men's health, female pulmonary adenocarcinoma is compareed with women's health.Wherein, due to miR-
205, the p value of miR-206 and miR-155-5p is greater than 0.05, is not included in experiment in next step and investigates.
The diagnostic of embodiment 2:ROC curve evaluation blood plasma difference microRNA
The principle of ROC curve evaluation assessment:
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden index, ROC,
AUC etc..Evaluation for diagnostic test, first it will be appreciated that the true classification of subject, i.e., which belongs to healthy group, which belongs to
In disease group.The standard for dividing health group and disease group is exactly goldstandard (the histopathogenic diagnosis method generally acknowledged in such as the application).
For the disease group and healthy group determined by goldstandard, following situation can be divided into using the result that diagnostic test detects:
Positive (True Positive, TP);Diagnostic test test positive (consistent with goldstandard result);
Negative (True Negative, TN);Diagnostic test is detected as negative (consistent with goldstandard result);
False positive (False Positive, FP): diagnostic test test positive (inconsistent with goldstandard result);
False negative (False Negative, FN): diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be indicated with following table:
Susceptibility=A/ (A+C) of diagnostic test;Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically
Property is it can be concluded that diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.Disease example is examined in the representative of susceptibility height
Break few for negative number, rate of missed diagnosis is low;The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With diagnosis possible in diagnostic test
Dividing value calculates corresponding susceptibility and specificity as diagnostic points, according to above table.Then, using susceptibility as ordinate,
1- specificity is abscissa, the susceptibility of each point when each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point
Smoothed curve is obtained, which is ROC curve.Diagnostic points are arranged much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, and the size of area under the curve AUC shows
The size of diagnostic test accuracy.Intrinsic standard of the area AUC (ROCAUC) as diagnostic test Authentic Assessment under ROC curve
Exactness index has been commonly recognized, and complete unworthy diagnostic test AUC is 0.5, and ideal diagnostic test AUC is 1, and general
Think that diagnostic value is lower when ROC AUC is between 0.5~0.7 for a diagnostic test, is diagnosed when between 0.7~0.9
It is worth medium, at 0.9 or more, diagnostic value is higher.
1, when single blood plasma difference microRNA distinguishes disease group and control group for diagnosing, the method for drafting of ROC curve
By the content of 13 blood plasma difference microRNA above-mentioned in all samples of test set after internal reference standardizes, marked
Quasi-ization value draws ROC curve using each possible standardized value as diagnostic points according to the method described above.
13 blood plasma difference microRNA, which are individually diagnosed, distinguishes andropathy example group VS. men's health control group, women case
Under the ROC curve of group VS. women's health control group at area AUC and best cut-off value susceptibility and specificity such as following table institute
Show:
The above results show the 10 blood plasma differences for passing through microRNA chip and confirming through real-time fluorescence quantitative RT-PCR
MicroRNAs be individually used for diagnosis distinguish male's adenocarcinoma of lung and men's health control or female pulmonary adenocarcinoma and women's health control
Diagnostic is lower, and for AUC between 0.5~0.7, diagnostic value is lower.
2, ROC curve drafting side when multiple blood plasma difference microRNAs joints distinguish disease group and control group for diagnosing
Method
The content of (male and female respectively there are 7) blood plasma difference microRNA 7 above-mentioned in all samples of test set is passed through
After internal reference standardization, standardized value is obtained, using male's adenocarcinoma of lung sample as group 1, men's health volunteer's sample is as group
Other 2 (or female pulmonary adenocarcinoma sample, as group 1, women's health volunteer's sample is as groups 2), to above-mentioned 7 blood plasma difference
Standardized value of any number of blood plasma difference microRNAs in two groups of samples carries out dualistic logistic regression in microRNAs, obtains
To dualistic logistic regression equation.Then, the standardized value of any number of blood plasma difference microRNAs of this in each sample is substituted into should
The regressand value of each sample can be obtained in dualistic logistic regression equation, using possible regressand value as diagnostic points, according to above-mentioned side
Method draws ROC curve.Diagnosis curve when the ROC curve is any number of blood plasma difference microRNAs Combining diagnosis, it is bent
Susceptibility and specificity can embody any number of blood plasma difference microRNAs' at area AUC and best cut-off value under line
Combining diagnosis efficiency.
The result shows that: this 5 blood plasma of miR-224-3p, miR-146a-5p, miR-564, miR-615-5p, miR-601 are poor
Different microRNAs joint distinguishes diagnostic highest when andropathy example group VS. men's health control group for diagnosing, and best
Susceptibility and specificity are high at cut-off value;miR-224-3p,miR-146a-5p,miR-648,miR-523-5p,miR-
Examining when this 5 blood plasma difference microRNAs joints of 758-5p distinguish women case group VS. women's health control group for diagnosing
Disconnected efficiency highest, and susceptibility and specificity are high at best cut-off value.As a result such as following table and Fig. 1 and Fig. 2:
Applicant further has rated 5 joint differences that andropathy example group and men's health control group are distinguished in diagnosis
MicroRNAs is to the diagnostic of women case group and women's health control group, and area AUC is only 0.586 under ROC curve;Shen
It asks someone also further to have rated 5 microRNAs pairs of difference of joints that women case group and women's health control group are distinguished in diagnosis
The diagnostic of male's case group and men's health control group, area AUC is only 0.614 under ROC curve.The result is also further
It proves, adenocarcinoma of lung has apparent gender differences, this may be related with the pathogenesis difference of different sexes adenocarcinoma of lung, internal generation
Thanking to network, there is also notable differences, treat with a certain discrimination when being diagnosed, and different sexes can just be obtained using different diagnosis indexes
Obtain high-accuracy.
Embodiment 3: the accuracy of further verifying blood plasma difference microRNAs Combining diagnosis is concentrated in verifying
One, experimental material
With embodiment 1.
Two, experimental method and result
1, the method for the extraction of blood plasma total serum IgE and real-time fluorescence quantitative PCR is the same as embodiment 1.
2, it concentrates in verifying, is volunteered verifying collection male's adenocarcinoma of lung and men's health based on above-mentioned dualistic logistic regression equation
5 blood plasma difference microRNAs (miR-224-3p, miR-146a-5p, miR-564, miR-615-5p, miR- in person's sample
601) standardized value of content makees dualistic logistic regression transformation, calculates 5 blood plasma difference microRNAs in male's sample
The logistic regression value of content.It is predicted as men's health volunteer lower than best cut-off value 0.568, is higher than best cut-off
Value 0.568 is predicted as male's adenocarcinoma of lung, finally calculates with 5 metabolic markers horizontal forecast male adenocarcinomas of lung and male
The accuracy rate of healthy volunteer's grouping.As a result such as Fig. 3,5 blood plasma difference microRNAs (miR-224-3p, miR- are based on
146a-5p, miR-564, miR-615-5p, miR-601) verifying collection sample in predictablity rate be 98.7%, only by 1 male
Sex-health volunteer's mistaken diagnosis is patients with lung adenocarcinoma, and accuracy rate is very high.
3, it concentrates in verifying, is volunteered verifying collection female pulmonary adenocarcinoma and women's health based on above-mentioned dualistic logistic regression equation
5 blood plasma difference microRNAs (miR-224-3p, miR-146a-5p, miR-648, miR-523-5p, miR- in person's sample
758-5p) standardized value of content makees dualistic logistic regression transformation, calculates 5 blood plasma differences in women sample
The logistic regression value of microRNAs content.It is predicted as women's health volunteer lower than best cut-off value 0.604, is higher than most
Good cut-off value 0.604 is predicted as female pulmonary adenocarcinoma, finally calculates with 5 metabolic markers horizontal forecast women lungs
The accuracy rate of gland cancer and women's health volunteer grouping.As a result such as Fig. 4,5 blood plasma difference microRNAs (miR-224- are based on
3p, miR-146a-5p, miR-648, miR-523-5p, miR-758-5p) verifying collection sample in predictablity rate be
98.5%, it is only patients with lung adenocarcinoma by 1 women's health volunteer's mistaken diagnosis, accuracy rate is very high.
Embodiment 4: the preparation of diagnostic reagent and diagnostic kit
Above-described embodiment shows that miR-224-3p, miR-146a-5p, miR-564, miR-615-5p, miR-601 combine
Male's adenocarcinoma of lung is distinguished in diagnosis and the accuracy of men's health volunteer is high, and susceptibility and high specificity can be based on miR-224-
3p, miR-146a-5p, miR-564, miR-615-5p, miR-601 production are for patients with lung adenocarcinoma in sieving and diagnosis male population
Diagnostic reagent or diagnostic kit.It include miR-224-3p primer, probe in the diagnostic reagent or diagnostic kit;miR-
146a-5p primer, probe;MiR-564 primer, probe;MiR-615-5p primer, probe;MiR-601 primer, probe.Primer tool
Body includes primer after primer and quantitative PCR before reverse transcription primer, quantitative PCR.Certainly, it is normal also to contain PCR reaction for diagnostic kit
Enzyme and reagent, such as reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water and Taq enzyme etc. and standard items and/or right
According to product.The design of primer and probe is conventional technical means in the art, and following table is a kind of design of primer and probe, can also be set
Count into other sequences.
Above-described embodiment shows that miR-224-3p, miR-146a-5p, miR-648, miR-523-5p, miR-758-5p join
It closes diagnosis and distinguishes female pulmonary adenocarcinoma and the accuracy height of women's health volunteer, susceptibility and high specificity, miR- can be based on
224-3p, miR-146a-5p, miR-648, miR-523-5p, miR-758-5p production are for lung in sieving and diagnosis female group
The diagnostic reagent or diagnostic kit of adenocarcinoma patients.Include miR-224-3p primer in the diagnostic reagent or diagnostic kit, visit
Needle;MiR-146a-5p primer, probe;MiR-648 primer, probe;MiR-523-5p primer, probe;MiR-758-5p primer,
Probe.Primer specifically includes before reverse transcription primer, quantitative PCR primer after primer and quantitative PCR.Certainly, diagnostic kit also contains
There is PCR to react common enzyme and reagent, such as reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water and Taq enzyme etc., Yi Jibiao
Quasi- product and/or reference substance.The design of primer and probe is conventional technical means in the art, and following table is that one kind of primer and probe is set
Meter, can also be designed to other sequences.
One group of blood plasma microRNAs marker provided by the invention can accurately be used for lung gland in sieving and diagnosis male population
Cancer patient, high sensitivity, high specificity, realize male population in patients with lung adenocarcinoma without intervention diagnosis;It is provided by the invention another
One group of blood plasma microRNAs marker can accurately be used for patients with lung adenocarcinoma in sieving and diagnosis female group, high sensitivity, specifically
Property it is strong, realize female group in patients with lung adenocarcinoma without intervention diagnosis.
SEQUENCE LISTING
<110>nine mourning hall Pharmaceutical Technology Co., Ltd of Nanjing
<120>blood plasma microRNAs is used to prepare the use of the diagnostic reagent of patients with lung adenocarcinoma in sieving and diagnosis female group
On the way
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<170> PatentIn version 3.3
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<212> DNA
<213>homo sapiens
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aaaauggugc ccuagugacu aca 23
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<212> DNA
<213>homo sapiens
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ugagaacuga auuccauggg uu 22
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<212> DNA
<213>homo sapiens
<400> 3
aggcacggug ucagcaggc 19
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<212> DNA
<213>homo sapiens
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gggggucccc ggugcucgga uc 22
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<212> DNA
<213>homo sapiens
<400> 5
uggucuagga uuguuggagg ag 22
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<212> DNA
<213>homo sapiens
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aagugugcag ggcacuggu 19
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<212> DNA
<213>homo sapiens
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cucuagaggg aagcgcuuuc ug 22
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<212> DNA
<213>homo sapiens
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gaugguugac cagagagcac ac 22
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<212> DNA
<213>artificial sequence
<400> 9
gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgactg tagtca 56
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<211> 56
<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacaa cccatg 56
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<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacgc ctgctg 56
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<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacga tccgag 56
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<211> 56
<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacct cctcca 56
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<211> 56
<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacac cagtgc 56
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<211> 56
<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacca gaaagc 56
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<211> 56
<212> DNA
<213>artificial sequence
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gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacgt gtgctc 56
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<212> DNA
<213>artificial sequence
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acactccagc tgggaaaatg gtgccctagt g 31
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<212> DNA
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acactccagc tgggtgagaa ctgaattcca t 31
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<212> DNA
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acactccagc tgggaggcac ggtgtcagca g 31
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<212> DNA
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acactccagc tggggggggt ccccggtgct c 31
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<212> DNA
<213>artificial sequence
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acactccagc tgggtggtct aggattgttg g 31
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<212> DNA
<213>artificial sequence
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acactccagc tgggaagtgt gcagggcact g 31
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<212> DNA
<213>artificial sequence
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acactccagc tgggctctag agggaagcgc t 31
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<212> DNA
<213>artificial sequence
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acactccagc tggggatggt tgaccagaga g 31
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<212> DNA
<213>artificial sequence
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cgccgcagtg cgtgtcgtgg agt 23
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<212> DNA
<213>artificial sequence
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cgtatcca 8