CN104131113B - A kind of miRNA detection kit and application thereof - Google Patents
A kind of miRNA detection kit and application thereof Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The present invention relates to the application of miR-148, miR-22, miR-185 and miR-221 conbined usage in diagnosis of esophageal; The application of miR-148 in the change of prediction esophageal carcinoma tumor size; The application of miR-148, miR-185 and miR-22 conbined usage in prediction esophageal carcinoma metastases; One or both in miR-221 or miR-148 are predicting the application in patient with esophageal carcinoma survival rate; The application of miR-148, miR-22, miR-185, miR-221 and miR-39 conbined usage in preparation esophagus cancer miRNA detection kit, wherein miR-39 is as internal reference; And corresponding detection kit.MiRNA detection kit detection of the present invention is quick and convenient, accuracy rate is high, can realize esophageal carcinoma early diagnosis and outcome prediction.
Description
Technical field
The present invention relates to testing product and miR-148, miR-22, miR-185 or miR-221 application in esophageal carcinoma early diagnosis and outcome prediction of a kind of miRNA, belong to biological technical field.
Background technology
China is one of Esophageal Cancer area in the world, and sickness rate even occupies the first in the world, and every annual is died of illness about 150,000 people.Once clinical diagnosis, be often in middle and advanced stage, within 5 years, survival rate is lower than 20%; And 5 years survival rates can reach 70% ~ 90% after early stage Operation on Esophageal Cancer.Therefore, early diagnosis and prevention are in advance the primary study directions of the esophageal carcinoma.Now, clinical diagnosis transfers to a greater extent according to Molecular Detection result by depending on pathological diagnosis mode traditionally, and Molecular Detection more becomes the necessary detection means of early clinical diagnosis; Find specific molecular marked compound from gene level, accurate rapid detection is carried out to mark and has become one of important means of tumour early prevention and treatment.Increasing evidence display, microRNA(miRNA) abnormal expression take part in generation and the development of the esophageal carcinoma, can be used as a kind of novel molecular marked compound.
MicroRNA is extensively present in animals and plants and virus, is the noncoding single strand RNA molecule that a class length is about 20 ~ 24 Nucleotide, by the mode negative regulation target gene that miRNA shears and arrestin is translated, participates in the adjustment of various biological signal path.Research shows, the diagnosis of miRNA and tumour, by stages, prognosis etc. is closely related.MiRNA is found independently to be present in outside cell, and not destroy by RNA degrading enzyme (endogenousRNase) endogenic in blood, therefore in blood, tumour-specific miRNA can be used as the diagnosis marker of disease.In addition in tumour radiotherapy field, in basis and clinical study, all show some miRNA can reflect tumour radiotherapy susceptibility, and closely related with tumor hypoxia.The expression of the special miRNA of the esophageal carcinoma in analyzing blood, filter out high specificity, highly sensitive, the miRNA that can be used as specific tumors molecular marker, thisly utilize miRNA molecule to mark to carry out medical diagnosis on disease, predictive disease occur and the Noninvasive testing mode of prognosis for setting up the systems such as esophageal carcinoma early screening and outcome prediction and exploitation related products provides opportunity.
In view of the vital role of miRNAs in the process of the generation of the esophageal carcinoma, development, invasion and attack, transfer, also more and more draw attention at the diagnosis of the esophageal carcinoma, the research field miRNA such as treatment and prognosis.The expression that GuoY etc. study 51 routine esophageal squamous cell carcinoma tissue miRNA finds, miR-25, miR-424 and miR-151 up-regulated, miR-100, miR-99a, miR-29c and miR-140 be down-regulated expression then, the miRNA of these abnormal expressions accurately can distinguish esophageal squamous cell carcinoma and healthy tissues (see foreign language literature GuoY, ChenZL, ZhangLetal.DistinctivemicroRNAprofilesrelatingtopatients urvivalinesophageal-3-squamouscellcarcinoma.CancerRes, 2008, 68 (1): 26-33.) research of AkagiI etc. show miR-21 and miR-205 in esophageal squamous cell carcinoma tissue high expression level (see foreign language literature AkagiI, MiyashitaM, IshibashiOetal.Relationshipbetweenalteredexpressionlevel sofMIR21, MIR143, MIR145, andMIR205andclinicopathologicfeaturesofesophagealsquamou scellcarcinoma.2011, 24(27), 523-30).Kurashige etc. have detected miRNA change in 71 routine patients with esophageal squamous cell carcinoma serum, display miR-21 in all esophageal squamous cell carcinoma samples high expression level (see foreign language literature KurashigeJ, KamoharaH, WatanabeM, etal.SerummicroRNA-21isanovelbiomarkerinpatientswithesop hagealsquamouscellcarcinoma.JSurgOncol, 2012,106 (2): 188-192).
How the achievement in these esophageal carcinoma fundamental research fields above-mentioned is applied to rapidly clinical, needs accurately to check and approve the most effective molecular marked compound detected, and set up fast and simple application system, guide related products exploitation.
Summary of the invention
The object of this invention is to provide a kind of detect quick and convenient, accuracy rate is high, the miRNA detection kit of esophageal carcinoma early diagnosis and outcome prediction can be realized, and one or more in miR-148, miR-22, miR-185 or miR-221 application in esophageal carcinoma early diagnosis and outcome prediction is provided.
The present invention is a kind of technical scheme solving the problems of the technologies described above proposition: the application of miR-148, miR-22, miR-185, miR-221 and miR-39 conbined usage in preparation esophagus cancer miRNA detection kit, wherein miR-39 is as internal reference.
The concrete technical scheme proposed based on above-mentioned application the present invention is: a kind of miRNA detection kit, its detection system comprises reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system, comprises respectively for forward primer and the reverse universal primer of miR-148, miR-22, miR-185, miR-221 in described quantitative fluorescent PCR reaction system; Described internal reference system comprises internal reference miRNA-39 and the forward primer for miR-39; For the nucleotide sequence of the forward primer of described miR-148 as shown in SEQIDNo.1; For the nucleotide sequence of the forward primer of described miR-22 as shown in SEQIDNo.2; For the nucleotide sequence of the forward primer of described miR-185 as shown in SEQIDNo.3; For the nucleotide sequence of the forward primer of described miR-221 as shown in SEQIDNo.4; For the nucleotide sequence of the forward primer of described miR-39 as shown in SEQIDNo.5; The nucleotide sequence of described reverse universal primer is as shown in SEQIDNo.6.
Reagent for preparing described quantitative fluorescent PCR reaction system comprises SYBRGreen mixed solution, forward primer liquid, oppositely universal primer liquid and pure water; Reagent for preparing described reverse transcription reaction system comprises poly A polymerase, ThermoScript II mixed solution, reverse transcription buffer and nuclease free pure water.
The cumulative volume of above-mentioned quantitative fluorescent PCR reaction system is 10 μ l; Also DNA profiling is comprised in described quantitative fluorescent PCR reaction system; Wherein, the volume of described SYBRGreen mixed solution is 5 μ l, and the volume of described forward primer liquid is 1 μ l, and the volume of described reverse universal primer liquid is 1 μ l, described DNA profiling 1 μ l, and all the other are pure water; The working concentration of described forward primer liquid is 2 μMs, and the working concentration of described reverse universal primer liquid is 2 μMs; Described DNA profiling is the five times of acquisitions of the reacted product dilution of described reverse transcription reaction system.
The cumulative volume of above-mentioned reverse transcription reaction system is 12.5 μ l; MiRNA sample is also comprised in described reverse transcription reaction system; Wherein, the volume of described poly A polymerase is 5 μ l, and the volume of described ThermoScript II mixed solution is 5 μ l, and the volume of described reverse transcription buffer is 2.5 μ l, and the add-on of described miRNA sample is 20ng, and all the other are nuclease free pure water; The working concentration of described poly A polymerase is 2.5U/ μ l; Containing internal reference miRNA-39 in described miRNA sample.
The internal reference miRNA-39 liquid of above-mentioned internal reference miRNA-39 to be the volume added in extracting miRNA sample be 2 μ l, its working concentration is 0.2 μM.
The present invention is the another kind of technical scheme solving the problems of the technologies described above proposition: the application of miR-148, miR-22, miR-185 and miR-221 conbined usage in diagnosis of esophageal.
Judgment formula is: Y
1=9.270 × A
miR-185-0.665 × B
miR-221-1.078 × C
miR-148-0.082 × D
miR-22-29.771.
Wherein A
miR-185it is the expression amount (2 of miR-185 before radiotherapy
-△ △ CTvalue), B
miR-221it is the expression amount (2 of miR-221 before radiotherapy
-△ △ CTvalue), C
miR-148it is the expression amount (2 of miR-148 before radiotherapy
-△ △ CTvalue), D
miR-22it is the expression amount (2 of miR-22 before radiotherapy
-△ △ CTvalue).
Work as Y
1during value >0.5, be then judged as patient with esophageal carcinoma.
The present invention is the another kind of technical scheme solving the problems of the technologies described above proposition: the application of miR-148 in the change of prediction esophageal carcinoma tumor size.
Judgment formula is: Y
2the expression amount (2 of miR-148 after=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-148 before-radiotherapy
-△ △ CTvalue).
Work as Y
2during >-4.4850, be judged as that curative effect is better; Otherwise, be judged as unsatisfactory curative effect.
The present invention is the another kind of technical scheme solving the problems of the technologies described above proposition: the application of miR-148, miR-185 and miR-22 conbined usage in prediction esophageal carcinoma metastases.
Judgment formula (regression equation) is: Y
3=0.05A
miR-185+ 0.09C
miR-148+ 0.02D
miR-22-3.122.
Wherein A
miR-185it is the expression amount (2 of miR-185 before radiotherapy
-△ △ CTvalue), C
miR-148it is the expression amount (2 of miR-148 before radiotherapy
-△ △ CTvalue), D
miR-22it is the expression amount (2 of miR-22 before radiotherapy
-△ △ CTvalue).
Work as Y
3during >0.4409, be judged as that metastases incidence is high; Otherwise, be judged as that metastases incidence is low.
The present invention is the another kind of technical scheme solving the problems of the technologies described above proposition: one or both in miR-221 or miR-148 are predicting the application in patient with esophageal carcinoma survival rate.
Judgment formula is:
Y
4the expression amount (2 of miR-148 before=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-148 after-radiotherapy
-△ △ CTvalue).
Y
5the expression amount (2 of miR-221 before=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-221 after-radiotherapy
-△ △ CTvalue).
By ROC tracing analysis, work as Y
4<4.25 or Y
5during <-2.37, after being judged as radiotherapy in esophageal carcinoma patients, survival rate is higher; Otherwise survival rate is lower.
The present invention has positive effect:
(1) miR-148, miR-22, miR-185 and miR-221 are as independence or combination molecule marker, through excessive data clinical verification experiment, first Application, in the exploitation of above-mentioned miRNA detection kit, achieves 1) miR-148, miR-22, miR-185 and miR-221 be as the application in combination molecule mark in early days diagnosis of esophageal; 2) miR-148 is as the application of independent molecule marker in prediction Radiotherapy of Esophageal Cancer curative effect (tumor size change); 3) miR-148, miR-22 and miR-185 are as the application of combination molecule mark in prediction Metastasis of Esophageal Carcinoma incidence; 4) application of miR-148 or miR-221 in prediction patient with esophageal carcinoma survival rate.
(2) miRNA detection kit of the present invention have selected high specificity, these four miRNA markers of highly sensitive miR-148, miR-22, miR-185 and miR-221 combine, be developed to detection kit, detect quick and convenient, that accuracy rate is high, a test kit realizes four aspects application, meet the detection demand of different tumour patient, range of application is extremely wide, high through clinical verification predictablity rate.
(3) miRNA detection kit of the present invention is for the internal reference as quantitative reaction in serum, factor widely different in different sample, the stable exogenous RNA sequence miR-39(of special introducing derives from nematode) as internal reference contrast RNA, and optimization design is for the internal reference forward primer of miR-39, greatly improve the accuracy carrying out relative quantification when miRNA detects.
(4) test kit of the present invention uses real time fluorescence quantifying PCR method, the change of the miRNA expression level of patient with esophageal carcinoma and Normal group in research serum, the equal high expression level of miR-148, miR-22, miR-185 and miR-221 can predict the generation of the esophageal carcinoma well, carries out auxiliary diagnosis; The change of the measurable tumor size of the change of expression amount before and after miR-148 radiotherapy, assessment Radiotherapy of Esophageal Cancer curative effect; Expression amount before miR-148, miR-185 and miR-22 radiotherapy may be used for the incidence predicting esophageal carcinoma metastases; Expression amount before miR-221 or miR-148 radiotherapy can predict the survival rate of patient with esophageal carcinoma.MiRNA detection kit of the present invention, by detecting the expression amount of miR-148, miR-22, miR-185 and miR-221, makes the early diagnosis of the esophageal carcinoma and outcome prediction become feasible.
Accompanying drawing explanation
Fig. 1 is the average expression amount chart of the radiotherapy pre-neoplastic patient four kinds of miRNA adopting test kit of the present invention to detect;
Fig. 2 adopts the expression amount of tumour patient miR-148 and the chart of radiotherapy effect dependency before and after the radiotherapy that detects of test kit of the present invention;
Fig. 3 is the expression amount of radiotherapy pre-neoplastic patient four kinds of miRNA and the chart of metastases dependency that adopt test kit of the present invention to detect;
Fig. 4 is the expression amount of radiotherapy pre-neoplastic patient miR-148 and the chart of patient's survival rate dependency that adopt test kit of the present invention to detect;
Fig. 5 is the expression amount of radiotherapy pre-neoplastic patient miR-221 and the chart of patient's survival rate dependency that adopt test kit of the present invention to detect;
Fig. 6 adopts the expression amount of tumour patient miR-148 and the chart of patient's survival rate dependency before and after the radiotherapy that detects of test kit of the present invention;
Fig. 7 adopts the expression amount of tumour patient miR-221 and the chart of patient's survival rate dependency before and after the radiotherapy that detects of test kit of the present invention.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is that following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, person skilled in art can make some nonessential improvement and adjustment according to the invention described above content to the present invention.In following embodiment, if not specially show, reagent used is analytical pure, and agents useful for same all can obtain from commercial channel.The experimental technique of unreceipted actual conditions in literary composition, the condition described in " Molecular Cloning: A Laboratory guide " book that the Science Press that conveniently condition is write as J. Pehanorm Brooker etc. usually publishes for 2002, or according to the condition that manufacturers advises.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.
Embodiment 1
One, the composition of test kit.
The miRNA detection kit of the present embodiment, comprises the reagent for preparing reverse transcription reaction system, for preparing reagent and the internal reference system of PCR system.
1, internal reference system:
The reagent of internal reference system comprises internal reference miRNA-39 liquid and the forward primer liquid for miR-39.
Internal reference miRNA-39 liquid is existing worm cel-miR-39(AccessionNumber:AJ487564) solution, for the synthesis of prompt base (Shanghai) trade Co., Ltd in the English Weihe River, be encapsulated in a bottle, the concentration of preparation is 20 μMs, totally 10 μ l, working concentration is 0.2 μM.
Forward primer fluid-tight for miR-39 is contained in a bottle, and volume is 100 μ l, and working concentration is 2 μMs.
2, reverse transcription reaction system:
Reagent for preparing reverse transcription reaction system comprises poly A polymerase (polyApolymerase, supplier: GeneCopoeia, article number: Cat#A02030B), ThermoScript II mixed solution (RTaseMix, supplier: GeneCopoeia, article number: Cat#C02020B), reverse transcription buffer (5 × PAP/RTbuffer, supplier: GeneCopoeia, article number: Cat#C02022B) and nuclease free pure water (RNaseandDNasefreeH
2o, supplier: GeneCopoeia, article number: Cat#C10230A).Reagent for preparing reverse transcription reaction system encapsulates by bottle, is configured to reverse transcription reaction system according to a certain percentage during use, and reverse transcription reaction system is 12.5 μ l/ time, and the volume of encapsulation is the consumption of 100 times, as shown in table 1.
Table 1 reverse transcription reaction system component table
3, PCR reaction system:
Reagent for preparing PCR reaction system comprises SYBRGreen mixed solution (2 × SYBRGreenMix, supplier: Hangzhou BIOER Technology Co., Ltd, article number: Cat#B254C1), forward primer liquid (Fprimer, synthetic), oppositely universal primer liquid (UniversalAdaptorPCRPrimer, synthetic) and pure water (H
2o).Reagent for preparing PCR reaction system encapsulates by bottle, is mixed with PCR reaction system according to a certain percentage during use, and PCR reaction system is 10 μ l/ time, and the volume of encapsulation is the consumption of 100 times, as shown in table 2.
Table 2PCR reaction system component table
Forward primer for preparing PCR reaction system is respectively the forward primer for miR-148, the forward primer for miR-22, the forward primer for miR-185 and the forward primer for miR-221.Four kinds of forward primers for preparing PCR reaction system separately encapsulate by bottle.Prepare the nucleotide sequence of the forward primer of PCR reaction system, internal reference forward primer and reverse universal primer, as shown in table 3, synthesize in Shanghai Sheng Gong biotechnology company limited.
Table 3 forward primer and reverse universal primer mark sheet
Two, the using method of test kit.
The concrete detecting step of the miRNA detection kit of the present embodiment is as follows:
1, the extraction of miRNA.
RN24-BLOODmisi whole blood (liquid sample) Microrna rapid extraction test kit (RN24) of Ai Delai bio tech ltd, Beijing is adopted to carry out miRNA extraction.First the first step to specifications: every 0.25ml liquid sample adds the lysis buffer (Lysisbuffer) of 0.75ml; Then second step to specifications: sample concuss is mixed, hatches under 15 ~ 30oC condition and decompose completely to make ribosome for 5 minutes; In sample solution, add 2 μ l working concentrations is afterwards 0.2 μM of internal reference miRNA-39 liquid, as reaction internal reference; Then the 3rd step and later step are carried out to specifications.
Pass through the Ratio control sample quality measuring concentration and OD260/OD280 after extracting miRNA sample, finally add the obtained peak optimization reaction result of ratio between 1.8 ~ 2.0 of the sample OD260/OD280 in reverse transcription reaction system.
2, reverse transcription reaction.
(1) reverse transcription reaction system: get the polyApolymerase of 5 μ l, the RTaseMix of 5 μ l and the 5 × PAP/RTbuffer of 2.5 μ l and mix; Add the miRNA sample of extracting again, the add-on of miRNA sample controls as 20ng; Finally add appropriate RNaseandDNasefreeH
2o, makes the reaction cumulative volume of reverse transcription be 12.5 μ l.
(2) reverse transcription program: 37 DEG C, 1h; 85 DEG C, 5min; 4 DEG C of refrigerations are stand-by.
3, quantitative fluorescent PCR reaction.
(1) quantitative fluorescent PCR reaction system: get the 2 × SYBRGreenMix of 5 μ l, wherein a kind of Fprimer of 1 μ l, the template cDNA of the UniversalAdaptorPCRPrimer of 1 μ l and 1 μ l mixes, finally add appropriate H
2the reaction cumulative volume that O makes PCR react is 10 μ l.Wherein, the product dilution 5 times of reverse transcription obtains by template cDNA.
(2) quantitative fluorescent PCR reaction and solubility curve routine analyzer:
1）95℃，10min；
2）95℃，10s；
3）60℃，20s；
4）72℃，10s；
5) the 2nd is repeated) step is to the 4th) step 40 circulation;
6）95℃，15s；
7）60℃，60s；
8）95℃，15s。
Three, the Testing index of test kit and judging criterion.
1, the miRNA of auxiliary diagnosis Patients With Carcinoma of Esophagus.
A, Testing index.
Extract the miRNA in serum before 60 routine Patients With Carcinoma of Esophagus radiotherapies, and the miRNA in 60 routine Healthy People (normal control) serum.The expression amount before radiotherapy of miR-148, miR-22, miR-185 and miR-221 is detected by the miRNA detection kit of the present embodiment.
Can find by analysis, the expression amount of miR-22, miR-148, miR-185, miR-221 is wherein compared all in high expression level with Healthy People, as shown in Figure 1.
P value=0.000<0.01 pole the significant difference of the T check analysis through between group: miR-22, the P value=0.004<0.01 pole significant difference of miR-148; P value=0.000<0.01 pole the significant difference of miR-185; P value=0.000<0.01 pole the significant difference of miR-221, as shown in table 4.
Table 4 four kinds of miRNA expression amount T check analysis
B, judging criterion.
Found by Logistic regression equation matching four indexs, the sensitivity after matching and specificity are all higher than independent index.By data analysis, obtaining judgment formula (regression equation) is:
Y
1=9.270×A
miR-185-0.665×B
miR-221-1.078×C
miR-148-0.082×D
miR-22-29.771。
Wherein A
miR-185it is the expression amount (2 of miR-185 before radiotherapy
-△ △ CTvalue), B
miR-221it is the expression amount (2 of miR-221 before radiotherapy
-△ △ CTvalue), C
miR-148it is the expression amount (2 of miR-148 before radiotherapy
-△ △ CTvalue), D
miR-22it is the expression amount (2 of miR-22 before radiotherapy
-△ △ CTvalue).
Work as Y
1during value >0.5, be then judged as patient with esophageal carcinoma.
2, relevant to Curative Effect of Esophagus Carcinoma miRNA.
The change of 2.1miRNA expression amount and the dependency of curative effect.
A, Testing index.
MiRNA before extracting 60 routine Patients With Carcinoma of Esophagus radiotherapies and after radiotherapy in serum, detect miR-22, miR-148, miR-185, miR-221 expression amount before and after radiotherapy by the miRNA detection kit of the present embodiment, be divided into corresponding expression rising group and express reduction group.
By RECIST the standard of curative effect evaluation:
(1) complete incidence graph (CR, completeresponse), all target foci disappearances.
(2) partial rcsponse (PR, partialresponse), the most major diameter sum of target focus compares with baseline state, at least reduces 30%.
(3) lesion growth (PD, progressivedisease), the most major diameter sum of the minimum target focus that the most major diameter sum of target focus is recorded to after starting with treatment compares, and increases by 20%, or occurs one or more new focus.
(4) pathology stable (SD, stabledisease), between partial rcsponse and progression of disease.
Judge the curative effect of above-mentioned 60 routine Patients With Carcinoma of Esophagus and divide into groups, and calculating corresponding miRNA expression amount rising group or CR, PR and SD number with correspondence in expression amount group of decreased, or PD number.The judgement of CR, PR, SD, PD relates to the size of tumour.
Research finds that only having miRNA-148 to express between rising group and miRNA-148 expression reduction group in above-mentioned four kinds of miRNA exists sex differernce evident in efficacy, as shown in Fig. 2 and table 5.Illustrate that before and after miRNA-148 radiotherapy, the change of expression amount is relevant with the curative effect of radiotherapy.
The expression amount change of table 5miRNA-148 and the dependency of radiotherapy effect
B, judging criterion.
By data analysis, obtaining judgment formula is: Y
2the expression amount (2 of miR-148 after=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-148 before-radiotherapy
-△ △ CTvalue).
Work as Y
2during >-4.4850, be judged as that curative effect is better; Otherwise, be judged as unsatisfactory curative effect.
The expression amount of 2.2miRNA and the dependency of metastases.
A, Testing index.
Extract the miRNA in serum before 60 routine Patients With Carcinoma of Esophagus radiotherapies, detected the expression amount of miR-22, miR-148, miR-185 and miR-221 by the miRNA detection kit of the present embodiment.
Whether above-mentioned tumour patient is shifted by tumour after radiotherapy and is divided into two groups: metastases group and tumour do not shift group.Metastases group and tumour do not shift group radiotherapy before miR-22, miR-148, miR-185, miR-221 expression amount as shown in Figure 3.
Find that miRNA-148, miR-185, miR-22 do not shift group in metastases group and tumour and there is pole significant difference by T check analysis, the i.e. P value=0.00078<0.01 of miR-148, P value=the 0.0136<0.01 of the P value of miR-185=0.000986<0.01, miR-22; And there is not significant difference in miRNA-221, the P value=0.0748>0.05 of miR-221.
B, judging criterion.
Found by Logistic regression equation matching two indices, the sensitivity after matching and specificity are all higher than independent index.By data analysis, obtaining judgment formula (regression equation) is:
Y
3=0.05A
miR-185+0.09C
miR-148+0.02D
miR-22-3.122。
Wherein A
miR-185it is the expression amount (2 of miR-185 before radiotherapy
-△ △ CTvalue), C
miR-148it is the expression amount (2 of miR-148 before radiotherapy
-△ △ CTvalue), D
miR-22it is the expression amount (2 of miR-22 before radiotherapy
-△ △ CTvalue).
Work as Y
3during >0.4409, be judged as that metastases incidence is high; Otherwise, be judged as that metastases incidence is low.ROC area under the curve is 0.784.
The expression amount of 2.3miRNA and the dependency of survival rate.
A, Testing index.
MiRNA before extracting 60 routine Patients With Carcinoma of Esophagus radiotherapies and after radiotherapy in serum, detects miR-22, miR-148, miR-185, miR-221 expression amount before and after radiotherapy by the miRNA detection kit of the present embodiment.
As shown in Figure 4 and Figure 5, the expression amount before analysis finds miR-221, miR-148 radiotherapy and patient's survival rate have significant dependency.Application Cox survival curve analysis display: the P value=0.039<0.05 of miR-148 is that significantly the P value=0.001<0.01 of miR-221 is extremely remarkable.The median getting each miRNA is that standard is divided into groups, and obtains two survival curves respectively, finds the equal <0.05 of P value of two curves, illustrates that the expression amount before miRNA radiotherapy can predict radiotherapeutic effect.
As shown in Figure 6 and Figure 7, by finding the analysis of the miRNA expression amount change before and after radiotherapy: the expression amount after miR-148 and miR-221 radiotherapy declines, and the survival time of patient is long, and radiotherapeutic effect is relatively good; After miR-148 and miR-221 radiotherapy, expression amount rises, and the survival time of patient is short, then the weak curative effect of radiotherapy.Its significance is respectively: P value=0.005 of miR-148, and P value=0.016 of miR-221 is extremely remarkable.
B, judging criterion.
By data analysis, obtaining judgment formula is:
Y
4the expression amount (2 of miR-148 before=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-148 after-radiotherapy
-△ △ CTvalue).
Y
5the expression amount (2 of miR-221 before=radiotherapy
-△ △ CTvalue) expression amount (2 of miR-221 after-radiotherapy
-△ △ CTvalue).
By ROC tracing analysis, work as Y
4<4.25 or Y
5during <-2.37, then after being judged as radiotherapy in esophageal carcinoma patients, survival rate is higher; Otherwise survival rate is lower.
Four, the specificity analysis of test kit.
1,308 routine patient suspected's serum miRNA are extracted, adopt the miRNA detection kit of the present embodiment to carry out the joint-detection of the expression amount of miR-148, miR-22, miR-185 and miR-221, be applied to predictablity rate in the early diagnosis of the auxiliary esophageal carcinoma and reach 91.27%.
2, miRNA before and after 119 routine Patients With Carcinoma of Esophagus radiotherapies is extracted, adopt the miRNA detection kit of the present embodiment to carry out the detection of the expression amount of miR-148 before and after radiotherapy, the predictablity rate for Patients With Carcinoma of Esophagus radiotherapy effect (tumor size change) reaches 68.72%;
3, miRNA before extraction 119 routine Patients With Carcinoma of Esophagus radiotherapies, adopt the miRNA detection kit of the present embodiment to carry out the joint-detection of miR-148, miR-22 and miR-185 expression amount before radiotherapy, the predictablity rate whether easily shifted for Patients With Carcinoma of Esophagus tumour reaches 81.45%;
4, extract miRNA before and after 119 routine Patients With Carcinoma of Esophagus radiotherapies, adopt the miRNA detection kit of the present embodiment to carry out the detection of miR-148 before and after radiotherapy, the predictablity rate for patient with esophageal carcinoma survival rate reaches 77.35%.Adopt the miRNA detection kit of the present embodiment to carry out the detection of miR-221 before and after radiotherapy, the predictablity rate for patient with esophageal carcinoma survival rate reaches 71.28%.
Obviously, above-described embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And these belong to spirit institute's apparent change of extending out of the present invention or change and are still among protection scope of the present invention.
Claims (10)
- The application of 1.miR-148, miR-22, miR-185 and miR-221 conbined usage in the test kit of preparation diagnosis of esophageal.
- The application of 2.miR-148 in the test kit of preparation prediction esophageal carcinoma tumor size change.
- The application of 3.miR-148, miR-185 and miR-22 conbined usage in the test kit of preparation prediction esophageal carcinoma metastases.
- One or both in 4.miR-221 or miR-148 are preparing the application in the test kit predicting patient with esophageal carcinoma survival rate.
- The application of 5.miR-148, miR-22, miR-185, miR-221 and miR-39 conbined usage in preparation esophagus cancer miRNA detection kit, wherein miR-39 is as internal reference.
- 6. a miRNA detection kit, its detection system comprises reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system, it is characterized in that: comprise respectively for forward primer and the reverse universal primer of miR-148, miR-22, miR-185, miR-221 in described quantitative fluorescent PCR reaction system; Described internal reference system comprises internal reference miRNA-39 and the forward primer for miR-39; For the nucleotide sequence of the forward primer of described miR-148 as shown in SEQIDNo.1; For the nucleotide sequence of the forward primer of described miR-22 as shown in SEQIDNo.2; For the nucleotide sequence of the forward primer of described miR-185 as shown in SEQIDNo.3; For the nucleotide sequence of the forward primer of described miR-221 as shown in SEQIDNo.4; For the nucleotide sequence of the forward primer of described miR-39 as shown in SEQIDNo.5; The nucleotide sequence of described reverse universal primer is as shown in SEQIDNo.6.
- 7. miRNA detection kit according to claim 6, is characterized in that: the reagent for preparing described quantitative fluorescent PCR reaction system comprises SYBRGreen mixed solution, forward primer liquid, oppositely universal primer liquid and pure water; Reagent for preparing described reverse transcription reaction system comprises poly A polymerase, ThermoScript II mixed solution, reverse transcription buffer and nuclease free pure water.
- 8. miRNA detection kit according to claim 7, is characterized in that: the cumulative volume of described quantitative fluorescent PCR reaction system is 10 μ l; Also DNA profiling is comprised in described quantitative fluorescent PCR reaction system; Wherein, the volume of described SYBRGreen mixed solution is 5 μ l, and the volume of described forward primer liquid is 1 μ l, and the volume of described reverse universal primer liquid is 1 μ l, described DNA profiling 1 μ l, and all the other are pure water; The working concentration of described forward primer liquid is 2 μMs, and the working concentration of described reverse universal primer liquid is 2 μMs; Described DNA profiling is the five times of acquisitions of the reacted product dilution of described reverse transcription reaction system.
- 9. miRNA detection kit according to claim 7, is characterized in that: the cumulative volume of described reverse transcription reaction system is 12.5 μ l; MiRNA sample is also comprised in described reverse transcription reaction system; Wherein, the volume of described poly A polymerase is 5 μ l, and the volume of described ThermoScript II mixed solution is 5 μ l, and the volume of described reverse transcription buffer is 2.5 μ l, and the add-on of described miRNA sample is 20ng, and all the other are nuclease free pure water; The working concentration of described poly A polymerase is 2.5U/ μ l; Containing internal reference miRNA-39 in described miRNA sample.
- 10. miRNA detection kit according to claim 7, is characterized in that: the internal reference miRNA-39 liquid of described internal reference miRNA-39 to be the volume added in extracting miRNA sample be 2 μ l, its working concentration is 0.2 μM.
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| CN109897899B (en) * | 2019-03-01 | 2023-11-03 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Marker for prognosis judgment of locally advanced esophageal squamous carcinoma and application thereof |
| CN111593109A (en) * | 2020-03-24 | 2020-08-28 | 上海交通大学医学院 | Application of exosome miRNA biomarker and kit for rapidly diagnosing tuberculosis |
| CN111778338B (en) * | 2020-08-06 | 2021-08-03 | 南京医科大学 | Application of circular RNA biomarker |
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