CN109897855A - A kind of serum miRNA marker and its application in the cancer of pancreas early diagnosis that pancreatitis mediates - Google Patents

Abstract

It is SEQ ID NO.1 the invention discloses the sequence of blood serum designated object miR-301a a kind of, and application of the kit for preparing as molecular marked compound of miR-301a marker in the cancer of pancreas early diagnosis and curative effect monitoring that pancreatitis mediates, the kit of miR-301a marker preparation can conveniently detect the expression of miRNA, without other customized special primer sequence, by detecting pancreatitis, Pancreas cancer patients blood plasma, the expression of miR-301a in Pancreatic Adenocarcinoma and pancreatic carcinoma, compare the relationship between miR-301a and known pancreatitis conventional sign object with Pearson correlation check analysis, miR-301a is assessed by Receiver operating curve (ROC), miR-301a With CA199 joint-detection diagnosis of pancreatic cancer and cancer of pancreas by stages in distinguishing ability, potential value of the blood plasma miR-301a marker in the cancer of pancreas early diagnosis that pancreatitis mediates is inquired into, to provide new theory and experimental basis from miRNA level for the early diagnosis of cancer of pancreas and targeted therapy.

Description

A kind of serum miRNA marker and its cancer of pancreas early diagnosis in pancreatitis mediation In application

Technical field

The invention belongs to technical field of molecular biology, and in particular to a kind of blood serum designated object miR-301a is situated between in pancreatitis Application in the cancer of pancreas early diagnosis led and curative effect monitoring.

Background technique

Cancer of pancreas grade malignancy is high, is worst one of the tumour of digestive system tumor China prognosis, and the World Health Organization is public The statistics of cloth shows that the disease incidence of whole world cancer of pancreas occupies the 13rd in malignant tumour, but its lethality is the 4th Position.In China, cancer of pancreas is the usually not apparent clinical symptoms of the 7th big cancer correlation fatal disease Early pancreatic carcinoma and body Sign, has the characteristics that early diagnostic rate is low, curative effect is not good enough, poor prognosis, and operation is the essential therapeutic arsenals of current treatment cancer of pancreas, But Resection Rate only has 10%~20%, cancer of pancreas easily occurs lymphatic metastasis and invades big blood vessel, 95% cancer of pancreas Patient has been progressive stage in diagnosis, and survival rate is lower than 5% within average 5 years, and the cause of disease of cancer of pancreas is not yet completely clear, in recent years It is wrapped more and more researches show that being formed by inflammatory microenvironment by Secretion of Inflammatory Factors around pancreatic cancer cell, body is scorching The generation of disease and cancer of pancreas, development and Prognostic significance are close.During human pancreatic carcinoma cell formation, inflammatory cell promotes Tumour growth generates the microenvironment for being conducive to human pancreatic carcinoma cell growth, promotes vascularization.

Early detection and intervention are the biology marks for improving the Key Strategy of Pancreas cancer patients final result, therefore searching out cancer of pancreas Will object and molecular target are the important directions for improving survival, and CA199 is that clinically most common cancer of pancreas is related at present Testing index is of great significance in the diagnosis of cancer of pancreas and relapse monitoring after operation, but its early diagnosis sensibility and Specificity is undesirable, and the expression of CA199 does not increase in about 30% Pancreas cancer patients, and CA199 is upper with pole in application Big limitation, searching is sensitiveer for cancer of pancreas, and special molecular marker is the direction of current most prospect.

MicroRNA (miRNA) is the non-coding microRNA of about 22 nucleotide of a kind of length of discovered in recent years, The expression of target gene is adjusted by making target mrna degradation or inhibiting its translation, in the occurrence and development of wide participation to tumour.Mesh Preceding to have found more than 2500 kinds of mankind miRNA, although these miRNA only account for the 1%-3% of human genome, they regulate and control Human gene more than 92%.Current research finds in Serum of Cancer Patients there are tissue specificity miRNA, and highly stable； Since serum specimen is easy to get and is easy to be received by patient, the expression of miRNA in patients serum is detected, disease will be become The tool of disease diagnosis, treatment and Index for diagnosis.Since miRNA is in the expression of post-transcriptional level adjusting gene, more The early discovery, early diagnosis and early treatment for being conducive to tumour, will have extensive potential applicability in clinical practice.

The detection method of research miRNA mainly has Northernblotting, in situ hybridization, microarray core both at home and abroad at present Chip technology, miRNA deep sequencing and Real-Time Fluorescent Quantitative PCR Technique.Northernblotting is that more classical detection is each The amount and size and the experimental method for estimating its abundance of RNA in tissue and organ, but Northern blotting is to the need of sample The amount of asking is higher, there are problems that certain mismatch hybridization, complex for operation step, is not suitable for high throughput analysis；In situ hybridization is detection MiRNA time and the most common method of tissue specificity, but miRNA molecule is too small, be to traditional hybridization in situ technique into one Step is improved, in order to avoid cause result inaccurate；Microarray chip technology is able to achieve the high throughput analysis of miRNA, but chip detects Technology is more demanding to the purity Coriolis mass of miRNA, and in addition the stability and repeatability of chip technology information quality are poor. MiRNA deep sequencing can the transcript profile to a species carry out the analysis of careful overall picture, but its original number for generating large amount of complex According to, need relevant bioinformatics technique to explain, it is costly, it is difficult to be widely applied；Real-Time Fluorescent Quantitative PCR Technique can be with The super-sensitive miRNA for detecting low expression, especially when using Taqman sonde method, specificity is higher, and is suitable for height Flux screening, but miRNA detection kit used in the round pcr sold on the market are all not equipped with a whole set of reverse transcription and draw Object and quantitative upstream and downstream primer.A kind of high sensitivity is found, easy to operate and low-cost detection method is current MiRNA urgent problem to be solved in clinical tumor detection, on the other hand, only with a kind of serum miRNA as cancer of pancreas It is insufficient as a result, will be great if combining miRNA with other kinds of tumor markers that marker can have specificity The accuracy of diagnosis is improved, if marker and energy of the serum miRNA as early screening of cancer of pancreas unconventionality expression can be filtered out The kit for developing corresponding ease of Use, the early stage diagnosis and treatment to cancer of pancreas, it will bring great benefit.

Generally acknowledged inflammation and tumor related genes include NF- κ B, STAT3, signal transducer and cell factor at present, are led to Accelerate the deterioration and development of tumour after normal NF- κ B, STAT3 activation, and miR-301a be just involved in wherein play it is important Effect, mechanism of action of this above-mentioned miR-301a in cancer of pancreas imply miR-301a possible as cancer of pancreas early diagnosis, The molecular marker of curative effect monitoring and the target spot of biological therapy.

Chinese granted patent CN1028766B be related to a kind of serum/plasma miRNA marker relevant to cancer of pancreas and its Using the marker is the combination of miR-451 and miR-409-3p, and the application of related kit needs to provide two kinds of markers pair The 4 kinds of specific primers answered, testing cost are high.On the other hand it has announced Chinese patent CN108103198A and has disclosed a kind of and pancreas The relevant blood plasma miRNA marker of cancer auxiliary diagnosis and its application, Research of predicting markers miR122-5p, miR-125b-5p, One of miR-192-5p, miR-193b3p, miR-221-3p and miR-27b-3p or a variety of, needed for being provided in kit One or more specific primers, by Exiqon miRNAqPCR panel chip and the absolute quantitation method based on qRT-PCR, But chip detection is higher to the purity requirement of miRNA, and the stability of chip technology information quality and repeatability are poor.This Invention provides the kit that completely can conveniently detect miRNA-301a, without other customized special primer sequence Column, the early diagnosis for the cancer of pancreas of pancreatitis mediation provides new direction and experimental basis, and kit is to the pure of miRNA It is low to spend quality opposite chip technology, it is cheap that testing cost also compares other methods, is conducive to marketing application.

Summary of the invention

In order to solve the problems in the prior art, the purpose of the present invention is to the cancer of pancreas early stage for disclosing a kind of pancreatitis mediation examines Disconnected miRNA marker and its application.Discovery miR-301a in pancreatic cancer models passes through inhibition NF- κ B's in early-stage study Inhibitor Nkrf promotes the translation of miR-301a after activating NF- κ B, NF- κ B to activate, and forms the logical of a negative-feedback regu- lation Road, miR-301a can also activate STAT3 signal path, generate an inflammatory reaction microenvironment.

To achieve the above object, the technical solution adopted by the present invention is that:

The present invention discloses the inflammation mediated cancer of pancreas of one kind and early diagnoses relevant marker miR-301a and one MiRNA-301a marker is applied to the miRNA detection kit that inflammation mediated cancer of pancreas early diagnoses, and kit is by reversing Reagent, qPCR reagent composition are recorded,

The reverse transcription reagents include: 10 μM of miRNA reverse transcriptase primers, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × RT Buffer, enzyme preparation；

The qPCR reagent includes: 10 μM of miRNA qPCR upstream primers, 10 μM of miRNA qPCR downstream primers, 10 μM U6 qPCR upstream primer, 10 μM of U6 qPCR downstream primers, 2 × UltraSYBR Mixture, distilled water.

10 μM of miRNA reverse transcriptase primers in the reverse transcription reagents, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × RT Buffer, enzyme preparation volume ratio be 0.5~2:0.5~2:15~31:4~10:1.25~5.

In the reverse transcription reagents, 10 μM of miRNA reverse transcriptase primers, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × RT Buffer, enzyme preparation volume ratio be 1:1:16.75:5:1.25.

In the qPCR reagent, 10 μM of miRNA qPCR upstream primers, 10 μM of miRNA qPCR downstream primers, 10 μM U6 qPCR upstream primer, 10 μM of U6 qPCR downstream primers, 2 × UltraSYBR Mixture, distilled water volume ratio be 1: 1:1:1:20:8。

In the kit, miRNA reverse transcriptase primer sequence is SEQ IDNo.2, and U6 reverse transcriptase primer sequence is SEQ ID No.3, miRNA-301a upstream primer sequence are that SEQ ID No.4, miRNA-301a downstream primer sequence is SEQ ID No.5, U6 qPCR upstream primer sequence is that SEQ ID No.6 and U6 qPCR downstream primer sequence is SEQ ID No.7.

The present invention provides a kind of miRNA-301a detection kit in the cancer of pancreas early diagnosis that Diagnosis of Pancreatic inflammation mediates Application, the expression of miR-301a in serum is detected by miRNA-301a detection kit, as identification cancer of pancreas Independent hazard factor.

Compared with prior art, the beneficial effects of the present invention are:

(1) present invention finds the miRNA-301a marker that a kind of sequence is SEQ ID NO.1, miR- is also disclosed The correlation that 301a marker is expressed in pancreatitis, cancer of pancreas, the cancer of pancreas early diagnosis mediated for pancreatitis and curative effect prison It surveys and new direction is provided；

(2) the present invention provides a kind of miRNA-301a markers to be applied to what the cancer of pancreas that pancreatitis mediates early diagnosed Detection kit, without other customized special primer sequence；

(3) expression of miR-301a is detected in pancreatitis, cancer of pancreas by application miR-301a detection kit Difference in patients blood plasma, Pancreatic Adenocarcinoma and pancreatic carcinoma, while comparing pancreatitis and different cancer of pancreas trouble by stages The expression situation of change of miR-301a in person's blood plasma examines (Pearson correlation using Pearson correlation Tests the correlation of miRNA-301a and the common inflammatory factor of cancer of pancreas) are analyzed, while using Receiver operating curve (ROC) assessment miR-301a, miR-301a and CA199 joint-detection diagnosis of pancreatic cancer and cancer of pancreas by stages in identification energy Power analyzes miR-301a expression data in blood plasma by ROC curve, it can be deduced that pancreatitis is converted into pancreatic cancer risk Judgment criteria, the early diagnosis and targeted therapy mediated for pancreatitis provide new theory and experimental basis, such as analysis data It obtains, when the expression of miR-301a is greater than pancreatitis group cutoff value 4.7, cancer of pancreas people at highest risk can be diagnosed as.

Detailed description of the invention

Fig. 1 normal control and pancreatitis, miR-301a relative expression quantity in Pancreas cancer patients blood plasma；

MiR-301a relative expression quantity in Fig. 2 Pancreatic Adenocarcinoma and cancer beside organism；

MiR-301a relative expression quantity in Fig. 3 pancreatic carcinoma；

Fig. 4 Pancreas cancer patients tissue and blood plasma miR-301a expression quantity compare；

Fig. 5 Pancreas cancer patients preoperative and postoperative blood plasma miR-301a expression quantity compares；

Fig. 6 Pancreas cancer patients blood plasma miR-301a level and reagent volume ratios different in kit reverse transcription reaction system Correlation；

The correlation of Fig. 7 Pancreas cancer patients blood plasma miR-301a level and IL-6；

The correlation of Fig. 8 Pancreas cancer patients blood plasma miR-301a level and TNF-α；

The ability that Fig. 9 ROC curve analysis miR-301a diagnoses Pancreas cancer patients；

Specific embodiment

Technical solution of the present invention is clearly and completely described below, it is clear that described embodiment is only this hair Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having All other embodiment obtained under the conditions of creative work is made, shall fall within the protection scope of the present invention.

Embodiment 1, embodiment 2, implementation column 3 and embodiment 4 provide a kind of miRNA detection kit, including following components:

Mentioned reagent box composition can be used for quantitative fluorescent PCR react 100 times, saved at -20 DEG C, the above reagent source by Above-mentioned each Reagent Company provides.

The present invention provides a kind of miRNA detection kit answering in the cancer of pancreas early diagnosis that pancreatitis mediates It is with, specific embodiment,

Embodiment 1: using in mentioned reagent box detection pancreatitis, Pancreas cancer patients blood plasma, Pancreatic Adenocarcinoma and cell line MiR-301a expression

1, sample is collected

Clinical definite is collected, not yet carrying out 70 of any treatment has the non-small cell type cancer of pancreas of pancreatitis medical history to suffer from The plasma specimen of person is shown in Table 1, wherein I/II phase patient 23, and III-IV phase patient 47 is collected simultaneously 85 Pancreatitis Patients Plasma specimen with 47 healthy normal persons is as control.In 70 Pancreas cancer patients, it is collected into blood after the treatment of 15 pairings Starch sample.All samples after 4 DEG C of centrifugation 15min, isolate blood plasma, set -80 DEG C and freeze through 1000g.Match in addition, collecting 15 Cancerous tissue and cancer beside organism's sample to Pancreas cancer patients, set -80 DEG C and freeze.70 Pancreas cancer patients average ages are 58.62 ± 6.45, wherein men and women is respectively 36 and 34, and 47 normal control average ages are 56.7 ± 5.2, and men and women is respectively 25 Hes 22, two groups form upper no notable difference at age and gender.

Table 1 is included in the Pancreas cancer patients situation list of research

2, experimental method

2.1 plasma specimen RNA are extracted:

Blood plasma RNA is extracted according to the mirVana PARIS kit operating procedure of Ambion company:

1. EP pipe is numbered, under room temperature environment, the denature liquid (denatured lysis liquid) of 280 μ L is added in every pipe；

2. above-mentioned each EP is added in 280 μ L sample blood plasma to manage, and mix well immediately；

3. the Acid-Phenol Chloroform (phenol chloroform mixed liquor) of 560 μ L is added in every pipe, 30- is mixed well 60s；

4. and being centrifuged 10min in room temperature environment 13000rpm, it is seen that obvious layering；

5. the liquid being layered respectively is taken in certain volume supernatant to new EP pipe, it is sure not to be drawn onto middle layer；

6. being mixed well to the room temperature dehydrated alcohol of above-mentioned 1.25 times of volumes of each Guan Zhongjia；

7. the mixed liquor in 5. is transferred in Filter Cartridge pillar, 8000rpm is centrifuged 30s, abandons waste liquid afterwards；

8. 700 μ L washing lotions 1 are added, 8000rpm is centrifuged 15s, abandons waste liquid afterwards；

9. 500 μ L washing lotions 2 are added, 8000rpm is centrifuged 15s, abandons waste liquid afterwards, repeats step 8；

10. dallying, 10000rpm is centrifuged 1min, Filter column is put into new EP pipe, is added what 50 μ L were preheated through 95 DEG C DEPC water stands 2min, then 8000rpm, and the RNA of 30s elution, extraction is tested for reverse transcription.

RNA is extracted in 2.2 tissues and cell line:

1. 1mL Trizol reagent is added into the tissue or cell after grinding；

2. in every 1mL Trizol reagent plus 0.2mL chloroform, covering tightly sample tube cover, is exerted oneself shaking test tube 15s with hand, make it It mixes well, is stored at room temperature 5min, rear 12000rmp, 4 DEG C of centrifugation 10min；

3. upper strata aqueous phase is transferred in new 1.5mL EP pipe, isometric isopropanol is added, is placed at room temperature for 20min, after 12000rmp, 4 DEG C of centrifugation 10min；

4. carefully outwelling supernatant, precipitating is left and taken, the ethyl alcohol oscillation washing RNA precipitate for the pre-cooling 75% for adding 1mL now to match is primary, 7500g afterwards, 4 DEG C of centrifugation 5min；

5. carefully outwelling supernatant, precipitating is set superclean bench and is dried；

6. the DEPC water in Guan Zhongjia 30-40 μ L dissolves, the RNA of extraction is tested for reverse transcription.

2.3 RNA purity and Concentration Testing: detecting absorbance, and ratio calculated under 260nm and 280nm wavelength, determines The purity and concentration of RNA takes appropriate RNA to carry out subsequent experimental.

2.4 TOYOBO, 15 μ L reverse transcription reaction system

EP pipe is set and is marked, according to it is above-mentioned it is shown by each reagent be added EP pipe in, whole process is set to be subtracted as far as possible on ice The degradation of few RNA, finger, which plays tube wall, makes institute's reagent adding 5~6s of of short duration centrifugation after mixing, by the security protection of EP pipe in PCR instrument, It is 37 DEG C of lasting 15min, 98 DEG C of lasting 5min, last 4 DEG C of preservations that program, which is arranged,.

In the kit, miRNA-301a reverse transcriptase primer sequence is SEQ IDNo.2, and U6 reverse transcriptase primer sequence is SEQ ID No.3。

The reaction of 2.5 quantitative PCRs:

Sample after the completion of reverse transcription is taken out, 3 secondary orifices of every group of setting, get out quantitative PCR reaction reagent, 20 μ L are fixed Measure PCR reaction system

In the kit, miRNA-301a upstream primer sequence is SEQ ID No.4, miRNA-301a downstream primer sequence It is SEQ that be classified as SEQ ID No.5, U6 qPCR upstream primer sequence, which be SEQ ID No.6 and U6 qPCR downstream primer sequence, ID No.7。

2.6 set eight connecting legs matched with quantitative PCR instruments, according to it is above-mentioned it is shown by each reagent be added eight connecting legs in, Pipe lid is covered tightly, of short duration centrifugation 5-6s makes institute's reagent adding uniformly mix and accumulate in bottom, to be detected.ABI StepOne Plus PCR instrument reaction condition, first 95 DEG C of initial denaturation 10min, start the cycle over reaction later, 95 DEG C of denaturation 10s, 57 DEG C of annealing 20s, and 72 DEG C extend 15s, in total recycle 40 times.

2.7 will test result is analyzed after copying out in instrument with CD, internal reference U6, first with experimental group CT value subtracts the CT value of the U6 internal reference of respective sample, as △ CT, then subtracts each other experimental group △ CT and control group △ CT, as △ △ CT finally calculates the relative expression quantity that 2- △ △ Ct is detected miRNA.

3 experimental results:

3.1 normal controls of quantitative PCR detection 47,85 Pancreatitis Patients and 70 Pancreas cancer patients blood plasma miR- The expression of 301a, is shown in Fig. 1, the results show that normal control blood plasma miR-301a relative expression quantity is 2.77 ± 2.45, pancreatitis is suffered from Person's blood plasma miR-301a is 6.86 ± 2.71, and Pancreas cancer patients blood plasma miR-301a is 7.82 ± 6.76, Pancreas cancer patients blood plasma The expression of miR-301a is apparently higher than Normal group and pancreatitis group (P < 0.01).

3.2 quantitative PCR detections 15 are horizontal to miR-301a in Pancreatic Adenocarcinoma and cancer beside organism, see Fig. 2, in cancer beside organism MiR-301a relative expression quantity is 1.4 ± 0.96, and miR-301a relative expression quantity is 4.74 ± 3.31 in cancerous tissue, in cancerous tissue MiR-301a level is apparently higher than cancer beside organism (P=0.03).Further compare 5 kinds of pancreatic carcinomas and pancreas normal cell The expression of miR-301a in H6C7 is shown in Fig. 3, and the expression of miR-301a is above H6C7 in 5 kinds of pancreatic carcinomas.It is above-mentioned The result shows that miR-301a in Pancreatic Adenocarcinoma and pancreatic carcinoma in high expression, and with the pancreas of 5 same patients The expression quantity of adenocarcinoma tissue and Pancreas cancer patients blood plasma detection miR-301a shows, expression no significant difference between the two, See Fig. 4.

The pretherapy and post-treatment blood plasma miR-301a of 3.3 Pancreas cancer patients of quantitative PCR detection 15 is horizontal, sees Fig. 5, pretherapy and post-treatment blood The relative expression quantity of slurry miR-301a is respectively 4.64 ± 2.31 and 1.3 ± 0.66, after treatment in blood plasma miR-301a expression Compared with (P < 0.01) is substantially reduced before treatment, illustrate that the highly expressed miR-301a of Pancreas cancer patients blood plasma is decreased obviously after treating, Prompt blood plasma miR-301a level can be used as the monitoring index during treatment of pancreatic cancer.

Embodiment 2, embodiment 3, embodiment 4: using miR-301a table in mentioned reagent box detection Pancreas cancer patients blood plasma Up to level

What the present embodiment 2, embodiment 3 and embodiment 4 provided a kind of early diagnoses applied to inflammation mediated cancer of pancreas Kit, the difference from embodiment 1 is that 10 μM of miRNA reverse transcriptase primers, 10 μM in reverse transcription (RT-PCR) reaction system The volume ratio that U6 reverse transcriptase primer, nuclease-free water, 5 × RT Buffer and enzyme preparation are added is different, is shown in Table 2:

Reagent volume in 2 reverse transcription reaction system of table

The expression of 63 Pancreas cancer patients blood plasma miR-301a of quantitative PCR detection, is shown in Fig. 6, use 10 μM of embodiment 1 MiRNA reverse transcriptase primer, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × RT Buffer, enzyme preparation volume ratio be 1:1:16.75:5:1.25 detects miR-301a expression highest in Pancreas cancer patients blood plasma.

Embodiment 5: Pancreas cancer patients blood plasma miR-301a level and pancreatitis correlation factor IL-6, the correlation of TNF-α Analysis

1. analysis method: carry out Pearson correlation inspection with 19.0 software of SPSS, analysis blood plasma miR-301a level with The correlation of pancreatitis marker.

2. experimental result: the correlation of analysis blood plasma miR-301a level and common pancreatitis marker IL-6 and TNF-α, See Fig. 7, Fig. 8, by Pearson correlation check analysis the results show that Pancreas cancer patients blood plasma miR-301a and IL-6 and The coefficient R of TNF-α is respectively 0.35 and 0.65, and the equal < 0.01 of P illustrates blood plasma miR-301a level and IL-6 and TNF-α With certain correlation, Carcinoembryonic Antigen CEA and cancer of pancreas mark known to blood plasma miR-301a level and the public are further illustrated Will object CA199 has certain correlation, and circulation miR-301a is expected to mark as the Non-Invasive early stage of inflammation mediated cancer of pancreas Will object.

Ability of the embodiment 6:ROC tracing analysis blood plasma miR-301a level to Pancreas cancer patients antidiastole

1. analysis method: using SPSS Software on Drawing ROC curve, and area AUC under calculated curve.

2. experimental result: analysis blood plasma miR-301a level is shown in Fig. 9 to the value of Pancreas cancer patients antidiastole, table 3, The results show that detection CA199, blood plasma miR-301a and joint CA199 and blood plasma miR-301a are to 70 diagnosis of pancreatic cancer Line Integral is not 0.665,0.731,0.859 under ROC curve, and sensibility and specificity is respectively 48.6%, 76.5%, 76.1% With 85.2%, 63.6%, 88.1%；Prompt blood plasma miR-301a level plays a significant role in the tentative diagnosis of cancer of pancreas, And the detection of joint CA199 and blood plasma miR-301a can greatly improve value of the cancer of pancreas in early diagnosis.

3 ROC curve of table analyzes blood plasma miR-301a to the ability of Pancreas cancer patients antidiastole

	Area under AUC	95%CI	Sensibility (%)	Specific (%)	P value
						CA199	0.665	0.556-0.774	48.6	85.2	0.005
miR-301a	0.731	0.633-0.829	76.5	63.6	6.76×10-5
						CA199 combines miR-301a	0.859	0.781-0.937	76.1	88.1	6.12×10-10

The Pancreas cancer patients of pancreatitis medical history and the blood plasma miR- of pancreatitis patient are detected by the miRNA kit of offer 301a expression, obtains with ROC curve, and the risk judgment foundation that pancreatitis patient suffers from cancer of pancreas is, when miR-301a's When expression is greater than pancreatitis group cutoff value 4.7, cancer of pancreas people at highest risk can be diagnosed as.

It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding And modification, the scope of the present invention is defined by the appended.

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Claims (6)

1. the miRNA marker in a kind of serum, which is characterized in that sequence is SEQ ID NO.1.

2. a kind of application of miRNA marker described in claim 1, which is characterized in that the miRNA marker is used to prepare pancreas The kit for the cancer of pancreas early diagnosis that adenositis mediates, the kit are made of reverse transcription reagents, qPCR reagent,

The reverse transcription reagents include: 10 μM of miRNA-301a reverse transcriptase primers, 10 μM of U6 reverse transcriptase primers, nuclease frees Water, 5 × RT Buffer, enzyme preparation；

The qPCR reagent includes: 10 μM of miRNA-301a upstream primers, 10 μM of miRNA-301a downstream primers, 10 μM of U6 QPCR upstream primer, 10 μM of U6 qPCR downstream primers, 2 × UltraSYBR Mixture, distilled water.

3. the application of miRNA marker according to claim 2, which is characterized in that the kit is in use, described 10 μM MiRNA-301a upstream primer sequence is SEQ ID No.4, and 10 μM of miRNA-301a downstream primer sequences are SEQ ID No.5.

4. the application of the miRNA marker according to Claims 2 or 3, which is characterized in that the kit is in use, described 10 μM of miRNA-301a reverse transcriptase primers, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × reverse transcriptions in reverse transcription reagents Buffer, enzyme preparation volume ratio be 0.5~2:0.5~2:15~31:4~10:1.25~5.

5. the application of miRNA marker according to claim 4, which is characterized in that the kit is in use, the reverse Record 10 μM of miRNA-301a reverse transcriptase primers in reagent, 10 μM of U6 reverse transcriptase primers, nuclease-free water, 5 × reverse transcription buffering Liquid, enzyme preparation volume ratio be 1:1:16.75:5:1.25.

6. the application of miRNA marker according to claim 2, which is characterized in that the kit is in use, the qPCR 10 μM of miRNA-301a upstream primers, 10 μM of miRNA-301a downstream primers, 10 μM of U6qPCR upstream primers, 10 μ in reagent M U6 qPCR downstream primer, 2 × UltraSYBR Mixture, distilled water volume ratio be 1:1:1:1:20:8.