CN102676524B - Molecular marker miR-147a of breast cancer - Google Patents

Abstract

The invention provides a molecular marker miR-147a of breast cancer and use thereof in a diagnostic reagent for diagnosing breast caner. Content of miR-147a in serum of breast cancer patient is raised compared with that of a normal person. After the patient receives operation, the content of miR-147a in serum falls back to normal level. The invention further provides a diagnostic reagent kit for diagnosing breast caner. By utilizing the molecular marker miR-147a of breast cancer to diagnose breast caner, the characteristics of simplicity in operation, conveniently-obtained materials, safety, no invasiveness, high specific, high sensitivity and easiness for screening in a large scale are achieved.

Description

Mammary cancer molecular marker miR-147a

Technical field

The present invention relates to the diagnosing tumor field, relate in particular to a kind of mammary cancer molecular marker miR-147a, it can be applicable to high-risk breast cancer crowd's screening, the evaluation of mammary cancer, the monitoring of breast cancer treatment situation and the fields such as monitoring of Prognosis in Breast Cancer.

Background technology

MicroRNA(miRNA) be in recent years study hotspot, it is a kind of strand microRNA that extensively is present in the eukaryote, do not have an encoding function, but the flank region that it can be incorporated into gene order checks or suppresses the translation of said target mrna, has conservative property, timing and the tissue specificity of height.Mitchell in 2008 etc. at first find many miRNA stable existences in blood plasma, and wherein the level of miR-141 can be used as the prostate cancer diagnosis mark.Colorectal carcinoma diagnosis marker miR-92a and liver injury monitoring mark miR-122 have been found again in research subsequently.

Mammary cancer is one of modal malignant tumour of women, and sickness rate is in rising trend in the whole world over nearly 50 years, and its sickness rate accounts for the 7-10% of the various malignant tumours of whole body, and the morbidity crowd is rejuvenation trend.At present, the clinical diagnosis mode of mammary cancer mainly contains following several: breast molybdenum target is taken the photograph sheet, infrared galactophore scanning, living tissue pathologic finding, oestrogenic hormon and progesterone receptor and is measured and ultrasonoscopy.Breast molybdenum target takes the photograph sheet and has simply, makes things convenient for, without the characteristics such as wound and expense be low, be one of prefered method of checking of mammary cancer, its size, number, position, density, edge, form, the form that has or not calcification and calcification, size, number, substep and halo on every side by showing lump, skin change etc. provide locates and qualitative sign and judge the character of pathology; Its limitation is: (1) when patient's corpus mamma enriches and pathology overlapping, overall picture that can not lesions showed, even false negative appears, and easily fail to pinpoint a disease in diagnosis for the little cancer kitchen range near the wall of the chest and compactness mammary gland (2), and (3) can not provide clear and definite etiologic diagnosis sometimes.Infrared galactophore scanning is easy and simple to handle, directly perceived, not damaged, with mammography in nonpalpable breast complementary effect is arranged, traveling substep and the caliber that can directly show the mammary gland blood vessel change, so but because it can not show that the internal structure of tumour and surrounding tissue diagnostic accordance rate are very low, the cancer kitchen range of mammary gland top and the nearly wall of the chest is easy to fail to pinpoint a disease in diagnosis.The living tissue pathologic finding is traumatic because of it, complicacy can not be as the means of primary dcreening operation, but its gold standard that to be mammary cancer make a definite diagnosis, and general and Imaging Technology is used in conjunction, and all should obtain the pathological diagnosis foundation before the patient is treated.In the human breast carcinoma tissue, there is 60～70% tissue to have estrogen receptor (ER) and/or progesterone receptor (PR), its existence is relevant with diagnosis, treatment and judging prognosis, and desired mammary cancer pathology report should comprise ER, PR and HER-2(human epidermal growth factor acceptor-2 at least in the breast cancer diagnosis standard of Ministry of Health's issue); Its limitation is mainly manifested in more greatly: (1) technology and equipment requires high, specimen amount is large, therefore be difficult to satisfy the demand to measuring the less breast carcinoma of early stage of non-Operated Specimens or lump, (2) acceptor is to warm extremely unstable, sample is essential fresh, stores transportation inconvenience, acceptor skewness in (3) tumor tissues, edge content is high than central part, draws materials therefore need to mix.Ultra sonic imaging has higher misdiagnosis rate to the good Malignant mass of real property that some lack typical sign, and can not find the lump that 5mm is following.

Studies show that in recent years, mammary cancer is closely related with miRNA, and they may participate in the generation of tumour, so also corresponding effect may be arranged to the treatment of tumour.The miRNA kind relevant with mammary cancer more and the effect differ, there are some researches show that the miRNA that raises comprises miR-26a in the patient with breast cancer, miR-29b, miR-375, miR-92, mir-183, miR-197 etc., the miRNA of downward modulation comprises miR-145, miR-497, miR-339-5p, miR-22, miR-125b etc.Although carried out some researchs in this field, all there is deficiency the accuracy of existing miRNA mark, susceptibility aspect, in clinical and research, still there is the demand of seeking more accurate and sensitive mammary cancer miRNA mark.

Summary of the invention

The invention provides the relevant miRNA of a kind of new mammary cancer as the molecular marker of mammary cancer, this miRNA is miR-147a, and its sequence is 5 '-GUGUGUGGAAAUGCUUCUGC-3 ' (SEQ ID NO:1).

The contriver finds, the content that the content of miR-147a in blood serum of patients with human breast carcinoma is compared in normal human serum raises, and after the patient underwent surgery, the miR-147a content in its serum fell back to normal level, this shows, existing of the miR-147a of increase and tumour is closely related.

On the basis of above-mentioned discovery, the invention provides the purposes of miR-147a in the diagnostic reagent of preparation diagnosing mammary cancer.

Particularly, described diagnostic reagent passes through to detect the content of miR-147a in subject's serum, and compares diagnosing mammary cancer with normal level.In a specific embodiment, detect miR-147a content in subject's serum with the method for quantitative PCR.

The present invention also provides a kind of diagnostic kit of diagnosing mammary cancer, and it comprises:

(1) serum total RNA extraction reagent,

(2) RNA adds polyA reagent,

(3) RT-PCR reagent,

(4) quantitative PCR reagent.

Wherein, comprise the specificity forward primer of mammary cancer molecular marker miR-147a in the quantitative PCR reagent, preferably, its sequence is 5 '-GTGTGTGGAAATGCT-3 ' (SEQ ID NO:7).

In a specific embodiment, comprise in the described serum total RNA extraction reagent that sequence is the External Control-1 of 5 '-CAACCUCCUAGAAAGA-3 ' (SEQ ID NO:2); Described RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of 5 '-UGAGCAACGCGAACAA-3 ' (SEQ ID NO:3); Comprise in the described RT-PCR reagent sequence be 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) RT-Primer(wherein, V is A or C or G, N is A or T or C or G); Comprise in the described quantitative PCR reagent that sequence is respectively general reverse primer UPM-short and the UPM-long of 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:5) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:6).

Use mammary cancer molecular marker miR-147a diagnosing mammary cancer of the present invention to have simple to operate, draw materials conveniently, safely without wound, high specific, high sensitivity and the characteristics that are easy to a large amount of examinations.

Embodiment

1. the extraction of total RNA in the serum

Extract before 5 patient with breast cancer's arts and 7 days each 2ml blood of postoperative, extract simultaneously 5 each 2ml of normal human blood as normal control, carry out centrifugally after the blood coagulation, get at last the RNase/DNase-free centrifuge tube that upper serum 1ml places 1.5ml.

Use RNA to extract test kit (Beijing Quanto Biotechnology Co., Ltd.) and in serum, extract total RNA, add 1 μ l(20nM in per 250 μ l serum) sequence is that External Control-1(Shanghai of 5 '-CAACCUCCUAGAAAGA-3 ' (SEQ ID NO:2) is given birth to worker's biotechnology company limited and synthesized) monitor the extraction quality of RNA in the serum.The total RNA that extracts uses Thermo NanoDrop2000c to measure concentration.

2. the miRNA in the three-step approach detection by quantitative serum

(1) add the polyA tail:

I. prepare the reaction solution that adds the polyA tail in the PCR pipe (Axygen company, 200 μ l) without the RNA enzyme, system is 20 μ l.Add 1 μ l(20nM in per 20 μ l systems) to be that worker's biotechnology company limited is given birth in External Control-2(Shanghai of 5 '-UGAGCAACGCGAACAA-3 ' (SEQ ID NO:3) synthetic for sequence) monitor tailing and the reverse transcription quality of miRNA.

(annotate: the RNA volume of adding is determined by the concentration of RNA, x=500ng/RNA concentration, and this tests the product that employed enzyme is Beijing Quanto Biotechnology Co., Ltd..）

Ii will be equipped with the PCR pipe that configures reaction solution and put into 37 ℃ in PCR instrument (Thermo) and hatched 1 hour.

(2) RT-PCR obtains the cDNA strand:

I. add 0.5 μ l(0.5ng/ μ l in the reaction solution that obtains to (1)) to be that worker's biotechnology company limited is given birth in RT-Primer(Shanghai of 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) synthetic for sequence), hatch 5min for 70 ℃, be put into immediately on ice at least 2min of ice bath.

Ii prepares inverse transcription reaction liquid:

(annotate: used product is the product of Beijing Quanto Biotechnology Co., Ltd..）

Iii. the solution that i and ii is obtained mixes, and hatches 70 ℃ of insulation 15min behind the 50min for 50 ℃, puts cooled on ice, obtains cDNA.

Iv. the product that iii is obtained is diluted to the reverse transcription product that contains the total RNA of 1ng in the 1 μ l system ,-20 ℃ of preservations after the packing.

(3) qPCR detection by quantitative:

I. in 2ml EP pipe (Axygen company), prepare reaction solution:

(annotate: UPM-long, UPM-short are all synthetic in Invitrogen company, and all the other reagent are all from Beijing Quanto Biotechnology Co., Ltd.

Green PCR Master Mix.）

Wherein, the sequence of general reverse primer UPM-short and UPM-long is respectively 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:5) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:6).

After the reaction solution that ii configures is fully put upside down mixing, be distributed in the point end PCR plate of 96 holes (Axygen company) every hole 18 μ l.

Iii. using the volley of rifle fire (Eppendoff company, 1-10 μ l) adding sequence is the miR-147a specificity forward primer (Invitrogen company is synthetic) of 5 '-GTGTGTGGAAATGCT-3 ' (SEQ ID NO:7), every hole 2 μ l(10 μ M).

Iv. seal with special-purpose pad pasting (ABI company) after adding 10 μ l paraffin oil fluid-tights.

V. put into the ABI7900PCR instrument, program setting is:

Vi. draw melting curve, check the specificity of primer, program setting is: 95 ℃ of 15s, 60 ℃ of 15s, 95 ℃ of 15s.

3. adopt Array Tools4.1.0 to carry out data analysis

Before can recording patient with breast cancer's art with aforesaid method, postoperative 7 days and normal people organize that the average Ct value of miR-147a is respectively 29.24,33.40 and 32.94 in each sample serum, the result shows that miR-147a relative content in the serum before operation in patients significantly raises, and operative results is to normal level.

With 2 of classics in the qPCR detection^{-Δ Ct}Mode represent the level (Δ Ct is Ct value poor of target miRNA and External Control-1) of purpose miRNA in the serum.Compare with the content of miR-147a in the normal control serum, the level of miR-147a is its 13.00 times (2 in the front serum of patient with breast cancer's art^{-(29.24-32.94)}), significant difference; And behind the operation in patients in the serum miR-147a content be 0.73 times (2 of normal control^{-(33.40-32.94)}), difference is not remarkable.

＜110〉Beijing Quanto Biotechnology Co., Ltd.

＜120〉mammary cancer molecular marker miR-147a

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<170> PatentIn version 3.3

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Claims (10)

1. Mammary cancer molecular marker miR-147a the preparation diagnosing mammary cancer diagnostic reagent in purposes.
2. 2. the described purposes of claim 1, wherein, the sequence of described mammary cancer molecular marker miR-147a is shown in SEQ ID NO:1.
3. 3. the described purposes of claim 1, wherein, described diagnostic reagent is by detecting the content of miR-147a in subject's serum, and compares diagnosing mammary cancer with normal level.
4. 4. the described purposes of claim 3 wherein, detects miR-147a content in subject's serum with the method for quantitative PCR.
5. 5. the diagnostic kit of a diagnosing mammary cancer, it comprises:

   (1) serum total RNA extraction reagent,

   (2) RNA adds polyA reagent,

   (3) RT-PCR reagent,

   (4) quantitative PCR reagent;

   Wherein, the specificity forward primer that comprises the molecular marker of mammary cancer described in the claim 1 miR-147a in the quantitative PCR reagent.
6. 6. the described diagnostic kit of claim 5 wherein, comprises in the described serum total RNA extraction reagent that sequence is the External Control-1 of SEQ ID NO:2.
7. 7. the described diagnostic kit of claim 5, wherein, described RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of SEQ ID NO:3.
8. 8. the described diagnostic kit of claim 5 wherein, comprises in the described RT-PCR reagent that sequence is the RT-Primer of SEQ ID NO:4.
9. 9. the described diagnostic kit of claim 5 wherein, comprises in the described quantitative PCR reagent that sequence is respectively general reverse primer UPM-short and the UPM-long of SEQ ID NO:5 and 6.
10. 10. the described diagnostic kit of claim 5, wherein, the sequence of the specificity forward primer of described mammary cancer molecular marker miR-147a is shown in SEQ ID NO:7.