CN102676524A - Molecular marker miR-147a of breast cancer - Google Patents
Molecular marker miR-147a of breast cancer Download PDFInfo
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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
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) is a hot research in recent years; 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, sequential property 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 in research subsequently again.
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 kinds: 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.Characteristics such as breast molybdenum target is taken the photograph sheet and had simply, makes things convenient for, no wound and expense are low; Be one of prefered method of mammary cancer inspection, its size, number, position, density, edge, form, the form that has or not calcification and calcification, size, number, substep and halo on every side through showing lump, skin change etc. provide locatees and qualitative sign and judge the character of pathology; Its limitation is: (1) when patient's mammary gland abundant glandular and pathology overlapping; Overall picture that can not lesions showed; Even false negative appears, and fail to pinpoint a disease in diagnosis for the little cancer kitchen range near the wall of the chest and compactness mammary gland easily (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; Take the photograph sheet with molybdenum target the complementary effect is arranged; The traveling substep and the caliber that can directly show the mammary gland blood vessel change; So but, the cancer kitchen range of the mammary gland top and the nearly wall of the chest is easy to fail to pinpoint a disease in diagnosis because of it can not show that the internal structure of tumour and surrounding tissue diagnostic accordance rate are very low.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 iconography technical battery usefulness 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 ERs (ER) and/or progesterone receptor (PR); It exists situation 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 mainly shows: (1) technology and equipment requires high, and specimen amount is big, so to measuring non-excision sample or the less breast carcinoma of early stage of lump is difficult to satisfy the demand; (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 so need to mix.Ultra sonic imaging has higher misdiagnosis rate to the good pernicious lump of real property that some lack typical sign, and can not find the lump below the 5mm.
Research in recent years shows that mammary cancer is closely related with miRNA, and they possibly participate in the generation of tumour, so tumor treatment is also had corresponding effect.The miRNA kind relevant with mammary cancer more and the effect differ, existing research is illustrated in the miRNA that raises among the patient with breast cancer and comprises miR-26a, 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.Though carried out some researchs in this field, all there is deficiency the accuracy of existing miRNA mark, susceptibility aspect, in clinical and research, still exists and seek more accurately and the demand of sensitive mammary cancer miRNA mark.
Summary of the invention
The invention provides the molecular marker of the relevant miRNA of a kind of new mammary cancer as 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 patient with breast cancer's serum 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 that 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, said diagnostic reagent passes through to detect the content of miR-147a in testee's serum, and compares diagnosing mammary cancer with normal level.In a specific embodiment, use the method for quantitative PCR to detect miR-147a content in testee's serum.
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 said serum total RNA extraction reagent that sequence is the External Control-1 of 5 '-CAACCTCCTAGAAAGA-3 ' (SEQ ID NO:2); Said RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:3); Comprise in the said RT-PCR reagent that sequence is the RT-Primer (wherein, V is A or C or G, and N is A or T or C or G) of 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4); Comprise in the said quantitative PCR reagent that sequence is respectively general reverse primer UPM-short and the UPM-long of 5 '-CTCACACGACTCACGACAC-3 ' (SEQ IDNO:5) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ IDNO:6).
It is simple to operate to use mammary cancer molecular marker miR-147a diagnosing mammary cancer of the present invention to have, and draws materials conveniently safety no 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 5 each 2ml of normal human blood simultaneously, carry out centrifugally after the blood coagulation, get the RNase/DNase-free centrifuge tube that upper serum 1ml places 1.5ml at last as normal control.
Use RNA to extract test kit (Beijing Quanto Biotechnology Co., Ltd.) and in serum, extract total RNA, add the extraction quality that the External Control-1 that 1 μ l (20nM) sequence is 5 '-CAACCTCCTAGAAAGA-3 ' (SEQ ID NO:2) (it is synthetic that worker's biotechnology ltd is given birth in Shanghai) monitors RNA in the serum in per 250 μ l serum.The total RNA that extracts uses Thermo NanoDrop 2000c to measure concentration.
2. the miRNA in the three-step approach detection by quantitative serum
(1) add the polyA tail:
I. in the PCR pipe (Axygen company, 200 μ l) of no RNA enzyme, prepare the reaction solution that adds the polyA tail, system is 20 μ l.The External Control-2 (it is synthetic that worker's biotechnology ltd is given birth in Shanghai) that adds 1 μ l (20nM) sequence in per 20 μ l systems and be 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:3) monitors tailing and the reverse transcription quality of miRNA.
(annotate: the RNA volume of adding is confirmed by the concentration of RNA, x=500ng/RNA concentration, and this tests the product that employed enzyme is Beijing Quanto Biotechnology Co., Ltd..）
Ii., the PCR pipe that configures reaction solution will be housed to be put into 37 ℃ in PCR appearance (Thermo) and hatched 1 hour.
(2) RT-PCR obtains the cDNA strand:
I. in the reaction solution that (1) obtains, add the RT-Primer that 0.5 μ l (0.5ng/ μ l) sequence is 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4) (it is synthetic that worker's biotechnology ltd is given birth in Shanghai); Hatch 5min for 70 ℃, be put on ice ice bath 2min at least immediately.
Ii. prepare 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
Green PCR Master Mix of Beijing Quanto Biotechnology Co., Ltd..）
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).
Ii. after the reaction solution that configures is fully put upside down mixing, divide in the 96 holes point end PCR plate of packing into (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 Yellow Protopet 2A fluid-tights.
V. put into ABI 7900 PCR detection by quantitative appearance, program setting is:
Vi. draw melting curve, the specificity of inspection primer, program setting is: 95 ℃ of 15s, 60 ℃ of 15s, 95 ℃ of 15s.
3. adopt Array Tools 4.1.0 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 patient's art significantly raises, and operative results is to normal level.
The mode of classical 2-Δ Ct is represented the level (Δ Ct is Ct value poor of target miRNA and External Control-1) of purpose miRNA in the serum in detecting with qPCR.Compare with the content of miR-147a in the normal control serum, the level of miR-147a is its 13.00 times of (2-in the preceding serum of patient with breast cancer's art
(29.24-32.94)), significant difference; And miR-147a content is 0.73 times of (2-of normal control in patient's postoperative serum
(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, its sequence is shown in SEQ ID NO:1.
The said mammary cancer molecular marker of claim 1 miR-147a the preparation diagnosing mammary cancer diagnostic reagent in purposes.
3. the said purposes of claim 2, wherein, said diagnostic reagent is through detecting the content of miR-147a in testee's serum, and compares diagnosing mammary cancer with normal level.
4. the said purposes of claim 3 wherein, uses the method for quantitative PCR to detect miR-147a content in testee's serum.
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 said mammary cancer molecular marker of claim 1 miR-147a in the quantitative PCR reagent.
6. the said diagnostic kit of claim 5 wherein, comprises in the said serum total RNA extraction reagent that sequence is the External Control-1 of SEQ ID NO:2.
7. the said diagnostic kit of claim 5, wherein, said RNA adds and comprises in the polyA reagent that sequence is the External Control-2 of SEQ ID NO:3.
8. the said diagnostic kit of claim 5 wherein, comprises in the said RT-PCR reagent that sequence is the RT-Primer of SEQ ID NO:4.
9. the said diagnostic kit of claim 5 wherein, comprises in the said quantitative PCR reagent that sequence is respectively the general reverse primer UPM-short and the UPM-long of SEQ ID NO:5 and 6.
10. the said diagnostic kit of claim 5, wherein, the sequence of the specificity forward primer of said mammary cancer molecular marker miR-147a is shown in SEQ ID NO:7.
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| CN101389770A (en) * | 2006-01-05 | 2009-03-18 | 俄亥俄州立大学研究基金会 | Microrna-based methods and compositions for the diagnosis, prognosis and treatment of solid cancers |
| CN101842484A (en) * | 2007-09-14 | 2010-09-22 | 俄亥俄州立大学研究基金会 | Mirna expression in human peripheral blood microvesicles and uses thereof |
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| MARIANA LAGOS-QUINTANA等: "Identification of tissue-specific microRNAs from mouse", 《CURRENT BIOLOGY》 * |
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