CN102827943A - Application of miRNA (micro ribonucleic acid) 320a in prostate cancer serological diagnostic kit - Google Patents
Application of miRNA (micro ribonucleic acid) 320a in prostate cancer serological diagnostic kit Download PDFInfo
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- CN102827943A CN102827943A CN2012103516836A CN201210351683A CN102827943A CN 102827943 A CN102827943 A CN 102827943A CN 2012103516836 A CN2012103516836 A CN 2012103516836A CN 201210351683 A CN201210351683 A CN 201210351683A CN 102827943 A CN102827943 A CN 102827943A
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Abstract
The invention relates to an application of an miRNA (micro ribonucleic acid, miR-320a, shown in SEQIDNO: 1) in the prostate cancer serological diagnostic kit, in particular to an application of reagents detecting miR-320a of a blood sample in the preparation of the object prostate cancer diagnostic kit, wherein the reagents are reagents specific to the miRNA (miR-320a) and the precursor of the miRNA; and particularly, the reagents are a specific probe detecting the miR-320a and the precursor, and a polymerase chain reaction (PCR) primer. The invention also relates to a prostate cancer diagnostic kit which contains a PCR reactive reagent, a miR-320a specific primer, a nematode miR-39 reference oligonucleotide sequence, a nematode miR-39 specific primer, a fluorescent dye SYBR-GREEN1 and an operating manual. When the detection result shows that the level of the miR-320a or precursor in the object blood is higher than that in the control serum, the object is shown to suffer from prostate cancer, thus the kit can be effectively used in the auxiliary diagnosis of clinical prostate cancer.
Description
Technical field
The present invention relates to a kind of Microrna (micro RA, miRNA, the miR) application of 320a in prostate cancer serodiagnosis test kit.Belong to technical field of biomedical materials.
Background technology
Prostate cancer (prostate carcinoma, PCa) be American-European countries male sex common cancer and major causes of death it
Along with the westernization of prolongation of China's average life span and mode of life, the PCa sickness rate is increases trend year by year, and its is classified as one of fastest-rising tumour of China's 21 century.On the basis of the miRNAs sequence of 19 up-regulateds in prostate cancer tissue that this project filters out, further detect its content in clinical serum sample in previous work, seek human prostate metastasis of cancer patient's Serology biological mark.
Prostate cancer diagnosis Study of indexes work at present mainly concentrates on the following aspects: (I) PSA hypotype and other kallikrein (like serum KLK2); (II) DNA biomarker comprises some epigenetics biomarkers (methylating like GSTPi), gene fusion (merging like TMPRSS2:ERG) and heterozygosity disappearance etc.; (III) RA biomarker is like RA PCA3 (once being called as DD3) and Alpha-Methyl acyl-CoA racemase (Alpha-methylacyl CoA racemase, the AMACR) expression of mRA in prostate cancer of noncoding prostate-specific; (IV) protein molecular mark, like PSA, prostatic specific membrane antigen (prostate-specifc membrane antigen, PSMA), PSCA (prostatestem cell antigen, PSCA) etc.Although many candidates' biological markers is constantly coming to light, index and the method for Shang Wuxin are applied to clinical detection.
MiRAs is a kind of little RAs of endogenous of not proteins encoded newly, and they are bringing into play important regulation through arrestin matter translation process aspect the biological functions such as cell proliferation, apoptosis and differentiation.Through means such as the little array of miRAs, Northern blot and PCR in real time, in broad variety people tumour, had been found that the phenomenon of some miRAs up-regulateds and downward modulation.There is research to report that miRA has brought into play important effect in mammary cancer and hepatoma Metastasis.In recent years discover that have other tissue-derived miRNAs in human plasma and the serum, they can exist with a kind of form of resisting endogenous RA enzymic activity.The content of measuring tumour source miRNAs in blood plasma or the serum can be used as one of means of lesion detection.In recent years there is the investigator to report that the level of miRA from serum and some other body fluid can diagnose some to be in early stage disease.Mitchell in 2008 report, miR-141 is higher 46 times than control group in prostate cancer group patients serum, and its sensitivity is 60%, and specificity is 100%.Can be for the further investigation of miRAs in the serum and to seek the disease biomarker new unique perspective is provided.
The research report is arranged, and miR-320a is frequent high expression level in most people's tumours such as liver cancer, carcinoma of the pancreas, bladder cancer, glioblastoma multiforme, thyroid papillary carcinoma, melanoma.It through targeting in cancer suppressor gene such as p27, p57, PTEN, preceding antiapoptotic factors Bim, Bmf etc. suppress apoptosis, promote cell growth and migration, participate in the incidence and development process of tumour.Present result of study prompting miRA-320a possibly be a kind of carcinogenic miRNA, is playing the part of vital role in the generation and the developmental stage of human kinds of tumors.
Summary of the invention
The purpose of this invention is to provide the reagent that detects miR-320a in the blood is used for the serodiagnostic test kit of prostate cancer in preparation application.
Heteroplastic transplantation model and new-generation sequencing technology that the present invention utilizes us to invent; Transcription group to miRNAs in transfer and the non-metastasized prostate cancer xenograft tissues system has been carried out deep research; The preliminary grade malignancy relevant (Watahiki et al., PLOS One 2011) of finding miR-320a and prostate cancer.
The method that we have optimized from serum the method for extracting miRNA and have detected miRNA with real-time quantitative PCR; Put and collected the pathological data and the serum sample of over one hundred part of patients with prostate cancer in order; 19 miRNA sequences that in the metastasized prostate cancer tissue, raise to filtering out with heteroplastic transplantation model have been carried out the detection of clinical serum sample, disclosed miR-320a in patients with prostate cancer serum with tissue in content obviously raise.Has important application prospects in the serology clinical diagnosis scheme of this invention to exploitation Chinese population prostate cancer.
The present invention at first provides the application of the test kit that a kind of reagent that detects Microrna miR-320a in the blood sample and precursor thereof is used in preparation object prostate cancer diagnosis and regimen are selected; Said reagent is that Microrna miR-320a and precursor thereof are had specific reagent, specifically: the gene chip that miR-320a and precursor thereof are had the PCR primer and the hybridization probe of detection specificity and utilize this probe preparation.
The sequence of said miR-320a is shown in SEQ ID NO:1;
In a preference, said Auele Specific Primer is selected from sequence shown in the SEQ ID NO:2;
In a preference, said specific probe is selected from sequence shown in the SEQ ID NO:5.
The present invention comes diagnosis object whether to suffer from prostate cancer through the content that detects miR-320a in the blood.
In a preference, through real time quantitative PCR method miR-320a is measured, comprising: the RNA in the object blood sample is extracted in (1), and the nematode miR-39 oligonucleotide of interpolation synthetic is as object of reference; (2) utilize reverse transcription method to synthesize cDNA; (3) content of miR-320a in the employing real time quantitative PCR method test sample; (4) content with nematode miR-39 carries out stdn as reference.If miR-320a content is compared obvious rising with the normal control group in the object serum, then diagnose this object to suffer from prostate cancer.
The level of Microrna miR-320a or its precursor is higher than the level in the control serum in detected result demonstration object blood, then points out said object to suffer from prostate cancer.
Secondly, the present invention provides a kind of prostatic cancer diagnostic reagent kit simultaneously, and the target nucleic acid that this test kit detects is the miR-320a with polynucleotide sequence shown in the SEQ ID NO:1, specifically comprises:
(1) pcr reagent; Comprise warm start Taq DAN polysaccharase (2.5U/ul) and reaction buffer thereof, dNTP (10mM);
(2) miR-320a Auele Specific Primer; In a preference, said miR-320a Auele Specific Primer is selected from sequence shown in the SEQ IDNO:2;
(3) nematode miR-39 is with reference to oligonucleotide sequence: 5 '-UCACCGGGUGUAAAUCAGCUUG-3 ' (SEQIDNO:3);
(4) nematode miR-39 Auele Specific Primer: 5 '-CCGGGTGTAAATCAGCTTGAAA-3 ' (SEQ ID NO:4);
(5) optical dye SYBR-GREEN 1;
(6) working instructions.
Advantage of the present invention and beneficial effect:
Utilize the reagent that provides in this test kit, the RNA that knows in conjunction with this area researchist extracts reagent and general miRNA reverse transcription reagent, and the present invention can detect the content of miR-320a in the serum specifically, is effective to the auxiliary diagnosis of clinical prostate cancer.
Description of drawings
Fig. 1 is PCR in real time amplification curve and the solubility curve of confidential reference items miRNA and purpose miRNA, and visible solubility curve is unimodal, and specific peak occurs nothing but;
Fig. 2 detects miR-320a at normal people and patients with prostate cancer expression of serum amount post height=x-+SE for qRT-PCR;
Fig. 3 is miR-320a relative expression quantity ROC tracing analysis in patients with prostate cancer serum.
Fig. 4 is miR-320a FISH photo (200 *) A:Normal prostate B:PCa-1 (gleasonscore:6 branch) C:PCa-2 (gleason score:9 branch) of prostata tissue.
Embodiment
Embodiment 1
The present invention at first provides the application of the test kit that a kind of reagent that detects Microrna miR-320a in the blood sample and precursor thereof is used in preparation object prostate cancer diagnosis and regimen are selected; Said reagent is that Microrna miR-320a (shown in SEQ ID NO:1) and precursor thereof are had specific reagent, specifically: miR-320a and precursor thereof are had the PCR primer and the probe of detection specificity and use the gene chip of this probe preparation.
Described Auele Specific Primer is selected from sequence shown in the SEQ ID NO:2;
Described specific probe is selected from sequence shown in the SEQ ID NO:5.
Embodiment 2, prostatic cancer diagnostic reagent kit
The target nucleic acid that test kit according to the invention detects is the miR-320a with polynucleotide sequence shown in the SEQ ID NO:1, specifically comprises:
(1) pcr reagent; Comprise warm start Taq DAN polysaccharase (2.5U/ul) and reaction buffer thereof, dNTP (10mM);
(2) miR-320a Auele Specific Primer; In a preference, said miR-320a Auele Specific Primer is selected from sequence shown in the SEQ IDNO:2;
(3) nematode miR-39 is with reference to oligonucleotide sequence: 5 '-UCACCGGGUGUAAAUCAGCUUG-3 ' (SEQIDNO:3);
(4) nematode miR-39 Auele Specific Primer: 5 '-CCGGGTGTAAATCAGCTTGAAA-3 ' (SEQ ID NO:4);
(5) optical dye SYBR-GREEN 1;
(6) working instructions.
Embodiment 3, diagnosis effect experiment
1, utilize the heteroplastic transplantation model that we invent (be about to prostate cancer tissue and be transplanted in the kidney peplos of immunity shortcoming mouse,
The pathological characters height correlation of resulting tissue system and clinical tumor) and new-generation sequencing technology, to transitivity and non-
MiRNAs in the metastatic prostate cancer xenograft tissues system (specimens from pri that comes from same patients with prostate cancer)
Transcription group carried out deep research.Through relatively we identify the miRNAs of 104 differential expressions, remove
Some known miRNAs and their shaped body (isomiRs), some is still undiscovered new
miRNAs。Wherein 21 miRNAs have come to light relevantly with the generation of prostate cancer, and 5 miRNAs
Be reported in prostate cancer and play a role in shifting, this also proved from the side research means that we adopt effectively
The property.Except those have been proved to be with prostate cancer shifts relevant miRNAs, we have also found 36 so far
The miRNAs that is not in the news as yet till the present (Watahiki et al., PLOS One 2011), this wherein possibly exist potential
The miRNAs that can be used as Chinese population patients with prostate cancer Serology biological mark.
2, the extraction of micro-miRNA in the serum
Because the content of miRNA in serum seldom, in experiment in earlier stage, we have consulted a large amount of documents, have attempted pure
Change post test kit extraction method and liquid phase Trizol extraction method and extract miRNA.Find the miRNA's of Trizol method extraction
Amount and purity more better (protein content of miRNA and salt content are still less).We carry out in the Trizol method
The optimization of two aspects: it is more to contain protein content in (1) serum, can strengthen the content of Trizol, so that contained egg
White matter obtains sufficient sex change; And carry out repeatedly extracting with phenol/chloroform/primary isoamyl alcohol, so that contained protein is filled
The removal that divides; (2),, be not easy deposition down adding isopropanol precipitating in this step because the miRNA molecular weight is very little
Come, the prolongation ST that can be an amount of, increase the yield of miRNA so that miRNA precipitates fully.3, the detection method of micro-miRNA in the serum
Adopt the synthetic cDNA of All-in-OneT miRNA First-Strand cDNA Synthesis Kit reverse transcription.
According to characteristics and the pcr amplification principle of miRNA, adopt the test kit of being invented at MJ Opticon II quantitative PCR
Detect the content of miR-320a and nematode miR-39 on the appearance.The relative expression quantity of miRNA uses 2-Δ Δ Ct method to calculate.4, detect the content of miRNAs in patients with prostate cancer serum of up-regulated in prostate cancer tissue
(1) our over one hundred part of clinical data and patient history letter that the second uropoiesis institute of affiliated hospital of Medical University Of Tianjin is provided
Breath etc. has carried out housekeeping, has filtered out 35 routine normal peoples, and 49 routine high classification (7-9 branch) prostate cancers are suffered from
Person's case history, the low classification prostate gland patient medical record of 13 examples, and obtain its blood sample.
(2) these clinical samples are carried out miRNA and extract, QRT-PCR detects and obtains following result:
From collected serum sample, extract RNA, utilize the PCR primer that is specific to miR-320a, adopt the expression level of this miRNA of method detection of reverse transcription-real-time quantitative PCR.The employed PCR primer sequence to be detected (Fig. 1) that can increase special, effectively.Resulting data are carried out independent sample T check analysis with SPSS software to it, and miR-320a expresses rising (Fig. 2) in patients with prostate cancer serum.
The relative expression multiple of table 1 miR-320a in normal group and prostate cancer group serum
(3) sensitivity and specificity analyses
SPSS software is carried out the ROC curve tracing to the qRT-PCR data of miR-320a, and analyze sensitivity and specificity that it detects prostate cancer group serum: miR-320a is 1.809 o'clock in threshold value, and sensitivity and specificity are respectively 67.1% and 74.3%.
5.miR-320a the expression level in prostata tissue
This experiment is to utilize the oligonucleotide probe that is selected from the digoxigenin labeled of sequence shown in the SEQ ID NO:5, and the fluorescence in situ hybridization technique that adopts this area colleague technician to know has detected the expression of miR-320a in prostate cancer tissue.At first the ripe miRA in specific probe and the paraffin organization section is hybridized; Hatch with the anti digoxin antibody of rhodamine mark afterwards; Dye nuclear with DAPI, thereby make fluorescence labels on the crossbred band, detection can be to its location or quantitative analysis under fluorescent microscope.
Hybridization through this probe detects confirmation, and miR-320a signal than fluorescent probe in healthy tissues in prostate cancer tissue is more, stronger, confirms that miR-320a expresses rising (Fig. 4) in prostate cancer tissue.
Claims (3)
1. the reagent of Microrna miR-320a and precursor thereof is used for the application to the test kit of object prostate cancer diagnosis and regimen selection in preparation in the detection blood sample; Said reagent is that Microrna miR-320a and precursor thereof are had specific reagent, specifically: the gene chip that miR-320a and precursor thereof are had the PCR primer and the probe of detection specificity and utilize this probe preparation.
2. the application described in claim 1 is characterized in that, the sequence of said miR-320a is shown in SEQ ID NO:1.
3. a prostatic cancer diagnostic reagent kit is characterized in that the target nucleic acid that this test kit detects is the miR-320a with polynucleotide sequence shown in the SEQ IDNO:1, specifically comprises:
(1) pcr reagent;
(2) miR-320a Auele Specific Primer is shown in SEQ ID NO:2;
(3) nematode miR-39 is with reference to oligonucleotide sequence, shown in SEQ ID NO:3;
(4) nematode miR-39 Auele Specific Primer is shown in SEQ ID NO:4;
(5) optical dye SYBR-GREEN 1;
(6) working instructions.
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Citations (4)
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| US20080076674A1 (en) * | 2006-07-06 | 2008-03-27 | Thomas Litman | Novel oligonucleotide compositions and probe sequences useful for detection and analysis of non coding RNAs associated with cancer |
| 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 |
| WO2009143379A2 (en) * | 2008-05-21 | 2009-11-26 | Fred Hutchinson Cancer Research Center | Use of extracellular rna to measure disease |
| CN101987197A (en) * | 2009-07-31 | 2011-03-23 | 中国科学院上海生命科学研究院 | Method and reagent for inhibiting invasiveness of cancer cells |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20080076674A1 (en) * | 2006-07-06 | 2008-03-27 | Thomas Litman | Novel oligonucleotide compositions and probe sequences useful for detection and analysis of non coding RNAs associated with cancer |
| WO2009143379A2 (en) * | 2008-05-21 | 2009-11-26 | Fred Hutchinson Cancer Research Center | Use of extracellular rna to measure disease |
| CN101987197A (en) * | 2009-07-31 | 2011-03-23 | 中国科学院上海生命科学研究院 | Method and reagent for inhibiting invasiveness of cancer cells |
Non-Patent Citations (3)
| Title |
|---|
| PATRICK S. MITCHELL ET AL.: "Circulating microRNAs as stable blood-based markers for cancer detection", 《PNAS》, 29 July 2008 (2008-07-29), pages 10513 - 10518 * |
| XI CHEN ETAL: "Characterizaiton of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases", 《CELL RESEARCH》, 2 September 2008 (2008-09-02), pages 997 * |
| ZHIHUA TAO ET AL: "Characterization of the Small RNA Transcriptomes of Androgen Dependent and Independent Prostate Cancer Cell Line by Deep Sequencing", 《PLOS ONE》, 30 November 2010 (2010-11-30) * |
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