CN102816862A - Application of miRNA (Micro Ribonucleic Acid) 222 to prostatic cancer serological diagnosis kit - Google Patents
Application of miRNA (Micro Ribonucleic Acid) 222 to prostatic cancer serological diagnosis kit Download PDFInfo
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- CN102816862A CN102816862A CN2012103516840A CN201210351684A CN102816862A CN 102816862 A CN102816862 A CN 102816862A CN 2012103516840 A CN2012103516840 A CN 2012103516840A CN 201210351684 A CN201210351684 A CN 201210351684A CN 102816862 A CN102816862 A CN 102816862A
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Abstract
The invention relates to application of a miRNA (miR-222 shown as SEQ ID NO: 1) to a prostatic cancer serological diagnosis kit, and in particular relates to application of a reagent for detecting miR-222 in a blood sample to preparation of a kit for diagnosing object prostatic cancers. The reagent has specificity to the micro RNA (Ribonucleic Acid) miR-222 and a precursor thereof, and is concretely a probe and a PCR (Polymerase Chain Reaction) primer for detecting the specificity of the microRNAmiR-222 and the precursor thereof. The invention further relates to a prostatic cancer diagnosis kit comprising a polymerase chain reaction reagent, a miR-222 specific primer, a nematode miR-39 reference oligonucleotide sequence, a nematode miR-39 specific primer and a fluorochrome SYBR-GREEN1 which are shown in the specification. When a detection result displays that the level of the miR-222 or the precursor thereof in an object blood is higher than that in a reference serum, the object is prompted to suffer from the prostatic cancers. The kit can be effectively used for auxiliary diagnosis of clinical prostatic cancers.
Description
Technical field
The present invention relates to a kind of Microrna (micro RNA, miRNA, miR) 222 application in prostate cancer serodiagnosis test kit.Belong to technical field of biomedical materials.
Background technology
(prostate carcinoma PCa) is one of American-European countries male sex common cancer and major causes of death to prostate cancer.
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) RNA biomarker is like RNA PCA3 (once being called as DD3) and Alpha-Methyl acyl-CoA racemase (Alpha-methylacyl CoA racemase, the AMACR) expression of mRNA in prostate cancer of noncoding prostate-specific; (IV) protein molecular mark, like PSA, prostatic specific membrane antigen (prostate-specific membrane antigen, PSMA), PSCA (prostatestem cell antigen, PSCA) [14] etc.Although many candidates' biological markers is constantly coming to light, index and the method for Shang Wuxin are applied to clinical detection.
MiRNAs is a kind of little RNAs 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 miRNAs, Northern blot and PCR in real time, in broad variety people tumour, had been found that the phenomenon of some miRNAs up-regulateds and downward modulation.There is research to report that miRNA 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 RNA 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 miRNA 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 miRNAs in the serum and to seek the disease biomarker new unique perspective is provided.
The research report is arranged, and miR-222 is frequent high expression level in most of human 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 miRNA-222 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-222 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-222 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-222 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-222 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-222 and precursor thereof are had specific reagent, specifically: the gene chip that miR-222 and precursor thereof are had the PCR primer and the probe of detection specificity and utilize this probe preparation.
The sequence of said miR-222 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-222 in the blood.
In a preference, through real time quantitative PCR method miR-222 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-222 in the employing real time quantitative PCR method test sample; (4) content with nematode miR-39 carries out stdn as reference.If miR-222 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-222 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-222 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-222 Auele Specific Primer; In a preference, said miR-222 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-222 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-222 at normal people and patients with prostate cancer expression of serum amount post height=x for qRT-PCR
-+ SE;
Fig. 3 is miR-222 relative expression quantity ROC tracing analysis in patients with prostate cancer serum.
Fig. 4 is miR-222FISH photo (200 *) A:Normal prostate B:PCa-1 (the gleason score:6 branch) C:PCa-2 (gleason score:9 branch) of prostata tissue.
Embodiment
The present invention at first provides the application of the test kit that a kind of reagent that detects Microrna miR-222 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-222 (shown in SEQ ID NO:1) and precursor thereof are had specific reagent, specifically: miR-222 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.
The target nucleic acid that test kit according to the invention detects is the miR-222 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-222 Auele Specific Primer; In a preference, said miR-222 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 ' (SEQID NO:3);
(4) nematode miR-39 Auele Specific Primer: 5 '-CCGGGTGTAAATCAGCTTGAAA-3 ' (SEQ ID NO:4);
(5) optical dye SYBR-GREEN 1;
(6) working instructions.
1, (be about to prostate cancer tissue is transplanted in the kidney peplos of immunity shortcoming mouse the heteroplastic transplantation model that utilizes us to invent; The pathological characters height correlation of resulting tissue system and clinical tumor) and new-generation sequencing technology, transitivity and the transcription group of the middle miRNAs of non-metastatic prostate cancer xenograft tissues system (specimens from pri that comes from same patients with prostate cancer) have been carried out deep research.Through relatively we identify the miRNAs of 104 differential expressions, except 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 have been reported in prostate cancer and have played a role in shifting, and this has also proved the validity of the research means that we adopt from the side.Except those have been proved to be with prostate cancer shifts relevant miRNAs; We have also found 36 miRNAs that are not in the news as yet up to now (Watahiki et al.; PLOS One 2011), this wherein possibly exist the miRNAs that potential 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 purification column test kit extraction method and liquid phase Trizol extraction method and have extracted miRNA.Find amount and the purity more better (protein content of miRNA and salt content are still less) of the miRNA that the Trizol method is extracted.We have carried out the optimization aspect two in the Trizol method: it is more to contain protein content in (1) serum, can strengthen the content of Trizol, so that contained protein obtains sufficient sex change; And carry out repeatedly extracting with phenol/chloroform/primary isoamyl alcohol, so that contained protein is removed fully; (2) because the miRNA molecular weight is very little, adding isopropanol precipitating in this step, be not easy to precipitate, the prolongation ST that can be an amount of,, miRNA increases the yield of miRNA so that precipitating fully.
3, the detection method of micro-miRNA in the serum
Adopt All-in-One
TMCDNA is synthesized in 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 on MJ Opticon II quantitative PCR appearance, to detect the content of miR-222 and nematode miR-39.The relative expression quantity of miRNA uses 2-
Δ Δ CtMethod is calculated.
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 that the second uropoiesis institute of affiliated hospital of Medical University Of Tianjin is provided and patient history information etc. have been carried out housekeeping; 28 routine normal peoples have been filtered out; 49 routine high classification (7-9 branch) patients with prostate cancer case histories; 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-222, 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-222 expresses rising (Fig. 2) in patients with prostate cancer serum.
The relative expression multiple of table 1miR-222 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-222, and analyze sensitivity and specificity that it detects prostate cancer group serum: miR-222 is 4.06 o'clock in threshold value, and sensitivity and specificity are respectively 40.3% and 88.6% (Fig. 3).
5.miR-222 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-222 in prostate cancer tissue.At first the ripe miRNA 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-222 signal than fluorescent probe in healthy tissues in prostate cancer tissue is more, stronger, confirms that miR-222 expresses rising (Fig. 4) in prostate cancer tissue.
Claims (3)
1. the reagent of Microrna miR-222 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-222 and precursor thereof are had specific reagent, specifically: the gene chip that miR-222 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-222 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-222 with polynucleotide sequence shown in the SEQ IDNO:1, specifically comprises:
(1) pcr reagent;
(2) miR-222 Auele Specific Primer is shown in SEQ ID NO:2;
(3) nematode miR-39 is with reference to oligonucleotide sequence, shown in SEQ IDNO:3;
(4) nematode miR-39 Auele Specific Primer is shown in SEQ IDNO:4;
(5) optical dye SYBR-GREEN 1;
(6) working instructions.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105505934A (en) * | 2016-01-11 | 2016-04-20 | 中国科学院北京基因组研究所 | Specificity miRNA related to M5 type acute myelogenous leukemia and application thereof |
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| WO2009143379A2 (en) * | 2008-05-21 | 2009-11-26 | Fred Hutchinson Cancer Research Center | Use of extracellular rna to measure disease |
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| WO2007081740A2 (en) * | 2006-01-05 | 2007-07-19 | The Ohio State University Research Foundation | Micrornarna-based methods and compositions for the diagnosis and treatment of solid cancers |
| WO2009058893A2 (en) * | 2007-10-29 | 2009-05-07 | University Of Southern California | Preventing hyaluronan-mediated tumorigenetic mechanisms using intronic rnas |
| WO2009143379A2 (en) * | 2008-05-21 | 2009-11-26 | Fred Hutchinson Cancer Research Center | Use of extracellular rna to measure disease |
| WO2012015765A2 (en) * | 2010-07-27 | 2012-02-02 | Genomic Health, Inc. | Method for using gene expression to determine prognosis of prostate cancer |
| CN101900733A (en) * | 2010-08-13 | 2010-12-01 | 中南大学 | Tumor marker colloidal gold immunochromatographic assay quantitative detection test paper and method for preparing same |
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| CN105505934A (en) * | 2016-01-11 | 2016-04-20 | 中国科学院北京基因组研究所 | Specificity miRNA related to M5 type acute myelogenous leukemia and application thereof |
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