CN105177173A - miRNA (microribonucleic acid) biomarkers and detection kit for ovarian cancer diagnosis - Google Patents
miRNA (microribonucleic acid) biomarkers and detection kit for ovarian cancer diagnosis Download PDFInfo
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Abstract
The invention discloses miRNA (microribonucleic acid) biomarkers and a detection kit for ovarian cancer diagnosis. The microRNA biomarkers are composed of the following microRNAs: has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p. The serological expression analysis on the five miRNAs has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p of ovarian cancer with obvious para-carcinoma tissue expression differences (the differential expression level is greater than 2 folds, and the CT value in RT-PCR (reverse transcription-polymerase chain reaction) is less than 30) indicates that the five miRNAs have stable expression in serum, the serum miRNA expression has favorable consistency with the tissue, the expressions of the has-miR-193b-3p, has-miR-155-5p and has-miR-145-5p are enhanced, and the expressions of the has-miR-132-3p and has-miR-143-3p are lowered. The five miRNAs can be used as biomarkers for ovarian cancer diagnosis, and the sensitivity and specificity of combined diagnosis are obviously higher than the sensitivity and specificity of single-miRNA diagnosis.
Description
Technical field
The present invention relates to biological detection, be specifically related to the microRNA biomarker for human ovarian cancer diagnosis and detection kit.
Background technology
Ovarian cancer is one of common three large malignant tumours of female reproductive system, and sickness rate occupy the 3rd.But owing to lacking effective method of early diagnosis and the resistance to chemotherapy of ovarian cancer, easily transfer and relapse, make the case fatality rate of ovarian cancer occupy the first place of gynecologic malignant tumor, 5 annual survival rates lower than 30%, the life and health of serious threat women.Although research has found and ovarian epithelial carcinoma (epithelialovariancarcinoma, EOC) vicious transformation and the relevant various molecular signaling pathways of resistance, but how to carry out early diagnosis and the prediction of chemotherapy side effect is remained to a significant challenge of EOC clinical treatment.
MicroRNA (miRNA) is the non-coding microRNA being about 18-25 Nucleotide of latest find, high conservative in evolution, quantity accounts for genomic 1%, what now generally believed miRNA and human diseases has close contacting, and it is found to be people and provides new approaches in gene level understanding cancer.MiRNA is transcribing or the expression of post-transcriptional level negative regulation protein coding gene: is combined by or approximate complete complementary complementary with its target gene mRNA non-fully, causes mRNA degraded or suppresses it to translate.The gene of human genome about 30% regulates and controls by miRNA, plays important biological regulation effect the growth of cell, propagation, differentiation and apoptosis etc. are many-sided.Research has confirmed the Abnormal regulation of miRNA and tumour is formed and evolving relations is close.The target gene majority of MiRNA is the gene of the biological effects such as participation is transcribed, signal transduction, tumour generation.Along with miRNA and deepening continuously of cancer are studied, find that to have more than be the initial stage that take part in formation of cancer to miRNA, also relate to disease condition change, susceptibility, patient's prognosis etc. many-side to medicine.Take advantage of a situation based on the gene therapy for cancer of miRNA subsequently and climb up stage, highlight very large researching value.
The research of MiRNA in ovarian cancer shows the researching value of miRNA in Diagnosis of Ovarian Cancer and application prospect.Increasing research shows, the change of microrna expression level is with the generation of EOC and develop relevant.MiRNA expresses imbalance, not only plays a significant role in the pathogenesis and tumoral character of ovarian cancer, the diagnosis of EOC, prognosis and to the prediction of therapeutic response in also have potential not yet clear and definite effect.Therefore, exploring that miRNA and ovarian cancer occur, shift, the dependency of resistance and recurrence and regulatory mechanism thereof, may be one of raising ovarian cancer curative effect, the means improving ovarian cancer patients prognosis.But, still more shallow to the understanding of miRNA autogenous control mechanism at present, and its research in ovarian cancer is also urgently deeply.MiRNA occurs with ovarian cancer, develop, shift and resistance etc. relevant.The miRNA molecule that separation and detection is relevant to tumour, illustrates its mechanism of action and clinical meaning, has great importance for raising early diagnosis of tumor and treatment level.MiRNA may become the potential target spot regulating ovarian cancer drug-resistant, and supposition patient reacts chemotherapeutics and the precocious marker of survival rate.Now widely accepted, miRNA can paracrine or internal secretion to local microenvironment or enter circulation, adjustment various biological function, comprises cancer cell growth.The miRNA circulated in serum, blood plasma or other body fluid not only may act on cell, also acts on other positions of health.Measure miRNA level in circulation, this simple, convenient, damage little detection method and have profound significance to the diagnosis of tumour patient, treatment and prognosis.
Summary of the invention
The object of the present invention is to provide the microRNA biomarker for ovarian cancer diagnosis and detection kit.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
One group of microRNA biomarker being used for human ovarian cancer and diagnosing, described microRNA biomarker is made up of following microRNA: has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p; The nucleotide sequence of described has-miR-193b-3p is as shown in SEQIDNO.1; The nucleotide sequence of described has-miR-155-5p is as shown in SEQIDNO.2; The nucleotide sequence of described has-miR-145-5p is as shown in SEQIDNO.3; The nucleotide sequence of described has-miR-132-3p is as shown in SEQIDNO.4; The nucleotide sequence of described has-miR-143-3p is as shown in SEQIDNO.5.
Further, described microRNA biomarker is human serum microRNA biomarker.
The purposes of described microRNA biomarker in the diagnosing tumor medicine of screening human ovarian cancer.
The combination of lineup's ovarian cancer diagnosis microRNA primer, probe, the combination of described microRNA primer, probe comprises: has-miR-193b-3p primer, probe; Has-miR-155-5p primer, probe; Has-miR-145-5p primer, probe; Has-miR-132-3p primer, probe; Has-miR-143-3p primer, probe.
Further, described microRNA primer to comprise before reverse transcription primer, quantitative PCR primer after primer and quantitative PCR.
Further, the reverse transcription primer sequence of has-miR-193b-3p is as shown in SEQIDNO.6, and before quantitative PCR, primer sequence is as shown in SEQIDNO.7, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-155-5p is as shown in SEQIDNO.8, and before quantitative PCR, primer sequence is as shown in SEQIDNO.9, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-145-5p is as shown in SEQIDNO.10, and before quantitative PCR, primer sequence is as shown in SEQIDNO.11, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-132-3p is as shown in SEQIDNO.12, and before quantitative PCR, primer sequence is as shown in SEQIDNO.13, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-143-3p is as shown in SEQIDNO.14, and before quantitative PCR, primer sequence is as shown in SEQIDNO.15, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The nucleotide sequence of each microRNA probe is as shown in SEQIDNO.17.
The purposes be combined in the diagnosing tumor medicine of preparation or screening human ovarian cancer of described human ovarian cancer diagnosis microRNA primer, probe.
A tumor diagnosis kit for human ovarian cancer, comprises described miRNA primer, the combination of probe.
Further, the tumor diagnosis kit of described human ovarian cancer also comprises reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2, DEPC water, Taq enzyme and standard substance and/or reference substance.
Advantage of the present invention:
By ovarian cancer and cancer beside organism's differential expression, obviously (differential expression amount is greater than 2 fold in the present invention, in RT-PCR, CT value is less than 30) 5 kinds of miRNAhas-miR-193b-3p, has-miR-155-5p, has-miR-132-3p, has-miR-145-5p and has-miR-143-3p carries out serology expression analysis, result shows these 5 kinds of miRNA stably express in serum, the expression of serum miRNA and tissue have good consistence, has-miR-193b-3p, has-miR-155-5p and has-miR-145-5p down-regulated expression, has-miR-132-3p and has-miR-143-3p up-regulated.These 5 kinds of miRNA can as the biomarker of ovarian cancer diagnosis, and the sensitivity of Combining diagnosis and specificity are significantly higher than sensitivity and specificity that single miRNA diagnoses.
Accompanying drawing explanation
Fig. 1-Fig. 5 shows has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p respectively separately for distinguishing the ROC curve of ovarian cancer sample and normal control;
Fig. 6 is shown as has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p and combines ROC curve for distinguishing ovarian cancer sample and normal control.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, such as, condition described in textbook and experiment guide, or according to the condition that manufacturer advises.
Embodiment 1: the screening of ovarian cancer tissue otherness miRNA
1 object and method
1.1 Specimen origin
After obtaining patient's informed consent, collecting Jiangsu Prov. People's Hospital, to turn out to be ovarian cancer patients Post operation sample 8 through pathology in June ,-2014 in April, 2013 right, comprise the cancer beside organism of distance cancerous tissue more than the 3 centimetres scopes of cancerous tissue sample and pairing, all patients all do not receive chemotherapy and radiotherapy in the preoperative.
1.2 key instrument equipment
Desk centrifuge: EppendorfMinispin (U.S.) Eppendorfcentrifuge5810R (U.S.)
Nucleic acid condensation instrument: Eppendorfconcentrator5301 (U.S.)
Ultraviolet spectrophotometer: DU640, balance (Beckman Products)
UV-crosslinked instrument: GSGENELINKERUVChamber (BIO-RAD Products)
Water-bath (Memert Products)
Hybridizing box, chip, chip scanner: LuxScan-10K/A (Capitalbio Products)
Horizontal shaker: TDK-2 (the sensible Science and Technology Ltd. in Beijing)
Gel image analyser: GDS-7600 (UPV product)
Bechtop (Memert Products)
Real-time fluorescence PCR instrument: ABIPRISM7500 (U.S.)
1.3 major experimental reagent
Trizol, glycogen (Invitrogen company); T4RNA ligase enzyme (NEB company); ControlRNA (Capitalbio company); 5P '-C-U-Cy3-3 ' 5P '-C-U-Cy5-3 ' (Dharmacon company); Ambion ' smiRNAIsolationKit (Ambion company); EDPC, first phthalein amine, Australia's phenol indigo plant (Sigma company); 20 × SSC, 10%SDS (PIERCE company); 50 × Dhardt ' s (ancient cooking vessel state); MOPS (Bocherigmer company); 5 × RTBuffer (Promega company of the U.S.), M-MLV reversed transcriptive enzyme: 200u/ μ l (Promega company of the U.S.); 4 × dNTP:10mMeach (Shanghai Sheng Gong biotechnology company limited); RNase inhibitors 4 0u/ μ l (the precious biotechnology company limited in Dalian).
The extraction of 1.4 tissue sample total serum IgE
Trizol single stage method extracts the total serum IgE in tissue sample.Concrete steps are as follows:
(1) equipment and the reagent such as mortar, even poly-device, spoon, scissors, tweezers, liquid nitrogen is prepared;
(2) mortar precooling: repeatedly add liquid nitrogen in mortar, at least 4-5 time, makes the abundant precooling of mortar;
(3) bring gloves, mouth mask, take out sample rapidly, weigh in analytical balance from liquid nitrogen container with tweezers, the tissue block weight once extracted is between 0.3-0.5g.Tissue block is put into and is ground with the mortar of Liquid nitrogen precooler, and grinding limit, limit adds liquid nitrogen, and whole process does not all make tissue block melt.
(4) after rough grinding, in Bechtop, moved into by the tissue of grinding rapidly in the hook dress device that Trizol reagent is housed with little spoon, add 1mlTrizol by 100mg tissue, homogenate is to the tissue samples particle that is invisible to the naked eye.
(5) transfer in 1.5ml centrifuge tube with dropper by homogenate, often pipe is put into about, more than room temperature is placed, is saved in-80 DEG C of refrigerators until analyze.
(6) homogenate sample normal temperature unfreezing, adds chloroform in the ratio of 0.2ml chloroform/1mlTrizol, vortex30 second, and room temperature places 3min, then 4 DEG C, the centrifugal 15min of 12000rpm.
(7) centrifugal rear solution layering, is divided into bottom phenol-chloroform, middle layer and upper strata aqueous phase.RNA only exists in aqueous phase, is transferred in another new 1.5ml centrifuge tube by supernatant, do not draw middle layer with sample injector.Add Virahol in the ratio of 0.5ml Virahol/1mlTrizol, mixing, room temperature places more than 10min, 12000rpm4 DEG C of centrifugal 15min.
(8) supernatant discarded, Virahol does not reflux too much, can be of short duration centrifugal again, with sample injector by remaining Virahol sucking-off, adds 1ml75% ethanol, vortex, 7500rpm4 DEG C of centrifugal 5min.
(9) discard 75% ethanol, seasoning 30min in stink cupboard, traditional vacuum is not dry, and RNA does not parch completely, in case can not dissolve completely, with DEPC water dissolution RNA, often pipe is dissolved in 30 μ l, 60-65 DEG C of hydrotropy 5min.
(10) RNA is quantitative, draws the RNA sample of 1 μ l, adds in 49 μ lDEPC water, blow and beat mixing up and down with sample injector, dilute 50 times.Blank by the DEPC water gauge note of dissolving RNA, draw 50 μ l and add in cuvette.OD value, ratio calculated OD260/OD280 ratio and RNA concentration is measured with ultraviolet spectrophotometer DU-640.
RNA concentration=OD260 × 40 μ g/ml × extension rate.
The separation and Extraction of miRNA in 1.5 total serum IgE
Get 50-100 μ g total serum IgE Ambion ' smiRNAIsolationKit and be separated miRNA, concrete steps are as follows:
(1) get 50-100 μ g total serum IgE and add EP pipe, be settled to appropriate volume.Add the Lysis/BindingBuffer of 5 times of volumes, mixing.
(2) add the miRNAHomogenateAdditive of 1/10th volumes, vibration mixing, is placed in and hatches 10 minutes on ice.
(3) 100% ethanol of 1/3rd volumes is added, fully mixed hook.
(4) above-mentioned mixed solution is added in chimney filter, centrifugal 1 minute of 5000rpm, collect filtrate (tiny RNA is in filtrate).
(5) in filtrate, add 100% ethanol of 2/3rds volumes, fully mix.
(6) change chimney filter, filtered by the mixture of step (5), centrifugal 1 minute of 5000rpm, discards filtrate, and collection tube continues to use.
(7) be placed in collection tube by chimney filter, with the miRNAwashingsolution1 filter wash pipe of 700 μ l, centrifugal 1 minute of 5000rpm, discards filtrate.
(8) with the miRNAwashingsolution2 filter wash pipe of 500 μ l, centrifugal 1 minute of 5000rpm, discards filtrate; One time is washed again with miRNAwashingsolution2; By chimney filter together with centrifugal 1 minute of collection tube 10000rpm, liquid residual in removing chimney filter.
(9) elutionsolution is heated to 95 DEG C; Change a new collection tube, the elutionsolution of 50 μ l95 DEG C is added filter, close the cover, incubated at room 2 minutes; Centrifugal 1 minute of 10000rpm, collect filtrate, tiny RNA is in filtrate.Repeating step (9).
1.6miRNA cDNA microarray
1.6.1miRNA chip
Mammals miRNA chip V3.0 is for people 677, rat 292, mouse 461 ripe microRNA, miRNA three due to people, rat and mouse has common sequence, get the union of three, devise altogether 924 probes (sangermiRNA database: miRNABase10.0).These probes with chip point sample instrument Smart-ArrayTM (CapitalbioCorp, Beijing, China) o'clock built in a 75 × 25mm, on the slide glass of chemically modified.The sample of point on chip also comprises U6, tRNA of people as interior mark; Probe corresponding to 30 bases longs RNA of 8 artificial preparations as the external standard (Zip5, Zip13, Zip15, Zip21, Zip23, Zip25, Y2, Y3) of chip, Hex as point sample positive control, 50%DMSO is as hybridization negative control.
1.6.2miRNA the fluorescent mark of sample
Concrete steps are as follows:
(1) fluorescent mark of miRNA: get 2-5 μ g cancerous tissue miRNA through polyethylene glycol precipitation, with 0.1mgATP, 50mMHEPES, 3.5mMDDT, 20mMMgCl
2, the 5P '-C-U-Cy3-3 ' (from Dharmacon company) of 10mg/mlBSA, 10%DMSO, 500ngCy3 mark and the T4RNA ligase enzyme of 20 units, blow and beat with rifle head, mix gently.Tinfoil parcel sample hose, 0 DEG C of labeled reactant 2 hours.In like manner mark cancer beside organism miRNA with Cy5.MiRNA mixing after two kinds of marks.
(2) purifying of miRNA: add DEPC water in the sample after above-mentioned fluorescent mark, polishing to 100 μ l, add 3mmol/L sodium-acetate (pH5.2) the 10 μ l of 1/10th volumes-20 DEG C of precoolings, glycogen 10 μ g, 2.5 times of volume dehydrated alcohols, leave standstill 1 hour in-20 DEG C.
(3) rinsing of miRNA precipitation: supernatant discarded, adds 75% ethanol 800 μ l of-20 DEG C of precoolings, centrifugal 5 minutes of 12000rpm at fully mixing latter 4 DEG C.Repeat 2 times.Supernatant discarded, in atmosphere dry 10min, for chip hybridization after drying up.
1.6.3miRNA chip hybridization
Concrete steps are as follows:
(1) RNA is dissolved in (15% methane amide 2.4 μ l in 16 μ l hybridization solutions; 0.2%SDS3.2 μ l; 3 × SSC2.4 μ l; 50 × Denhardt ' s1.6 μ l, DEPC process water 6.4 μ l).
(2) constant-temperature metal bath is heated, and dissolves, vibration, mixing.
(3) take out, to place in-20 DEG C of refrigerators 10 minutes, make it to lower the temperature.
(4) 95 DEG C of distortion 3min.
(5) cool rapidly on ice, be all added drop-wise on Mammals microRNA chip V3.0, add a cover silication cover glass.
(6) humidity is kept, 42 DEG C of hybridized overnight, usual more than 16 hours through the filter paper that distilled water is moistening after sterilization in hybridizing box.
(7) after hybridization terminates, first rinsing 4 minutes in about the 42 DEG C liquid shaking tables containing 0.2%SDS, 2 × SSC, then wash 4 minutes containing room temperature in 0.2 × SSC liquid shaking table at room temperature, slide is placed in pipe, and namely 1600rpm can be used for scanning after within centrifugal 1 minute, drying.
1.6.4 core body scanner uni data processing
Chip Luxscan10K/A twin-channel laser scanner (Capitalbio company) scans.Data are extracted and are adopted Luxscan3.0 image analysis software (Capitalbio company) to chip image analysis, picture signal to be converted into numerary signal.
1.7miRNA chip results realtimeRT-PCR verifies
1.7.1miRNArealtimeRT-PCR design of primers
Special stem ring primer (reverse transcription primer sequence) about 56 Nucleotide, its 5 ' 48 nucleotide sequences held are fixing, form the structure of a stem ring, and its 3 ' 8 Nucleotide held are just complementary with microRNA.Forward primer (before PCR primer) is about 30-31 Nucleotide, to have an appointment 16-17 Nucleotide and corresponding microRNA complementation, and the Tm value remaining 14 Nucleotide is higher than 65 degrees Celsius at 3 ' end.General reverse primer (after PCR primer) is about 23nt, wherein the loop-stem structure of 18 corresponding specific reverse primer of Nucleotide, and the Tm value of 5 ' holds 5 Nucleotide is higher than 65 degrees Celsius.
1.7.2miRNArealtimeRT-PCR
1.7.2.1cDNA reverse transcription synthesis
Tissue sample total serum IgE reverse transcription synthesis cDNA, reaction system is as follows:
| Component | Volume (unit: μ l) |
| Total serum IgE template | 1 μ g (calculating volume according to concentration) |
| Stem-loop RT primer(500nM) | 1 |
| 5×RT Buffer | 2 |
| 100mM DTT | 1 |
| dNTPs(10mM each) | 0.5 |
| RNase inhibitor (40U/ μ l) | 0.1 |
| M-MLV(200U/μl) | 1 |
| Add DEPC water | To 10 μ l |
Reaction conditions: 16 DEG C, 30min; 42 DEG C, 60min; 85 DEG C, 5min; 4 DEG C, hold.
1.7.2.1miRNArealtimeRT-PCR reaction
MiRNArealtimeRT-PCR reaction system is as follows:
| Component | Volume (unit: μ l) |
| SYBR R Premix Ex Taq TM(2×) | 12.5 |
| Forward primer 10 μMs | 0.5 |
| Reverse universal primer μM | 0.5 |
| ROX Reference Dye Ⅱ(50×) | 0.5 |
| DNA profiling (diluting 10 times) | 2 |
| DEPC process water | To 25 |
Reaction conditions: 95 DEG C of denaturation 10min; 95 DEG C of 5s, 60 DEG C of 34s × 40.
1.8 statistical analysis
The result of gene chip screening adopts Cluster3.0 and SignificanceAnalysisofMicroarrys (SAM, version2.1) to analyze.Data with (± represent, compare between group adopt inspection, adopt software carry out analyzing and processing, for difference has statistical significance.RealtimeRT-PCR data represent with (x ± s), compare and adopt t inspection between group, adopt SPSS17.0 software to carry out analyzing and processing, and P < 0.05 has statistical significance for difference.
2 results
2.1miRNA gene chip the selection result
MiRNA chip is utilized to analyze 924 kinds of miRNA expressions in the cancer beside organism of 8 pairs of ovarian cancer tissues and pairing, filter out the miRNA that 20 species diversity are expressed altogether, wherein has-miR-132-3p, has-miR-497, has-miR-143-3p up-regulated, has-miR-381, has-miR-145-5p, has-miR-199b-5p, has-miR-155-5p, has-miR-335, has-miR-99a, has-miR-193b-3p, has-miR-125b, has-let-7b, has-miR-10a, has-miR-126, has-miR-30a, has-miR-141, has-miR-125a-5p, has-miR-19b, has-miR-26a, rno-miR-324-3p down-regulated expression.
The realtimeRT-PCR checking of 2.2miRNA gene chip the selection result
With miRNArealtimeRT-PCR, the result of chip examination is verified, filter out the miRNA that 9 species diversity are expressed altogether, wherein, has-miR-132-3p, has-miR-143-3p and has-miR-497 up-regulated, has-miR-155-5p, has-miR-145-5p, has-miR-381, has-miR-497, has-miR-99a, has-miR-193b-3p down-regulated expression.To wherein different expression obvious 5 kinds of miRNA, has-miR-193b-3p, has-miR-155-5p, has-miR-132-3p, has-miR-145-5p and has-miR-143-3p carry out further serology expression analysis.
Embodiment 2: the serological analysis of otherness miRNA in ovarian cancer tissue
1 object and method
1.1 Specimen origin
After obtaining patient's informed consent, collect Jiangsu Prov. People's Hospital example in year March 165 in May, 2013 to 2014 through the clear and definite ovarian cancer patients of pathological diagnosis and 120 routine Healthy People limosis vein blood in early morning 6ml in contrast.All ovarian cancer patients are first patient diagnosed, do not carry out performing the operation, radiotherapy and chemotherapeutic treatment before getting blood.120 routine normal healthy controls groups for not suffer from malignant tumour and other diseases, the simultaneously healthy population of age-matched.
1.2 serum sample collection and process
Extracting early morning limosis vein blood 6ml is placed in not containing the pipe of antithrombotics, leaves standstill 30 minutes, in centrifugal 15 minutes of 4 DEG C of 1300g (or 4000rpm), gets the every 300 μ l of upper serum and divides and be filled to RNase-freeEP pipe and be placed in-80 DEG C of refrigerator storage.
1.3 key instrument equipment
Eppendorfcentrifuge (U.S.); Horizontal laminar flow clean bench (memert company); Ultraviolet spectrophotometer nano (Thermo science and technology); Water-bath (memert company); Real-time PCR instrument CFX96 (German BIO-RAD); Vortex whirlpool concussion instrument (U.S. SI); Ultralow Temperature Freezer (Thermo science and technology); MilliQ water purifior (Synthesis company).
1.4 major experimental reagent
Blood total serum IgE rapid extraction test kit (Beijing hundred Imtech); Lysate RLS; Protein liquid removal RE; Rinsing liquid RW; RNase-freeH
2o; 70% ethanol; RNase-free adsorptivity RA; MiRcutemiRNAcDNA first chain synthetic agent box (TIANGEN); E.coliPoly (A) Polymerase (5U/ml); 10 × Poly (A) PolymeraseBuffer; 5 × rATPSolution; 10 × RTPrime; 10 × RTBuffer; SuperPuredNTPMixture; Rnasin; QuantRTase; RNase – FreeddH
2o; MiRcutemiRNA fluorescence quantitative detection kit (TIANGEN); 2 × miRNApremix (SYBRROX); Reverseprimer; 50 × ROXReferenceDye; Chloroform (Sigma company).
1.5 design of primers
1.6 experimental technique
1.6.1 serum sample Total RNAs extraction
(1) every 250 μ l serum add 750 μ l lysate RLS, blow and beat sample several times with sample loading gun, the final volume ratio always 3:1 of lysate RLS and liquid sample.
(2) mixed by sample concuss, incubated at room temperature decomposes to make ribosome for 5 minutes completely.
Under (3) 4 DEG C of conditions, centrifugal 10 minutes of 12000rpm, carefully gets supernatant and proceeds in the centrifuge tube of new RNasefree.
(4) every milliliter of RLS adds 0.2ml chloroform, covers tightly sample hose lid, concuss 15 seconds ambient temperatare puts 3 minutes.
(5) in centrifugal 10 minutes of 4 DEG C of 12000rpm, sample can be divided into three layers: lower floor's organic phase, and the colourless aqueous phase in middle layer and upper strata, RNA is present in aqueous phase.The capacity of aqueous phase layer is approximately 70% of added RLS volume, and aqueous phase is transferred in new pipe, carries out next step operation.
(6) add 1 times of volume 70% ethanol, put upside down mixing (now may occur precipitation).The solution obtained proceeds to (adsorption column is enclosed within collection tube) in RA post together with may precipitating.
(7) centrifugal 45 seconds of 10000rpm, discards waste liquid, adsorption column is recovered collection tube again.
(8) add 500 μ l protein liquid removal RE, centrifugal 45 seconds of 12000rpm, discards waste liquid.
(9) add 700 μ l rinsing liquid RW, centrifugal 60 seconds of 12000rpm, discards waste liquid.
(10) add 700 μ l rinsing liquid RW, centrifugal 60 seconds of 12000rpm, discards waste liquid.
(11) put back in sky collection tube by adsorption column RA, centrifugal 2 minutes of 12000rpm, removes rinsing liquid as far as possible, in order to avoid residual ethanol suppresses downstream reaction in rinsing liquid.
(12) adsorption column RA is taken out, put into a RNasefree centrifuge tube, the RNasefreewater that 30 μ l heat in advance in 65 DEG C of water-baths is added in the middle part of adsorption film, room temperature places 2 minutes, centrifugal 1 minute of 12000rpm, the solution obtained is rejoined in centrifugal adsorbing column, centrifugal 1 minute.
1.6.2cDNA transcribe
After serum sample Total RNAs extraction, adopt and add poly A tract Poly (A) at miRNA3 ' end, re-use the general reverse transcriptase primer of oligo (dT)-universaltag and carry out reverse transcription reaction, cDNA first chain that final generation miRNA is corresponding.
1.6.2.1miRNA3 ' end carries out Poly (A) process
(1) on ice precooling RNasefree reaction tubes in add following reagent to cumulative volume 20 μ l (finally adding E.coliPoly (A) Polymerase).
| Component | Volume (μ l) | Final concentration |
| Total serum IgE | 2 μ g can be reached | |
| E.coli Poly(A)Polymerase | 0.4 | 2U |
| 10×Poly(A)Polymerase Buffer | 2 | 1× |
| 10×rATP solution | 4 | 1× |
| RNase free ddH 2O | - | - |
| Cumulative volume | 20 | - |
(2) pipettor mixes the reaction solution of above-mentioned preparation gently, of short duration centrifugal after 37 DEG C reaction 60 minutes, continue experiment.
1.6.2.2Poly the miRNA that (A) modifies carries out reverse transcription reaction, carries out the preparation of reaction solution according to following table component.
| Component | Volume (μ l) |
| Poly (A) reaction solution | 2 |
| 10×stem-loop RT Prime | 2 |
| 10×RT Buffer | 2 |
| Super Pure dNTP Mixture | 1 |
| Rnasin | 1 |
| Quant RTase | 0.5 |
| RNase-Free ddH 2O | 11.5 |
| Cumulative volume | 20 |
1.6.3realtimeRT-PCR
(1) room temperature melts 2 × miRNApremix (SYBR) and Reverseprimer.
(2) 2 × miRNApremix (SYBR) is turned upside down mix gently, avoid bubbling, then re-use after light gentle centrifugation.
(3) reagent is placed on ice, and according to following table preparation reaction volume.
| Component | 50 μ l systems | Final concentration |
| 2×miRNA premix(SYBR) | 25 | 1× |
| Forward primer | - | 200nM |
| Reverse primer | 1 | 200nM |
| MiRNA first chain cDNA | - | - |
| ddH 2O | To 50 μ l | - |
PCR response procedures is arranged according to following table
| Circulation | Temperature (DEG C) | Time | Content |
| 1× | 94 | 2min | Starting template sex change |
| 40-45× | 94 | 20s | Template denaturation in PCR circulation |
| 60 | 34s | Annealing, extension |
1.6.4PCR data processing
Pcr amplification result CT value represents, the implication of CT value is cycle number when fluorescent signal reaches set threshold value in PCR reaction solution.The relative expression of sample goal gene leads (RQ) and adopts △ △ CT method to calculate,
(cycle number when CT represents that the real-time fluorescence intensity of reaction is significantly greater than background value, △ CTsample=CTsample – CTU6sample, △ CTcontrol=CTcontrol – CTU6control, △ △ CT=△ CTsample-△ CTcontrol).
1.7 statistical analysis
SPSS17.0 statistical software is adopted to carry out data processing, measurement data represents with (x ± s), compare between group and adopt t inspection, the relation of serum miRNA relative expression quantity and ovarian cancer clinical pathologic characteristic adopts MannWhiney to check and KruskalWallis checks.P < 0.05 has statistical significance for difference.
2 results
The realtimeRT-PCR of 2.1 serum target miRNA detects
This research has carried out quantitative analysis to target miRNA expression in 165 routine ovarian cancer patients and 120 routine normal healthy controls group serum, and adopt U6 as interior mark, result shows miRNA stably express in serum, adopts U6 reliable and stable as internal reference.
Target miRNA expression in 2.2 ovarian cancer patients and control group serum
Control group compares with ovarian cancer group has-miR-155-5p, has-miR-132-3p, has-miR-145-5p, has-miR-143-3p and has-miR-193b-3p relative expression quantity, and difference all has statistical significance (P < 0.05).
Data see the following form.
| Group | Number of cases | has-miR-143-3p | has-miR-155-5p | has-miR-132-3p | has-miR-145-5p | has-miR-193b-3p |
| Control group | 120 | 1.13±0.22 | 1.03±0.29 | 1.18±0.26 | 0.90±0.12 | 0.94±0.17 |
| Ovarian cancer | 165 | 2.86±0.25 | 0.58±0.32 | 2.88±0.23 | 0.75±0.14 | 0.76±0.21 |
| T class value | - | 2.552 | 2.694 | 2.548 | 2.371 | 2.395 |
| P value | - | 0.011 | 0.012 | 0.01 | 0.010 | 0.011 |
The present invention utilizes miRNA chip to analyze miRNA express spectra in the cancer beside organism of 8 pairs of ovarian cancer tissues and pairing, filter out the miRNA that 20 species diversity are expressed altogether, real-time fluorescence quantitative RT-PCR is verified chip results, filter out the miRNA that 9 species diversity are expressed altogether, wherein has-miR-132-3p, has-miR-143-3p and has-miR-497 up-regulated, has-miR-155-5p, has-miR-145-5p, has-miR-381, has-miR-497, has-miR-99a, has-miR-193b-3p down-regulated expression.Tumour be the accumulative result of a series of molecular biology behavior change, relate to all respects such as cell cycle, apoptosis, proliferation and differentiation, the change that a series of miRNA therefore should be had to express participates in the generation of tumour, this hypothesis of the result verification of gene chip, the expression of multiple miRNA there occurs change.
Obviously (differential expression amount is greater than 2 fold to differential expression, in RT-PCR, CT value is less than 30) 5 kinds of miRNA, has-miR-193b-3p, has-miR-155-5p, has-miR-132-3p, has-miR-145-5p and has-miR-143-3p carry out further serology expression analysis.Result shows miRNA stably express in serum, the expression of serum miRNA and tissue have good consistence, has-miR-193b-3p, has-miR-155-5p and has-miR-145-5p down-regulated expression, has-miR-132-3p and has-miR-143-3p up-regulated.This result shows, these 5 kinds of miRNA can as the biomarker of ovarian cancer diagnosis.
Embodiment 3: Receiver operating curve (ROC) analyzes
Build ROC curve and compare the diagnosis capability that 5 serum miRNA distinguish human ovarian cancer patients and normal healthy controls.5 miRNAROC area under curve (AUC) are respectively: has-miR-193b-3p, 0.840 (95% fiducial interval: 0.760-0.920); Has-miR-155-5p, 0.819 (95% fiducial interval: 0.736-0.902); Has-miR-145-5p, 0.814 (95% fiducial interval: 0.734-0.894); Has-miR-132-3p, 0.843 (95% fiducial interval: 0.765-0.920); Has-miR-143-3p, 0.738 (95% fiducial interval: 0.644-0.832).Under the cutoff value of the best, sensitivity and the specificity of miRNA are as follows: has-miR-193b-3p, are respectively 83.3% and 82.7%; Has-miR-155-5p, is respectively 64.8% and 94.2%; Has-miR-145-5p, is respectively 90.7% and 61.5%; Has-miR-132-3p, is respectively 70.4% and 82.5%; Has-miR-143-3p, is respectively 67.3% and 74.1%.The AUC that these 5 miRNA join together can reach 0.973, and sensitivity and specificity are respectively 92.3% and 90.7%, are obviously better than single miRNA.This result shows, has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p join together to detect ovarian cancer to have very high sensitivity and specificity.
Embodiment 4: human ovarian cancer diagnosis test kit
Above-described embodiment shows, has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p join together to detect ovarian cancer to have very high sensitivity and specificity, therefore, can make based on has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p the test kit being used for human ovarian cancer diagnosis.This test kit comprises has-miR-193b-3p primer, probe; Has-miR-155-5p primer, probe; Has-miR-145-5p primer, probe; Has-miR-132-3p primer, probe; Has-miR-143-3p primer, probe.Primer specifically to comprise before reverse transcription primer, quantitative PCR primer after primer and quantitative PCR.Certainly, also reversed transcriptive enzyme, damping fluid, dNTPs, MgCl should be comprised in this test kit
2, DEPC water, Taq enzyme and standard substance and/or reference substance.Following table is the one design of primer and probe.
The design of primer and probe is this area routine techniques means, can be designed to other sequences.The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (9)
1. one group is used for the microRNA biomarker of human ovarian cancer diagnosis, it is characterized in that, described microRNA biomarker is made up of following microRNA: has-miR-193b-3p, has-miR-155-5p, has-miR-145-5p, has-miR-132-3p and has-miR-143-3p; The nucleotide sequence of described has-miR-193b-3p is as shown in SEQIDNO.1; The nucleotide sequence of described has-miR-155-5p is as shown in SEQIDNO.2; The nucleotide sequence of described has-miR-145-5p is as shown in SEQIDNO.3; The nucleotide sequence of described has-miR-132-3p is as shown in SEQIDNO.4; The nucleotide sequence of described has-miR-143-3p is as shown in SEQIDNO.5.
2. the microRNA biomarker for human ovarian cancer diagnosis according to claim 1, is characterized in that: described microRNA biomarker is human serum microRNA biomarker.
3. the purposes of the microRNA biomarker described in claim 1 or 2 in the diagnosing tumor medicine of screening human ovarian cancer.
4. the combination of lineup's ovarian cancer diagnosis microRNA primer, probe, is characterized in that, the combination of described microRNA primer, probe comprises: has-miR-193b-3p primer, probe; Has-miR-155-5p primer, probe; Has-miR-145-5p primer, probe; Has-miR-132-3p primer, probe; Has-miR-143-3p primer, probe.
5. the combination of human ovarian cancer diagnosis microRNA primer according to claim 4, probe, is characterized in that: described microRNA primer to comprise before reverse transcription primer, quantitative PCR primer after primer and quantitative PCR.
6. the combination of human ovarian cancer diagnosis microRNA primer according to claim 5, probe, it is characterized in that: the reverse transcription primer sequence of has-miR-193b-3p is as shown in SEQIDNO.6, before quantitative PCR, primer sequence is as shown in SEQIDNO.7, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-155-5p is as shown in SEQIDNO.8, and before quantitative PCR, primer sequence is as shown in SEQIDNO.9, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-145-5p is as shown in SEQIDNO.10, and before quantitative PCR, primer sequence is as shown in SEQIDNO.11, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-132-3p is as shown in SEQIDNO.12, and before quantitative PCR, primer sequence is as shown in SEQIDNO.13, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The reverse transcription primer sequence of has-miR-143-3p is as shown in SEQIDNO.14, and before quantitative PCR, primer sequence is as shown in SEQIDNO.15, and after quantitative PCR, primer sequence is as shown in SEQIDNO.16; The nucleotide sequence of each microRNA probe is as shown in SEQIDNO.17.
7. the purposes be combined in the diagnosing tumor medicine of preparation or screening human ovarian cancer of the arbitrary described human ovarian cancer diagnosis microRNA primer of claim 4-6, probe.
8. a tumor diagnosis kit for human ovarian cancer, is characterized in that: the combination comprising the arbitrary described miRNA primer of claim 4-6, probe.
9. the tumor diagnosis kit of human ovarian cancer according to claim 8, is characterized in that: also comprise reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2, DEPC water, Taq enzyme and standard substance and/or reference substance.
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