CN104911248A - Micro RNA combination used for II and III stage colorectal cancer diagnosis and prognosis as well as application thereof - Google Patents
Micro RNA combination used for II and III stage colorectal cancer diagnosis and prognosis as well as application thereof Download PDFInfo
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Abstract
The invention discloses a micro RNA combination used for II and III stage colorectal cancer diagnosis and prognosis as well as an application thereof. The miRNA composition comprises any two to three of miR-17-3p, miR-106a and miR-145. A kit comprises a TaqMan probe combination of the above miRNA. According to the invention, serum is easily obtained without other tissue, the application belongs to the noninvasive inspection; serum miRNA reflects the integral pathology and physiology case of the body, and can be taken as a diagnosis marker for increasing the detection accuracy; the kit simplifies a TaqMan probe database, making cost and production time are reduced, pertinency and practicality are increased, detection sensitivity and singularity are increased; the combination, the method and the kit can be used for early detection of colorectal cancer, and can be used for prediction of II and III stage colorectal cancer lifetime and chemotherapy benefit.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of micro RNA combination for II, III phase diagnosis of colorectal carcinoma and prognosis and application thereof.
Background technology
In China, colorectal cancer is as the third-largest tumour, and its sickness rate and mortality ratio rise in recent years year by year.Many problem demanding prompt solutions are still had at present in colorectal cancer clinical position.The first, still lack effectively for the Noninvasive biomarker of disease early diagnosis.Fecal occult blood testing and colonoscopy are the method for early diagnosis of comparatively generally acknowledging at present, and multinomial large sample clinical research data display, carries out examination and can reduce colorectal cancer incidence rate and mortality ratio in the crowd more than 50 years old.Fecal occult blood inspection is used widely because of easy to operate, with low cost, but muting sensitivity and specific degree make its clinical value limited.Colonoscopy is the gold standard of current early diagnosis colorectal cancer, but operation easier is large, spend high and complication risk to cause patient dependence poor, is difficult to the conventional means becoming Mass screening.The second, still lack effective Index for diagnosis and chemotherapeutic efficacy evaluation index.At present clinically Main Basis based on the TNM of clinicopathological parameters (as tumor size, nodus lymphoideus transferring rate number, distant metastasis, organizational hierarchy etc.) by stages as judgement and the guiding clinical treatment of disease prognosis, but judge that patient's prognosis exists larger heterogeneity according to this by stages, also can not predicting tumors patient to the reaction of chemotherapy.
The ratio making newly to send out nonmetastatic disease in colorectal cancer case that improves constantly of diagnostic techniques increases greatly, in an annual new colorectal cancer patients of statistics from U.S. display, II, III phase patient ratio exceedes half, for this part patient, surgical operation and adjuvant chemotherapy are essential therapeutic arsenals.For the local advanced patient (AJCC III phase) of lymph node positive, radical surgery and adjuvant chemotherapy can significantly improve existence to have multinomial large sample clinical trial to prove, become standard care.It is generally acknowledged, II phase patients after radical Post operation is cured, still there is dispute in postoperative adjuvant chemotherapy of whether carrying out, think that patient exists high risk factor (as T4 at present, vascular invasion, histological is poor, blocks, perforation etc.) indicate poor prognosis and may benefit from chemotherapy, but its meaning of these clinicopathological parameters not yet obtains large sample data test.Research show II, III phase colorectal cancer patients still have after standard care 20% ?30% occur recurrence, wherein 40 ?50% occur in primary tumor excision latter 1 year in, 80% occurs in primary tumor excised in 3 years, recurred latter 5 years survival rates lower than 1%.Therefore, for this part patient, find effective prognosis and recurrence prediction index seems particularly important.
MiRNA (microRNA, miRNA) is the non-coding strand small ribonucleic acid molecules that a class is about 19 to 23 Nucleotide, and its major function is level modulation after participation genetic transcription.By holding with said target mrna 3 ', non-translated sequence is complementary wholly or in part to be combined miRNA, causes said target mrna to be degraded or transcribes rear Translational repression thus the expression of regulation and control target gene.MiRNA participates in 1/3 human protein coding gene regulation and control, and it is relevant with multiple physiological activity, as grown, cell proliferation, apoptosis, cytodifferentiation etc.; Also develop closely related, as diabetes, cardiovascular disorder, tumour etc. with the generation of numerous disease.Nearest research display
[1-3], miRNA plays cancer suppressor gene or the effect of oncogene sample in tumorigenesis process.Confirm, some the miRNA expression level in tumor tissues and tumour cell there occurs change, and relative to healthy tissues, some miRNA expression amounts significantly rise or decline.Many cancerous tissues such as lung cancer, mammary cancer and the esophageal carcinoma etc. organize the expression of some tissue specificity miRNA, and prompting miRNA has tumor tissue specificity.In addition, the expression of miRNA is also relevant with tumor differentiation degree and prognosis.A large amount of result of study discloses, and miRNA can be used as new tumor markers, and provides novel targets for the gene therapy of tumour.But tissue sample is drawn materials and is belonged to traumatic detecting, and cannot realize early diagnosis and the Index for diagnosis of tumour.Nearest report display, also abundant, stable miRNA is there is in human serum/blood plasma, some tumours comprise miRNA express spectra and normal people in nonsmall-cell lung cancer, colorectal cancer, prostate cancer, ovarian cancer and Pancreas cancer patients serum, and there were significant differences, all has express spectra special separately.
Still do not utilize non-invasive at present, the blood preparation easily of drawing materials detects the specificity miRNA combination and detection kit that judge that and prognosis relevant to staging of colorectal cancer is correlated with.
Summary of the invention
Object of the present invention is just the specific variations by studying II, III serum in patients with colorectal miRNA, filter out one group with normal population differential expression and serum miRNA closely-related with tumor load, and utilize the TaqMan probe of these miRNA to prepare the test kit being applicable to clinic diagnosis purposes, for staging of colorectal cancer diagnosis and Postoperative determination provide specific intermediate result and High altitude means rapidly.
Below the detailed description to summary of the invention of the present invention and technical scheme:
For detecting a miRNA combination for II, III phase colorectal cancer, this miRNA combination comprise following miRNA:miR ?17, miR ?106a, miR ?one or more in 145; Preferably by miR ?17, miR ?106a and miR ?any two or three in 145 form; Further preferred by miR ?17 and miR ?106a form.
The screening method of above-mentioned miRNA combination comprises the following steps (see Fig. 1):
(1) collect 20 routine operation consent II, III colorectal cancer patients pooled serums respectively, 20 routine Post operation II, III colorectal cancer patients pooled serums, normal people's pooled serum of 20 example ages and gender matched, extracts total serum IgE;
(2) adopt low density chip technology (TaqMan low density Array technology), miRNA ripe in above-mentioned total serum IgE is detected, filters out the miRNA of differential expression between 3 groups of samples;
(3) with real time fluorescence quantifying PCR method (TaqMan probe method), above-mentioned sample is detected one by one, from the miRNA sifted out at the beginning of low density chip method, sift out the remarkable miRNA of differential expression again;
(4) with quantifying PCR method, sample in enormous quantities is verified one by one further, filter out the significant one group of miRNA of stable differential expression.
Specifically, above-mentioned screening method comprises the following steps: (1) collects normal people and the perioperative pooled serum of II, III colorectal cancer patients respectively, extracts total serum IgE; (2) use low density chip technology to detect above-mentioned RNA, primary dcreening operation goes out perioperatively and miRNAs more significant with non-cancer control group differential expression; (3) be cDNA by the RNA reverse transcription of extracting, adopt quantitative fluorescent PCR (TaqMan probe method) to carry out multiple sieve and checking to the miRNA just sifted out, pick out perioperatively and stablize with non-cancer control group, one group of miRNA of specifically expressing.
The detection method that the present invention uses can be selected from: RT ?PCR method, low density chip technology (TaqMan low density Array technology), Real ?one or more in time PCR method.Such as, in serum, the detection method of miRNA molecule comprises the following steps:
(1) Trizol reagent (Invitrogen company) is used to extract serum total serum IgE;
(2) cDNA is obtained by RNA reverse transcription reaction;
(3) TaqMan probe method is utilized to carry out PCR reaction;
(4) agarose gel electrophoresis of PCR primer is carried out;
(5) EB dyeing after under ultraviolet lamp observations.
Present invention also offers a kind of miRNA TaqMan probe composition for colorectal cancer early diagnosis and/or Prognosis, it comprise miR ?17, miR ?106a and miR ?the TaqMan probe of any two or three miRNA in 145, preferably by miR ?17 and miR ?the TaqMan probe of 106a two kinds of miRNA form, or by miR ?17, miR ?106a and miR ?the TaqMan probe of 145 3 kinds of miRNA form, each probe sequence is preferably as follows shown in table further:
In addition, the present invention also provides a kind of test kit for diagnosis of colorectal carcinoma and/or prognosis.
In a specific embodiments of the present invention, mentioned reagent box comprises Taq enzyme and dNTP, and magnesium chloride, 10 × PCR damping fluid be not (containing Mg
2+) and comprise miR ?17, miR ?106a and miR ?the TaqMan probe (ABI company provides, and is exclusively used in miRNA fluorescent quantitation) of any one or multiple miRNA in 145.
Mentioned reagent box can preferably include in following TaqMan probe one or more, preferably include further miR ?17 and miR ?the TaqMan of 106a two kinds of miRNA, or comprise miR ?17, miR ?106a and miR ?the TaqMan of 145 3 kinds of miRNA.
Colorectal cancer of the present invention comprises II, III phase colorectal cancer.
The TaqMan probe combination of miRNA combination of the present invention or its correspondence, and the test kit containing described TaqMan probe combination can be used among staging of colorectal cancer detection and Index for diagnosis.By detecting the expression amount of miRNA of the present invention in patients serum and healthy population serum, if the two has the differential expression of significance, then illustrate that patient suffers from II, III phase colorectal cancer.By can judge the prognosis situation of patient to the detection by quantitative of miRNA above-mentioned in serum during patient.
Beneficial effect of the present invention:
First, relatively other organizes and more easily obtains serum, compared with colon biopsy, belongs to woundless testing, is very easy to the use of healthcare givers, more alleviate the misery of patient;
Secondly, the miRNA reflection in serum be pathology, the physiological conditions of animal economy, its detected result has accurate and detailed directive significance;
3rd, the miRNA combination filtered out is significantly higher than the tumor markers CEA (carcinomebryonic antigen) used clinically at present to the specificity of diagnosis of colorectal carcinoma and sensitivity, substantially increase the accuracy of diagnosis.In addition, serum miRNA detects and to reflect in disease generating process regulation and control state after genetic transcription on a molecular scale, and provides potential target spot for the treatment of colorectal cancer.
4th, the colorectal cancer serum miRNA combination that the present invention filters out is not quite similar with the miRNA of special change in other several Serum of Cancer Patients reported so far, point out miRNA combination of the present invention not only colorectal cancer and non-cancer normal people can be made a distinction, also separate with other tumor areas simultaneously.
5th, the colorectal cancer serum miRNA combination that the present invention filters out with not only colorectal cancer and non-cancer normal people can be made a distinction, be also simultaneously that the judgement of postoperative recurrence of colorectal cancer is offered help.
6th, the TaqMan probe that test kit of the present invention comprises is the one group of serum miRNA TaqMan probe expressing significant difference under colorectal cancer and standard state filtered out based on low density chip technology, quantitative PCR technique equally, sensitivity and the specificity of detection can be increased like this, improve detection level.
In sum, the detected result of special miRNA combination provided by the invention, as the intermediate result of Colorectal Cancer Diagnosis, contributes to doctor carries out colorectal cancer diagnosis in conjunction with clinical symptom, medical history or other inspection messages.Simultaneously, it is significant that this serological index is used for II, III phase patient had postoperative recurrent tumor Prognostic Factors, more clinical postoperative recurrence prediction index mostly are histopathology at present, to draw materials difficulty, and the relation between these index and palindromias does not obtain large sample checking, lacks strong evidence.We are by filtering out the serology molecular marker of effective predictive disease recurrence to patients serum's sample miRNA at Liang Ge center, solve the clinical major issue faced at present, by going out easy recurrence crowd at disease initial screening, thus carry out closer follow up a case by regular visits to and to the treatment more strengthened.In addition, the discovery of these miRNAs for further molecule Mechanism Study between research miRNA and postoperative recurrence of colorectal cancer, analyzes the effect of miRNA in recurrence, realize the treatment of colorectal cancer of target miRNA and realizes the molecular diagnosis of tumour and targeted therapy has epochmaking clinical application potentiality and value.
Accompanying drawing explanation
Fig. 1 is main flow figure of the present invention.
The miRNAs diagnosis index of Fig. 2 tumor load.
A scheme .miR ?145 expressions in non-cancer, preoperative colorectal cancer and postoperative serum in patients with colorectal; B scheme .miR ?17 ?the expression of 3p in non-cancer, preoperative colorectal cancer and postoperative serum in patients with colorectal; C scheme .miR ?the expression of 106a in non-cancer, preoperative colorectal cancer and postoperative serum in patients with colorectal.
Fig. 3 II, III phase colorectal cancer is correlated with the ROC tracing analysis of miRNAs.
A schemes. the diagnostic analysis between preoperative colorectal cancer patients and normal people; B. the diagnostic analysis that colorectal cancer patients is preoperative and postoperative.
The existence correlation analysis of expression level in two central sample of Fig. 4 miR-17-3p and miR-106a.
A scheme .miR ?17 ?the existence correlation analysis of 3p in Tianjin tumour hospital sample; B scheme .miR ?the existence correlation analysis of 106a in the sample of Tianjin; C schemes the existence correlation analysis of .miR ?17 ?3p in Xiang Ya hospital sample; D schemes the existence correlation analysis of .miR ?106a in Xiang Ya hospital sample; E schemes the existence correlation analysis of .miR ?17 ?3p in Liang Ge hospital mixing sample; F schemes the existence correlation analysis of .miR ?106a in Liang Ge hospital mixing sample.
The correlation analysis of Fig. 5 miR-17-3p and miR-106a and colorectal carcinoma chemotherapy income.
A scheme .miR ?17 ?3p and miR ?the benefited correlation analysis of 106a and II, III phase colorectal cancer patients chemotherapy; B scheme .miR ?17 ?3p and miR ?the benefited correlation analysis of 106a and II phase colorectal cancer patients chemotherapy; C scheme .miR ?17 ?3p and miR ?the benefited correlation analysis of 106a and III phase colorectal cancer patients chemotherapy.
Embodiment
The invention will be further elaborated by the following examples.
The collection of embodiment 1 sample
We are from Tianjin tumour hospital, and Xiang Ya hospital have collected the serum of 100 routine II phases, 75 routine III phase colorectal cancer tumour patients altogether, under specifying information is shown in:
Table 1 sample information table
Embodiment 2 II, III phase different miRNA express spectra primary dcreening operation
Initial screening stage, by respectively to before 20 example II, III phase surgery for colorectal carcinomas, the pooled serum (sample is from hospital general of Nanjing Military Command) of Post operation pooled serum (sample is from Tumour Hospital Attached To Tianjin Medical Univ.) and 20 routine normal controls carries out low density chip detection, just sifts out the miRNA of one group of differential expression.Found that, have the miRNAs (referring to table 2,3,4) that 126 kinds exist differential expression.
The miRNAs of differential expression between the normal people sifted out at the beginning of table 2 low density chip and II, III phase colorectal cancer patients operation consent
II, III phase colorectal cancer patients of sifting out at the beginning of table 3 low density chip is performed the operation the miRNAs of forward and backward differential expression
The normal people sifted out at the beginning of table 4 low density chip and II, III phase colorectal cancer patients perform the operation forward and backward between the miRNAs of differential expression
Embodiment 3: the realtime fluorescent quantitative PCR experiment (TaqMan probe method) of miRNA in serum
Adopt quantifying PCR method to verify one by one batch sample, from the miRNA sifted out at the beginning of low density chip sequence measurement, filter out the remarkable miRNA of differential expression.
The experimental principle of quantitative PCR (real ?time PCR) and experimental procedure with RT ?PCR the same.Instrument uses ABI Prism 7300 quantitative real time PCR Instrument.
(1) phenol/chloroform method extracts serum total serum IgE
A) getting 100ul sample joins in 300ul water, add 200ul (PH=4.7 ?5.5) acidic phenol after abundant mixing, concuss mixes, and room temperature adds 200ul chloroform after leaving standstill 2min, again fully shake, room temperature leaves standstill the centrifugal 15min of 12000g room temperature after 5min
B) careful Aspirate supernatant (about 400ul) joins in 800ul Virahol, then adds the 3M sodium-acetate 40ul of PH=5.2, fully mixing Hou ?20 DEG C of standing 30min, 16000g, 4 DEG C of centrifugal 20min
C) after fully abandoning supernatant, add 75%DEPC ?ethanol 1ml, gentle inversion for several times, 16000g, 4 DEG C of centrifugal 10min
D) fully abandon supernatant, after drying at room temperature, add 40ul DEPC water dissolution precipitation, obtain RNA sample
(2) cDNA sample is prepared: RNA sample obtained in the previous step is obtained cDNA by RNA reverse transcription reaction.The reaction system of reverse transcription comprises 2 μ l 5 × AMV buffer, 1 μ l 10mM each dNTP mixture (Takara company), 0.5 μ lAMV (Takara company), 4ulDEPC water, 0.5 μ l reverse primer (ABI company) and 2ul RNA sample.Reactions steps is 16 DEG C and hatches 30min, and 42 DEG C of reaction 30min, hatch 5 minutes for 85 DEG C
(3) qRT ?PCR: with 20ul system carry out qRT ?PCR, system comprises 14.77ul H
2o, 2 μ l 10 × PCR buffer (Takara company), 1.2 μ l 25mM MgCl
2(Takara company), 0.4 μ l 10mM each dNTP mixture (Takara company), 0.3ul Taq enzyme (Takara company), 0.33ul TaqMan probe (ABI company), 1ul cDNA.Instrument used is ABI Prism 7300 quantitative real time PCR Instrument, and the reaction conditions of PCR is: within 95 DEG C, 5 minutes, carry out 1 circulation → 95 DEG C, 15 seconds, within 60 DEG C, 1 minute, carry out 40 circulations.
(4) preparation of typical curve: by the 10nM miR of synthesis ?16 ripe body RNA (Takara company) become cDNA by identical reverse transcription system reverse transcription in (2).By gained miR ?16 cDNA use water by 10 times of gradient dilutions 100 times, 1000 times, 10000 times, 100000 times, 1000000 times, obtain one group of cDNA.This group cDNA is pressed identical qRT ?PCR system in (3) and in same PCR plate, carries out quantitative fluorescent PCR with the cDNA in (3)
(5) data analysis and process: increase and terminate to set unified threshold value according to amplification curve afterwards, then according to standard substance miR ?16 concentration and CT value drawing standard curve corresponding to each concentration, and calculate the absolute content of the miRNA measured needed for this sample according to the CT value of this typical curve and sample to be tested.
Embodiment 4: quantifying PCR method sieves again to the serum miRNA just sifted out
With quantifying PCR method (TaqMan probe method), one group of experimenter's sample is detected one by one, from the 22 kinds of miRNA sifted out at the beginning of low density chip method, filter out the significant miRNA of differential expression (concrete steps are see embodiment 2) between 20 routine Normal groups and 20 routine perioperatively II, III phase colorectal cancer patients.Found that, (absolute content method for expressing is means standard deviation to have 3 kinds of miRNA to there are differences between 3 groups of samples in the 22 kinds of miRNA just sifted out, p<0.001), these 3 kinds of miRNA be miR ?106a, miR ?17 ?3p and miR ?145 (see table 5).
The miRNA that table 5 quantifying PCR method sifts out again
1colorectal cancer patients perioperatively differential expression
2differential expression between colorectal cancer patients and normal control
Embodiment 5: quantifying PCR method is to the clinical verification of the serum miRNA sifted out again
Sift out again 3 kinds of miRNA are verified with quantifying PCR method further in clinical samples in enormous quantities, filters out stable, the significant miRNA of differential expression.We have chosen 65 examples from perioperative II, III phase Patients with Colorectal Cancer serum of Tianjin tumour hospital and 40 routine non-cancer control serums, 90 examples from II, III phase Patients with Colorectal Cancer serum of Xiang Ya hospital and 70 routine non-cancer control serums.The result as shown in Figure 2, miR in the 3 kinds of miRNA sifted out again ?17 ?3p and miR ?the expression amount of 106a in II, III phase preoperative serum in patients with colorectal in enormous quantities to be significantly higher than the postoperative patient of normal people and correspondence, miR ?145 then significantly lower than normal people and postoperative patient.
Embodiment 6: the miRNA clinical value assessment filtered out
Draw area (AUC) under ROC curve and calculated curve and assess the miRNA that filters out to the accuracy of diagnosis of colorectal carcinoma, and compare with the current tumor markers CEA used clinically.Result as shown in Figure 3, miRNA ?145, miRNA ?106a and miRNA ?17 ?3p 3 kinds of miRNA be used for the diagnosis of colorectal cancer, the sensitivity of diagnosis is 83%, specific degree is 80%, AUC is 0.85, is obviously better than the detection (susceptibility 60 ?70%) of change of serum C EA.Utilize serology miRNA to express pedigree, be expected to for colorectal cancer early diagnosis, comparatively traditional diagnosis method advantage is remarkable.In addition contact survival of patients data to find, miR ?17 ?the level of 3p and 106a relevant to palindromia, high-level miR ?17 ?3p and miR ?106a patient point out and high-levelly indicate poor prognosis, more easily there is palindromia (Fig. 4 and Fig. 5).
Embodiment 7: for detecting the test kit of serum miRNA
Be based on low density chip technology and quantitative PCR technique measured result for detecting manufacture craft and the operating process of the serum miRNA test kit of the esophageal carcinoma, following TaqMan probe is combined: SEQ ID collects NO:1 ~ 3 the colorectal cancer detection kit of preparation specific T aqMan probe combinations in PCR kit (RT ?PCR or Real ?time PCR) respectively.
The concrete composition (every part of sample) of described test kit is as follows:
The concrete operations flow process of prepared test kit is as follows:
(1) collect the serum sample of experimenter, after extracting RNA, reverse transcription prepares cDNA sample;
(2) according to above-mentioned formula application of sample;
(3) carry out PCR reaction, condition is 95 DEG C of 5min, 95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
First by low density chip technology primary dcreening operation, then one group of large serum miRNA of expression amount difference degree under disease and normal physiological condition is screened further by quantitative PCR technique, as the TaqMan probe detecting colorectal cancer.This test kit comprises serum miRNA TaqMan probe provided by the present invention, the reagent such as Taq enzyme and dNTP.The value of described test kit is with specific TaqMan probe combine detection serum miRNA expression amount, not only can improve specificity, accuracy that colorectal cancer detects, contribute to the early discovery of this disease, the judgement of colorectal cancer lifetime and treatment prognosis can also be used for.Therefore, this test kit drops into practice use and the Diagnosis and Treat of disease can be pushed to a new high degree.
Claims (9)
1. for detecting a miRNA combination for II, III phase colorectal cancer, it is characterized in that this miRNA combination comprises in following miRNA:miR ?17, miR ?106a, miR ?145 one or more.
2. miRNA combination according to claim 1, it is characterized in that this miRNA combination by miR ?17, miR ?106a and miR ?any two or three in 145 form.
3. miRNA combination according to claim 2, it is characterized in that this miRNA combination by miR ?17 and miR ?106a form.
4., for detecting a miRNATaqMan probe compositions for II, III phase colorectal cancer, it is characterized in that this probe combinations comprises the TaqMan probe of miRNA according to any one of claims 1 to 3.
5. TaqMan probe composition according to claim 4, is characterized in that described TaqMan probe composition is made up of any two or three TaqMan probe in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
6. the application of the miRNA combination according to any one of claims 1 to 3 in preparation II, III phase diagnosis of colorectal carcinoma and/or prognostic agent or instrument.
7. the application of the TaqMan probe composition according to any one of claim 4 or 5 in preparation II, III phase diagnosis of colorectal carcinoma and/or prognostic agent or instrument.
8., for a detection kit for II, III phase diagnosis of colorectal carcinoma and/or prognosis, it is characterized in that this test kit comprises the TaqMan probe composition described in claim 4 or 5.
9. application according to claim 8, is characterized in that described test kit also comprises Taq enzyme, dNTP, magnesium chloride and do not contain Mg
2+10 × PCR damping fluid.
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