CN110229899A - For colorectal cancer early diagnosis or the plasma markers object combination of prognosis prediction - Google Patents

For colorectal cancer early diagnosis or the plasma markers object combination of prognosis prediction Download PDF

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CN110229899A
CN110229899A CN201910533068.9A CN201910533068A CN110229899A CN 110229899 A CN110229899 A CN 110229899A CN 201910533068 A CN201910533068 A CN 201910533068A CN 110229899 A CN110229899 A CN 110229899A
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mirna
colorectal cancer
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徐学虎
蒋析文
刘学娟
朱小亚
刘悦
张丽妹
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Third Affiliated Hospital of Guangzhou Medical University
Daan Gene Co Ltd of Sun Yat Sen University
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Daan Gene Co Ltd of Sun Yat Sen University
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Abstract

The invention discloses the plasma markers object combinations for colorectal cancer early diagnosis or prognosis prediction.Specifically, present invention obtains the miRNA of unconventionality expression obvious in plasma of colorectal cancer, i.e. miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183.The present invention has found that blood plasma miRNA combination can be as colorectal cancer early diagnosis or Index for diagnosis marker by studying differential expression of these blood plasma miRNAs in each phase colorectal cancer patients, Colon and rectum adenoma patients and healthy population.

Description

For colorectal cancer early diagnosis or the plasma markers object combination of prognosis prediction
Technical field
The invention belongs to biotechnologys and medical domain, specifically, the present invention relates to early diagnose for colorectal cancer Or the plasma markers object combination of prognosis prediction.
Background technique
Colorectal cancer (colorectal cancer, CRC) is one of most common malignant tumour, the CRC in terms of global range Disease incidence women ranked second position, and male ranked third position, and the overall CRC death rate is number two.It is estimated that 2018 will have 1,800,000 New colorectal cancer case and 881,000 people is dead, accounts for about the 1% of cases of cancer and death toll.To the year two thousand thirty, global CRC hair Sick rate is estimated will to increase by 60%.Cancer in China statistical data in 2015 indicates that male CRC disease incidence occupies the 5th, and women occupies the 4th Position, total disease incidence and the death rate are respectively positioned on the 5th, and tumor in digestive tract overall incidence rises year by year.It is early according to Document system After phase CRC Canonical management, 5 year life cycle had 15%~20% CRC just to have found that liver turns in diagnosis up to 90% It moves, remaining 60% also discovery recurrence or transfer in subsequent therapeutic process.The key of CRC treatment is that early early diagnosis of sieving early is controlled, It is come out in CRC in adenoma stage screening and treatment in time is to improve the effective means of CRC patient survival rate.80% colorectal cancer It is to be evolved by adenomatoid polyp, there are 10~15 years benign tumour developing stage before canceration, which is colorectal cancer Prevention and early diagnosis provide strong opportunity, which also allows colorectal cancer that can pass through early screening as few in number The malignant tumour to reduce the incidence of disease with the death rate.The morbidity of colorectal cancer can be reduced by being identified by colorectal cancer screening and cutting off adenoma Rate is up to 53%.In this stage, operation method almost can achieve the effect for curing disease, hold the time of Adenoma canceration, know Other early lesion is the key that the colorectal cancer diagnosis and treatment first step.However, only about 59% just receives knot after more than 50 years old Enteroscopy.In addition, being II phase or advanced stage when 60%~70% PATIENTS WITH LARGE BOWEL is found.With it is treatment-related bad anti- CRC should be caused to be easy recurrence and transfer with poor prognosis.Chemotherapy resistance is a big obstacle of advanced stage CRC treatment, and DISTANT METASTASES IN The main reason for local recurrence being CRC patient postoperative death.Understand risk factor relevant to CRC survival rate, helps to sieve Select easy to recur and transfer high-risk patient.Tumor markers can be used for the screening of infantile tumour patient, reflection patient undergos surgery, Curative effect, assessment recurrence and DISTANT METASTASES IN situation after chemicotherapy or Biological target therapy, turn the clinical treatment of CRC, recurrence Shifting, prognosis evaluation are significant.
Early screening means currently used for colorectal cancer have: fecal occult blood detects (chemical method and immunization), Sigmoidoscope Inspection, the detection of multiple target point excrement, questionnaire risk assessment, CT simulate Sigmoidoscope, excrement PKM2 Protein Detection, blood plasma ctDNA Sept99 DNA methylation assay etc..Fecal occult blood detects chemical method because sensibility is poor, the control in terms of diet is required when sampling, as a result By multifactor interference, Brenner etc. analyzes nearly 40,000 people screening crowd, and compare Sigmoidoscope as a result, the inspection of chemical method excrement is failed to pinpoint a disease in diagnosis Rate is up to 90% and 75% respectively in progressive stage adenoma and colorectal cancer.So being no longer proposed use；Immunochemistry Method FIT is 79% to the sensibility of colorectal cancer screening, and specificity is 94%.It is mainly the widest in screening with colloid gold test paper General application.As can annual conventional 1 immuno-chemical method FIT can also reach screening purpose in practical application.But its screening is not Foot be it is low to the sensibility of progressive stage adenoma, only 20%~30%.In addition, because the Sampling of excrement causes to abandon inspection rate It is high.The most sensitive means of colonoscopy enteron aisle lesion examining, can intuitive entire intestinal wall situation, in addition to flat and depressed lesion and rare Outside " interval cancer " the more difficult discovery seen, most polyp lesion is not easy to fail to pinpoint a disease in diagnosis, and totally sees screening crowd disease under asymptomatic Sigmoidoscope Become recall rate, up to 0.5%~1.8% in colorectal cancer, 14.8% is up in progressive stage adenoma, the totality of polyposis intestinalis Recall rate can be higher than 1/3.But it is needed before colonoscopy by stringent and the INTESTINAL CLEANSING of pain, and technical requirements are high, wound The factors such as risk is higher, which largely hinder colonoscopy, becomes the first-selected instrument of colorectal carcinoma early screening, especially For the status that China's medical resource lacks, Sigmoidoscope still needs to consider carefully as primary dcreening operation.The detection of multiple target point excrement utilizes excrement Just middle exfoliated tumor cells DNA molecular detection combine immunization FIT, further improve the screening sensibility of intestinal cancer, into The duration of an exhibition screening sensibility of adenoma can also be enhanced to 54%.The U.S. has been applied to Silent cerebral infarction colorectal carcinoma early diagnosis sieve It looks into, the screening period is recommended as 1 time/1 year or 3 years.The major defect of this method is exactly that price is higher.Questionnaire risk assessment sieve It looks into, is inquiry and the presence for assessing high risk factor, is proposed by Zhejiang University Zheng Shu professor team, be included into country within 2007 and defend The standard scheme that planning commission's cancer early diagnosis is early controlled.This method sharpest edges be it is simple and easy, the screening for improving public group is suitable It should rate.But in questionnaire can evaluation factor venture influence degree it is on the weak side, to the screening sensibility and specificity of advanced colorectal rectal neoplasm Also not high.Other are used for the means of colorectal carcinoma early diagnosis screening, because there are various defects also not to be recommended extensively.
In conclusion accomplishing the morning discovery to colorectal cancer people at highest risk, intervenes in time, clinically still lack more accurate Early warning means or Biological indicators.
Summary of the invention
The object of the present invention is to provide a kind of plasma markers objects for colorectal cancer early diagnosis or prognosis prediction to combine.
First aspect present invention provides miRNA or the purposes of its detection reagent, is used to prepare colorectal cancer early diagnosis Or the kit of prognosis prediction, the miRNA are selected from the group: miR-224-5p, miR-301a-3p, miR-145-5p, miR- 193b-3p or combinations thereof.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p and miR-193b-3p.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p, miR-145-5p, miR- 193b-3p；Preferably, the miRNA further includes miR-1183.
In another preferred example, the kit contains miR-224-5p, miR-301a-3p, miR-145-5p, miR- 193b-3p, miR-1183, or combinations thereof be used as positive control.
In another preferred example, the detection sample of the detection kit includes blood (blood plasma) or tissue.
In another preferred example, the detection reagent is selected from the group: antibody, primer, probe, sequencing library, nucleic acid chip, Or combinations thereof.
In another preferred example, the detection reagent includes primer, antisense nucleic acid, probe or chip.
Second aspect of the present invention, provides a kind of miRNA chip, and the miRNA chip includes:
Solid phase carrier；And
The oligonucleotide probe being orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to It is selected from the group in target miRNA, the target miRNA: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b- 3p, or combinations thereof.
In another preferred example, the oligonucleotide probe contains:
Complementary combined area；And/or
The bonding pad being connected with solid phase carrier.
In another preferred example, the target miRNA includes miR-224-5p, miR-301a-3p and miR-193b-3p.
In another preferred example, the target miRNA include miR-224-5p, miR-301a-3p, miR-145-5p, MiR-193b-3p and miR-1183.
Third aspect present invention provides a kind of kit, and the kit contains miRNA or its detection reagent, described MiRNA is selected from the group: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, or combinations thereof；Also, institute Stating kit further includes specification, and it is pre- for colorectal cancer early diagnosis or prognosis that the kit is indicated in the specification It surveys.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p and miR-193b-3p.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p, miR-145-5p, miR- 193b-3p and miR-1183.
In another preferred example, the specification indicates the following contents:
When the level of miR-224-5p, miR-301a-3p and/or miR-193b-3p in the sample measured and healthy sample This level difference is significant, then prompting the detection sample is colorectal cancer high-risk patient sample.
In another preferred example, the specification indicates the following contents:
In the sample measured, compared with healthy sample,
MiR-224-5p level significantly improves；
MiR-301a-3p level significantly reduces；And/or
MiR-193b-3p level significantly improves；
Then prompting the detection sample is colorectal cancer high-risk patient sample.
In another preferred example, the specification records a regression model, establishes logit (P)=- 1.052+86.495 × (miR-193b-3p)+30.897 × (miR-224-5p) -24.876 × (miR-301a-3p), wherein miR-193b-3p, The expression of miR-224-5p and miR-301a-3p is measured by internal reference of miR-30a-5p, and logit (P) value is aobvious in model Show that there are significant differences for detection sample and healthy sample, then prompting the detection sample is colorectal cancer high-risk patient sample.
In another preferred example, the specification indicates the following contents:
As miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p in the sample measured and/or The level of miR-1183 and the level difference of healthy sample are significant, then prompting the detection sample is colorectal carcinoma high-risk patient sample This.
In another preferred example, in the sample measured, compared with healthy sample,
MiR-224-5p level significantly improves；
MiR-301a-3p level significantly reduces；
MiR-193b-3p level significantly improves；
MiR-145-5p level significantly reduces；And/or
MiR-1183 level is significantly reduced or is improved；
Then prompting the detection sample is colorectal carcinoma high-risk patient sample.
In another preferred example, in the sample measured, compared with Colon and rectum adenoma sample,
MiR-224-5p level significantly improves；
MiR-301a-3p level significantly reduces；
MiR-193b-3p level significantly improves；
MiR-145-5p level significantly reduces；And/or
MiR-1183 level significantly improves；
Then prompting the detection sample is colorectal cancer high-risk patient sample.
In another preferred example, the specification records a regression model, establishes logit (P)=- 2.338+7.166 ×(miR-145-5p)+173.161×(miR-193b-3p)-27.658×(miR-1183)+46.071×(miR-224- 5p) -58.807 × (miR-301a-3p), wherein miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p with And the expression of miR-1183 is measured by internal reference of miR-30a-5p, logit (P) value display detection sample and knot are straight in model There are significant differences for enteric adenoma sample, then prompting the detection sample is colorectal cancer high-risk patient sample.
Fourth aspect present invention provides a kind of isolated miRNA, and the miRNA is selected from:
(i) miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p or its group It closes, or
(i i) is mutual with miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p or miR-193b-3p The miRNA of benefit, or combinations thereof.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p and miR-193b-3p.
In another preferred example, the miRNA includes miR-224-5p, miR-301a-3p, miR-145-5p, miR- 193b-3p and miR-1183.
In another preferred example, the miRNA includes miR-375-3p, miR-135b-5p, miR-224-5p, miR- 125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183.
In another preferred example, the miRNA is selected from the group:
(a) SEQIDNO.:1-8 it is any shown in sequence；
(b) any sequence in 8 complementary series complementary with any shown sequence of SEQIDNO.:1-8；Or
(c) combination from (a) or (b), and the sequence from (a) is not complementary with the complementary series from (b) mutually.
Fifth aspect present invention, provide a kind of separation or artificial constructed precursor miRNA, the precursor miRNA It can be sheared in people's cell and be expressed as miRNA described in fourth aspect present invention.
Sixth aspect present invention, provides a kind of isolated polynucleotides, and the polynucleotides can be transcribed by people's cell At precursor miRNA, the precursor miRNA can be sheared and be expressed as described in fourth aspect present invention in people's cell miRNA。
In another preferred example, the polynucleotides have structure shown in Formulas I:
SeqIt is positive-X-SeqReversely
Formulas I
In Formulas I,
SeqIt is positiveFor the nucleotide sequence that can be expressed as the miRNA in people's cell；
SeqReverselyFor with SeqIt is positiveIt is substantially complementary or the nucleotide sequence of complete complementary；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positiveAnd SeqReverselyNot mutually It mends；
And structure shown in Formulas I forms secondary structure shown in Formula II after being transferred to people's cell:
In Formula II, SeqIt is positive、SeqReverselyIt is as defined above and states with X,
| | it indicates in SeqIt is positiveAnd SeqReverselyBetween the base pair complementarity relationship that is formed.
In another preferred example, the measurement is realized by real-timePCR.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1: each miRNA expression in 60 plasma samples, in figure:
(1) * indicates P < 0.05；* indicates P < 0.005；* * indicates P < 0.001
(2) every group is designated as on column compared with normal group；Black line indicates two groups and compares；Gray line indicates three groups or four groups and compares
(3) (20) N: normal group 20；Ad (20): Colon and rectum adenoma group 20；CRC (20): colorectal cancer group 20；0- II (10): II phase of 0- early carcinoma group 10；III-IV (10): III-IV phase early carcinoma group 10.
Situation is verified in expression of each miRNA of Fig. 2 in 158 plasma samples, in figure:
(1) * indicates P < 0.05；* indicates P < 0.005；* * indicates P < 0.001
(2) every group is designated as on column compared with normal group；Black line indicates two groups and compares；Gray line indicates three groups or four groups and compares
(3) (50) N: normal group 50；Ad (50): Colon and rectum adenoma group 50；CRC (50): colorectal cancer group 50；0- II (28): II phase of 0- early carcinoma group 28；III-IV (30): III-IV phase early carcinoma group 30.
Diagnostic potential of Fig. 3 A:ROC tracing analysis blood plasma miRNA to colorectal cancer.
Diagnostic potential of Fig. 3 B:ROC tracing analysis blood plasma miRNA to Colon and rectum adenoma.
Identification potential of Fig. 3 C:ROC tracing analysis blood plasma miRNA to colorectal cancer and adenoma.
Identification potential of Fig. 3 D:ROC tracing analysis blood plasma miRNA to Colon and rectum early carcinoma and late cancer.
The ROC curve analysis of Fig. 4: two built-up patterns.
Fig. 5: logit equation and combined diagnostic, in figure:
N be it is normal, M be Colon and rectum adenoma, T is colorectal cancer.
Detailed description of the invention
The present invention after extensive and in-depth study, will in plasma of colorectal cancer sample unconventionality expression it is obvious and more constant MiRNA be determined as Primary Study object, the expression of the colorectal cancer early diagnosis and prognosis prediction marker with 30a-5p and 30e-5p is that internal reference is detected, and is had found and the closely related miRNA of Colorectal Cancer.It is finally obtained and is tying The miRNA of obvious unconventionality expression, i.e. miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b- in carcinoma of the rectum blood plasma 3p and miR-1183.The present invention is by studying these blood plasma miRNAs in each phase colorectal cancer patients, Colon and rectum adenoma patients and being good for Differential expression in Kang Renqun finds that blood plasma miRNA combination can early diagnose marker as colorectal cancer.It is basic herein On complete the present invention.
Specifically, present invention application real time fluorescence quantifying PCR method is altogether in 78 each phase CRC patients, 70 Colon and rectum glands It is verified in tumor patient and 70 healthy volunteer's blood plasma, detects the expression of above-mentioned 9 candidate targets, observation difference is faced Bed feature plasma sample in express spectra difference, and analyze plasma specific miRNA combination colorectal cancer early diagnosis and Value in prognosis prediction.After verified, the present invention obtains specific plasma 3-miRNA combination, i.e. miR-224-5p, miR- The susceptibility of 301a-3p and miR-193b-3p, this group of biomarker combinations detection colorectal cancer are 0.655, and specificity is 0.920, and can produce maximum Receiver operating curve (receiver operator characteristic curve, ROC area under the curve (area under the curve, AUC)) is 0.856.Specific plasma 5-miRNA combination is obtained, That is miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR-1183, this group of biomarker group The susceptibility for closing difference Colon and rectum adenoma and colorectal cancer is 0.720, specificity 0.680, and producible AUC is 0.930.Colorectal carcinoma herein includes benign adenomas and pernicious colorectal cancer.The progress of colorectal carcinoma Journey generally normal-adenoma-early carcinoma-late cancer therefore, blood plasma miR-224-5p, miR-301a-3p, miR-145-5p, MiR-193b-3p and miR-1183 can be used as the potential tumor markers of colorectal carcinoma, and the miRNA combination of building can be quasi- Colorectal cancer, Colon and rectum adenoma and normal healthy controls person are really distinguished, is miRNA as potential colorectal cancer and early diagnoses biology mark The existing Laboratory evidence of will object has carried out new effective supplement.
The purpose of the present invention is to provide a kind of early diagnosis of colorectal cancer and prognosis prediction markers, develop related reagent Box.
Colorectal cancer early diagnosis of the present invention and prognosis prediction marker are made of multiple nucleic acid molecules, described Multiple nucleic acid molecules include coding blood plasma miR-375-3p, miR-135b-5p, miR-224-5p, miR-125b-5p, miR- The nucleic acid molecules of 301a-3p, miR-145-5p, miR-193b-3p and miR-1183；The nucleic acid molecules are at least one Differential expression in blood plasma is compareed at least one in target blood plasma.Colorectal cancer early diagnosis of the present invention and prognosis prediction Kit, including colorectal cancer of the present invention early diagnosis and prognosis prediction marker.
The present invention provides at least one blood plasma miRNA for distinguishing Colon and rectum early lesion/colorectal cancer patients and health The kit of individual.At least one blood plasma miRNA is provided for predicting that colorectal cancer patients transfer or risk of recurrence or chemotherapy are anti- The kit answered.
The obvious and more constant miRNA of the unconventionality expression in plasma of colorectal cancer sample is determined as Primary Study by the present invention The expression of object, the colorectal cancer early diagnosis and prognosis prediction marker is with miR-30a-5p or miR-30e- 5p is that internal reference is detected, and preferably miR-30a-5p is that internal reference is detected.It has found closely related with Colorectal Cancer miRNA.The present invention selected 8 may in plasma of colorectal cancer obvious unconventionality expression miRNA, i.e. miR-375-3p, MiR-135b-5p, miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR-193b-3p and miR- 1183.The present invention is by studying these blood plasma miRNAs in each phase colorectal cancer patients, Colon and rectum adenoma patients and healthy population Differential expression, find blood plasma in above-mentioned miRNA combination can as colorectal cancer early diagnose marker.In combination with wherein The clinical and pathological data of colorectal cancer patients does clinically relevant analysis, finds this group of blood plasma miRNA and clinical stages, differentiation journey The indexs such as degree, lymphatic metastasis, DISTANT METASTASES IN, change of serum C EA expression are related, to find that this group of blood plasma miRNA can be made For colorectal cancer prognosis prediction marker.
MiRNA and its precursor
Small molecule RNA (microRNA, miRNA) is a kind of single-stranded tiny RNA of non-coding by about 19~25nt.With MiRNAs research deepen continuously, find extracellular miRNAs (cfmiRNAs) in various body fluid have well stablize Property, predominantly blood, there are also urine, saliva, sweat, cerebrospinal fluid etc..It is sent out rapidly in high-throughput techniques (such as microarray and sequencing) On the basis of exhibition, comprehensive cfmiRNAs express spectra is disclosed (predominantly circulation miRNA) in kinds cancer, it was found that more next More new having early diagnose and the miRNAs of prediction prognosis potential.But there is no blood plasma miRNA phase still in colorectal cancer The kit of pass lists.
The present invention provides a new class of miRNA found from people.As used herein, " miRNA " refers to one Kind RNA molecule is processed from the transcript that can form miRNA precursor.Mature miRNA usually has 18-26 nucleotide (nt) (more particularly about 19-22nt) is also not excluded for the miRNA molecule with other number nucleotide.MiRNA usually can quilt Northern trace detects.
The miRNA in people source can be separated from people's cell.As used herein, " separation " refers to substance from its original ring (if it is crude, primal environment is natural surroundings) is separated in border.Under the native state in active somatic cell Polynucleotide and polypeptide do not isolate and purify, but same polynucleotide or polypeptide are deposited together such as from native state Other substances in separate, then isolate and purify.
MiRNA can be processed from precursor miRNA (PrecursormiRNA, Pre-miRNA), the precursor miRNA It is can be folded into a kind of stable stem ring (hair clip) structure, the loop-stem structure length is generally between 50-100bp.Described Precursor miRNA can be folded into stable loop-stem structure, and the stem two sides of loop-stem structure include the two sequences being substantially complementary.Institute The precursor miRNA stated can be natural or artificial synthesized.
Precursor miRNA, which can be sheared, generates miRNA, and the miRNA can be at least part of the mRNA of encoding gene Sequence is substantially complementary.As used herein, it " is substantially complementary " and refers to that the sequence of nucleotide is enough complementations, it can be with one kind Foreseeable mode interacts, and such as forms secondary structure (such as loop-stem structure).In general, the core of two " being substantially complementary " Nucleotide sequence from each other at least 70% nucleotide be complementary；Preferably, at least 80% nucleotide is complementary； It is furthermore preferred that at least 90% nucleotide is complementary；It is further preferred that at least 95% nucleotide is complementary； Such as 98%, 99% or 100%.Generally, most 40 unmatched nucleosides be can have between two molecules complementary enough Acid；Preferably, there are most 30 unmatched nucleotide；It is furthermore preferred that having most 20 unmatched nucleotide；Into one Step is preferred, has most 10 unmatched nucleotide, such as has 1,2,3,4,5,8,9 unmatched nucleotide.
As used herein, " stem ring " structure is also referred to as " hair clip " structure, refers to a kind of nucleic acid molecule, can form one Kind includes the secondary structure of double-stranded region (stem), and the double-stranded region (is located at same by two regions of the nucleic acid molecule On one molecule) it is formed, the two sides of column double stranded section are divided in two regions；It further includes at least one " ring " structure, including incomplementarity Nucleic acid molecule, i.e. single-stranded regions.Even if two regions of the nucleic acid molecule are not complete complementary, the double-strand of nucleotide Part can also keep double-stranded state.For example, insertion, missing, substitution etc. can lead to not complementary or zonule of a zonule Itself forms the secondary structure of loop-stem structure or other forms, however, two regions can be still substantially complementary, and is being contemplated that Mode in interact, form the double-stranded region of loop-stem structure.Loop-stem structure be it is well-known to those skilled in the art, Usually after the nucleic acid for obtaining a nucleotide sequence with primary structure, those skilled in the art can determine the nucleic acid Whether loop-stem structure can be formed.
MiRNA of the present invention has the sequence as shown in SEQIDNO:1-8.In order to improve miRNA stability or Other properties can also add at least one protectiveness base, such as " TT " at least one end of the miRNA.
Antisense oligonucleotides
Provided miRNA sequence according to the present invention can be designed that its antisense oligonucleotides, antisense widow's core Thuja acid can lower the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotides (antisense- Oligonucleotides, AS-Ons or ASO) " be also known as " GEM 132 ", refer to length be about 18-26nt (more particularly About 19-22nt) DNA molecular or RNA molecule or its analog.
In the present invention, " antisense oligonucleotides " further includes using as based on nucleic acid lock or nucleic acid chains backbone modification The modified GEM 132 that the means such as technology obtain, the modification do not change the activity of antisense oligonucleotides substantially, more Goodly, described to modify the stability, activity or therapeutic effect that antisense oligonucleotides can be improved.Nucleic acid lock (lockednucleicacid, LNA) typically refers to connect 2 ' oxygen atoms of ribose and 4 ' carbon atoms by a methylene bridge The modification technique to get up.LNA can extend the serum half-life of miRNA, improve to target compatibility, reduce the range for effect of missing the target And degree.For the antisense drug that modification technique based on nucleic acid chain backbone develops in solubility, nuclease-resistant degradation etc. is big There is improvement, and is easy to largely synthesize.There are many backbone modification methods of oligonucleotides, including thio method, such as by deoxyribonucleoside Sour chain thio-modification is thio deoxynucleotide chain.This method is to replace the oxygen atom of the phosphate bond on DNA skeleton with sulphur atom In generation, can resist nuclease degradation.It should be understood that any the largely or entirely active of the antisense oligonucleotides that be able to maintain is repaired Decorations are included in the present invention.
As preferred embodiment of the invention, nucleic acid lock modification is carried out to antisense oligonucleotides；More preferably also carry out thio repair Decorations.
After antisense oligonucleotides of the present invention transfer into the human body, they can obviously lower related miRNA's Expression.
Polynucleotides construction
Provided miRNA sequence according to the present invention, can be designed can be processed to influence after being imported into accordingly The polynucleotides construction namely the polynucleotides construction of the miRNA of mRNA expression can raise accordingly in vivo The amount of miRNA.Therefore, the present invention provides a kind of isolated polynucleotides (construction), the polynucleotides (construction) Precursor miRNA can be transcribed by people's cell, the precursor miRNA can be sheared by people's cell and be expressed as the miRNA.
As a kind of preferred embodiment of the invention, the polynucleotides construction contains structure shown in Formulas I:
SeqIt is positive-X-SeqReversely
Formulas I
In Formulas I,
SeqIt is positiveFor the nucleotide sequence that can be expressed as the miRNA in cell, SeqReverselyFor with SeqIt is positiveSubstantially mutually The nucleotide sequence of benefit；Alternatively, SeqReverselyFor the nucleotide sequence that can be expressed as the miRNA in cell, SeqIt is positiveFor with SeqReverselyThe nucleotide sequence being substantially complementary；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positiveAnd SeqReverselyNot mutually It mends；
Structure shown in Formulas I forms secondary structure shown in Formula II after being transferred to cell:
In Formula II, SeqIt is positive、SeqReverselyIt is as defined above and states with X；
| | it indicates in SeqIt is positiveAnd SeqReverselyBetween the base pair complementarity relationship that is formed.
In general, the polynucleotides construction is located on expression vector.
Therefore, the invention also includes a kind of carriers, it contains the miRNA or the polynucleotides construction.Institute The expression vector stated is usually also containing promoter, replication orgin and/or marker gene etc..Side well-known to those having ordinary skill in the art Method can be used to construct the required expression vector of the present invention.These methods include recombinant DNA technology in vi, DNA synthetic technology, in vivo Recombinant technique etc..The expression vector preferably includes one or more selected markers, to provide for selecting to turn The phenotypic character of the host cell of change, such as kalamycin, gentamicin, hygromycin, amicillin resistance.
Chip
MicroRNA chip of expression spectrum usually contains up to several hundred a probes, covers a variety of microRNA, utilizes DNA double chain The principle of homologous complementary detects the content of contained various microRNA in sample in full-length genome level.It therefore, can be same Time detects the transcriptional level of the microRNA within the scope of full-length genome in sample to be tested.
Using miRNA sequence of the present invention, corresponding miRNA chip can also be prepared, and then studies its express spectra And the regulative mode of miRNAs.
On the other hand, the present invention also provides a kind of for analyzing the chip of miRNA express spectra, and the chip can be used for Distinguish colorectal cancer sample and normal sample.
The few core that the miRNA chip of the invention includes solid phase carrier and is orderly fixed on the solid phase carrier Thuja acid probe.
Specifically, can miRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, formed " oligonucleotide arrays "." oligonucleotide arrays " refer to addressable point (i.e. with distinctive, addressablely The position that location is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to need It wants, oligonucleotide arrays can be divided into multiple sub- battle arrays.
The various common used materials in genetic chip field, such as, but not limited to nylon membrane can be used in the solid phase carrier, through work Property group (such as aldehyde radical, amino) slide or silicon wafer, unmodified slide, plastic sheet etc. for modifying.
The conventional manufacturing method of biochip known in the art can be used in the preparation of the miRNA chip.For example, such as Fruit solid phase carrier is gone here and there using modification slide or silicon wafer, 5 ' ends of probe containing amido modified poly- dT, can be by oligonucleotides Probe is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence or array, Then it is fixed by standing overnight, so that it may obtain miRNA chip of the invention.If nucleic acid is without amido modified, system Preparation Method can also refer to:
" the gene diagnosis technology-on-radiation operation manual " of Wang Shenwu chief editor；J.L.erisi,V.R.Iyer,P.O.B ROWN.Exploringthemetabolicandgeneticcontrolofg eneexpressiononagenomicscale. Science,1997；278:680 and Ma Li people, Jiang Zhonghua chief editor Beijing biochip: Chemical Industry Press, 2000,1- 130。
On the other hand, the present invention also provides a kind of sides that miRNA express spectra in people's tissue is detected by miRNA chip Method, comprising steps of
(1) RNA sample for being isolated from people's tissue, the setting flag object on the RNA are provided；
(2) RNA of (1) is contacted with the chip, sends out the oligonucleotide probe on the RNA and solid phase carrier Raw hybridization reaction, to form " oligonucleotide probe-RNA " binary complex on solid phase carrier；
(3) marker for the binary complex that detection (2) is formed, so that it is determined that the expression of miRNA accordingly in people's tissue Spectrum.
Extracting the method for RNA from people's tissue is method well known to those skilled in the art, including Trizol method.
It is furthermore preferred that after isolating RNA sample in people's tissue tissue, being fitted to RNA sample in step (1) Work as processing, to be enriched with the RNA with certain length, the length (small fragment RNA) generally between 10-100.By above-mentioned After processing, subsequent hybridization is carried out using these small fragment RNAs, the accuracy of chip capture miRNA can be improved in this way.This field Personnel are convenient to isolate the RNA with certain fragment length, for example gel electrophoresis can be used to separate.
It is also method well known to those skilled in the art that RNA, which is marked, can be special with RNA by being added in hybridization The method for the marker that the opposite sex combines realizes that the marker is such as labelling groups.The labelling groups include but unlimited In: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and its derivative biomolecule (FITC etc.), other fluorescence point Sub (such as Cy3, Cy5), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc..These labels and its labeling method are all It is routine techniques well-known in the art.
When above-mentioned RNA is hybridized with miRNA chip, first miRNA chip and pre-hybridization buffer can be carried out Prehybridization.
Solid-phase hybridization between RNA and miRNA chip of the present invention is carried out according to the classical way of this field, ability Domain general staff is empirically easy to determine related buffer, probe and concentration of specimens, prehybridization temperature, hybridization temperature with timely Between equal optimum condition.Or it is also referred to described in " Molecular Cloning:A Laboratory guide ".
Then the acquisition of information such as position, intensity according to marking signal on miRNA chip wait for measurement information.If amplified production It is marked with fluorophor, fluorescence detection device (such as laser confocal scanner Scanarray3000) can also directly be used to obtain To measurement information.
Detection kit
The present invention also provides a kind of kit, miRNA or its detection reagent of the invention are contained in the kit (e.g., chip).The kit can be used for detecting the express spectra of miRNA；Or it is used for Diagnose Rectal Cancer, it is preferred that described Also containing the marker for labeled RNA sample, and substrate corresponding with the marker in kit.
In addition, may also include in the kit for various reagents needed for extracting RNA, PCR, hybridization, colour developing etc., Including but not limited to: extract, amplification liquid, hybridization solution, enzyme, comparison liquid, developing solution, washing lotion, antibody etc..
In addition, may also include operation instructions and/or chip image analysis software in the kit.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment
Sample process
1998 are strictly observed by " mankind's inheritance resources management Tentative Measures " issued by the State Council and 2005 " the Chinese mankind Genetic resources disease flesh tissue compiles Techniques of preserving regulation " and as benchmark.It collects -2019 years 01 month 2017 02 Third affiliated hospital, Guangzhou medical university gastrointestinal surgery, endoscope center, 78 knots of gastroenterology and medical center are straight during month Patients with bowel cancer, 70 Colon and rectum adenoma patients and 70 normal healthy controls persons peripheric venous blood, colorectal cancer patients are through pathology Inspection is made a definite diagnosis, and does not include the diagnosis that other tumor diseases or great, chronic disease etc. may influence this research target, sample For patient without any Radiotherapy chemotherapy, all patients have complete clinical, pathological data before acquiring.Male 43, women 35； It is age 25-90 years old, 65 years old average；TNM stage, II phase of 0- 38, III-IV phase are carried out in accordance with the TNM stage standard of NCCN guide 40.Colon and rectum adenoma patients be included in standard be the clear adenomatous polyp of pathological diagnosis or tubulose/villiform/Serrated adenoma or High/low rank intraepithelial neoplasia (cin), and do not include that tumor disease or great, chronic disease etc. may influence examining for this research target It is disconnected.Male 39, women 31；It is age 22-88 years old, 59 years old average.Healthy person is included in standard to exclude to have tumor disease history, again Big or chronic disease history may influence the disease of this research target with other.Wherein male 34, women 36；Age 19-68 It is year, 41 years old average.This research has passed through the audit approval of third affiliated hospital, Guangzhou medical university Medical Ethics Committee, owns The acquisition of sample obtains the informed consent of patient.
Test main material
1, key instrument
Gene-amplificative instrament: day is won in TC-96/G/H (b), Hangzhou.
Fluorescence quantitative PCR instrument: LightCycler480II, Roche.
2, main agents
Serum/plasma miRNA serum extracts separating kit, DP503, Tiangeng.
All-in-OneTM miRNA First-Strand cDNA Synthesis Kit:QP014, GeneCopoeia.
HieffTM qPCR SYBR Green Master Mix (No Rox): 11201ES08, the holy biology of assist.
Primer: deep fast biosynthesis.
Experimental procedure
1, RNA is extracted
Plasma sample 200uL is taken, content is operated to specifications, and last elution volume is 35uL.
1) sample treatment: being added 900 μ l lysate MZA in every 200 μ l serum or blood plasma, oscillator oscillation mixes 30sec To complete
Homogenate, is mixed by inversion.If you need to use detection is outer to join (CR100-01), please after complete homogenate, be mixed by inversion it It is preceding to add
Enter (1 μ l is added in 1 μM of concentration).
2) it is placed at room temperature for 5min, so that nucleic acid-protein compound is kept completely separate.
3) 200 μ l chloroforms are added, covers pipe lid, acutely vibrates 15sec, be placed at room temperature for 5min.
4) 12,000rpm (~13,400 × g), is centrifuged 15min by 4 DEG C, and sample can be divided into three layers: the organic phase of yellow, white Color
Middle layer and colourless water phase, RNA mainly in water phase, water phase are transferred in new pipe, carry out next step operation.
5) measure transfer liquid volume, be slowly added to transfer 2 times of volumes of liquid dehydrated alcohol (such as: the transfer liquid of 500 μ l adds 1ml is anhydrous
Ethyl alcohol), mix (at this time it is possible that precipitating).Obtained solution and precipitating are transferred to adsorption column together MiRelute, room
Temperature places 2min, room temperature 12, and 000rpm (~13,400 × g) is centrifuged 30sec, efflux is discarded after centrifugation, retains Absorption
Column miRelute.
6) 700 μ l protein liquid removal MRD (please first check whether and ethyl alcohol has been added), room temperature are added into adsorption column miRelute It stands
2min, room temperature 12,000rpm (~13,400 × g) are centrifuged 30sec, abandon waste liquid.
7) 500 μ l rinsing liquid RW (please first check whether and ethyl alcohol has been added) are added into adsorption column miRelute, room temperature is quiet 2min is set,
Room temperature 12,000rpm (~13,400 × g) are centrifuged 30sec, abandon waste liquid.
8) it is primary to repeat step 7.
9) room temperature 12,000rpm (~13,400 × g) are centrifuged 2min, abandon collecting pipe.
2, cDNA is synthesized
1) reagent of defrosting miRNA qRT-PCR Detection Kit, upper and lower gentle inversion are mixed, are put after of short duration centrifugation It sets stand-by on ice.
2) miRNA inverse transcription reaction liquid is prepared on ice, and following component is added extremely in the RNase-free reaction tube of pre-cooling 25 μ L of total volume
Ingredient Additional amount Final concentration
Total RNA 50ng
2.5U/μl PolyA Polymerase 1μL
RTase Mix 1μL
5×Reaction Buffer 5μL 1×
DEPC handles water to 25μL
3) reverse transcription reaction: mixing reaction Mix, is incubated for 1 hour for 37 DEG C after of short duration centrifugation.
4) reaction: 85 DEG C of inactivation treatment 5min is terminated, -20 DEG C of preservation reverse transcription products are stand-by after diluting 8 times with aqua sterilisa.
3, quantitative PCR detection
1) Mix is melted at 4 DEG C, gently turning upside down mixing and carries out of short duration centrifugation.
2) reaction solution in following table is prepared on ice
3) reaction tube is subjected to of short duration centrifugation, it is ensured that all reaction solutions are in reacting hole bottom.
4) it is reacted using following procedure:
4, primer sequence
5, it statisticallys analyze
Using 5.0 software of Primer Premier and SPSS16.0 statistical analysis.The data normal state of blood plasma miRNA point When cloth, two groups of data are compared using t check analysis；When data partial velocities or heterogeneity of variance, two comparison among groups of plasma sample and Clinicopathologic features correlation analysis is examined using Mann-Whitney U, and 3 comparison among groups are examined using Kruskal-Wallis.It adopts Multiplicity is carried out with logistic binary regression and establishes logit equation.With Receiver Operating Characteristics (receiver Operator characteristic, ROC) tracing analysis blood plasma miRNA is to the diagnostic of colorectal cancer and adenoma.With P < 0.05 has statistical significance for difference.
Experimental result
1, clinical case profile
It collects and arranges 218 (78 colorectal cancers, 70 Colon and rectum adenomas and 70 normal healthy controls) samples sources persons Clinical pathologic characteristic data (table 1).
The clinical case profile of 1.218 samples sources persons of table
2, the relationship between the expression and clinical pathologic characteristic of blood plasma miRNA
Using the expression and the phase of clinicopathologic features of miRNA in 78 colorectal cancer patients blood plasma of t check analysis Guan Xing, colorectal cancer patients blood plasma miR-1183 expression and the pathological staging (P=0.015) and N of patient divide as the result is shown Phase (P=0.018) significant correlation；Blood plasma miR-301a-3p expression is by stages (P=0.000) significant related to the M of patient； Blood plasma miR-193b-3p expression and the change of serum C EA expression (P=0.028) of patient are significant related；Blood plasma miR-145- 5p expression and the M of patient (P=0.001) significant related (table 2) by stages.Other correlation analysis have no significant difference.Table The miRNA of bright difference may take part in the pernicious process of colorectal cancer, have as the latent of colorectal carcinoma prognosis biomarker Energy.
Using the expression and gender, age and serum of miRNA in 70 Colon and rectum adenoma patients' blood plasma of t check analysis The correlation of CEA expression, the as the result is shown gender (P=of Colon and rectum adenoma patients blood plasma miR-301a-3p expression and patient 0.038) significant related to the age (P=0.004)；Blood plasma miR-224-5p (P=0.045) and miR-145-5p (P=0.043) Expression is significant related (table 3) to patient age.Other correlation analysis have no significant difference.Show blood plasma miR-301a- 3p, blood plasma miR-224-5p and miR-145-5p expression may be influenced by the gender of patient and age distribution.Equally use t The expression and the correlation of gender and age of miRNA, is surveyed as the result is shown in 70 healthy volunteer's blood plasma of check analysis The gender and age of blood plasma miRNA expression and patient are not significantly related to (table 4).
The correlation of table 2.miRNA expression and clinicopathologic features in plasma of colorectal cancer
Table 2- is continuous
The correlation of table 3.miRNA expression and clinicopathologic features in Colon and rectum adenoma blood plasma
Table 3- is continuous
The correlation of table 4.miRNA expression and clinicopathologic features in normal healthy controls person's blood plasma
Table 4- is continuous
3, expression detection of 7 targets in 60 plasma samples of wheel
Real-time fluorescence quantitative PCR detects 60 (colorectal cancer, Colon and rectum adenoma and each 20 of normal healthy controls) plasma samples Middle miR-301a-3p, miR-145-5p, miR-193b-3p, miR-125b-5p, miR-224-5p, miR-375-3p, 135b- The expression of 5p.MiR-301a-3p, miR-145-5p, miR-193b-3p, miR-125b-5p, miR-224- as the result is shown 5p has significant statistical difference between each group, and miR-375-3p, 135b-5p difference are unobvious.So five of significant difference Target can enter two wheel large sample verifyings.(partial results are shown in Fig. 1).
4, expression verifying of 6 targets in two 158 plasma samples of wheel
MiR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR- are screened from a wheel result Five targets of 193b-3p enter two wheel verifyings and form 6 targets altogether in addition plus the miR-1183 for having verified that significant meaning Mark enters two wheel large sample verifyings.Real-time fluorescence quantitative PCR detects 158 (colorectal cancers 58, Colon and rectum adenoma 50 and strong Health compares 50) expression of 6 targets in plasma sample.The results show that miR-224-5p, miR-301a-3p, miR- Tetra- target significant differences of 145-5p, miR-1183.(partial results are shown in Fig. 2).
5, ROC curve analyzes single miRNA to the diagnostic value of colorectal cancer and adenoma
Two-wheeled data are merged, miR-224-5p, miR-125b-5p, miR-301a-3p, miR-145-5p, miR- are analyzed 193b-3p and miR-1183 is individually to the diagnostic value of colorectal cancer and adenoma.ROC curve analysis shows that, blood plasma miR-224- 5p, miR-301a-3p and miR-145-5p level can identify colorectal cancer patients and healthy person (Fig. 3 A).Blood plasma miR- 1183, miR-224-5p and miR-301a-3p level can identify Colon and rectum adenoma patients and healthy person (Fig. 3 B).Blood plasma miR- 1183, miR-224-5p and miR-301a-3p level can identify colorectal cancer and adenoma person (Fig. 3 C).Blood plasma miR-1183 water It is flat to identify Colon and rectum early carcinoma and late cancer (Fig. 3 D).By the area under the curve AUC of the above ROC curve, 95% confidence Section CI, sensibility sensitivity, specificity specificity and p value statistics are in table 1.
Fig. 3 A ROC curve analyzes blood plasma miRNA to the diagnostic potential of colorectal cancer
Fig. 3 B ROC curve analyzes blood plasma miRNA to the diagnostic potential of Colon and rectum adenoma
Fig. 3 C ROC curve analyzes blood plasma miRNA to the identification potential of colorectal cancer and adenoma
Fig. 3 D ROC curve analyzes blood plasma miRNA to the identification potential of Colon and rectum early carcinoma and late cancer
Diagnostic value of the single miRNA of table 5 to colorectal cancer and adenoma
Note: cutoff value takes youden index maximum value.
6, the multiplicity of miRNA, built-up pattern are established and diagnostic potential detects
MiRNA target is put into all significant targets after the differential expression detection in all types of plasma samples Logistic binary regression, screening can be with the target of independent effect Colon and rectum adenoma and cancer.And logit equation is established, it carries out ROC curve is analyzed after combination.Logit equation and combined diagnostic are summarised in lower Fig. 5.As a result it indicates, first equation generation Table miR-224-5p, miR-301a-3p, miR-193b-3p can be used as the independentpredictor of CRC generation, by these three The area under the curve 0.856 that the combination that miRNA is constituted diagnoses colorectal cancer, 95%CI is 0.781~0.930, sensitive Property and specificity be 0.655 and 0.92 (A in Fig. 4) respectively；Second equation represent miR-145-5p, miR-224-5p, MiR-301a-3p, miR-193b-3p and miR-1183 can identify adenoma and cancer, and the combination being made of five miRNA is to gland The area under the curve 0.930 that tumor and cancer are identified, 95%CI are 0.881~0.979, and sensibility and specificity is respectively 0.72 and 0.68 (B in Fig. 4).These data show the two built-up patterns to colorectal carcinoma diagnosis valence with higher Value, and detect efficiency and be higher than current early sieve and auxiliary diagnosis means.It is analyzed from grouped comparison, miR-224-5p, miR- 301a-3p, miR-193b-3p may have apparent Guide in cancer group, may take part in human colorectal by normal-gland The occurrence and development of tumor-carcinoma stage.
Fig. 5 .logit equation and combined diagnostic
Detection verifying
By 100 detection samples (including 30 colorectal cancer samples, the healthy samples of 30 Colon and rectum adenoma samples and 40 This), according to the above method carry out miRNA marker expression detection, according to the present invention shown in miRNA marker table Up to the classification of horizontal judgement sample, double-blind comparative study is carried out.
The result shows that the combination including three miRNA of the invention can effectively identify colorectal cancer sample, sensitivity Up to 70% (21/30), up to 92.9% (65/70), repeatability has good stability specificity.Including five miRNA's of the invention Combination can effectively distinguish adenomas sample and colorectal cancer sample, and sensitivity is reached up to 76.7% (23/30), specificity 73.3% (22/30), repeatability have good stability.
The present invention relates to the combinations that a variety of blood plasma miRNAs are constituted to early diagnose and Index for diagnosis marker as colorectal cancer And related kit.It is detected compared to traditional fecal occult blood, sensibility improves, especially bright to the sensibility of progressive stage adenoma It is aobvious to improve, and sample without stringent diet control, it is substantially reduced to abandon inspection rate.Compared with colonoscopy, without warp before screening Stringent and pain INTESTINAL CLEANSING is crossed, and technical requirements are substantially reduced, wound risk is very small, and Sigmoidoscope is universal as head sieve Property it is low, will be big if after the kit that can be related to through the invention carries out the primary dcreening operation of hypersensitivity, then making a definite diagnosis under Sigmoidoscope is carried out The big recall rate for improving Large bowel disease.Compared with the multiple target point excrement for being newly included in screening guide detects, the present invention in sensibility and In the comparable situation of specificity, also have it is cheap, it is easy to operate, the advantages such as be easy to carry out.It is sieved compared to questionnaire risk assessment Cha Fa, blood plasma miRNA molecule of the present invention can assess the raising of venture influence degree, overcome the sieve of advanced colorectal rectal neoplasm Look into the not high disadvantage of sensibility and specificity.In conclusion accomplishing the morning discovery to colorectal cancer people at highest risk, intervene in time, Clinically still lack more accurately early warning means or Biological indicators.And kit of the present invention is that a kind of sensibility is special Property it is good, cheap, it is easy to operate, be easy to carry out detection means, it is also right except early screening/diagnosis to CRC is valuable The Index for diagnosis of CRC has preferable meaning.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>attached third hospital, Guangzhou medical university
Da'an Gene Company, Zhongshan University
<120>for colorectal cancer early diagnosis or the plasma markers object combination of prognosis prediction
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tagcagcacg taaatattgg cg 22
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 24
ggtgatggtg agagtgggca 20
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial)
<400> 25
gctgtcaacg atacgctacg taac 24

Claims (10)

  1. The purposes of 1.miRNA or its detection reagent, which is characterized in that be used to prepare colorectal cancer early diagnosis or prognosis prediction Kit, the miRNA are selected from the group: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p or its group It closes.
  2. 2. purposes as described in claim 1, which is characterized in that the miRNA include miR-224-5p, miR-301a-3p and miR-193b-3p；Preferably, the miRNA includes miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b- 3p；Preferably, the miRNA further includes miR-1183.
  3. 3. a kind of miRNA chip, which is characterized in that the miRNA chip includes:
    Solid phase carrier；And
    The oligonucleotide probe being orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to mesh Mark miRNA, the target miRNA is selected from the group: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, Or combinations thereof.
  4. 4. a kind of kit, which is characterized in that the kit contains miRNA or its detection reagent, and the miRNA is selected from down Group: miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, or combinations thereof；Also, the kit also wraps Specification is included, the kit is indicated in the specification for colorectal cancer early diagnosis or prognosis prediction.
  5. 5. kit as claimed in claim 4, which is characterized in that the miRNA includes miR-224-5p, miR-301a-3p And miR-193b-3p.
  6. 6. kit as claimed in claim 4, which is characterized in that the miRNA include miR-224-5p, miR-301a-3p, MiR-145-5p, miR-193b-3p and miR-1183.
  7. 7. kit as claimed in claim 4, which is characterized in that the specification indicates the following contents:
    When the level of miR-224-5p, miR-301a-3p and/or miR-193b-3p in the sample measured and healthy sample Level difference is significant, then prompting the detection sample is colorectal cancer patients sample.
  8. 8. a kind of isolated miRNA, which is characterized in that the miRNA is selected from:
    (i) miR-224-5p, miR-301a-3p, miR-145-5p, miR-193b-3p, or combinations thereof, or
    (ii) miRNA complementary with miR-224-5p, miR-301a-3p, miR-145-5p or miR-193b-3p or its group It closes.
  9. 9. a kind of separation or artificial constructed precursor miRNA, which is characterized in that the precursor miRNA can be in people's cell It shears and is expressed as miRNA according to any one of claims 8.
  10. 10. a kind of isolated polynucleotides, which is characterized in that the polynucleotides can be transcribed into precursor miRNA by people's cell, The precursor miRNA can be sheared in people's cell and be expressed as miRNA according to any one of claims 8.
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