CN103898219A - Serum miRNA marker for detecting primary biliary cirrhosis, marker composition and application thereof - Google Patents
Serum miRNA marker for detecting primary biliary cirrhosis, marker composition and application thereof Download PDFInfo
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Abstract
The invention relates to a serum miRNA marker for distinguishing primary biliary cirrhosis and normal human, a marker composition and application thereof, and belongs to the technical field of biology. The serum miRNA marker for detecting primary biliary cirrhosis is one of the following has-miRNA: has-miR-122, has-miR-34a and has-miR-141. A miRNA probe for detecting primary biliary cirrhosis includes one of the following sequences: SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3. With the adoption of the serum miRNA marker, the primary biliary cirrhosis can be found early, and non-invasive detection can be carried out quickly.
Description
Technical field
The present invention relates to the invention belongs to biological technical field, relate to a kind of for distinguishing primary biliary cirrhosis and normal people's serum miRNA mark and mark combination and application.
Background technology
Primary biliary cirrhosis (primary biliary cirrhosis, PBC) be a kind of chronic inflammation, progressive intrahepatic cholestasis systemic autoimmune venereal disease, there is anti-mitochondrial antibody (antimitochondrial antibody with serum, AMA), liver portal vein peripheral lymphocyte infiltrates, little bile duct specificity is destroyed is feature, finally develop into liver cirrhosis and liver failure [1,2].PBC diagnosis at present mainly depends on peripheral blood AMA antibody and hepatic pathology.Although AMA is for PBC diagnostic sensitivity Da Gaoda 90%, this antibody also can be present in other autoimmune diseases, and specificity is not high, and in PBC patient, usually has AMA negative patient yet; Secondly, this antibody also with disease activity degree, prognosis non-correlation; Moreover the patient of but the AMA positive normal for early stage liver biochemical indexes or AMA feminine gender but patient that bile duct enzyme increases must just can clarify a diagnosis by liver puncture, because often making to diagnose, more traumatic, risk and cost be very limited.Thereby at present only in serum specific autoantibody oneself through meeting clinical demand, actively find the new disease marker relevant to disease diagnosis and prognosis, can contribute to clinical diagnosis and treatment level raising [3].
MiRNA (microRNA) is a class endogenous non-coding strand microRNA, and length is 18～24 Nucleotide.MiRNA is prevalent in the mankind's body fluid, and stable in properties can detection by quantitative, and has significant disease specific.In recent years research shows, the body fluid miRNA such as blood, saliva, urine, milk and cerebrospinal fluid detect the diagnosis and prognosis judgement of the disease such as judgement and oral carcinoma, bladder cancer and Alzheimer thatch disease that can be applicable to the physiological statuss such as gestation.Body fluid specificity miRNA detect clinical meaning and application prospect cause and show great attention to, miRNA likely replaces as the microRNA of a class non-coding modulability biomarker that traditional differential protein is representative.Body fluid miRNA rich content and stable in properties, possesses the potential quality that becomes excellent biomarker, and have the characteristic that the traditional biological mark take protein as representative does not possess, prepare and be easy to accurate quantification without antibody, may overcome the bottleneck of preparing of antigen-antibody class biomarker.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of and can be used in the early discovery of primary biliary cirrhosis and carry out serum miRNA mark and mark combination and the application of non-invasive detection fast.
Serum miRNA mark for detection of primary biliary cirrhosis of the present invention is the one in following three kinds of has-miRNA: has-miR-122, has-miR-34a, has-miR-141.
A kind of combination of the serum miRNA mark for detection of primary biliary cirrhosis of the present invention comprises above-mentioned three kinds of has-miRNA.
MiRNA probe for detection of primary biliary cirrhosis of the present invention, has the one in following sequence: SEQ ID N0.1, SEQ ID N0.2, SEQ ID N0.3.
MiRNA probe combinations for detection of primary biliary cirrhosis of the present invention, comprises following sequence: SEQ ID N0.1, SEQ ID N0.2, SEQ ID N0.3.
The application in the reagent of preparation detection primary biliary cirrhosis of the above-mentioned miRNA probe for detection of primary biliary cirrhosis or probe combinations.
Test kit for detection of primary biliary cirrhosis of the present invention, comprises above-mentioned miRNA probe or probe combinations for detection of primary biliary cirrhosis, also comprises Taq enzyme, magnesium chloride and PCR damping fluid.
The present invention can realize the early discovery of primary biliary cirrhosis and non-invasive detection rapidly.
Accompanying drawing explanation
Fig. 1 is that two groups of mappable reads data of the embodiment of the present invention are carried out length analysis chart;
Fig. 2 is that 22 of the embodiment of the present invention are at the miRNA of two groups of differential expressions cluster analysis figure;
Fig. 3 is that normal healthy controls group and the primary biliary cirrhosis group of the embodiment of the present invention verified respectively miR-122, miR-141 and miR-34a expression level figure;
Fig. 4 is the ROC graphic representation of embodiment of the present invention mark miRNA-122;
Fig. 5 is the ROC graphic representation of embodiment of the present invention mark miRNA-34a;
Fig. 6 is the ROC graphic representation of embodiment of the present invention mark miRNA-141;
Fig. 7 is the ROC graphic representation of embodiment of the present invention mark combination.
Embodiment
Research object
Control group is the healthy volunteer in the court's health check-up; Experimental group is PBC patient; basic condition is in table 1; grind according to the U.S.'s hepatopathy primary biliary cirrhosis guide that the association that makes internal disorder or usurp (American Association for the Study of Liver Diseases, AASLD) 2009 formulates: in following 3, meet 2 or above person and can be diagnosed as the reflection bile such as PBC:ALP and swim long-pending biochemical indicator rising; Anti-mitochondrial antibody (AMA) positive; On pathology, there is the infringement of the destructive cholangitis of apyetous and interlobular bile duct.Exclusion standard: local obstruction of bile duct or EHBO in liver; Viral hepatitis, alcoholic liver disease, drug induced hepatic injury, the long-pending disease of Gestation period hepatic bile trip; Autoimmune hepatitis, primary sclerosing cholangitis; Merge other autoimmune disorders, or other autoimmune disorder liver injuries; Systemic disease is with liver injury.
Table 1 screens difference miRNA serum specimen basic condition
Experimental technique
Sample collecting: patient outpatient service first or be in hospital time gather fresh blood 5ml (Anticoagulation without heparin) in Medical blood pigging, put upside down and mix gently up and down, immediately whole blood is placed in to 4OC ice chest and preserves, and in 2h 4000g, centrifugal 10min.Upper serum is transferred in 1.5ml centrifuge tube (RNase free) to 13000g, centrifugal 2min.Finally supernatant liquor is transferred to (RNase free) in 2ml spiral cover conical centrifuge tube, each pipe sucks 250ul serum and carries out packing, discards hemocyte precipitation.Be placed in-800C preserves for a long time.
Total RNA extracts and quality inspection: the extracting of serum RNA adopts LCS TRK1001 test kit (LC Sciences) process specifications to carry out.The miRNA that randomly draws two stably express in serum extracts quality as the total RNA of standard detection, is respectively hsa-miR-16, hsa-miR-192.Then respectively get 2 μ l, the reverse transcriptase primer of answering take above-mentioned primer pair respectively reverse transcription (reaction system as 10 μ l); Take the cDNA in 1 μ l/ hole as template, carry out realtimePCR, reaction system is 20 μ l, multiple Kong Weisan, does primer NTC(template simultaneously and replaces with water).Then detect hsa-miR-16 and hsa-miR-192PCR expansion curve, solubility curve and CT value.
Library construction: use Illumina Truseq Small RNA Preparation kit test kit reference reagent box specification sheets Illumina ' s TruSeq Small RNA Sample Preparation Guide to build little RNA library.Total RNA link 5, joint and 3, through the cDNA library of RT-PCR amplification formation microRNA, separates through 6%TBE sex change gel electrophoresis after joint, length range is cut to glue at the microRNA of 147bp and reclaim.
S-generation order-checking: after generating DNA bunch after cDNA is purified on Illumina ' s Cluster Station, upper machine (Illumina GAIIx) checks order.Pass through Illumina ' s Sequencing Control Studio software version2.8 (SCS v2.8) software real-time analysis order-checking picture and use Illumina's Real-Time Analysis version1.8.70 (RTA v1.8.70) to extract base-calling.The original series extracting utilizes ACGT101-miR v4.2 (LC Sciences) software analysis, generate RawData database, remove the non-pure sequence producing due to the optics Digital Signal Processing of sample preparation, order-checking chemistry and processing and order-checking instrument simultaneously.Remaining sequence (length 15 and 32bases) is divided into groups according to families, generates mappableReads.The miRbase database of Mappable sequence and latest edition and order-checking species gene group are carried out sequence alignment, identify the miRNA that these species are known; Find new 5p or 3p miRNA sequence simultaneously, identify and have been reported in other nearly source species, brand-new miRNA sequence in these species.Wherein Mappable sequence can with Rfam(ie rRNA, tRNA, snRNA, snoRNA and others), being all removed on Repbase and mRNA sequence alignment.In addition,, in order to guarantee to screen high-quality gene result, we are less than 10 gene knockout reads number and fall.The miRNAs of differential expression between ultimate analysis experimental group and control group.
By above-mentioned sequencing analysis, normal healthy controls group obtains 4,691,800 original series after elementary analysis, after removing redundancy, remains 881,742, accounts for 18.79% of total sequence; PBC group obtains 944,362 original series, and 462,263 can aligned sequences, accounts for 48.95%.Two groups of mappable reads data are carried out to length analysis and see Fig. 1.The length of visible miRNAs concentrates on 19nt.Account for 38.85%.
Differential expression and new discovery miRNAs:
PBC group and CTL group are carried out to both differential expressions of comparison after data normalization.P<0.05 indicates significant difference.Find altogether the miRNAs of 143 variant significancees, wherein up-regulated has 105, down-regulated expression have 38.Known has 137, and newfound have 6.Be respectively has-miR-3665-p5, hsa-mir-3960-p3, hsa-mir-4508-p5, hsa-mir-7641-2-p5, hsa-mir-7641-2-p3, PC-3p-755_3750.To above-mentioned diversity sequence comparing difference multiple, find that >2 miRNAs doubly has 22, wherein that difference multiple maximum is miR-122, and it is higher 8.76 times than normal group in PBC patient, secondly miR-34a is higher 7.26 times than normal group, and miR-141 is higher 4.98 times than normal group; MiR-26b is the miRNA of only downward, than 2.49 times (in table 2) of Normal group decline.
Table 2 is compared with normal healthy controls group, the sequence of differential expression multiple
Above-mentioned miRNA differential expression carries out cluster analysis, uses Cluster3.0 to analyze, as Fig. 2
.miRNAs qRT-PCR checking
The miRNAs of above-mentioned 12 differences is carried out to preliminary identification respectively in 32 PBC, 18 CTL samples.Found that the expression amount of miR-122 in PBC significantly raises, rising multiple is 21.24 times, and p<0.001, apparently higher than the difference multiple of sequencing result.MiR-34a and miR-141 difference and order-checking differ and are respectively 5.51 and 5.43 times, and p<0.001(is shown in Fig. 2).
MiR-122, the miR-141 going out with final election and miR-34a carry out clinical further checking
Reaction system as quantitative fluorescence PCR: 1 μ lcDNA0.3 μ lTaq enzyme, 0.33 μ lTaqMan probe (provided by ABI, be exclusively used in miRNA fluorescent quantitation), 1.2 μ l25mMgcl2, the various dNTP mixtures of 0.4 μ l2.5mM, 2 μ l10 × PCR damping fluids and 14.77 μ lDEPC water.MiR-122, the miR-141 TaqMan probe sequence corresponding with miR-34a is respectively SEQ ID N0.1, SEQ ID N0.2, SEQ ID N0.3.Its RT-PCR reaction conditions be 95 ℃ 15 minutes, then circulate, cycling condition is: 94 ℃ 15 seconds, 55 ℃ 30 seconds, 70 ℃ 34 seconds, 40 circulations.
Patient's situation of clinical verification: PBC organizes 124 examples, CTL organizes 38 examples, in table 3
Table 3 is verified the comparison of PBC group and normal control Serum markers expression level
Two groups of qRT-PCR Clinical results of CTL and PBC are in table 4, and non-ginseng Mann-Whitney checks
Table 4qRT-PCR Clinical results
| miRNA | CTL（n=38） | PBC（n=124） | P |
| hsa-miR-122 | 25.34±12.45 | 642.23±269.23 | 1.34×10-5 |
| hsa-miR-34a | 142.27±57.89 | 342.34±53.34 | 3.34×10-3 |
| hsa-miR-141 | 349.23±190.23 | 1361.12±231.67 | 5.21×10-4 |
Utilize Medcalc to draw ROC curve, area under calculated curve (AUC), has-miR-122, has-miR-34a, the AUC of has-miR-141 is respectively 0.895,0.911,0.722, and the AUC of three's combination is 0.913.See Fig. 4-7, can find out, this group has-miR-122, has-miR-34a, has-miR-141 all can effectively come PBC and normal human serum difference, and the susceptibility of this miRNA combination reaches 89.5%, and specificity reaches 84.6%.
For detection of the test kit of PBC patients serum miRNA
Manufacture craft and operating process for detection of the serum miRNA test kit of PBC are based on Illumina high-flux sequence and quantitative PCR technique gained, by following TaqMan probe combinations: SEQ, N0.1, SEQ, N0.2, in SEQ, N0.3 a kind of or three kinds collect respectively the primary biliary cirrhosis detection kit of preparing specific T aqMan probe combinations in PCR test kit.
The concrete composition (detecting every kind of miRNA) of described test kit is as follows:
The concrete operations flow process of prepared test kit is as follows:
Collect experimenter's serum sample, after extraction RNA, transcribe preparation cDNA sample;
According to above-mentioned formula application of sample, various miRNA measure respectively, in the time measuring, use the specific TaqMan probe of miRNA separately;
Carry out PCR reaction, 95 ℃ 15 minutes, then circulate, cycling condition is: 94 ℃ 15 seconds, 55 ℃ 30 seconds, 70 ℃ 34 seconds, 40 circulations.
The value of this test kit is that specific TaqMan probe combinations detects serum miRNA expression amount, can distinguish normal people and PBC serum, contributes to the early discovery of PBC.
Sequence table
The 3rd the People's Hospital of <110> Zhengjiang City
<120> is for detection of serum miRNA mark and mark combination and the application of primary biliary cirrhosis
<130> 2014
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> RNA
<213> artificial sequence
<400> 1
uggaguguga caaugguguu ug 22
<210> 2
<211> 22
<212> RNA
<213> artificial sequence
<400> 2
<210> 3
<211> 22
<212> RNA
<213> artificial sequence
<400> 3
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Claims (6)
1. for detection of the serum miRNA mark of primary biliary cirrhosis, the one in following three kinds of has-miRNA: has-miR-122, has-miR-34a, has-miR-141.
2. the combination of the serum miRNA mark for detection of primary biliary cirrhosis, comprises following three kinds of has-miRNA:has-miR-122, has-miR-34a, has-miR-141.
3. for detection of a miRNA probe for primary biliary cirrhosis, there is the one in following sequence: SEQ ID N0.1, SEQ ID N0.2, SEQ ID N0.3.
4. for detection of a miRNA probe combinations for primary biliary cirrhosis, comprise following sequence: SEQ ID N0.1, SEQ ID N0.2, SEQ ID N0.3.
5. the miRNA probe of the detection primary biliary cirrhosis of claim 3 or 4 or the probe combinations application in the reagent of preparation detection primary biliary cirrhosis.
6. for detection of a test kit for primary biliary cirrhosis, comprise the miRNA probe for detection of primary biliary cirrhosis of claim 3 or the miRNA probe combinations for detection of primary biliary cirrhosis of claim 4; Also comprise Taq enzyme, magnesium chloride and PCR damping fluid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828859A (en) * | 2018-02-02 | 2018-03-23 | 济南大学 | A kind of detection miRNA 122 biological sensor and its preparation method and application |
| WO2019086041A1 (en) * | 2017-11-06 | 2019-05-09 | 高雄医学大学 | Method for assessing risk of liver diseases or cancer by means of micro-ribonucleic acid |
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| WO2008054828A2 (en) * | 2006-11-01 | 2008-05-08 | The Ohio State University Research Foundation | Microrna expression signature for predicting survival and metastases in hepatocellular carcinoma |
| WO2009066291A2 (en) * | 2007-11-21 | 2009-05-28 | Rosetta Genomics Ltd. | Micrornas expression signature for determination of tumors origin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008054828A2 (en) * | 2006-11-01 | 2008-05-08 | The Ohio State University Research Foundation | Microrna expression signature for predicting survival and metastases in hepatocellular carcinoma |
| WO2009066291A2 (en) * | 2007-11-21 | 2009-05-28 | Rosetta Genomics Ltd. | Micrornas expression signature for determination of tumors origin |
Non-Patent Citations (2)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019086041A1 (en) * | 2017-11-06 | 2019-05-09 | 高雄医学大学 | Method for assessing risk of liver diseases or cancer by means of micro-ribonucleic acid |
| CN107828859A (en) * | 2018-02-02 | 2018-03-23 | 济南大学 | A kind of detection miRNA 122 biological sensor and its preparation method and application |
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