CN104232637A - microRNA molecular marker of liver cirrhosis and use thereof - Google Patents
microRNA molecular marker of liver cirrhosis and use thereof Download PDFInfo
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Abstract
The invention provides microRNA molecular marker combination of liver cirrhosis. The combination comprises more than one microRNA molecules of nucleic acid with sequence numbers of 1-24 and 54. The invention further provides use of preparing a liver cirrhosis diagnostic reagent. Compared with the content of the microRNA molecular marker of a hepatitis B patient in plasma/serum, the content of the microRNA molecular marker of a liver cirrhosis patient in plasma/serum is remarkably different, so that the liver cirrhosis patient and the hepatitis B patient can be effectively differentiated. When the expression level of the microRNA molecular marker reaches the level of liver cirrhosis, liver cirrhosis can be diagnosed. The invention further provides a diagnostic reagent kit for liver cirrhosis. The microRNA molecular marker of liver cirrhosis provided by the invention is used for guiding diagnosis of liver cirrhosis and has the characteristics of being simple in operation, safe, free from damage, high in specificity, high in sensitivity and easy to screen massively.
Description
Technical field
The present invention relates to the diagnostic field of liver cirrhosis, relate in particular to one group of liver cirrhosis microRNA molecule mark combination, it can be applicable to the diagnosis to liver cirrhosis, pass through liver cirrhosis diagnosis, find in time and treat liver cirrhosis, Morbidity control for liver cirrhosis is very helpful, can effectively lapse to, and avoids liver cirrhosis to the development of liver cancer.
Background technology
MicroRNA (miRNA) is study hotspot in recent years, it is a kind of strand microRNA be extensively present in eukaryote, do not have an encoding function, but the flank region that it can be incorporated into gene order checks or suppresses the translation of said target mrna, there is the conservative property of height, timing and tissue specificity, played vital role in the field such as clinical diagnosis, Treatment and Prognosis evaluation of the various diseases such as tumour, hemopathy, virus infection at present.
China is hepatitis B big country, and HBVer accounts for the 8-10% of total population, and wherein about have 25% to develop into chronic hepatitis, liver cirrhosis according to estimates, about 10% develops into hepatocellular carcinoma (HCC).Have data to show, a chronic hepatitis B patients average medical fee of a year is exactly approximately more than 1100 yuan, if but patient's disease progression develops into hepatocellular carcinoma, the average cost of a year is almost Renminbi more than 40,000.Therefore our country is annual because of chronic hepatitis B or because of the hepatitis b virus infected relative disease occurred later, as liver cirrhosis, liver cancer, annual direct economic loss is up to 1,123 hundred million Renminbi, the infection of hepatitis B virus brings serious Health hazard to our people as can be seen here, and directly causes the serious financial loss of our country.
The biopsy of current liver cirrhosis clinical diagnosis means histopathology, FibroScan, CDFI, CT, gastroscope, serological index etc.Histopathology biopsy judges hepatic fibrosis, degree of cirrhosis " gold standard " at present, but liver wears location single-point, limitation of drawing materials, and is not enough to reflect liver integral status, clinical about have 25% patient due to pathological diagnosis deviation by mistaken diagnosis, fail to pinpoint a disease in diagnosis.FibroScan (Transient elastography) is based on ultrasonic examination, judges hepatic fibrosis and liver cirrhosis classification by measuring liver hardness value.Fibroscan detect have that method is simple, painless, quick, patient compliance is good, without the need to being in hospital, reproducible, be beneficial to advantages such as carrying out dynamic follow-up observation.But in inflammation active period, ALT and TBIL fluctuation will affect the stability of detected value, the factors such as around ascites, liver great vessels, fat lesion and intercostal space be narrow all can limit the application of FibroScan.CDFI is as the indagation means of diagnosis liver cirrhosis, in wide clinical application, but because it can not be corresponding with respective organization pathological change, the subjective judgement error of manual operation in addition, makes ultrasonicly still have larger limitation as liver cirrhosis classification, staging diagnosis standard.CT all can provide more accurate quantization parameter to liver organization structure, density, form and hemodynamic responses, for liver cirrhosis classification, staging diagnosis provide Precise Diagnosis index, but to the difference of early stage liver cirrhosis and hepatic fibrosis, still lack the corresponding standard between diagnosis index with pathological staging and perfect scanning sequence.Gastroscope has higher diagnostic value for the varix being positioned at Gastric and esophageal mucosa layer, judge whether esophagus fundus ventricularis varication exists and classification by gastroscopy, Cirrhotic Portal Hypertension degree can be judged, but check comparatively painful, some patients cannot tolerate, and part early stage liver cirrhosis patient can not have varix and fail to pinpoint a disease in diagnosis, the index of the judgement state of an illness that neither be ideal.The more direct serodiagnostic markers of hepatic fibrosis of current clinical application comprises: serum type III procollagen amino terminal peptide (PIIINP), Serum Laminin (LN), Serum hyaluronic acid (HA), serum I V collagen (IV2C) etc., they and hepatic fibrosis and degree of cirrhosis dependency bad, liver cirrhosis cannot be diagnosed accordingly.In sum, the clinical application of current monotechnics or index all can not diagnose liver cirrhosis progress extent accurately, in time, make that liver is still depended on to the staging diagnosis of liver cirrhosis and wear biopsy pathology standard, clinical in the urgent need to one/mono-prescription just, timely, noninvasive hepatic fibrosis, liver cirrhosis grading diagnosis index.
Research in recent years shows, hepatitis b virus infected, chronic hepatitis B, liver cirrhosis are closely related with microRNA, microRNA by acting on virus itself or acting on immunity system thus affect disease process, therefore also can may have corresponding effect to the diagnosis of liver cirrhosis.More to hepatitis b virus infected relevant microRNA kind and act on and differing, there are some researches show that after hepatitis b virus infected, in patient body, microRNA express spectra can change.Although carried out some researchs in this field, but the microRNA of liver cirrhosis patient expresses change and does not also further investigate at present, clinical with in research, the microRNA mark that effectively can judge liver cirrhosis need be found, especially for liver cirrhosis and hepatitis B being distinguished the microRNA mark come.
Summary of the invention
One aspect of the present invention provides the combination of a kind of liver cirrhosis microRNA molecule mark, it comprises more than one and is selected from following microRNA nucleic acid molecule: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:54.
Preferably, described more than one are 2-18 kind, more preferably 3-10 kind, further preferred 3-8 kind, most preferably 4 kinds.
Preferably, described liver cirrhosis microRNA molecule mark combination is containing SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:15 and SEQ ID NO:54.
The invention provides the molecular marker of one group of new microRNA relevant to liver cirrhosis as liver cirrhosis, be made up of one group of microRNA, its sequence be selected from SEQ ID NO:1 ~ SEQ ID NO:24 as follows and SEQ ID NO:54 respectively more than one.
| MicroRNA title | MicroRNA sequence |
| miR-106b-5p | uaaagugcugacagugcagau |
| miR-122-3p | aacgccauuaucacacuaaaua |
| miR-122-5p | uggagugugacaaugguguuug |
| miR-146a-5p | ugagaacugaauuccauggguu |
| miR-15a-5p | uagcagcacauaaugguuugug |
| miR-16-1-3p | ccaguauuaacugugcugcuga |
| miR-185-5p | uggagagaaaggcaguuccuga |
| miR-186-5p | caaagaauucuccuuuugggcu |
| miR-18a-5p | uaaggugcaucuagugcagauag |
| miR-192-5p | cugaccuaugaauugacagcc |
| miR-21-5p | uagcuuaucagacugauguuga |
| MiR 1 | ugucaguuuigucaaauacccca |
| miR-22-3p | aagcugccaguugaagaacugu |
| miR-26a-5p | uucaaguaauccaggauaggcu |
| miR-27a-3p | uucacaguggcuaaguuccgc |
| miR-29c-3p | uagcaccauuugaaaucgguua |
| miR-32-5p | uauugcacauuacuaaguugca |
| miR-378a-5p | cuccugacuccagguccugugu |
| miR-381-3P | uauacaagggcaagcucucugu |
| miR-433 | aucaugaugggcuccucggugu |
| miR-450b-5p | uuuuugcaauauguuccugaaua |
| miR-455-3p | gcaguccaugggcauauacac |
| miR-663 | cuugguucagggagggucccca |
| miR-92a-3p | uauugcacuugucccggccugu |
| miR-451a | aaaccguuaccauuacugaguu |
In a preferred embodiment, described at least one microRNA contrasts differential expression in plasma/serum with at least one at least one target plasma/serum.
Preferably, described target plasma/serum is from liver cirrhosis patient, and described contrast plasma/serum is from hepatitis B patient.
In the embodiment be more preferably, the expression of described at least one microRNA at least one target plasma/serum contrasts with at least one compared with the expression in plasma/serum and is raised, and/or the expression of microRNA wherein at least one target plasma/serum contrast in plasma/serum expression with at least one compared with lowered.
Contriver finds, in liver cirrhosis, the content of microRNA molecule mark in liver cirrhosis patient blood plasma/serum compares hepatitis B patient, there is significant difference, can effectively distinguish liver cirrhosis and hepatitis B, as can be seen here, the disease of the infection of liver cirrhosis microRNA molecule marker expression level and hepatitis B virus, hepatitis B, liver cirrhosis is closely related.
On the basis of above-mentioned discovery, the invention provides liver cirrhosis microRNA molecule mark and be combined in the purposes prepared in the diagnostic reagent of liver cirrhosis.Preferably, described liver cirrhosis microRNA molecule mark combination is containing SEQIDNO:3, SEQIDNO:4, SEQIDNO:15 and SEQ IDNO:54.
Therefore, a second aspect of the present invention provides a kind of liver cirrhosis microRNA molecule mark and is combined in the purposes prepared in liver cirrhosis diagnosis reagent.Preferably, liver cirrhosis and hepatitis B make a distinction by described diagnosis.
Particularly, described diagnostic reagent learns the content of liver cirrhosis microRNA molecule mark in blood plasma by detecting subject, and liver cirrhosis is diagnosed compared with hepatitis B patient mean level (ML), wherein, the content of liver cirrhosis microRNA molecule mark in liver cirrhosis patient blood plasma/serum compares hepatitis B patient, there is significant difference, preferably, this species diversity can be that the latter raises than the former otherness, also can be that otherness reduces.In a specific embodiment, the method for quantitative PCR is used to detect liver cirrhosis microRNA molecule mark content in subject's blood plasma.
A third aspect of the present invention provides a kind of diagnostic kit, and it comprises the specific forward primer of described liver cirrhosis microRNA molecule mark combination.
Preferably, described specific forward primer comprises the primer that more than one are selected from following sequence: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQIDNO:35, SEQIDNO:36, SEQIDNO:37, SEQIDNO:38, SEQID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ IDNO:45, SEQ IDNO:46, SEQ IDNO:47, SEQ IDNO:48, SEQ IDNO:55.
Described diagnostic kit also comprises further:
(1) plasma/serum total RNA extraction reagent,
(2) RNA adds polyA reagent,
(3) RT-PCR reagent,
(4) quantitative PCR reagent,
Wherein, quantitative PCR reagent comprises the specific forward primer of described liver cirrhosis microRNA molecule mark combination.
Preferably, described blood plasma total RNA extraction reagent comprises the external control sequence-1 (External Conirol-1) that sequence is SEQ ID NO:49, described RNA adds polyA reagent and comprises the external control sequence-2 (External Control-2) that sequence is SEQ ID NO:50, and described RT-PCR reagent comprises the reverse transcription primer (RT-Primer) that sequence is SEQ ID NO:51.
Preferably, described quantitative PCR reagent comprises general reverse primer UPM-short and UPM-long that sequence is respectively SEQ ID NO:52 and 53.
4th aspect of the present invention provides the purposes of described diagnostic kit for the diagnosis of liver cirrhosis, and preferably, described purposes is in order to distinguish the diagnosis of liver cirrhosis and hepatitis B.
Quantitative PCR reagent of the present invention comprises the specific forward primer of liver cirrhosis microRNA molecule mark, preferably, and its sequence (SEQ ID NO:25 ~ SEQ ID NO:48 and, SEQ ID NO:55) as follows.
| MicroRNA title | Primer sequence |
| miR-106b-5p | GTGCTGACAGTGCAGA |
| miR-122-3p | GAACGCCATTATCACACT |
| miR-122-5p | GTGGAGTGTGACAATGGT |
| miR-146a-5p | GAGAACTGAATTCCATGGGT |
| miR-15a-5p | GCAGCACATAATGGTTTGT |
| miR-16-1-3p | AGTATTAACTGTGCTGCTG |
| miR-185-5p | GAGAGAAAGGCAGTTCCTG |
| miR-186-5p | CAAAGAATTCTCCTTTTGGG |
| miR-18a-5p | AGGTGCATCTAGTGCAGA |
| miR-192-5p | TGACCTATGAATTGACAG |
| miR-21-5p | GCTTATCAGACTGATGTTG |
| miR-223 | TGTCAGTTTGTCAAATACC |
| miR-22-3p | AAGCTGCCAGTTGAAGAA |
| miR-26a-5p | CAAGTAATCCAGGATAGGC |
| miR-27a-3p | TCACAGTGGCTAAGTTCC |
| miR-29c-3p | GCACCATTTGAAATCGGT |
| miR-32-5p | GTATTGCACATTACTAAG |
| miR-378a-5p | TGACTCCAGGTCCTGTG |
| miR-381-3P | CAAGGGCAAGCTCTCTG |
| miR-433 | CATGATGGGCTCCTCGG |
| miR-450b-5p | GTTTTGCAATATGTTCCTG |
| miR-455-3p | GGCAGTCCATGGGCATA |
| miR-663 | AGGCGGGGCGCCGCGGGA |
| miR-92a-3p | TGCACTTGTCCCGGC |
| miR-451a | ACCGTTACCATTACTGAG |
In a specific embodiment, described blood plasma total RNA extraction reagent comprises the External Control-1 that sequence is 5 '-CAACCTCCTAGAAAGAGTA-3 ' (SEQ ID NO:49); Described RNA adds polyA reagent and comprises the External Control-2 that sequence is 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:50); Described RT-PCR reagent comprise sequence be the RT-Primer of 5 '-CAGTGGTATCAACG-CACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:51) (wherein, V is A or C or G, N is A or T or C or G); Described quantitative PCR reagent comprises general reverse primer UPM-shorr and UPM-long that sequence is respectively 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:52) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ IDNO:53).
Liver cirrhosis microRNA molecule mark of the present invention is used to diagnose liver cirrhosis to have simple to operate, safe hurtless measure, high specific, high sensitivity and be easy to the feature of a large amount of examination.
Accompanying drawing explanation
Fig. 1 describes and comprises the present invention for determining miR-106b-5p in the preferred microRNA of at least one liver cirrhosis plasma/serum, miR-122-3p, miR-122-5p, miR-146a-5p, miR-15a-5p, miR-16-1-3p, miR-185-5p, miR-186-5p, miR-18a-5p, miR-192-5p, miR-21-5p, miR-223, miR-22-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-32-5p, miR-378a-5p, miR-381-3P, miR-433, miR-450b-5p, miR-455-3p, miR-663, the Logic Regression Models of miR-92a-3p and miR-451a and miRNA combination.
Fig. 2 describe comprise the present invention for determine miR-122-5p, miR-146a-5p, miR-27a-3p and miR-451a and microRNA combination at least one liver cirrhosis plasma/serum Logic Regression Models.
Embodiment
As described herein, microRNA or miRNA has its its ordinary meaning (such as seeing CN10294310SA) in the art.That is, microRNA represents the RNA molecule from genetic site, and it is from the transcript processing that can form partial rna precursor microRNA structure.Ripe microRNA normal length is 20,21,22,23,24 or 25 Nucleotide, although the Nucleotide of other number also can exist, and such as 18,19,26,27 or 28 Nucleotide.
As described herein, " more than one " represent between 2-18 kind, preferred 2-15 kind, more preferably 3-10 kind, more preferably 4-8 kind, most preferably 4 kinds.
As described herein, diagnosis of hepatitis b was according to Chinese Medical Association's viral hepatitis guideline of prevention and treatment in 2000, specific as follows: the hepatitis course of disease exceedes half a year, or original hepatitis B or HBsAg carry history, this is again because again there is hepatitis symptom, sign and dysfunction of liver in same cause of disease, but do not have the patient that liver cirrhosis shows, diagnosable is chronic hepatitis B.
As described herein, liver cirrhosis diagnosis was according to Chinese Medical Association's viral hepatitis guideline of prevention and treatment in 2000, specific as follows: there is hepatitis B virus chronic infection medical history, hepatic fibrosis is filled the air in iconography prompting, regeneration knot circle is formed, other performances can have splenomegaly, hypersplenism, esophagus fundus ventricularis varication, and gold standard is that pathologic finding finds regenerated nodule.
The present invention sets forth technical scheme of the present invention further by specific embodiments and the drawings, but one of ordinary skill in the art will appreciate that: following embodiment and embodiment are intended to set forth the present invention, and should not be construed as and limit the present invention by any way.
Embodiment 1: plasma sample collection and preparation
During in March ,-2014 in June, 2012,150 plasma samples of patient meeting the definition of above liver cirrhosis and 150 plasma samples meeting the definition of above hepatitis B are gathered from Beijing YouAn Hospital, Capital Medical University in advance.
Gather peripheric venous blood 10ml, EDTA anti-freezing, plasma collection flow process is as follows: whole blood sample to be placed in whizzer 4 DEG C, the centrifugal 15min of 1,500-3,000g.With 200 μ L liquid-transfering guns, upper plasma is transferred in the sterile centrifugation tube of 1.5mL without RNA enzyme carefully.Mark is carried out to each sample.Plasma sample must be put in very low temperature (-80 DEG C) refrigerator in 4 hours and preserve.
The extraction of total serum IgE in embodiment 2. blood plasma
Use RNA to extract test kit (Beijing Quanto Biotechnology Co., Ltd.) and extract total serum IgE in blood plasma, in every 250 μ l blood plasma, add the extraction quality that External Control-1 (synthesis of Shanghai Sheng Gong biotechnology company limited) that 1 μ l (20nM) sequence is 5 '-CAACCTCCTAGAAAGAGTA-3 ' (SEQ ID NO:49) monitors RNA in blood plasma.The total serum IgE extracted uses Thermo NanoDrop2000c to measure concentration.
MicroRNA in embodiment 3. three-step approach detection by quantitative blood plasma
(1) polyA tail is added:
I. in without the PCR pipe (Axygen company, 200 μ l) of RNA enzyme, prepare the reaction solution adding polyA tail, system is 20 μ l.Tailing and reverse transcription quality that External Control-2 (synthesis of Shanghai Sheng Gong biotechnology company limited) that 1 μ l (20nM) sequence is 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:50) monitors microRNA is added in every 20 μ l systems.
(note: the enzyme that this experiment uses is the product of Beijing Quanto Biotechnology Co., Ltd..)
Ii. put into PCR instrument (Thermo) 37 DEG C hatch 1 hour by being equipped with the PCR pipe configuring reaction solution.(2) RT-PCR obtains cDNA strand:
I. the RT-Primer (synthesis of Shanghai Sheng Gong biotechnology company limited) that 0.5 μ l (0.5ng/ μ l) sequence is 5'-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:51) is added in the reaction solution obtained to (1), hatch 5min for 70 DEG C, be put into ice bath at least 2min on ice immediately.
Ii. inverse transcription reaction liquid is prepared:
(note: product used is the product of Beijing Quanto Biotechnology Co., Ltd..)
Iii. the solution mixing obtained by i and ii, hatches 70 DEG C of insulation 15min after 50min, puts cooled on ice, obtain cDNA for 50 DEG C.
-20 DEG C of preservations after the product packing that iii obtains by iv.
(3) qPCR detection by quantitative:
I. preparation reaction solution in (Axygen company) is managed at 2mi EP:
(note: UPM-long, UPM-short all in the synthesis of Invitrogen company, all the other reagent are all from Beijing Quanto Biotechnology Co., Ltd.
green PCR Master Mix.)
Wherein, the sequence of general reverse primer UPM-short and UPM-long is respectively 5'-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:52) and 5'-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:53).
Ii., after the reaction solution configured fully puts upside down mixing, be distributed in 96 hole point end PCR plate (Axygen company), every hole 18 μ l.
Iii. the volley of rifle fire (Eppendoff company is used, 1-10 μ l) add the liver cirrhosis microRNA molecule mark specific forward primer (synthesis of Invitrogen company) that sequence is SEQ ID NO:25 to 48 and SEQ ID NO:55 respectively, every hole 2 μ l (10 μMs).
Iv. seal with special pad pasting (ABI company) after adding 10 μ l paraffin oil fluid-tights.
V. put into ABI7900PCR instrument, program setting is:
Vi. draw melting curve, check the specificity of primer, program setting is: 95 DEG C of 15s, 60 DEG C of 15s, 95 DEG C of 15s.
Embodiment 4: interpretation of result:
ArrayTools4.1.0 is adopted to carry out data analysis.
About RT-PCR method and 2
-Δ Δ Ct method is shown in (the Analysis ofrelative gene expression data using real4ime quantitative PCR and the2 such as Kenneth J Livak
-△ △ Ct method.Methods25,402-408 (2001), introduce for reference herein in full.
According to embodiment, carried out the hepatitis B microRNA molecule marker expression level detection of 150 routine liver cirrhosis patients and 150 routine hepatitis B patients, the miRNA expressed that finds differences has 28.
And wherein between hepatitis B and liver cirrhosis significant difference have 25, the results are shown in Table 1.
Table 1
| Detect thing | DDCt_A-B | P. value _ A-B |
| miR-106b-5p | 0.91664 | 2.79E-05 |
| miR-122-3p | 1.067348 | 0.000258 |
| miR-122-5p | 1.976136 | 3.29E-11 |
| miR-146a-5p | 1.31619 | 1.32E-09 |
| miR-15a-5p | 0.653437 | 0.000472 |
| miR-16-1-3p | 0.732137 | 0.000254 |
| miR-185-5p | 0.905477 | 1.05E-05 |
| miR-186-5p | 1.054347 | 7.01E-06 |
| miR-18a-5p | 0.907725 | 2.85E-05 |
| miR-192-5p | 1.120112 | 9.07E-06 |
| miR-21-5p | 1.225066 | 1.89E-09 |
| miR-223 | 1.443635 | 3.02E-08 |
| miR-22-3p | 1.067446 | 8.32E-10 |
| miR-26a-5p | 1.504132 | 2.75E-05 |
| miR-27a-3p | 1.320157 | 4.57E-09 |
| miR-29c-3p | 1.360216 | 2.83E-09 |
| miR-32-5p | 0.93285 | 1.43E-05 |
| miR-378a-5p | 0.693924 | 0.000235 |
| miR-381-3P | 1.090251 | 2.16E-09 |
| miR-433 | 1.229113 | 1.56E-06 |
| miR-450b-5p | 0.673866 | 0.004524 |
| miR-455-3p | 1.612232 | 1.17E-06 |
| miR-663 | 0.918747 | 4.46E-06 |
| miR-92a-3p | 0.812931 | 4.04E-06 |
| miR-451a | 0.550826 | 0.019445 |
Utilize Spss mapping software, with True Positive Rate (sensitivity) be ordinate zou, false positive rate (1-specificity) is for X-coordinate, draw ROC (receiver operating characteristic) curve, wherein 25 relevant miRNA and miRNA combination the results are shown in Figure 1, wherein miRNA combination AUC (area under curve) reaches 0.866, and this shows that liver cirrhosis microRNA molecule mark of the present invention can effectively distinguish liver cirrhosis and hepatitis B patient.
According to above experimental result, to miR-106b-5p in 40 routine experimenter's blood plasma, miR-122-3p, miR-122-5p, miR-146a-5p, miR-15a-5p, miR-16-1-3p, miR-185-5p, miR-186-5p, miR-18a-5p, miR-192-5p, miR-21-5p, miR-223, miR-22-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-32-5p, miR-378a-5p, miR-381-3P, miR-433, miR-450b-5p, miR-455-3p, miR-663, the expression level of miR-92a-3p and miR-451a combination detects, according to miRNA expression level, prediction experimenter is liver cirrhosis or hepatitis B patient.Blind Test the results are shown in as following table 2.
Table 2
| ? | Liver cirrhosis | Hepatitis B |
| Sample number | 20 | 20 |
| Result | 18 routine interpretations are correct, and 2 routine interpretations are hepatitis B | 14 routine interpretations are correct, and 6 routine interpretations are liver cirrhosis |
| Predictablity rate | 90％ | 70％ |
Embodiment 5
Utilize Spss mapping software, with True Positive Rate (sensitivity) be ordinate zou, false positive rate (1-specificity) is for X-coordinate, draw ROC (receiver operating characteristic) curve, miR-122-5p, miR-146a-5p, miR-27a-3p, miR-451a of wherein 4 relevant miRNA are analyzed, it the results are shown in Figure 2, visible in figure, wherein AUC (area under curve) reaches 0.785, shows that liver cirrhosis lapses to microRNA molecule mark and can effectively distinguish liver cirrhosis and hepatitis B.
According to above experimental result, the expression level of miR-122-5p, miR-146a-5p, miR-27a-3p, miR-451a combination in 40 routine experimenter's blood plasma is detected, according to miRNA expression level, prediction experimenter is liver cirrhosis patient or hepatitis B patient.Blind Test the results are shown in as following table 3.
Table 3
| ? | Liver cirrhosis | Hepatitis B |
| Sample number | 20 | 20 |
| Result | 17 routine interpretations are correct, and 3 routine interpretations are hepatitis B | 16 routine interpretations are correct, and 4 routine interpretations are liver cirrhosis |
| Predictablity rate | 85％ | 80％ |
Claims (10)
1. a liver cirrhosis microRNA molecule mark combination, it comprises more than one and is selected from following microRNA nucleic acid molecule: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQIDNO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQIDNO:54, preferably, described more than one are 2-18 kind, more preferably 3-10 kind, preferred 3-8 kind further, most preferably 4 kinds, preferably, described liver cirrhosis microRNA molecule mark combination is containing SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:15 and SEQ ID NO:54.
2. described in claim 1, liver cirrhosis microRNA molecule mark is combined in the purposes prepared in liver cirrhosis diagnosis reagent.
3. purposes according to claim 2, wherein, described diagnostic reagent by detecting the content of the liver cirrhosis microRNA molecule mark described in subject's plasma/serum, and judges whether liver cirrhosis occurs compared with hepatitis B patient mean level (ML).
4. the purposes described in Claims 2 or 3, wherein, uses the method for quantitative PCR to detect the liver cirrhosis microRNA molecule mark content described in subject's blood plasma.
5. a diagnostic kit for liver cirrhosis, is characterized in that: the specific forward primer comprising liver cirrhosis microRNA molecule mark combination described in claim 1.
6. diagnostic kit according to claim 5, it is characterized in that described specific forward primer comprises the primer that more than one are selected from following sequence: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQIDNO:47, SEQ ID NO:48, SEQ ID NO:55.
7. diagnostic kit according to claim 6, it also comprises further:
(1) blood plasma/blood disappears total RNA extraction reagent,
(2) RNA adds polyA reagent,
(3) RT-PCR reagent,
(4) quantitative PCR reagent,
Wherein, quantitative PCR reagent comprises the specific forward primer of liver cirrhosis microRNA molecule mark combination described in claim 6.
8. diagnostic kit described in claim 7, wherein, described plasma/serum total RNA extraction reagent comprises the external control sequence-1 (External Control-1) that sequence is SEQ ID NO:49, wherein, described RNA adds polyA reagent and comprises the external control sequence-2 (External Control-2) that sequence is SEQ ID NO:50, wherein, described RT-PCR reagent comprises the reverse transcription primer (RT-Primer) that sequence is SEQ IDNO:51.
9. diagnostic kit described in claim 7, wherein, described quantitative PCR reagent comprises general reverse primer UPM-short and UPM-long that sequence is respectively SEQ ID NO:52 and 53.
10. the diagnostic kit according to any one of claim 5 to 9 is used for the purposes of the diagnosis of liver cirrhosis, and preferably, described purposes is in order to distinguish the diagnosis of liver cirrhosis and hepatitis B.
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| CN106909767B (en) * | 2015-12-21 | 2021-11-05 | 北京旷博生物技术股份有限公司 | System for classifying hepatitis B-related cirrhosis |
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| CN105420405B (en) * | 2015-12-30 | 2018-12-07 | 山东农业大学 | The cow's serum microRNA molecule of milk cow Fatty Liver Disease and cow in perinatal period related metabolic diseases label |
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