CN104232636A - Hepatitis B microRNA molecular marker composition and application thereof - Google Patents
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Abstract
The invention provides a hepatitis B microRNA molecular marker composition and application thereof in preparing a hepatitis B infection diagnosis and/or prognosis evaluation kit. The hepatitis B microRNA molecular marker composition comprises more than one microRNA molecule as shown in SEQ NO. 1-18. The invention also provides a diagnostic kit for instructing the infection diagnosis and/or prognosis evaluation of hepatitis B. The hepatitis B microRNA molecular marker and the diagnostic kit which are provided by the invention have the characteristics of easiness for operation, safety, no injury, high specificity, high sensitivity and easiness for large-scale screening in instructing the infection diagnosis and/or prognosis evaluation of a hepatitis B patient.
Description
Technical field
The present invention relates to diagnosis of hepatitis b and medication field, relate in particular to one group of hepatitis B blood plasma microRNA molecule mark combination, and this combination is used for the purposes of hepatitis B patient being carried out to Infect And Diagnose and prognosis evaluation, instruct the Infect And Diagnose of hepatitis B patient, Scientific Usage of Drugs and drug withdrawal, both reduce patient medication misery, also reduce the waste of medical resource.
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.
The HBVer of China accounts for the 7-10% of total population, calculate with this, about there are 9,500 ten thousand people's Hepatitis B carriers in the whole nation, wherein hepatitis B patient nearly 3,000 ten thousand, in HBVer, 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.
Chronic hepatitis B diagnostic field bottleneck problem mainly lacks morbid state system evaluation index.Clinical diagnosis means main at present comprise immune indexes, viral index, biochemical indicator.Immune indexes (HBsAg, HBsAb, HBeAg, HBeAb, HBcAb) reflects the humoral immune reaction of body after HBV infection, whether prompting body infects HBV and postinfectious state (cause of disease removing/chronicity), but cannot differentiate the immune tolerant phase of chb, immune clearance stage, low virus replication phase and reactivation stage; Virus index (HBV-DNA) quantitatively/qualitative detection all points out body inner virus loading levels, but because all showing high virus load in patient body when immunological tolerance and immune clearance stage, therefore virus load also can not be used for differentiating above-mentioned clinical state; In biochemical indicator, the serum enzyme such as ALT/AST prompting compromised liver function situation, be used for differentiating chb immune tolerance and immune clearance phase, but liver is reticent organ, just there will be serum enzyme exception when compromised liver function more than 70%.Therefore, although clinically triplicity comprehensively to be analyzed at present the morbid state of chronic hepatitis B together, because of its respective limitation, cannot system, evaluate chb morbid state in time.Bring the many hot and difficult issues in chronic hepatitis B diagnosis and treatment thus, as when chb starts antiviral therapy, the validity of how to evaluate antiviral therapy and the when problem such as drug withdrawal.
Research in recent years shows, hepatitis b virus infected, chronic hepatitis B is closely related with miRNA, miRNA by acting on virus itself or acting on immunity system thus affect therapeutic process, therefore also can may have corresponding effect to the diagnosis of hepatitis B medication.Some correlative studys have been carried out in this field, such as, as as described in patent application CN201110462133, CN102002491A, CN102002490A/CN102002492A and CN102776185B, describe the miRNA marker that liver cancer is relevant, but it is well-known, although diagnosing liver cancer and hepatitis B mark used can overlap, not identical.Up to now, miRNA expression before and after hepatitis B patient medication changes and whether this expression change is relevant to the Treatment and Prognosis of hepatitis B does not also study at present, is necessary to find curative effect after can effectively judging hepatitis B medication in clinical studies, instructs the miRNA marker of clinical drug withdrawal.
Summary of the invention
One aspect of the present invention provides the combination of a kind of hepatitis B molecular marker, 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 and SEQ ID NO:18.Preferably, described hepatitis B molecular marker combination is containing SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12 and SEQ ID NO:16.
Described hepatitis B molecular marker combination preferably include 2-18 kind, more preferably 3-10 kind, most preferably 4 kinds be selected from the microRNA nucleic acid molecule of miR-1, miR-106b-5p, miR-122-3p, miR-122-5p, miR-146a-5p, miR-20b-3p, miR-21-5p, miR-223, miR-22-3p, miR-25-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-381-3P, miR-433, miR-451a, miR-455-3p, miR-663.
The miRNA that new hepatitis B provided by the invention is correlated with and the molecular marker combined as hepatitis B thereof, containing following specificity miRNA, miR-1, miR-106b-5p, miR-122-3p, miR-122-5p, miR-146a-5p, miR-20b-3p, miR-21-5p, miR-223, miR-22-3p, miR-25-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-381-3P, miR-433, miR-451a, miR-455-3p and miR-663, its sequence respectively (see SEQ ID NO:1 to SEQ ID NO:18) as follows.
| MicroRNA title | MiRNA sequence |
| miR-1 | uggaauguaaagaaguauguau |
| miR-106b-5p | uaaagugcugacagugcagau |
| miR-122-3p | aacgccauuaucacacuaaaua |
| miR-122-5p | uggagugugacaaugguguuug |
| miR-146a-5p | ugagaacugaauuccauggguu |
| miR-20b-3p | acuguaguaugggcacuuccag |
| miR-21-5p | uagcuuaucagacugauguuga |
| miR-223 | ugucaguuugucaaauacccca |
| miR-22-3p | aagcugccaguugaagaacugu |
| miR-25-3p | cauugcacuugucucggucuga |
| miR-26a-5p | uucaaguaauccaggauaggcu |
| miR-27a-3p | uucacaguggcuaaguuccgc |
| miR-29c-3p | uagcaccauuugaaaucgguua |
| miR-381-3P | uauacaagggcaagcucucugu |
| miR-433 | aucaugaugggcuccucggugu |
| miR-451a | aaaccguuaccauuacugaguu |
| miR-455-3p | gcaguccaugggcauauacac |
| miR-663 | cuugguucagggagggucccca |
In a preferred embodiment, described at least one miRNA contrasts differential expression in plasma/serum with at least one at least one target plasma/serum.
Preferably, described target plasma/serum is from hepatitis B patient, and described contrast plasma/serum is from healthy individuals.
In the embodiment be more preferably, the expression of described at least one miRNA at least one target plasma/serum contrasts with at least one compared with the expression in plasma/serum and is raised, or the expression of miRNA wherein at least one target plasma/serum contrast in plasma/serum expression with at least one compared with lowered.
Contriver finds, the content of hepatitis B microRNA molecule mark in hepatitis B patient plasma/serum compares normal healthy controls crowd, there is significant difference, effectively can distinguish hepatitis B and normal healthy controls crowd, as can be seen here, the infection of this hepatitis B microRNA molecule mark and hepatitis B virus, treatment and prognosis are closely related.
On the basis of above-mentioned discovery, the invention provides hepatitis B microRNA molecule mark and preparing the purposes in diagnosis of hepatitis b reagent.
A second aspect of the present invention provides a kind of diagnosis of hepatitis b test kit, and it comprises above-mentioned hepatitis B molecular marker combination.
A third aspect of the present invention provides a kind of described hepatitis B molecular marker combination for the preparation of the purposes in hepatitis B prognosis evaluation reagent kit.Preferably, described hepatitis B molecular marker combination is containing SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12 and SEQ ID NO:16.
A fourth aspect of the present invention provides the test kit of a kind of plasma/serum for distinguishing at least one hepatitis B patient and at least one normal subjects's plasma/serum, and it comprises described hepatitis B molecular marker combination.
Particularly, described diagnostic reagent learns the content of hepatitis B microRNA molecule mark in plasma/serum by detecting subject, and with hepatitis B patient to compare with normal healthy controls crowd mean level (ML) diagnose chb patient morbid state, hepatitis B medication curative effect, wherein, the content of hepatitis B microRNA molecule mark in hepatitis B patient plasma/serum compares normal healthy controls crowd, there is significant difference.In a specific embodiment, the method for quantitative PCR is used to detect hepatitis B microRNA molecule mark content in subject's blood plasma.
A fifth aspect of the present invention provides a kind of diagnostic kit instructing hepatitis B infection diagnosis, prognosis evaluation, and it comprises:
(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 primer sets of described hepatitis B molecular marker.
Wherein, quantitative PCR reagent comprises the specific forward primer of hepatitis B microRNA molecule mark, preferably, corresponding to following specificity miRNA:miR-1, miR-106b-5p, miR-122-3p, miR-122-5p, miR-146a-5p, miR-20b-3p, miR-21-5p, miR-223, miR-22-3p, miR-25-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-381-3P, miR-433, miR-451a, miR-455-3p and miR-663, the sequence of its corresponding primer is shown in SEQ ID NO:18 to SEQ ID NO:36 respectively.
Preferably, described plasma/serum total RNA extraction reagent comprises the external control sequence-1 (External Control-1) that sequence is SEQ ID NO:37.Preferably, described RNA adds polyA reagent and comprises the external control sequence-2 (External Control-2) that sequence is SEQ ID NO:38.Preferably, described RT-PCR reagent comprises the reverse transcription primer (RT-Primer) that sequence is SEQ ID NO:39.Preferably, described quantitative PCR reagent comprises general reverse primer UPM-short and UPM-long that sequence is respectively SEQ ID NO:40 and 41.Preferably, the sequence of the specific forward primer of described hepatitis B microRNA molecule mark is selected from one or more in sequence shown in SEQ ID NO:18 to SEQ ID NO:36.Preferably, the specific forward primer of described hepatitis B molecular marker combination contains SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:30 and SEQ ID NO:34.
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:37); Described RNA adds polyA reagent and comprises the External Control-2 that sequence is 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:38); Described RT-PCR reagent comprise sequence be the RT-Primer of 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:39) (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-short and UPM-long that sequence is respectively 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:40) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:41).
Hepatitis B microRNA molecule mark diagnosis of hepatitis b of the present invention is used 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-1, miR-106b-5p, miR-122-3p in the preferred microRNA of at least one hepatitis B plasma/serum, miR-122-5p, miR-146a-5p, miR-20b-3p, miR-21-5p, miR-223, miR-22-3p, miR-25-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-381-3P, miR-433, the Logic Regression Models of miR-451a, miR-455-3p and miR-663 and microRNA combination.
Fig. 2 describe comprise the present invention for determine preferred miR-122-5p, miR-146a-5p, miR-27a-3p and miR-451 and microRNA combination at least one hepatitis B plasma/serum Logic Regression Models.
Embodiment
As described herein, miRNA has its its ordinary meaning (such as seeing CN102943108A) in the art.That is, miRNA represents the RNA molecule from genetic site, and it is from the transcript processing that can form partial rna precursor miRNA structure.Ripe miRNA 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, 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.
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 meeting normal healthy controls 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.
Embodiment 2: the extraction of total serum IgE in 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:37) monitors RNA in blood plasma.The total serum IgE extracted uses Thermo NanoDrop2000c to measure concentration.
Embodiment 3: the miRNA in 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:38) monitors miRNA 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:39) is added in the reaction solution obtained to step (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.
Iv.-20 DEG C of preservations after product packing iii obtained.
(3) qPCR detection by quantitative:
I. preparation reaction solution in (Axygen company) is managed at 2ml 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:40) and 5 '-CTCACACGACTCACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:41).
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 hepatitis B microRNA molecule mark specific forward primer (synthesis of Invitrogen company) that sequence is SEQ ID NO:18 to SEQ ID NO:36, 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:
Array Tools4.1.0 is adopted to carry out data analysis.
About RT-PCR method and 2
-Δ Δ Ct method is shown in Kenneth J Livak (Analysis of relative gene expression data using real-time quantitative PCR and the2
- Δ Δ 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 normal healthy controls and 150 routine hepatitis B patients, the miRNA expressed that finds differences has 28.
And wherein between hepatitis B and normal healthy controls significant difference have 18, the results are shown in Table 1.
Table 1
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 18 relevant micRNA the results are shown in Figure 1, and by described 18 micRNA combination the results are shown in Figure 2, wherein AUC (area under curve) reaches 0.998, and this shows that hepatitis B microRNA molecule mark can effectively distinguish hepatitis B patient and normal healthy controls.
According to above experimental result, to miR-1, miR-106b-5p, miR-122-3p in 28 routine experimenter's blood plasma, miR-122-5p, miR-146a-5p, miR-20b-3p, miR-21-5p, miR-223, miR-22-3p, miR-25-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-381-3P, miR-433, miR-451a, the expression level of miR-455-3p and miR-663 combination detects, and according to miRNA expression level, prediction experimenter is normal healthy controls or hepatitis B patient.Blind Test the results are shown in as following table 2.
Table 2
| ? | Normal healthy controls | Hepatitis B |
| Sample number | 8 | 20 |
| Result | 8 routine interpretations are correct | 14 routine interpretations are correct, and 6 routine interpretations are liver cirrhosis |
| Predictablity rate | 100％ | 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 micRNA are analyzed, it the results are shown in Figure 2, visible in figure, wherein AUC (area under curve) reaches 0.999, shows that hepatitis B lapses to microRNA molecule mark and can effectively distinguish hepatitis B and normal healthy controls.
According to above experimental result, the expression level of miR-122-5p, miR-146a-5p, miR-27a-3p, miR-451a combination in 28 routine experimenter's blood plasma is detected, according to miRNA expression level, prediction experimenter is hepatitis B patient or normal health contrast.Blind Test the results are shown in as following table 3.
Table 3
| ? | Hepatitis B | Normal healthy controls |
| Sample number | 20 | 8 |
| Result | 16 routine interpretations are correct, and 4 routine interpretations are liver cirrhosis | 8 routine interpretations are correct |
| Predictablity rate | 80％ | 100％ |
Claims (10)
1. a hepatitis B molecular marker 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 and SEQ ID NO:18, preferably, described more than one are 2-18 kind, more preferably 3-10 kind, preferred 3-8 kind further, most preferably 4 kinds.。
2. hepatitis B molecular marker combination according to claim 1, it contains SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:12 and SEQ ID NO:16.
3. hepatitis B molecular marker combination according to claim 1 is for the preparation of the purposes in hepatitis B infection diagnosis and/or prognosis evaluation reagent kit.
4., for a test kit for the plasma/serum He at least one normal subjects's plasma/serum of distinguishing at least one hepatitis B patient, it is characterized in that the Auele Specific Primer comprising hepatitis B molecular marker according to claim 1 combination.
5. instruct a diagnostic kit for hepatitis B infection diagnosis and/or prognosis evaluation, it comprises:
(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 hepatitis B molecular marker combination described in claim 1.
6. diagnostic kit according to claim 5, wherein, described blood plasma total RNA extraction reagent comprises the external control sequence-1 (External Control-1) that sequence is SEQ ID NO:37.
7. diagnostic kit according to claim 5, wherein, described RNA adds polyA reagent and comprises the external control sequence 1 (External Control-2) that sequence is SEQ ID NO:38.
8. diagnostic kit according to claim 5, wherein, described RT-PCR reagent comprises the reverse transcription primer (RT-Primer) that sequence is SEQ ID NO:39.
9. diagnostic kit according to claim 5, wherein, described quantitative PCR reagent comprises general reverse primer UPM-short and UPM-long that sequence is respectively SEQ IDNO:40 and 41.
10. diagnostic kit according to claim 5, wherein, the sequence of the specific forward primer of described hepatitis B microRNA molecule mark contain be selected from sequence shown in SEQ ID NO:18 to SEQ ID NO:36 one or more, preferably containing SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:30 and SEQ ID NO:34.
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| CN107779504A (en) * | 2016-08-24 | 2018-03-09 | 北京旷博生物技术股份有限公司 | Colorectal cancer microRNA molecule mark and application thereof |
| CN109439757A (en) * | 2018-12-18 | 2019-03-08 | 首都医科大学附属北京佑安医院 | Application of the blood plasma excretion body miR-455-3p as early liver cancer diagnosis marker |
| CN110229877A (en) * | 2019-06-21 | 2019-09-13 | 广东药科大学附属第一医院 | A kind of application of miRNA in preparation prevention or diagnosis dyslipidemia TCM liver depression reagent |
| CN113186224A (en) * | 2021-04-29 | 2021-07-30 | 中国人民解放军陆军军医大学士官学校 | MicroRNA-27a with hepatitis B virus replication inhibition activity and application thereof |
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