CN112921079A - Early hepatic fibrosis diagnosis marker based on microRNA and kit - Google Patents
Early hepatic fibrosis diagnosis marker based on microRNA and kit Download PDFInfo
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Abstract
The invention discloses a microRNA-based early hepatic fibrosis diagnosis marker and a kit, which comprise nucleic acid molecules for coding the following microRNA molecule combinations: Has-miR-122; Has-miR-151-3 p. Furthermore, the level of the microRNA in the plasma of the PNALT hepatic fibrosis patient is higher than that of a healthy person and a PNALT hepatic fibrosis-free person. Further, the method for obtaining the PNALT hepatic fibrosis diagnosis combination consisting of the plasma microRNA comprises the following steps: the method comprises the following steps: high-throughput sequencing; step two: real-time quantitative fluorescent PCR verification; step three: establishing plasma microRNA combination capable of distinguishing hepatic fibrosis from non-fibrosis in a training group consisting of healthy people, PNALT patients without hepatic fibrosis and PNALT patients with hepatic fibrosis; step four: and (3) verifying the effect of the plasma microRNA combination established in the third step on diagnosing the hepatic fibrosis in two independent verification groups. The invention realizes that the condition of circulating microRNA is comprehensively and objectively reflected, increases multi-center verification, improves the perfection degree of contrast setting and is convenient for explaining that the determined marker is a specific diagnosis marker for early hepatic fibrosis.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a microRNA-based early hepatic fibrosis diagnosis marker and a kit.
Background
About 3.5-4 million people are Hepatitis B Virus (HBV) infected people all over the world, while Chinese Chronic Hepatitis B Virus (CHB) patients reach 1.2 million, and the pathogenesis progress of HBV-related liver diseases generally presents: the stages of HBV continuous infection → hepatic fibrosis → hepatocirrhosis → hepatocellular carcinoma, wherein hepatic fibrosis is the only reversible stage, and effective antiviral treatment can delay the occurrence of hepatic cirrhosis and hepatic carcinoma and even achieve the reversal of fibrosis, so that the accurate diagnosis of different stages of liver fibrosis is crucial to the intervention treatment of CHB infected patients, and is a major health problem to be solved urgently at present
Hepatic fibrosis is a pathophysiological process, which refers to abnormal hyperplasia of connective tissues in the liver caused by various pathogenic factors, any liver injury has a hepatic fibrosis process in the process of liver repair and healing, and if the injury factors cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time; fibrosis occurrence is an important factor for predicting chronic liver injury and disease progression, and detection of early occurrence of hepatic fibrosis is very important, and clinical diagnosis methods of hepatic fibrosis include two major categories, one is invasive examination, namely, liver puncture biopsy; another category is non-invasive tests, including serum molecular markers and imaging tests.
At present, biochemical detection indexes and imaging examination which are applied to clinic cannot completely detect and reflect the damaged state and early fibrosis degree of the liver of the part of people, so that missed diagnosis is easy to occur; although liver puncture is currently identified as the gold standard for examining the degree of liver injury and fibrosis, it is an invasive examination, and the compliance of patients is poor, and due to the small puncture specimen (limitation of material collection) and the diagnosis experience of pathologists, it is difficult to ensure the accuracy of examination results.
The body fluid samples such as blood and the like are easy to obtain, the clinical operability is strong, the wound is small, the stability of the circulating microRNA is good, and the detection is convenient, so that the circulating microRNA has the potential of being used as a disease noninvasive biomarker and is suitable for screening chronic hepatitis B carriers. However, the current research on circulating microRNA as a liver fibrosis diagnosis marker still has shortcomings: most researches only select circulating microRNA with maladjustment in hepatic fibrosis tissue expression reported in former worries as candidate indexes, and probably cannot comprehensively and objectively reflect the condition of the circulating microRNA; (2) part of research samples are few, and multi-center verification is lacked; (3) the control setting is imperfect, and the determined marker is difficult to be explained as a diagnostic marker specific to early hepatic fibrosis.
In summary, there is a need for a microRNA-based early hepatic fibrosis diagnosis marker and a kit to solve the disadvantages of the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a microRNA-based early hepatic fibrosis diagnosis marker and a kit, aiming at solving the problems that the condition of circulating microRNA cannot be comprehensively and objectively reflected, part of research samples are few, and the determined marker is difficult to explain as a specific diagnosis marker of early hepatic fibrosis.
In order to achieve the purpose, the invention provides the following technical scheme: a microRNA-based early hepatic fibrosis diagnosis marker comprises a nucleic acid molecule encoding the following combination of microRNA molecules:
Has-miR-122；
Has-miR-151-3p。
furthermore, the level of the microRNA in the plasma of the PNALT hepatic fibrosis patient is higher than that of a healthy person and a PNALT hepatic fibrosis-free person.
Further, the method for obtaining the PNALT hepatic fibrosis diagnosis combination consisting of the plasma microRNA comprises the following steps:
the method comprises the following steps: high-throughput sequencing;
step two: real-time quantitative fluorescent PCR verification;
step three: establishing plasma microRNA combination capable of distinguishing hepatic fibrosis from non-fibrosis in a training group consisting of healthy people, PNALT patients without hepatic fibrosis and PNALT patients with hepatic fibrosis;
step four: verifying the effect of the plasma microRNA combination determined in the step three on diagnosing the hepatic fibrosis in two independent verification groups;
step five: analyzing the effect of the plasma microRNA combination established in the step three on early hepatic fibrosis PNALT patients.
Further, the kit for diagnosing early hepatic fibrosis based on microRNA comprises reagents for detecting the levels of the following microRNA molecules in plasma respectively: Has-miR-122 and Has-miR-151-3 p.
Further, the diagnostic kit further comprises a plasma RNA extraction system and a reverse transcription system.
Further, the diagnostic kit further comprises an assay for assessing whether liver fibrosis is present.
Further, the diagnostic kit comprises LNA modified primers capable of respectively detecting the following microRNA levels: Has-miR-122 and Has-miR-151-3 p.
Further, the reagent for detecting the water in the plasma of the combination of the microRNA molecules is a real-time fluorescence quantitative PCR related reagent.
Further, the diagnostic kit also comprises a LAN modified primer for detecting the exogenous reference NC 67.
The invention has the beneficial effects that:
1. in the invention, the kit adopts high-throughput screening, multi-center verification and research in high risk groups, the effect of serum microRNA combination and the diagnostic kit are comprehensively evaluated, the potential application value of the developed kit in clinical diagnosis is ensured, and a reference method strategy is provided for the development of other disease biomarkers.
2. According to the invention, the circulating microRNA hepatic fibrosis diagnosis kit can reflect the disease state of chronic hepatitis B hepatic fibrosis in time, avoid complicated detection in the past, save time and labor cost, facilitate clinicians to adopt personalized prevention and treatment schemes in time, solve the problem that circulating microRNA can not be comprehensively and objectively reflected, increase multi-center verification, improve the perfection degree of contrast setting, and facilitate judgment and specification that a determined marker is a specific diagnosis marker for early hepatic fibrosis.
Drawings
Fig. 1 is a statistical representation of qRT-PCR validation results for candidate miRNAs differentially expressed by SPNALT patients of the present invention, wherein fig. 1 comprises: FIGS. 1-1 and 1-2.
FIG. 2 is a statistical schematic diagram of the validation results of the expanded sample plasma qRT-PCR of SPNALT patients according to the invention.
FIG. 3 is a schematic diagram of the area statistics under the curves of MiR-122-5p and miR-151-3pAUC of the present invention.
Detailed Description
As shown in fig. 1, 2 and 3, a microRNA-based early liver fibrosis diagnosis marker comprises nucleic acid molecules encoding the following combination of microRNA molecules:
Has-miR-122；
Has-miR-151-3p。
preferably, the level of microRNA in the plasma of PNALT liver fibrosis patients is higher than that of healthy people and PNALT liver fibrosis-free people.
Preferably, the method for obtaining the PNALT hepatic fibrosis diagnosis combination consisting of plasma microRNA comprises the following steps:
the method comprises the following steps: high-throughput sequencing;
step two: real-time quantitative fluorescent PCR verification;
step three: establishing plasma microRNA combination capable of distinguishing hepatic fibrosis from non-fibrosis (healthy people and PNALT hepatic fibrosis-free people) in a training group consisting of healthy people, PNALT hepatic fibrosis-free patients and PNALT hepatic fibrosis-free patients;
step four: verifying the effect of the plasma microRNA combination determined in the step three on diagnosing the hepatic fibrosis in two independent verification groups;
step five: analyzing the effect of the plasma microRNA combination established in the step three on early hepatic fibrosis PNALT patients.
The experimental results are analyzed and relevant statistics show that: in the first step, the inventor determines 18 candidate microRNAs through high-throughput qPCR Array screening, verifies the levels of the 18 candidate microRNAs in the plasma of a PNALT hepatic fibrosis patient in the second step, further detects the levels of the candidate microRNAs in a training group (sample amount is XX parts), establishes an optimal microRNA combination for distinguishing hepatic fibrosis from non-fibrosis, and further verifies that the plasma microRNA combination can distinguish liver cancer from non-cancer contrast in independent verification groups 1 and 2 (sample amounts are XX and XX parts respectively); meanwhile, compared with the traditional liver fibrosis screening means, such as PLT, the combination of plasma microRNA has better diagnosis effect in liver fibrosis patients.
Preferably, the kit for diagnosing early hepatic fibrosis based on microRNA comprises reagents for detecting the levels of the following microRNA molecules in plasma respectively: Has-miR-122 and Has-miR-151-3 p.
Preferably, the diagnostic kit further comprises a plasma RNA extraction system and a reverse transcription system.
Preferably, the diagnostic kit further comprises an assay for assessing whether liver fibrosis is present.
Preferably, the diagnostic kit comprises LNA modified primers that can detect the following microRNA levels, respectively: Has-miR-122 and Has-miR-151-3 p.
Preferably, the reagent for detecting the water in the plasma of the combination of microRNA molecules is a real-time fluorescence quantitative PCR related reagent.
Preferably, the diagnostic kit further comprises a LAN modified primer for detecting the exogenous reference NC67, and the level of the 2 target genes in the plasma sample to be detected is obtained by calibrating the exogenous reference NC67 (etaCt ═ C)target-Creference) And respectively assigning the microRNA level of the detection sample to be 1 or 0 according to the microRNA threshold value, so as to realize discretization.
For the diagnosis of liver cancer with a single microRNA discretization threshold XX, the diagnosis with the discretization threshold XX is non-fibrosis.
And further analyzing the combination of plasma microRNAs according to a logistic regression method to evaluate whether hepatic fibrosis exists or not:
Logit(p＝SPNALT)＝-0.351-0.597*has-miR-122-0.300*hsa-miR-151-3p。
logit (p) SPNALT XX is used as a diagnosis threshold, and hepatic fibrosis is diagnosed above XX, and non-liver cancer is diagnosed below XX.
Example 1
In FIG. 1, 11 miRNAs are selected from the group as candidate miRNAs differentially expressed in plasma of SPNALT patients, including miR-130-3P, miR-140-3P, miR-151a-3P, miR-320a, miR-320b, miR-361, miR-423-5P and miR-320a, which are low-expressed in plasma of SPNALT patients, and miR-122-5P, miR-7975 and miR-7977 are high-expressed, and the 11 miRNAs are preliminarily verified by Taqman probe QPCR method, which shows that the four miRNAs are differentially expressed in plasma of SPNALT patients, and are miR-122-5P, miR-151a-3P, miR-140-3P and miR-320b, respectively, and compared with NPNALT and the healthy group, the plasma expression levels of miR-122-5P and miR-151a-3P in the SPNALT group are obviously increased and decreased (P is 0.027 value: P, respectively) <0.05, P ═ 0.002< 0.05; p ═ 0.028 <0.05, P ═ 0.000<0.01), consistent with earlier chip screening results; however, plasma expression levels of miR-140-3P and miR-30d were significantly increased in the SPNALT group (P ═ 0.014<0.05, P ═ 0.003< 0.05; P ═ 0.026<0.05, P ═ 0.013<0.05), inconsistent with previous chip results screening, and seven miRNAs were not significantly different between the three groups for the remaining miR-130b-3P, miR-320a, miR-361-5P, miR-423-5P, miR-486-5P, miR-7975 and miR-7977 (P values: 0.824, P ═ 0.945, P ═ 0.647, P ═ 0.539, P ═ 0.545, P ═ 0.818 and P ═ 0.508, both P values were greater than 0.05).
In fig. 2, the QPCR verification was performed again on miR-122-5P, miR-151a-3P, miR-140-3P and miR-320B four miRNAs in the subsequent process by expanding the sample (30 samples per group), and the results showed that the Δ Ct value of miR-122 in the SPNALT group was significantly higher than that in the NPNALT group and the health group, P value was <0.05, the difference was statistically significant, and the Δ Ct value of miR-151-3P was significantly lower than that in the NPNALT group and the health group, respectively, and the difference was also statistically significant (P ═ 0.037, P ═ 0.001, P <0.05) (see fig. 2A and 2B); however, the Δ Ct values for the remaining two miRNAs (miR-140-3P and miR-320b) were compared two by two between three groups, with no statistical difference (P ═ 0.323, P ═ 0.255, P > 0.05) (see fig. 2C, 2D).
In fig. 3, using ROC curve analysis and evaluation of miR-122-5p and miR-151-3p as the accuracy for diagnosing SPNALT early fibrosis, the AUC (area under ROC curve) results for miR-122-5p and miR-151-3p were 0.901 (95% CI 1.85-0.825; 80% sensitivity; 87% specificity) (see fig. 3A) and 0.844 (95% CI, 0.46-0.745; 76.7% sensitivity; 87.3% specificity) (see fig. 3B), respectively.
Example 2
TABLE 1 basic characteristics of 18 differentially expressed miRNAs of the present application
| MiRNAs | Name | Foldchange | Pvalue |
| Up-regulated | miR-122-5p | 15.60390762 | 0.014086604 |
| miR-7975 | 2.385684592 | 0.012760626 | |
| miR-7977 | 2.765338185 | 0.024320926 | |
| miR-1260a | 2.086515206 | 0.010217115 | |
| miR-6125 | 3.002375671 | 0.032804494 | |
| Down-regaluted | miR-130b-3p | 0.48506793 | 0.035250824 |
| miR-140-3p | 0.48826817 | 0.029497252 | |
| miR-151a-3p | 0.38595192 | 0.006746703 | |
| miR-320a | 0.422711253 | 0.017810562 | |
| miR-320d | 0.287118213 | 0.031172524 | |
| miR-361-5p | 0.474121976 | 0.013009276 | |
| miR-423-5p | 0.362739062 | 0.012701571 | |
| miR-486-5p | 0.456280672 | 0.028838438 | |
| miR-320b | 0.338475206 | 0.022090953 | |
| miR-320e | 0.261952637 | 0.038086723 | |
| miR-324-3p | 0.428690312 | 0.006556401 | |
| miR-484 | 0.484917882 | 0.006419125 | |
| miR-6127 | 0.481246124 | 0.038785289 |
The miRNAs with complete genome differential expression among NPNALT, SPNALT and Healthy groups are analyzed by pairwise comparison of miRNAs chips, 18 miRNAs with differential expression are screened from 2549 miRNAs (the absolute value of Fold difference is more than 2, the P value is less than 0.05,5 miRNAs are high-expression and 13 low-expression (shown in table 1) by adopting a screening standard that the P value is less than 0.05, the Fold change absolute value is more than 2 and the chip signal value is more than 5), and the heat map analysis result shows that the 8 miRNAs of the NPNALT group and the Healthy group, the 10 miRNAs of the SPNALT group and the Healthy group and the 3 miRNAs of the SPNALT group and the Healthy group.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A microRNA-based early hepatic fibrosis diagnosis marker comprises a nucleic acid molecule encoding the following combination of microRNA molecules:
Has-miR-122；
Has-miR-151-3p。
2. the early hepatic fibrosis diagnosis marker based on microRNA of claim 1, wherein the level of the microRNA in the plasma of PNALT hepatic fibrosis patients is higher than that of healthy people and PNALT hepatic fibrosis-free people.
3. The early hepatic fibrosis diagnosis marker based on microRNA of claim 1, wherein the method for obtaining the PNALT hepatic fibrosis diagnosis combination composed of plasma microRNA comprises the following steps:
the method comprises the following steps: high-throughput sequencing;
step two: real-time quantitative fluorescent PCR verification;
step three: establishing plasma microRNA combination capable of distinguishing hepatic fibrosis from non-fibrosis in a training group consisting of healthy people, PNALT patients without hepatic fibrosis and PNALT patients with hepatic fibrosis;
step four: verifying the effect of the plasma microRNA combination determined in the step three on diagnosing the hepatic fibrosis in two independent verification groups;
step five: analyzing the effect of the plasma microRNA combination established in the step three on early hepatic fibrosis PNALT patients.
4. A microRNA-based early hepatic fibrosis diagnosis kit comprises reagents for detecting the levels of the following microRNA molecules in plasma respectively: Has-miR-122 and Has-miR-151-3 p.
5. The kit for diagnosing early hepatic fibrosis based on microRNA according to claim 4, wherein the kit further comprises a plasma RNA extraction system and a reverse transcription system.
6. The kit for diagnosing early hepatic fibrosis based on microRNA of claims 4 and 5, wherein the kit further comprises an analysis method for assessing whether hepatic fibrosis is present.
7. The early hepatic fibrosis diagnosis kit based on microRNA according to claim 4, wherein the diagnosis kit comprises LNA modified primers capable of detecting the following microRNA levels respectively: Has-miR-122 and Has-miR-151-3 p.
8. The kit for diagnosing early hepatic fibrosis based on microRNA of claim 4, wherein the reagent for detecting the water in the combination of microRNA molecules in plasma is a real-time fluorescence quantitative PCR related reagent.
9. The early hepatic fibrosis diagnosis kit based on microRNA of claim 4, wherein the diagnosis kit further comprises a LAN modified primer for detecting the exogenous reference NC 67.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170010264A1 (en) * | 2008-10-06 | 2017-01-12 | Gale W. Newman | Exosome-mediated diagnosis of hepatitis virus infections and diseases |
| WO2018231851A1 (en) * | 2017-06-13 | 2018-12-20 | Gilead Sciences, Inc. | Methods of treating liver fibrosis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170010264A1 (en) * | 2008-10-06 | 2017-01-12 | Gale W. Newman | Exosome-mediated diagnosis of hepatitis virus infections and diseases |
| WO2018231851A1 (en) * | 2017-06-13 | 2018-12-20 | Gilead Sciences, Inc. | Methods of treating liver fibrosis |
Non-Patent Citations (1)
| Title |
|---|
| JIN-LIN CHENG等: "Plasma miRNA-122-5p and miRNA-151a-3p identified as potential biomarkers for liver injury among CHB patients with PNALT", 《HEPATOLOGY INTERNATIONAL》 * |
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