Application of microRNA marker miR-122-5p in hepatic fibrosis diagnosis
Technical Field
The invention relates to the technical field of biomedicine, in particular to a hepatic fibrosis microRNA marker and application thereof in diagnosis of hepatic fibrosis.
Background
China is a big country with viral hepatitis, and particularly, the number of hepatitis B patients is large. Hepatitis B carriers account for approximately 8-10% of the general population, with approximately 25% developing chronic hepatitis and further developing hepatic fibrosis and cirrhosis. About 10% of the last cases developed hepatocellular carcinoma (HCC). The data show that the average medical cost of a chronic hepatitis B patient per year is about 1100 Yuan, while if the patient progresses to hepatocellular carcinoma, the average cost per year is as much as 4 million RMB. Hepatic fibrosis is a key stage in the process of chronic hepatitis-cirrhosis-liver cancer, and if hepatic fibrosis can be diagnosed at an early stage, the development of diseases can be reversed through treatment.
The hepatic fibrosis patient has no obvious symptoms, and the conventional index of blood has no obvious change. At present, no effective diagnosis means exists for the hepatic fibrosis clinically. Histopathological biopsy is the current 'gold standard' for judging the degree of liver fibrosis and cirrhosis, but the liver puncture location is single-point and limited in material collection, which is not enough to reflect the overall condition of the liver, and about 25% of patients are misdiagnosed and missed in clinic due to pathological diagnosis deviation. Fibroscan (instantaneous elastography) is based on ultrasound examination and determines liver fibrosis and cirrhosis grading by determining liver stiffness values. The detection method has the advantages of simplicity, no pain, rapidness, good patient compliance, no need of hospitalization, good repeatability, contribution to dynamic follow-up observation and the like. However, the application of Fibroscan is limited by factors such as liver inflammation condition, ascites, large blood vessels around the liver, fatty lesion, narrow intercostal space and the like. Resulting in a low diagnosis standard population rate of Fibroscan for hepatic fibrosis. In summary, clinical application of a single technique or index at present cannot accurately and timely diagnose the degree of hepatic fibrosis progression, so that hepatic fibrosis diagnosis still depends on pathological standards of liver biopsy, and a convenient, timely and noninvasive hepatic fibrosis diagnosis index is urgently needed in clinic.
microRNA (miRNA) is a research hotspot in recent years, is single-stranded small-molecule RNA widely existing in eukaryotes, has no coding function, can be combined with flanking regions of a gene sequence to inhibit translation of target mRNA, and has high conservation, time sequence and tissue specificity. At present, the kit plays an important role in the fields of clinical diagnosis, treatment, prognosis evaluation and the like of various diseases such as tumors, hematopathy, virus infection and the like. Although some research has been carried out in this field, the change of microRNA expression of hepatic fibrosis patients has not been studied deeply, and in clinical and research, there is a need to find microRNA markers that can effectively determine hepatic fibrosis, especially microRNA markers for distinguishing hepatic fibrosis from hepatitis.
Disclosure of Invention
The invention provides application of a microRNA nucleic acid molecule miR-122-5p (SEQ ID No.: 1) in preparation of a hepatic fibrosis diagnosis reagent. Preferably, said use is for differentiating between diagnosis of liver fibrosis and hepatitis.
Preferably, the diagnostic reagent determines whether hepatic fibrosis occurs by detecting the content of the hepatic fibrosis microRNA marker in the plasma/serum of a subject and comparing the level with the average level of hepatitis patients.
The method uses a quantitative PCR method or other methods to detect the hepatic fibrosis microRNA marker content in the plasma/serum of a subject.
The invention provides a hepatic fibrosis diagnosis kit in a second aspect, which comprises the following components in parts by weight: 1, specific reverse transcription primer, specific upstream primer, specific probe and qPCR detection downstream primer. Preferably, said SEQ ID No.: 1 is SEQ ID No.: 2, and/or the specific probe is SEQ ID No.: 3, and/or the specific upstream primer is SEQ ID No.: 4, and/or qPCR detection of the downstream primer as SEQ ID No.: 5.
the diagnostic kit further comprises:
(1) a reagent for extracting total RNA from blood plasma/blood serum,
(2) a reagent for reverse transcription is added into the reaction solution,
(3) and (4) quantifying PCR reagents.
Wherein the plasma/serum total RNA extraction reagent comprises an External Control sequence (External Control) with a sequence of 5'-UUUGGAUUGAAGGGAGCUCUA-3'; the reverse transcription reagent comprises a specific reverse transcription primer of a hepatic fibrosis microRNA marker; the quantitative PCR reagent comprises a specific probe and a specific forward primer of the hepatic fibrosis microRNA marker.
The hepatic fibrosis microRNA marker has the characteristics of simple operation, safety, no wound, high sensitivity, high specificity and easy mass screening.
Drawings
FIG. 1 is a graph showing ROC (receiver operating characteristic) curves in patients with liver fibrosis and hepatitis, using Spss mapping software, with true positive rate (sensitivity) as ordinate and false positive rate (1-specificity) as abscissa. The result of miR-122-5p shows that the AUC (area under the curve) reaches 0.863, which indicates that the hepatic fibrosis microRNA marker can effectively distinguish hepatic fibrosis and hepatitis patients.
Detailed Description
Micrornas or mirnas, as described herein, have their ordinary meaning in the art. That is, micrornas represent RNA molecules from genetic loci that are processed from transcripts that can form local RNA precursor microRNA structures. Mature micrornas are typically 20, 21, 22, 23, 24, or 25 nucleotides in length, although other numbers of nucleotides may be present, for example 18, 19, 26, 27, or 28 nucleotides.
According to the invention, the diagnosis of hepatitis B and hepatic fibrosis is based on the viral hepatitis prevention and treatment guidelines of the Chinese medical society of 2000, which are as follows: has a history of chronic infection of hepatitis virus, has shown hepatitis symptoms and liver function abnormality, and has been shown by imaging to locally or diffusely hepatic fibrosis, but has not shown patients with cirrhosis.
According to the invention, the hepatitis diagnosis is based on the viral hepatitis prevention and treatment guidelines of the Chinese medical society of 2000, which are as follows: the course of hepatitis is more than half a year, or the original hepatitis virus carrying history, this time, the same pathogen will again appear hepatitis symptoms, signs and liver function abnormalities, but patients without liver fibrosis/cirrhosis will be diagnosed as chronic hepatitis.
The hepatic fibrosis microRNA marker comprises a microRNA nucleic acid molecule: miR-122-5 p.
Name of microRNA
|
MicroRNA sequence
|
miR-122-5p
|
Uggagugugacaaugguguuug(SEQ ID NO.:1) |
In a preferred embodiment, said one microRNA is differentially expressed in at least one target plasma/serum compared to at least one control plasma/serum. The target plasma/serum is from hepatic fibrosis patients, and the control plasma/serum is from hepatitis patients.
The inventor finds that the content of the hepatic fibrosis microRNA marker in plasma/serum of a hepatic fibrosis patient is obviously different from that of a hepatitis patient, and the hepatic fibrosis and the hepatitis can be effectively distinguished. Therefore, the expression level of the hepatic fibrosis microRNA marker is closely related to the infection of hepatitis B virus, chronic hepatitis and the development of the course of hepatic fibrosis.
On the basis of the discovery, the invention provides the application of the hepatic fibrosis microRNA marker in preparing a hepatic fibrosis diagnostic reagent.
Specifically, the diagnostic reagent diagnoses hepatic fibrosis by detecting the content of hepatic fibrosis microRNA markers in serum/plasma of a subject and comparing the hepatic fibrosis microRNA markers with the average level of hepatitis patients. Wherein, the content of the hepatic fibrosis microRNA marker in the plasma/serum of the cirrhosis patients is obviously different from that of the hepatitis patients. In a specific embodiment, the quantitative PCR method is used to detect the hepatic fibrosis microRNA marker content in the serum/plasma of the subject.
The diagnosis kit comprises specific primers/probes of the hepatic fibrosis microRNA marker. The specific primer/probe comprises: miR-122-5p specific reverse transcription primers, specific upstream primers and specific probes.
The diagnostic kit further comprises:
(1) a reagent for extracting total RNA from blood plasma/blood serum,
(2) a reagent for reverse transcription is added into the reaction solution,
(3) the quantitative PCR reagent is added into the reaction kettle,
wherein the plasma/serum total RNA extraction reagent comprises an External Control sequence (External Control) with a sequence of 5'-UUUGGAUUGAAGGGAGCUCUA-3', and the reverse transcription reagent comprises a specific reverse transcription primer of a hepatic fibrosis microRNA marker; the quantitative PCR reagent comprises a specific probe and a specific forward primer of the hepatic fibrosis microRNA marker.
The technical scheme of the invention is further illustrated by the specific embodiment and the attached drawings, but the technical scheme can be understood by those skilled in the art: the following detailed description and examples are intended to illustrate the invention and should not be construed as limiting the invention in any way.
Example 1 plasma sample Collection and preparation
During the period of 1-2016 12 in 2014, 27 plasma samples of patients meeting the above definition of liver fibrosis and 27 plasma samples meeting the above definition of hepatitis were collected in advance from the Beijing Youtanan hospital affiliated with the university of capital medical.
Collecting 10ml of peripheral venous blood, performing EDTA anticoagulation, and collecting plasma as follows: the whole blood sample was centrifuged at 1,500-3,000g for 15min at 4 ℃ in a centrifuge. The supernatant plasma was carefully transferred to a 1.5mL RNase-free sterile centrifuge tube using a 200. mu.L pipette gun. Each sample is marked. It is important to preserve the plasma samples in an ultra-low temperature (-80 ℃) freezer within 4 hours.
Example 2 extraction of Total RNA from plasma
Total RNA was extracted from plasma using an RNA extraction kit (Beijing, Osbeckon Biotech Co., Ltd.) and the quality of RNA extraction from plasma was monitored by adding 1. mu.l (20nM) of an External Control sequence (External Control) having a sequence of 5'-UUUGGAUUGAAGGGAGCUCUA-3' (synthesized by King-rui Biotechnology Co., Ltd.) to 250. mu.l of plasma. The concentration of extracted total RNA was determined using Thermo NanoDrop2000 c.
Example 3 quantitative determination of MicroRNA in plasma
(1) Obtaining cDNA single strand by RT-PCR:
i. mu.l of microRNA solution was taken and 1.0. mu.l (4.0. mu.M) of specific reverse transcription primer (synthesized by Shanghai Biotechnology engineering Co., Ltd.) was added thereto.
Preparing a reverse transcription reaction solution:
(Note: all products used were products of Beijing Kuangbo Biotech Co., Ltd.)
And iii, mixing the solutions obtained by the i and the ii, incubating at 16 ℃ for 30min, incubating at 42 ℃ for 30min, incubating at 85 ℃ for 5min, and cooling to 4 ℃ to obtain the cDNA.
iv, subpackaging the product obtained in the step iii and storing at-20 ℃.
(2) quantitative qPCR detection:
i. preparing qPCR reaction liquid:
(Note: Each primer/probe was synthesized by Shanghai Biotechnology, Inc., 2 XqPCR Mix and ROX were provided by Baori doctor Biotechnology (Beijing) Inc., and the remaining reagents were from Beijing Kuangbo Biotechnology, Inc.)
ii, after the prepared reaction solution is fully mixed, putting the mixture into a quantitative PCR detector (such as ABI7900), and setting the program as follows:
example 4: and (4) analyzing results:
and performing data analysis by adopting OmicOffcie analysis software.
Methods for RT-PCR and 2-ΔΔCThe T method is described in Kenneth J Livak et al (Analysis of a responsive expression data using a real-time quantitative PCR and the 2-ΔΔCMethods 25, 402-408(2001), which is incorporated herein by reference in its entirety.
According to the embodiment, the expression level of microRNA markers of 27 hepatic fibrosis patients and 27 hepatitis patients is tested, and the results are shown in the following table.
Detection marker
|
DDCt
|
P value
|
miR-122-5p
|
2.93
|
4.61×10-7 |
The results of plotting ROC (receiver operating characterization) curves using the Spss plotting software with true positive rate (sensitivity) as ordinate and false positive rate (1-specificity) as abscissa are shown in FIG. 1. The AUC (area under the curve) of the miR-122-5p reaches 0.863, which shows that the hepatic fibrosis microRNA marker can effectively distinguish hepatic fibrosis and hepatitis patients.
On the ROC curve, since the john index (sensitivity + specificity-1) is the largest when Ct is 22.246, Ct is 22.246 as the cutoff value determined as a result. Namely:
when the Ct of the sample is more than or equal to 22.246, the sample is judged to be a hepatic fibrosis patient sample;
when the Ct of the sample is less than 22.246, the sample is judged to be a hepatitis patient sample.
According to the above determination method, the expression levels of miR-122-5p in the plasma of 27 patients with liver fibrosis and 27 patients with hepatitis are detected, and whether the subjects are patients with liver fibrosis or hepatitis B is predicted according to the miRNA expression levels. The results of the blind tests are shown in Table 2 below.
TABLE 2
|
Hepatic fibrosis
|
Hepatitis (HAV)
|
Number of samples
|
27
|
27
|
Results
|
22
|
23
|
Prediction accuracy
|
81.48%
|
85.19% |