CN110699450A

CN110699450A - Application of miRNA biomarker in diagnosis and prognosis of liver disease

Info

Publication number: CN110699450A
Application number: CN201910428949.4A
Authority: CN
Inventors: 郑振宜; 曾宗渊
Original assignee: Pu Xi (guangzhou) Biological Immune Technology Co Ltd
Current assignee: Pu Xi (guangzhou) Biological Immune Technology Co Ltd
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2020-01-17

Abstract

The invention relates to application of miRNA biomarkers in diagnosis and prognosis of liver diseases, belonging to the technical field of biotechnology and medicine. The miRNA biomarkers of the invention include: miR-15a and miR-16 a. According to a large amount of clinical case data, tissue and blood samples and clinical and laboratory data, the miR-15a and miR-16a are found to be not only in liver tissues, but also in peripheral blood, and more importantly, the early diagnosis efficiency of the liver cancer can be effectively improved, and the antiviral curative effect can be accurately evaluated with assistance. The biomarker can be applied to a kit for diagnosis and prognosis judgment of liver diseases, and the development of diagnosis and treatment technologies of liver diseases is greatly promoted.

Description

Application of miRNA biomarker in diagnosis and prognosis of liver disease

Technical Field

The invention relates to the technical field of biotechnology and medicine, in particular to application of miRNA biomarkers in diagnosis and prognosis judgment of liver diseases.

Background

Liver cancer is the second most common life cancer in the world, the death rate of liver cancer reaches 74.5 ten thousand every year, and the morbidity and mortality of liver cancer in China are always in the first place in the world, thus bringing heavy economic burden to China. Although liver cancer treatment methods are continuously developed and advanced, including surgical resection, liver transplantation, radiofrequency ablation, Transcatheter Arterial Chemoembolization (TACE), radiotherapy, chemotherapy and the like, since the liver is a silent organ, the onset of disease has obvious hiding property, and most patients have advanced once; meanwhile, the molecular mechanism of occurrence and development of liver cancer has the characteristics of complexity and diversity, and the factors cause the problems of low early diagnosis rate, high recurrence and metastasis, difficult prognosis judgment, insufficient diagnosis and evaluation indexes and the like of clinical diagnosis and treatment of liver cancer. Therefore, the search of the related markers which are helpful for the early diagnosis and prognosis of the liver cancer is of great significance for improving the overall diagnosis and treatment of the liver cancer.

More than 85% of liver cancer patients in China develop on the basis of chronic hepatitis B (chronic hepatitis B) infection, and at present, nearly 1 hundred million Hepatitis B Virus (HBV) infectors exist in China, wherein 3000 ten thousand patients with chronic hepatitis; the existing treatment method is an antiviral drug, and can inhibit virus replication to the utmost extent for a long time, thereby reducing the occurrence and development of liver cancer; however, since 90% of HBV infections in our country are transmitted from mother to infant or from young adults, there is a great difference in the response of patients with chronic hepatitis B to antiviral therapy, so that there is still a risk that patients undergoing antiviral therapy will develop liver cancer. Therefore, accurate assessment of antiviral efficacy also becomes a means of determining the risk of developing severe liver disease. Currently, the evaluation of antiviral efficacy mainly depends on the change of the viral index itself for prediction or evaluation. However, due to the diversity and complexity of chronic hepatitis B infection in China and the characteristics of host heredity, the change of virus indexes of part of patients in the antiviral treatment period is not enough to accurately reflect the pathological change condition of the liver; therefore, how to comprehensively evaluate the disease state by combining the viral index and the human body index is an urgent problem to be solved, so that the prognosis of the liver disease patient can be better improved.

The exosome is a nano-level lipid inclusion structure with the diameter of 30-100nm, and substances such as protein, mRNA (messenger ribonucleic acid), microRNA (micro RNA, miR) and the like are contained in the exosome, so that almost all types of cells can generate and release the exosome and the exosome is an important medium for intercellular communication; research proves that the exosome miRNA participates in the biological processes of tumorigenesis, growth, invasion and metastasis.

In recent years, the relevance of microRNA and occurrence and development of human diseases, particularly tumors is more and more emphasized, miR is non-coding RNA which is 21-25 nucleotides in length and widely exists in eukaryotes, and is firstly transcribed into initial miR containing a plurality of stem-loop structures in cell nucleus and processed into 70 nucleotide precursor miR under the action of nuclease; finally, the precursor RNA is moved to cytoplasm to form a double-stranded miR with 21 base pairs, and is combined with AGO2 protein to form a single-stranded miR; the single-chain miR reenters the mRNA of the target gene and is complementarily combined in the 3' UTR region of the mRNA, and the combination interferes the expression of the mRNA of the target gene by inhibiting the translation or degradation of the mRNA. Research already shows that miR can regulate multiple biological processes of gene expression, cell cycle, biological development and the like of malignant tumors, and multiple miRs are found in liver cancer and are related to disease states and prognosis; however, due to the diversity, complexity, multimechanistic nature and unknown nature of liver diseases (liver cancer and hepatitis), the mirs discovered so far fail to address the mechanisms underlying disease progression.

Disclosure of Invention

Based on this, it is necessary to provide an application of miRNA biomarkers in diagnosis and prognosis of liver diseases, wherein miR-15a and miR-16a are used as biomarkers, and can effectively improve early diagnosis efficiency of liver cancer in liver tissues and more importantly in peripheral blood and assist in accurate evaluation of antiviral efficacy.

Use of a biomarker for a miRNA for diagnosis and/or prognostic assessment of a liver disease, said miRNA comprising: miR-15a and miR-16 a.

The invention also discloses application of miRNA biomarkers in developing and/or preparing products with liver disease diagnosis and/or prognosis evaluation application, wherein the miRNA comprises: miR-15a and miR-16 a.

It is understood that the product may be a kit or an integrated detection device.

In one embodiment, the miRNA biomarker is used for developing and/or preparing a product for predicting anti-hepatovirus therapeutic effect; or the miRNA biomarker is applied to development and/or preparation of products for diagnosing liver cancer.

In one embodiment, the miRNA serves as a biomarker for peripheral blood biopsy.

The invention also discloses an application of the reagent for detecting the miRNA marker in the biological sample in the preparation of liver cancer diagnostic reagents or diagnostic equipment, wherein the miRNA comprises the following components: miR-15a and miR-16 a.

In one embodiment, the marker further comprises alpha-fetoprotein, and the liver cancer diagnostic reagent or the diagnostic device is used for performing liquid biopsy on peripheral blood. The diagnosis efficiency can reach 91 percent by jointly detecting miR-15a, miR-16a and alpha-fetoprotein (AFP) in peripheral blood, and is remarkably improved compared with single use.

The reagent can be a specific reagent designed for detecting miR-15a and miR-16a or alpha fetoprotein in a biological sample, and can also be a conventional reagent capable of realizing the detection of miR-15a and miR-16a or alpha fetoprotein in the biological sample and/or a combination of the specific reagents.

The invention also discloses a liver disease detection kit, which comprises a reagent for reverse transcription quantitative detection of miR-15a and miR-16 a.

In one embodiment, the following reverse transcription primers are included:

5’-CAGGTCCAGTTTTTTTTTTTTTTTVN，

the V is selected from A or G, and the N is selected from A or T.

In one embodiment, the kit further comprises the following qPCR primer pairs:

miR-15a-F:5’-TAGCAGCACATAATGGTTTGTG-3’

miR-15a-R:5’-AGGTCCAGTTTTTTTTTTTTTCAC-3’；

miR-16-F:5’-CAGTAGCAGCACGTAAATA-3’

miR-16-R:5’-CCAGTTTTTTTTTTTTTTCGCC-3’。

in one embodiment, the liver disease comprises liver cancer, hepatitis virus infection.

Compared with the prior art, the invention has the following beneficial effects:

according to the application of the miRNA biomarker in diagnosis and prognosis of liver diseases, a large amount of clinical case data, tissue and blood samples and clinical and laboratory data show that miR-15a and miR-16a not only can effectively improve the early diagnosis efficiency of liver cancer, but also can assist in accurately evaluating the antiviral curative effect in peripheral blood.

The biomarkers miR-15a and miR-16a are applied to a kit for diagnosis and prognosis of liver diseases, and the development of diagnosis and treatment technologies of the liver diseases is greatly promoted.

Drawings

FIG. 1 is a flow chart of the process for producing exosomes in example 1;

FIG. 2 is the expression of miR-15a (A) and miR-16a (B) in liver cancer and para-cancer tissues in example 1;

FIG. 3 shows the expression of miR-15a (A) and miR-16a (B) in hepatoma cells in example 1;

FIG. 4 is a correlation analysis of miR-15a (A) and miR-16a (B) expression and cancer-free survival and overall survival rate in example 1;

FIG. 5 is the expression of miR-15a (A) and miR-16a (B) in different liver disease states in example 1;

FIG. 6 is a graph of the correlation of miR-15a (A) and miR-16a (B) expression in example 1 with the clinical stage of liver cancer;

FIG. 7 is a graph showing the efficiency of miR-15a (A) and miR-16a (B) in diagnosing liver cancer in example 1;

FIG. 8 shows the effect of the miR-15a and miR-16a synthesizers on liver cancer cell cloning in example 2;

FIG. 9 shows the effect of miR-15a and miR-16a inhibitors on hepatoma cell cloning in example 2;

FIG. 10 is a graph of the effect of miR-15a and miR-16a synthases (A) and their inhibitors (B, C) on hepatoma cell proliferation in example 2;

FIG. 11 is a graph showing the effect of miR-15a (A) and miR-16a (B) synthases on hepatoma cell invasion in example 2 (400-fold magnification);

FIG. 12 is the correlation of miR-15a (A) and miR-16a (B) with HBVDNA levels in example 3;

FIG. 13 is a graph of the relevance of miR-15a (A) and miR-16a (B) to antiviral treatment in example 4;

FIG. 14 is the levels of miR15a (A) and miR-16a (B) in patients who turned HBVDNA negative and remained HBVDNA positive at 48 weeks of antiviral treatment in example 4;

FIG. 15 is the difference in HBeAg turning negative rate by 96 weeks for patients with baseline miR-15a high expression (miR-15a-H) and miR-15a low expression (miR-15a-L) in example 4 with antiviral treatment (A); difference in HBeAg negative conversion rate by 96 weeks for patients with baseline miR-16a high expression (miR-16a-H) and miR-16a low expression (miR-16a-L) antiviral treatment (B);

FIG. 16 is the difference in the decline of HBsAg for antiviral treatment of patients with high expression of miR-15a (miR-15a-H) and low expression of miR-15a (miR-15a-L) at baseline in example 4 (A); difference in decrease in antiviral HBsAg in subjects with high miR-16a expression (miR-16a-H) and low miR-16a expression (miR-16a-L) at baseline (B);

wherein: p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The starting materials and reagents used in the following examples are commercially available unless otherwise specified.

Example 1

miR-15a and miR-16a distinguish hepatitis and liver cancer and the effect of the liver cancer on early diagnosis of the liver cancer.

Research materials:

1. liver disease cases were enrolled and specimens:

the early-stage research of the invention brings 540 cases of liver diseases including 152 cases of hepatitis, 120 cases of severe liver fibrosis and 268 cases of liver cancer into the analysis in two three hospitals in Guangdong province; blood samples from all the above cases and liver tissues from 236 patients (79 non-liver cancer cases and 157 liver cancer cases) were obtained.

2. Liver cancer cell line: HepG2, SK-Hep-1, SMMC-7721, Bel7402, MHCC97L, MHCC97H, Huh7 and LM 3.

(II) research method:

1. extracting total RNA of liver tissue by conventional method.

2. Exosome extraction:

2.1 plasma, serum samples:

the anticoagulation tube was used to draw whole blood, gently mixed and then placed at 4 ℃ for the next step within 1 hour.

Centrifuging at 1900xg for 10 minutes at 4 ℃ to obtain supernatant as plasma; centrifuge again at 3000Xg for 15 minutes, carefully aspirate plasma, serum.

2.2 cell sample:

when the fusion degree of the cultured cells of the liver cancer cell strain reaches 70-80%, a serum-free culture medium is adopted to replace a cell culture medium, and then the cells are cultured for 24-28 h.

The cell culture supernatant was collected as a sample, centrifuged at low speed at 300Xg10 minutes to separate the cells, and the supernatant was left to be centrifuged at 200Xg for 10 minutes to remove dead cells.

The remaining supernatant was centrifuged at 10000Xg30 min to remove cell debris again.

2.3 exosome acquisition:

centrifuging the plasma/serum or cell sample at 100,000Xg for 70 min to obtain a crude exosome pellet

And (3) resuspending the exosome precipitate by PBS, and centrifuging for 70 minutes at 100,000Xg to obtain pure exosome.

The operational flow of the preparation of exosomes is shown in figure 1.

RT-qPCR detection

Unlike the conventional miRNA reverse transcription method, this example redesigns the miRNA reverse transcription method from a two-step method to a one-step method, which can increase the amplification efficiency and the purity of the target product.

The one-step method scheme is a tail method RT-qPCR detection scheme by adding Poly (A), and comprises the following specific steps:

(1) reverse Transcription (RT): poly (A) tailing and RT were done in one step:

A. add poly (a) tail to miRNA 3' using NEB e.

B. Reverse transcription primer: the reverse transcription primer is 5' -CAGGTCCAGTTTTTTTTTTTTTTTVN, wherein V is selected from A, C or G, N is selected from A, C, G or T, namely CAGGTCCAGTTTTTTTTTTTTTTT (SEQ ID No.1) sequence is added with VN terminal.

Reverse transcription was performed with the following reverse transcription primer sequences:

5’-CAGGTCCAGTTTTTTTTTTTTTTTAA(SEQ ID No.2)

5’-CAGGTCCAGTTTTTTTTTTTTTTTAC(SEQ ID No.3)

5’-CAGGTCCAGTTTTTTTTTTTTTTTAG(SEQ ID No.4)

5’-CAGGTCCAGTTTTTTTTTTTTTTTAT(SEQ ID No.5)

5’-CAGGTCCAGTTTTTTTTTTTTTTTCA(SEQ ID No.6)

5’-CAGGTCCAGTTTTTTTTTTTTTTTCC(SEQ ID No.7)

5’-CAGGTCCAGTTTTTTTTTTTTTTTCG(SEQ ID No.8)

5’-CAGGTCCAGTTTTTTTTTTTTTTTCT(SEQ ID No.9)

5’-CAGGTCCAGTTTTTTTTTTTTTTTGA(SEQ ID No.10)

5’-CAGGTCCAGTTTTTTTTTTTTTTTGC(SEQ ID No.11)

5’-CAGGTCCAGTTTTTTTTTTTTTTTGG(SEQ ID No.12)

5’-CAGGTCCAGTTTTTTTTTTTTTTTGT(SEQ ID No.13)

C. the reverse transcription reaction system (cDNA synthesis) was as shown in the following Table, and a 10. mu.l system was used.

TABLE 1 reverse transcription reaction System

Reagent	Lower limit of addition amount	Upper limit of addition amount
			RNA	100ng	1μg
10×poly(A)buffer	1μl	1μl
			ATP	0.1μl(0.1mM)	0.1μl(1mM)
dNTP	0.1μl(0.1mM)	0.1μl(1mM)
			Reverse transcription primer	1 μ M/strip	10 μ M/strip
RTase(160u/μl)	100units	160units
			Poly(A)ase(5u/μl)	1unit	5units

The above reagents were diluted to working concentrations and used with ATP concentration of 10mM and dNTP concentration of 10 mM.

Reaction conditions are as follows: after reaction at 42 ℃ for 1h, the reaction mixture was treated at 95 ℃ for 5 min.

(2) miR-15a qPCR primer design:

A. mature sequence: 5'-GCAGCACATAATGGTTTG-3' (SEQ ID No.14)

Reverse complement sequence: 5'-CAAACCATTATGTGCTGC-3' (SEQ ID No.15)

B.cDNA:5’-CAGGTCCAGTTTTTTTTTTTTTTTCACAAACCATTATGTGCTGCTA-3’(SEQ IDNo.16)

Qpcr primer pair:

miR-15a-F:5’-TAGCAGCACATAATGGTTTGTG-3’(SEQ ID No.17)

miR-15a-R:5’-AGGTCCAGTTTTTTTTTTTTTCAC-3’(SEQ ID No.18)

(3) miR-16qPCR primer design:

A. mature sequence: 5'-GCAGCACGTAAATATTGGCG-3' (SEQ ID No.19)

Reverse complement sequence: 5'-CCAATATTTACGTGCTGCTA-3' (SEQ ID No.20)

B.cDNA:5’-GGTCCAGTTTTTTTTTTTTTTCGCCAATATTTACGTGCTGCTA-3’(SEQ IDNo.21)

Qpcr primer pair:

miR-16a-F:5’-CAGTAGCAGCACGTAAATA-3’(SEQ ID No.22)

miR-16a-R:5’-CCAGTTTTTTTTTTTTTTCGCC-3’(SEQ ID No.23)

(III) experimental results:

1. results of RT-qPCR experiments in tissues.

In order to determine the expression levels of miR-15a and miR-16a in liver cancer tissues, the mRNA levels of miR-15a and miR-16a in cancer and para-cancer tissues are respectively detected by an RT-qPCR (reverse transcription-quantitative polymerase chain reaction) experiment, and the result is shown in figure 2.

As can be seen from FIG. 2, the levels of miR-15a and miR-16a in cancer tissues are significantly lower than in non-cancer tissues, and have statistically significant differences (P < 0.0001).

2. Results of RT-qPCR experiments in cells.

The expression levels of miR-15a and miR-16a were detected using normal liver cell lines (LO2, HL7702) and liver cancer cell lines (Bel7402, SMMC7721, MHCC97L, MHCC97H, HepG2, Huh7, LM3), respectively, and the results of the experiment are shown in FIG. 3.

As can be seen from FIG. 3, the expression of miR-15a and miR-16a in the liver cancer cell line is obviously lower than that of normal cells, and the difference is statistically significant.

3. And (4) carrying out prognosis analysis.

Taking the median of miR-15a and miR-16a levels of the patients, wherein the median is miR1-L and miR-16a-L when the median is lower than the median, and the median is miR1-H and miR-16a-H when the median is higher than the miR-15a and miR-16 a; then, the miR-15a and miR-16a levels and clinical data of a patient are analyzed, the survival of the patient is analyzed by using a Kaplan-Meier statistical method, and the result is shown in figure 4, wherein miR-15a-L is miR-15a low expression; the miR-15a-H is miR-15a high expression; miR-16a-L is miR-16a low expression; the miR-16a-H is high expression of miR-16 a.

From the results of FIG. 4, it is found that the low-expression patients of miR-15a and miR-16a have the survival which is obviously lower than that of the high-expression patients of miR-15a and miR-16 a; the result body indicates that the miR-15a and miR-16a levels are closely related to the prognosis of the liver cancer patient.

The results show that miR-15a and miR-16a are low in expression in liver cancer and liver cancer cell strains, and the low expression of the miR-15a and miR-16a is related to poor prognosis of hepatocellular carcinoma patients.

3. Liver cancer early diagnosis efficiency.

As the miR-15a and miR-16a levels are closely related to the growth, invasion, metastasis and prognosis of liver cancer patients, the invention further discusses the effect of miR-15a and miR-16a on early diagnosis of liver cancer.

The invention compares the level of miR-15a and miR-16a in the peripheral blood of hepatitis (152 cases), severe hepatic fibrosis (120), liver cancer (268), and the result is shown in figure 5, and figure 5 shows the expression of miR-15a (A) and miR-16a (B) in different liver disease states.

From the results in FIG. 5, it can be found that the expression levels of miR-15a and miR-16a in patients with liver diseases are hepatitis, severe liver fibrosis and liver cancer in sequence from high to low, which indicates that miR-15a and miR-16a decrease with the disease progression.

4. Staging results of liver cancer.

According to the international liver cancer clinical stage (Barcelona stage, BCLC), 268 cases of liver cancer are divided into 75 cases in A stage (early stage), 100 cases in B stage (middle stage) and 93 cases in C stage (late stage), the peripheral blood miR-15a and miR-16a levels of patients are detected, and the difference between the groups is compared. The results are shown in FIG. 6, and FIG. 6 shows the correlation between the expression of miR-15a (A) and miR-16a (B) and the clinical staging of liver cancer.

The results in FIG. 6 show that the levels of miR-15a and miR-16a in liver tissues are significantly lower than those of non-cancer tissues around cancer, the levels of miR-15a and miR-16a in different groups of cancer tissues are far different, the level of miR-15a and miR-16a is the highest in the early stage of liver cancer, the level of miR-16a is the lowest in the late stage of liver cancer, and the average number between the groups is significantly different.

5. And (3) combining with alpha fetoprotein to carry out peripheral blood detection on the liver cancer diagnosis result.

Alpha-fetoprotein (AFP) is a common indicator for diagnosing peripheral blood of liver cancer, however, AFP can only diagnose about 60% of liver cancer patients at most, while 40% of patients have normal AFP, especially early liver cancer patients, and the negative AFP reaches 80%. In order to investigate whether miR-15a and miR-16a are superior to AFP in diagnosing liver cancer, 120 liver cancer patients with negative AFP are analyzed, the results are shown in figure 7, and figure 7 is a schematic diagram of the diagnosis efficiency of miR-15a (A) and miR-16a (B), wherein A is the relative expression quantity of miR-15a, B is the relative expression quantity of miR-16a, and C is the sensitivity and specificity analysis result.

As a result, 78 liver cancer patients with remarkably reduced miR-15a and miR-16a are found compared with non-cancer patients, namely 60% of liver cancer patients with missed AFP diagnosis can be identified by miR-15a and miR-16 a. Sensitivity and specificity analysis are further carried out on the efficiency of miR-15a, miR-16a and AFP for diagnosing liver cancer, and the joint diagnosis efficiency of the miR-15a and the miR-16a can reach 91%.

Because miR-15a and miR-16a are peripheral blood indexes and are easy to obtain clinically compared with liver tissue specimens, the method is favorable for judging the disease state and predicting the liver cancer occurrence risk by detecting miR-15a and miR-16a of liver disease patients with AFP negative.

Example 2

The miR-15a and miR-16a play a role in proliferation, clone formation and cancer cell invasion of liver cancer cells.

Research materials:

1. liver disease cases were enrolled and sampled as in example 1.

2. In vitro chemical synthesis of a mimic organism endogenous miRNA preparation miR-15a mimics (synthesized by dharmacon).

3. In vitro chemical synthesis of a mimic organism endogenous miRNA preparation miR-16a mimics (synthesized by dharmacon).

miR-15a-Inhibitor (Inhibitor of miR-15a, miR-15a-INH) (synthesized by dharmacon).

miR-16a-Inhibitor (Inhibitor of miR-16a, miR-16a-INH) (synthesized by dharmacon).

(II) research method:

1. cell proliferation assay

MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide) cell proliferation assay was performed according to the conventional procedure.

2. Plate cloning experiments were performed according to the general procedure.

3. Invasion experiments of cancer cells were performed according to conventional procedures.

(III) experimental results:

1. the result of liver cancer cell growth and invasion and metastasis.

In order to further research the influence of miR-15a and miR-16a on the activity function of the liver cancer cells, the invention utilizes in-vitro chemical synthesis preparations miR-15a micic and miR-16a mics for simulating endogenous miRNAs of organisms, and inhibitors (miR-15a-INH) and miR-16a of miR-15a, and the influence of miR-15a and miR-16a on the growth of the liver cancer cells and the invasion and metastasis of the cancer cells is identified.

The experimental results are shown in fig. 8-11, fig. 8 shows the influence of the miR-15a and miR-16a synthesizers on the liver cancer cell clone, fig. 9 shows the influence of the miR-15a and miR-16a inhibitors on the liver cancer cell clone, and it can be seen from the graphs that the growth of the liver cancer cell (SMMC-7721) is obviously inhibited after the miR-15a and miR-16a synthesizers are added, and the growth of the liver cancer cell (SMMC-7721 and BEL-7404) is promoted after the miR-15a and miR-16a inhibitors are added.

FIG. 10 shows the effect of the miR-15a and miR-16a synthesizers (A) and their inhibitors (B, C) on the proliferation of hepatoma cells, in which the OD value is shown on the ordinate for representing the density of hepatoma cells (Huh 7).

FIG. 11 shows the influence of miR-15a (A) and miR-16a (B) synthesizers on liver cancer cell invasion, the cancer cell selected in the diagram is SK-Hep1 cell line, and the diagram shows that the miR-15a and miR-16a chemical synthesizers are added to obviously inhibit the cancer cell invasion.

In the above FIG. 8-11, miR-15a-mimics and miR-16a-mimics are chemical synthesizers of miR-15a and miR-16 a; miR-15a-INH and miR-16a-INH are inhibitors of miR-15a and miR-16 a.

The results show that the miR-15a mimics and the miR-16a mimics can obviously reduce the growth speed of the liver cancer cells, inhibit the invasion of the liver cancer cells and reduce the metastasis of the liver cancer cells; on the contrary, the inhibitors miR-15a-INH and miR-16a-INH can accelerate the growth speed of the liver cancer cells, promote the invasion and metastasis of the liver cancer cells and enhance the cancer cell clone forming capability, namely, the inhibitors miR-15a and miR-16a can inhibit the proliferation, clone forming and cancer cell invasion of the liver cancer cells.

Example 3

Correlation of miR-15a and miR-16a with virological indexes of patients with chronic hepatitis B.

Research materials:

the early research of the invention incorporates 500 hepatitis B infection cases in two three hospitals in Guangdong province for analysis.

(II) research method:

according to the method in example 1, the peripheral blood miR-15a and miR-16a level of hepatitis B infected persons is detected and analyzed to be related to HBVDNA, HBeAg and HBsAg titer.

(III) experimental results:

results shown in FIG. 12, FIG. 12 shows that miR-15a (A) and miR-16a (B) are correlated with HBVDNA level, and the results show that miR-15a and miR-16a are both in negative correlation with the virology indexes, namely the higher the virus titer is, the lower miR-15a and miR-16a are.

The results show that the miR-15a and the miR-16a have a prediction effect on the antiviral efficacy of chronic hepatitis B.

Example 4

The miR-15a and miR-16a are related to the antiviral efficacy of patients with chronic hepatitis B.

Research materials:

the preliminary study of the present invention included 230 patients with chronic hepatitis B in two hospitals in Guangdong province.

(II) research method:

the course of antiviral treatment was studied longitudinally and the observation indices were: HBVDNA negative conversion efficiency at 48 weeks of treatment; the HBeAg negative conversion rate at 96 weeks of treatment; HBsAg was reduced to 50% of baseline and miR-15a and miR-16a levels were simultaneously detected at different time points (0w, 12w, 24w, 48w, 72w, 96w) in patients with chronic hepatitis B using the antiviral drug, as described in example 1.

(III) experimental results:

the results of the correlation experiments of miR-15a and miR-16a with the progress of antiviral treatment are shown in FIG. 13, and miR-15a and miR-16a gradually increase along with the antiviral treatment; the result indicates that virus infection can inhibit miR-15a and miR-16a expression, and antiviral treatment can improve miR-15a and miR-16a dysregulation.

The negative conversion of miR-15a and miR-16a and antiviral treatment patients is shown in figure 14, and the results show that the miR-15a and miR-16a levels of the negative conversion patients are obviously higher than those of the patients still positive.

Taking the median of miR-15a and miR-16a levels of the patients, wherein the median is miR1-L and miR-16a-L when the median is lower than the median, and the median is miR1-H and miR-16a-H when the median is higher than the miR-15a and miR-16 a; the relationship between baseline miR-15a and miR-16a levels and antiviral treatment of the patient is then analyzed. Wherein, the miR-15a-L is miR-15a low expression; the miR-15a-H is miR-15a high expression; miR-16a-L is miR-16a low expression; the miR-16a-H is high expression of miR-16 a. These results demonstrate that viral replication capacity is inversely correlated with miR-15a and miR-16a expression levels.

The baseline miR-15a and miR-16a levels and HBeAg negative conversion rate conditions at 96 weeks after antiviral treatment are shown in FIG. 15, and the results show that the higher the baseline miR-15a and miR-16a levels are, the better the antiviral treatment effect is, and the higher the HBeAg negative conversion rate is.

The relation between the baseline miR-15a and miR-16a and the reduction speed of the HBsAg in antiviral treatment is shown in FIG. 16, and the results show that the higher the level of the baseline miR-15a and miR-16a is, the faster the HBsAg is reduced.

The above results demonstrate that baseline miR-15a and miR-16a levels prior to antiviral treatment can predict the extent of viral antigen decline.

The results of multifactor regression analysis suggest that miR-15a and miR-16a are independent prediction factors for HBVDNA turning negative when chronic hepatitis B is treated by antiviral therapy for 48 weeks (see Table 2)

TABLE 2 antiviral treatment 48 weeks multifactorial regression analysis

The above results indicate that baseline miR-15a and miR-16a levels are independent correlation factors in HBVDNA negative reversal for 48 weeks of antiviral treatment.

The results of the multifactor regression analysis also suggest that miR-15a and miR-16a are independent prediction factors for turning HBeAg negative for 96 weeks after chronic hepatitis B is treated by antivirus (Table 3)

TABLE 3 antiviral treatment 48 weeks multifactorial regression analysis

From the results, even if the levels of the miR-15a and miR-16a of patients with similar baseline virology indexes and liver function levels are different, the treatment curative effect is also obviously different, so that the miR-15a and miR-16a detection of hepatitis patients is helpful for evaluating the curative effect of antiviral treatment.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Application of miRNA biomarker in diagnosis and prognosis of liver disease

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Claims

1. Use of a miRNA biomarker for diagnosis and/or prognostic assessment of liver disease, wherein the miRNA comprises: miR-15a and miR-16 a.

Use of a miRNA biomarker for the development and/or manufacture of a product for diagnostic and/or prognostic evaluation of liver disease, wherein the miRNA comprises: miR-15a and miR-16 a.

3. The use according to claim 2, wherein the miRNA biomarker is used in the development and/or preparation of a product for predicting anti-hepatovirus efficacy;

or

The miRNA biomarker is applied to development and/or preparation of products for diagnosing liver cancer.

4. The use of claim 2, wherein the miRNA is used as a biomarker for peripheral blood biopsy.

5. The application of a reagent for detecting miRNA markers in a biological sample in the preparation of liver cancer diagnostic reagents or diagnostic equipment is characterized in that the miRNA comprises: miR-15a and miR-16 a.

6. The use of claim 5, wherein the marker further comprises alpha-fetoprotein, and the liver cancer diagnostic reagent or diagnostic device is used for performing a liquid biopsy on peripheral blood.

7. A kit for detecting liver diseases is characterized by comprising a reagent for reverse transcription quantitative detection of miR-15a and miR-16 a.

8. The liver disease detection kit of claim 7, comprising the following reverse transcription primers:

5’-CAGGTCCAGTTTTTTTTTTTTTTTVN，

the V is selected from A or G, and the N is selected from A or T.

9. The liver disease detection kit of claim 8, further comprising the following qPCR primer pairs:

miR-15a-F:5’-TAGCAGCACATAATGGTTTGTG-3’

miR-15a-R:5’-AGGTCCAGTTTTTTTTTTTTTCAC-3’；

miR-16-F:5’-CAGTAGCAGCACGTAAATA-3’

miR-16-R:5’-CCAGTTTTTTTTTTTTTTCGCC-3’。

10. the liver disease detection kit of claim 2, wherein the liver disease comprises liver cancer and hepatitis virus infection.