LU504821B1

LU504821B1 - Diagnostic marker for liver fibrosis in patient with hepatitis b, and method and use thereof

Info

Publication number: LU504821B1
Application number: LU504821A
Authority: LU
Inventors: Yifei Li; Xian Shen; Zhichao Lang; Yating Zhan; Lifan Lin; Jianjian Zheng
Original assignee: First Affiliated Hospital Wenzhou Medical Univ
Priority date: 2023-04-19
Filing date: 2023-07-27
Publication date: 2024-01-29
Also published as: CN117778552A

Abstract

The present disclosure discloses a diagnostic marker for liver fibrosis in a patient with hepatitis B, and a method and use thereof. In the present disclosure, based on bioinformatics analysis, a key diagnostic marker ribonucleotide reductase M2 (RRM2) for liver fibrosis is found. An expression level of RRM2 in serum of a patient to be tested can be detected to allow diagnosis of a stage of fibrosis in a patient with hepatitis B. Compared with liver fibrosis indexes APRI, FIB-4, and M2BPGi, RRM2 has high diagnostic efficiency, and can allow staging diagnosis of fibrosis in a patient with hepatitis B in which an alanine transaminase (ALT) level is normal or abnormal. The detection technique established by the present disclosure can be used to determine a course of liver fibrosis in a patient with hepatitis B. In addition, the diagnostic marker disclosed in the present disclosure may be used to prepare a reagent or a kit for diagnosing liver fibrosis in a patient with hepatitis B or monitoring progression of liver fibrosis in a patient with hepatitis B, and can also be used to prepare an antiviral drug.

Description

DESCRIPTION LU504821

DIAGNOSTIC MARKER FOR LIVER FIBROSIS IN PATIENT WITH HEPATITIS B, AND

METHOD AND USE THEREOF

TECHNICAL FIELD

The present disclosure relates to the fields of biotechnology and medicine, and in particular to a diagnostic marker for liver fibrosis in a patient with hepatitis B, and a use thereof.

BACKGROUND

It is well known that hepatitis B virus (HBV)-induced hepatopathy is one of the main sources for immune liver diseases. In addition, chronic hepatitis B (CHB) contributes to the development of immune-mediated liver damage, thereby leading to progressive liver fibrosis or liver cirrhosis. Apparently, patients with hepatitis B are at a relatively high risk of developing hepatocellular carcinoma (HCC). HBV is a partially double-stranded DNA virus that infects liver cells and replicates in nuclei. In patients with hepatitis B, HBV clearance and liver damage are primarily induced by adaptive immune responses. Due to a medical burden caused by a large population of patients with hepatitis B in China, dynamic real-time monitoring should be adopted to control the progression of fibrosis.

Alanine transaminase (ALT) activity is an important reference index for treatment selection and prognosis evaluation of patients with hepatitis B. However, serum ALT levels are not always consistent with a state of liver damage. 37% of patients with hepatitis B in which an ALT level is normal have significant fibrosis and inflammation.

Currently, many patients with hepatitis B in which an ALT level is normal often miss the optimal antiviral therapy opportunity during routine examination. Therefore, there is an urgent need to find novel promising biomarkers.

The traditional liver fibrosis staging mainly relies on detection of a histopathologic, serum marker, or imaging method, but a gold standard of the staging is still liver histopathological examination with a pathological tissue acquired through liver biopsy.

Although the traditional pathological examination has a specified accuracy, the traditional pathological examination is an invasive means, which brings various inconveniences to patients, and even causes complications and sequelae such as bleeding and infection.

Compared with pathological examination, the development of serum markers facilitates,594g24 the staging diagnosis of liver fibrosis and leads to stable results. The widespread use of liver damage markers ALT and aspartate aminotransferase (AST) indicates a potential of serological markers in staging of liver fibrosis. Therefore, the search of effective non-invasive clinical serological markers is urgently needed to monitor the progression of liver fibrosis.

Ribonucleotide reductase (RR) is a multisubunit enzyme, which is a key enzyme for

DNA synthesis and repair, and plays a key role in regulation of an overall rate of DNA synthesis. An active RR complex consists of R1 and R2 subunits of a polymer, namely,

RRM1 and RRM2. Studies have shown that an increase in RRM2 activity is associated with malignant transformation, cancer cell growth, tumorigenesis, and HBV DNA replication.

RRM2 is often expressed in a disease-specific manner and thus has a potential to be a biomarker. At present, the relevant content of RRM2 in liver fibrosis in patients with hepatitis B still needs to be further investigated, and the exploration of a role of RRM2 in the occurrence and development of liver fibrosis in patients with hepatitis B is conducive to the exploration of novel biomarkers for diagnosis of liver fibrosis in patients with hepatitis B or markers for monitoring treatment responses, which plays an important role in diagnosis, treatment, and prevention of liver fibrosis in patients with hepatitis B.

SUMMARY

An objective of the present disclosure is to provide a diagnostic marker for liver fibrosis in a patient with hepatitis B, and a use thereof. The present disclosure proposes that RRM2 can be used as a diagnostic marker for staging of liver fibrosis in a patient with hepatitis B, and an expression level of RRM2 in serum of a patient to be tested can be detected to allow early diagnosis for a patient with hepatitis B. The diagnostic marker provided in the present disclosure has excellent specificity, and can accurately identify a fibrosis stage of a patient with CHB in which an ALT level is normal or abnormal. The present disclosure has advantages such as reliability and supportability, and can allow rapid screening of liver fibrosis in a patient with CHB.

To solve the above technical problems, the present disclosure provides the following technical solutions: A diagnostic marker for liver fibrosis in a patient with hepatitis B is provided, where the diagnostic marker is RRM2.

A diagnostic method using the diagnostic marker for liver fibrosis in a patient witfh,504821 hepatitis B described above is provided, where an expression level of RRM2 in serum of a patient with hepatitis B is detected to identify a stage of liver fibrosis in the patient with hepatitis B.

A use of the diagnostic marker for liver fibrosis in a patient with hepatitis B described above in preparation of a reagent or a kit for diagnosing a stage of liver fibrosis in a patient with hepatitis B is provided.

In the use, the reagent or kit includes RRM2.

A use of the diagnostic marker for liver fibrosis in a patient with hepatitis B described above in preparation of a drug for preventing or treating liver fibrosis in a patient with

CHB is provided.

In the use, the drug includes RRM2.

Compared with the prior art, the present disclosure proposes that RRM2 can be used as a diagnostic marker for staging of liver fibrosis in a patient with hepatitis B, and an expression level of RRM2 in serum of a patient to be tested can be detected to allow early diagnosis of liver fibrosis for a patient with hepatitis B. The diagnostic marker provided in the present disclosure has excellent specificity, and can accurately identify a fibrosis stage of a patient with CHB in which an ALT level is normal or abnormal. The diagnostic marker of the present disclosure has high sensitivity, standards for a detection level of the diagnostic marker are easily formulated, and a detection method of the diagnostic marker is simple, has high diagnostic efficiency and guaranteed reliability, and can allow rapid screening of liver fibrosis in a patient with CHB.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows clustering analysis results of inmune-associated subtypes in patients with hepatitis B in Example 1 of the present disclosure;

FIG. 2 shows construction results of key modules in a protein-protein interaction (PPI) network in Example 2 of the present disclosure;

FIG. 3 shows association analysis results of a module core gene RRM2 with fibrosis grading and staging in Example 3 of the present disclosure;

FIG. 4 shows association analysis results of a serum RRM2 level with liver fibrosis staging in Example 4 of the present disclosure; and

FIG. 5 shows correlation analysis results of PD-L1, CTLA4, and LAG3 between an,504821

RRM2 low expression group and an RRM2 high expression group in Example 6 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure is further described below with reference to accompanying drawings and embodiments, but the present disclosure is not limited thereto.

Example 1 Clustering analysis of immune-associated subtypes in patients with hepatitis B

Transcriptome spectra and clinical relevant data of 124 patients with hepatitis B (GSE84044) were downloaded by the inventors of the present disclosure from the Gene

Expression Omnibus (GEO) (https://www.ncbi.nim.nih.gov/) database for analysis. An "ssGSEA" package was used to calculate an ssGSEA immune score for each patient and conduct clustering analysis. In addition, a distribution of 29 immune components in immune-associated subtypes was analyzed.

Experimental results were shown in FIG. 1. The results in FIG. 1 showed that the 124 patients with hepatitis B exhibited three different immune states (Immunity_H,

Immunity_M, and Immunity_L) during evaluation of the immune components, where levels of the 29 immune components in the Immunity_H group were higher than those in the Immunity_L group.

Example 2 Construction and functional enrichment analysis of key modules in a PPI network log fold change (FC)|>1 and P < 0.05 were used by the inventors of the present disclosure as standards for differential expression analysis among immune subtypes. To investigate an interaction between differential genes, the PPI network was generated through the STRING database (https://string-db.org/), and a minimum interaction score was 0.900. In Cytoscape software, an MCODE plugin was used to identify a key module.

Experimental results were shown in FIG. 2. The results in FIG. 2 showed that 391 differential genes were detected, a PPI network with 181 nodes and 1,206 edges was generated, and two key modules were identified.

Example 3 Association analysis of a module key gene RRM2 and fibrosis grading,504824 and staging

Grouping was conducted according to staging and grading, and expression levels of serum RRM2 in patients at different stages and grades in GSE84044 were subjected to differential analysis.

Experimental results were shown in FIG. 3. The results in FIG. 3 showed that RRM2 was positively correlated with a fibrosis grade and stage.

Example 4 Association analysis of serum RRM2 with a liver fibrosis stage

Blood samples were collected by the inventors of the present disclosure from 317, 146, and 179 patients with hepatitis B in the First Affiliated Hospital of Wenzhou Medical

University (FAHWMU), the Second Affiliated Hospital of Wenzhou Medical University (SAHWMU), and the First People's Hospital of Zunyi (FPHZ), respectively. Liver biopsy was conducted for each patient, and a liver tissue sample with a length of at least 2.0 cm was collected. The research of the present disclosure was approved by ethics committees of all centers; all patients signed written informed consent forms; and all experiments were consistent with principles of the Declaration of Helsinki (DoH).

A fibrosis stage of each liver sample acquired through liver biopsy was evaluated according to Ishak fibrosis scores (FO: no fibrosis; F = 3: significant fibrosis; and F = 5: liver cirrhosis). A blood sample was centrifuged at 1,000 g for 20 min in a 4°C centrifuge, and then upper serum was collected and stored at -80°C. Relevant clinical indexes such as age, sex, body mass index (BMI), ALT, AST, alkaline phosphatase (ALP),

Platelet (PLT), and HBV DNA of the patients with hepatitis B were measured.

Relevant clinical characteristics of the patients with hepatitis B were shown in Tab|@,504824 1.

FAHWMU SAHWMU FPHZ

Total Total Total patients ALT < 40 | patients ALT < 40 | patients ALT < 40 (n=317) (n=121) | (n=146) (n=53) (n=179) (n=80)

Age 145 35 45 9 110 31 (= 45) | (45.7%) (28.9%) | (30.8%) (17.0%) | (61.5%) (38.8%)

Sex 168 61 79 25 84 39 (male) | (53.0%) (50.4%) | (54.1%) (47.2%) | (46.9%) (48.8%)

BMI 21.82 21.92 20.85 20.92 22.27 22.04 (21.59, (21.54, (20.45, (20.23, (21.87, (21.44, (kg/m?) 22.05) 22.29) 21.26) 21.62) 22.66) 22.64)

RRM2 1.62 1.60 1.86 1.52 1.80 1.92 (10910 (1.48, (1.37, (1.70, (1.43, 1.61) (1.65, 1.95) (1.81, 2.04) pg/ml) 1.76) 1.83) 2.02)

M2BP 7.05 5.35 6.11 7.42 6.25 6.76

Gi (6.30, (4.32, (5.47, (6.97, 7.86) (5.58, 6.92) (6.30, 7.22) (ng/ml) 7.81) 6.38) 6.74)

ALT 62.40 31.60 64.45 30.53 63.59 31.86 un (59.20, (30.48, (59.18, (28.56, (58.42, (30.34, 65.61) 32.73) 69.71) 32.51) 68.76) 33.38)

AST 67.20 53.84 73.78 64.63 80.23 73.44 un (63.77, (48.45, (68.80, (57.28, (75.45, (64.95, 70.63) 59.24) 78.76) 71.98) 85.00) 81.93)

ALP 86.77 86.88 88.13 86.75 77.24 76.14 un (85.07, (83.98, (85.70, (82.09, (74.83, (72.44, 88.47) 89.79) 90.57) 91.40) 79.64) 79.85)

PLT 187.68 186.41 179.84 177.05 195.87 209.60 (x102/) (181.58, (175.52, | (171.08, (161.07, | (189.17, (198.42, x 193.78) 197.29) 188.60) 193.03) | 202.57) 220.78)

HBV 6.20 6.61 5.52 6.17 6.50 5.68

DNA (5.98, (6.18, (5.30, (6.04, 6.30) (6.26, 6.74) (5.52, 5.84) (l0g10 6.41) 7.04) 5.74)

Total Total Total patients ALT < 40 | patients ALT < 40 | patients ALT < 40 (n=317) (n=121) | (n=146) (n=53) (n=179) (n=80) _mj | || {|}

Fibrosi

CP RE

77 36 26 12 22 14 87 23 39 15 34 22 65 33 37 12 40 21 15 23 9 50 15 38 14 21 5 33 8

Table 1

A serum RRM2 level in each patient with hepatitis B was quantitatively determined with an enzyme-linked immunosorbent assay (ELISA) kit. Specific experimental steps were as follows: (a): Standard wells and a sample well were set. 50 uL of a standard was added to each of the standard wells at different concentrations. (b): 50 uL of a sample to be tested was added to the sample well, and no sample was added to a blank well. (c): 100 uL of a horseradish peroxidase (HRP)-labeled detection antibody was added to each one of the standard wells and the sample well other than the blank well, reaction wells were sealed with a sealing film, and an ELISA plate was incubated in a water bath or incubator at 37°C for 60 min. (d): A liquid in each well was discarded, the ELISA plate was pat-dried on absorbent paper, each well was filled with a washing liquid (350 pL) and allowed to stand for 1 min, the washing liquid was removed, and the ELISA plate was pat-dried on absorbent paper; and this washing process was repeated 5 times (the ELISA plate may be washed with a plate washer). LU504821 (e): 50 uL of each of substrates A and B was added to each well, and the ELISA plate was incubated at 37°C in the dark for 15 min. (f): 50 pL of a stop solution was added to each well, and within 15 min, an OD value of each well was determined at a wavelength of 450 nm. (9): With an OD value of a tested standard as x-coordinate and a concentration value of a standard as y-coordinate, a standard curve was plotted on coordinate paper or with related software, and a linear regression equation was acquired; and an OD value of a sample was substituted into the equation to calculate a concentration of the sample. (h): The correlation between an RRM2 level and a stage was analyzed.

Experimental results were shown in FIG. 4. The results in FIG. 4 showed that, with an increase in a stage of liver fibrosis, a serum RRM2 level in a patient with hepatitis B in which an ALT level was normal or abnormal gradually increased.

Example 5 Diagnostic values of serum RRM2, APRI, FIB-4, and M2BPGi

An ELISA kit was used by the inventors of the present disclosure to quantitatively determine serum RRM2 and M2BPGi levels in a patient with hepatitis B in which an ALT level was normal or abnormal, and experimental steps were the same as in Example 4.

APRI and FIB-4 scores are calculated according to the following formulas, respectively:

APRI = (AST/upper normal value/PLT count (10%L)) x 100; and

FIB-4 = (age x AST)/(PLT count (10%L) x ALT?)

The diagnostic values of serum RRM2, APRI, FIB-4, and M2BPGi were evaluated through receiver operator characteristic (ROC) curve analysis.

Results of the diagnostic values of serum RRM2, APRI, FIB-4, and M2BPGi wefg,504821 shown in Table 2.

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I ELLE (mr [ees Jom joww jomm

TT jrs jem ow [wm fom (vas jomm [ee ww orm (amer Jom ow jw ow (wæwer(sams fom (ave [mw (ea oem jomm [ees Jom jem (ame [wgome ome (oem mew jm om

I CL (ue [ees Jomm Jom jon wes om fom [mm (emser own (ave [ee ew orm jon jomw we oem jew ow (wma (amet ome Jom [ew [orm Joe Jom

Table 2

It can be seen from Table 2 that, in the First Affiliated Hospital of Wenzhou Medical

University (FAHWMU), the Second Affiliated Hospital of Wenzhou Medical University (SAHWMU), and the First People's Hospital of Zunyi (FPHZ), compared with APRI,

FIB-4, and M2BPGi, serum RRM2 has a high AUC value, that is, RRM2 can well diagnose a stage of liver fibrosis in a patient with hepatitis B.

Example 6 Correlation analysis of immune checkpoints between an RRM2 loW,504821 expression group and an RRM2 high expression group

A low-risk group and a high-risk group were divided by the inventors of the present disclosure according to a median serum RRM2 value in each cohort. PD-L1, CTLA4, and

LAGS in each group were then detected by polymerase chain reaction (PCR). Specific steps were as follows:

RNA of each of PD-L1, CTLA4, and LAG3 in serum was extracted with a Trizol kit of

Thermo, and a concentration of the extracted RNA was determined by a nucleic acid quantification instrument, which needed to make an A260/280 ratio be 1.8 to 2.0. cDNA was then synthesized with a RevertAid First Strand cDNA Synthesis Kit of Thermo.

An FASTA sequence of PD-L1 was as follows: >NM_001267706.2 Homo sapiens CD274 molecule (CD274), transcript variant 2, mRNA.

AGTTCTGCGCAGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTG

CAGGGCATTCCAGAAAGA

TGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCCCC

ATACAACAAAATCAA

CCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCA

GGCTGAGGGCTACCCC

AAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGAC

CACCACCACCAATTCCA

AGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACA

ACTAATGAGATTTTCTA

CTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCAT

CCCAGAACTACCTCTG

GCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTAT

GCCTTGGTGTAGCAC

TGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTG

GCATCCAAGATACAAA

CTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAATCCAGCATTGGAA

CTTCTGATCTTCAAGCA

GGGATTCTCAACCTGTGGTTTAGGGGTTCATCGGGGCTGAGCGTGACAAGAG

GAAGGAATGGGCCCGTGG

GATGCAGGCAATGTGGGACTTAAAAGGCCCAAGCACTGAAAATGGAACCTGG

CGAAAGCAGAGGAGGAGA

ATGAAGAAAGATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTE 504801

AAATGCCTGAGGGGCTC

ATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAG

CAAATCATCCATTGCTCA

TCCTAGGAAGACGGGTTGAGAATCCCTAATTTGAGGGTCAGTTCCTGCAGAAG

TGCCCTTTGCCTCCACT

CAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTCAGTGTTGGAACGGGA

CAGTATTTATGTATGAG

TTTTTCCTATTTATTTTGAGTCTGTGAGGTCTTCTTGTCATGTGAGTGTGGTTGT

GAATGATTTCTTTTG

AAGATATATTGTAGTAGATGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAA

TGATTTGCTCACAT

CTAGTAAAACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTA

TACATTGGAAGCATA

AAGATCAAACCGTTGGTTGCATAGGATGTCACCTTTATTTAACCCATTAATACTC

TGGTTGACCTAATCT

TATTCTCAGACCTCAAGTGTCTGTGCAGTATCTGTTCCATTTAAATATCAGCTTTA

CAATTATGTGGTAG

CCTACACACATAATCTCATTTCATCGCTGTAACCACCCTGTTGTGATAACCACTA

TTATTTTACCCATCG

TACAGCTGAGGAAGCAAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAG

ACCTCAGACTGCCACC

CACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCAATTCTTTTATT

CAAAAACCATTT

ATTAAGTGCCCTTGCAATATCAATCGCTGTGCCAGGCATTGAATCTACAGATGT

GAGCAAGACAAAGTAC

CTGTCCTCAAGGAGCTCATAGTATAATGAGGAGATTAACAAGAAAATGTATTATT

ACAATTTAGTCCAGT

GTCATAGCATAAGGATGATGCGAGGGGAAAACCCGAGCAGTGTTGCCAAGAG

GAGGAAATAGGCCAATGT

GGTCTGGGACGGTTGGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTT

ACAAAGAGAGGTCG

GTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAGCATTATATTTA

TTCCTGATTTGCC

TTTGCCATATAATCTAATGCTTGTTTATATAGTGTCTGGTATTGTTTAACAGTTCTG

TCTTTTCTATTTA LU504821

AATGCCACTAAATTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCC

ATGGGAGATGGTTG

GAAAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAACT

GCTACTGCCTTTCATT

CATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAAAGCACGTATT

TTTAAAATTTTTTT

CCTAAATAGTAACACATTGTATGTCTGCTGTGTACTTTGCTATTTTTATTTATTTTA

GTGTTTCTTATAT

AGCAGATGGAATGAATTTGAAGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGT

TTCTAAGTTATCTTTC

CCATAGCTTTTCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATAT

ACATTTAGACAA

CCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTATGTTTGC

TCAAAAGGAGACCCA

TGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAATCTTATTATT

AACTCTGTATGACAG

AATCATGTCTGGAACTTTTGTTTTCTGCTTTCTGTCAAGTATAAACTTCACTTTGA

TGCTGTACTTGCAA

AATCACATTTTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCC

TGTGCCAGAAAAGCCT

CATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACACAGCC

CTCCTAAGAGGCTTCCT

GGAGGTTTCGAGATTCAGATGCCCTGGGAGATCCCAGAGTTTCCTTTCCCTCT

TGGCCATATTCTGGTGT

CAATGACAAGGAGTACCTTGGCTTTGCCACATGTCAAGGCTGAAGAAACAGTG

TCTCCAACAGAGCTCCT

TGTGTTATCTGTTTGTACATGTGCATTTGTACAGTAATTGGTGTGACAGTGTTCT

TTGTGTGAATTACAG

GCAAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCC

TAACTCCTCCTTGTGG

TGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAATGGC

ATAGGCAGAGATGAT

ACCTAATTCTGCATTTGATTGTCACTTTTTGTACCTGCATTAATTTAATAAAATATT

CTTATTTATTTTG

TTACTTGGTACACCAGCATGTCCATTTTCTTGTTTATTTTGTGTTTAATAAAATGT, U504821

TCAGTTTAACATCC

CA

An FASTA sequence of CTLA4 was as follows: >NM_001037631.3 Homo sapiens cytotoxic T lymphocyte-associated protein 4 (CTLA4), transcript variant 2, mRNA.

GCTTTCTATTCAAGTGCCTTCTGTGTGTGCACATGTGTAATACATATCTGGGATC

AAAGCTATCTATATA

AAGTCCTTGATTCTGTGTGGGTTCAAACACATTTCAAAGCTTCAGGATCCTGAA

AGGTTTTGCTCTACTT

CCTGAAGACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAG

CGGCACAAGGCTCAGCT

GAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCAT

CCCTGTCTTCTGCAAA

GCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCG

CCAGCTTTGTGTGTGAGT

ATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGC

TGACAGCCAGGTGACTGA

AGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTC

CATCTGCACGGGCACC

TCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACAC

GGGACTCTACATCTGCA

AGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACC

CAGATTTATGTAATTGC

TAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACA

GAGCCAGAATGTGAAAA

GCAATTTCAGCCTTATTTTATTCCCATCAATTGAGAAACCATTATGAAGAAGAGA

GTCCATATTTCAATT

TCCAAGAGCTGAGGCAATTCTAACTTTTTTGCTATCCAGCTATTTTTATTTGTTTG

TGCATTTGGGGGGA

ATTCATCTCTCTTTAATATAAAGTTGGATGCGGAACCCAAATTACGTGTACTACAA

TTTAAAGCAAAGGA

GTAGAAAGACAGAGCTGGGATGTTTCTGTCACATCAGCTCCACTTTCAGTGAA

AGCATCACTTGGGATTA

ATATGGGGATGCAGCATTATGATGTGGGTCAAGGAATTAAGTTAGGGAATGGCA

CAGCCCAAAGAAGGAA LU504821

AAGGCAGGGAGCGAGGGAGAAGACTATATTGTACACACCTTATATTTACGTATG

AGACGTTTATAGCCGA

AATGATCTTTTCAAGTTAAATTTTATGCCTTTTATTTCTTAAACAAATGTATGATTA

CATCAAGGCTTCA

AAAATACTCACATGGCTATGTTTTAGCCAGTGATGCTAAAGGTTGTATTGCATATA

TACATATATATATA

TATATATATATATATATATATATATATATATATATATATATATATTTTAATTTGATAGTATTG

TGCATAG

AGCCACGTATGTTTTTGTGTATTTGTTAATGGTTTGAATATAAACACTATATGGCA

GTGTCTTTCCACCT

TGGGTCCCAGGGAAGTTTTGTGGAGGAGCTCAGGACACTAATACACCAGGTA

GAACACAAGGTCATTTGC

TAACTAGCTTGGAAACTGGATGAGGTCATAGCAGTGCTTGATTGCGTGGAATTG

TGCTGAGTTGGTGTTG

ACATGTGCTTTGGGGCTTTTACACCAGTTCCTTTCAATGGTTTGCAAGGAAGCC

ACAGCTGGTGGTATCT

GAGTTGACTTGACAGAACACTGTCTTGAAGACAATGGCTTACTCCAGGAGACC

CACAGGTATGACCTTCT

AGGAAGCTCCAGTTCGATGGGCCCAATTCTTACAAACATGTGGTTAATGCCATG

GACAGAAGAAGGCAGC

AGGTGGCAGAATGGGGTGCATGAAGGTTTCTGAAAATTAACACTGCTTGTGTT

TTTAACTCAATATTTTC

CATGAAAATGCAACAACATGTATAATATTTTTAATTAAATAAAAATCTGTGGTGGT

CGTTTTCCGGA

An FASTA sequence of LAG3 was follows: >NM_001414176.1 Homo sapiens lymphocyte activation gene 3 (LAGS), transcript variant 2, mRNA.

AGAGACCAGCAGAACGGCATCCCAGCCACGACGGCCACTTTGCTCTGTCTGC

TCTCCGCCACGGCCCTGC

TCTGTTCCCTGGGACACCCCCGCCCCCACCTCCTCAGGCTGCCTGATCTGCC

CAGCTTTCCAGCTTTCCT

CTGGATTCCGGCCTCTGGTCATCCCTCCCCACCCTCTCTCCAAGGCCCTCTCC

TGGTCTCCCTTCTTCTA

GAACCCCTTCCTCCACCTCCCTCTCTGCAGAACTTCTCCTTTACCCCCCACCC

CCCACCACTGCCCCCTT LU504821

TCCTTTTCTGACCTCCTTTTGGAGGGCTCAGCGCTGCCCAGACCATAGGAGAG

ATGTGGGAGGCTCAGTT

CCTGGGCTTGCTGTTTCTGCAGCCGCTTTGGGTGGCTCCAGTGAAGCCTCTC

CAGCCAGGGGCTGAGGTC

CCGGTGGTGTGGGCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCC

ACAATCCCCCTCCAGGATC

TCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGACAGTGG

CCCGCCCGCTGCCGCCCC

CGGCCATCCCCTGGCCCCCGGCCCTCACCCGGCGGCGCCCTCCTCCTGGGG

GCCCAGGCCCCGCCGCTAC

ACGGTGCTGAGCGTGGGTCCCGGAGGCCTGCGCAGCGGGAGGCTGCCCCT

GCAGCCCCGCGTCCAGCTGG

ATGAGCGCGGCCGGCAGCGCGGGGACTTCTCGCTATGGCTGCGCCCAGCCC

GGCGCGCGGACGCCGGCGA

GTACCGCGCCGCGGTGCACCTCAGGGACCGCGCCCTCTCCTGCCGCCTCCG

TCTGCGCCTGGGCCAGGCC

TCGATGACTGCCAGCCCCCCAGGATCTCTCAGAGCCTCCGACTGGGTCATTTT

GAACTGCTCCTTCAGCC

GCCCTGACCGCCCAGCCTCTGTGCATTGGTTCCGGAACCGGGGCCAGGGCC

GAGTCCCTGTCCGGGAGTC

CCCCCATCACCACTTAGCGGAAAGCTTCCTCTTCCTGCCCCAAGTCAGCCCCA

TGGACTCTGGGCCCTGG

GGCTGCATCCTCACCTACAGAGATGGCTTCAACGTCTCCATCATGTATAACCTC

ACTGTTCTGGGTCTGG

AGCCCCCAACTCCCTTGACAGTGTACGCTGGAGCAGGTTCCAGGGTGGGGCT

GCCCTGCCGCCTGCCTGC

TGGTGTGGGGACCCGGTCTTTCCTCACTGCCAAGTGGACTCCTCCTGGGGGA

GGCCCTGACCTCCTGGTG

ACTGGAGACAATGGCGACTTTACCCTTCGACTAGAGGATGTGAGCCAGGCCCA

GGCTGGGACCTACACCT

GCCATATCCATCTGCAGGAACAGCAGCTCAATGCCACTGTCACATTGGCAATCA

TCACAGTGACTCCCAA

ATCCTTTGGGTCACCTGGATCCCTGGGGAAGCTGCTTTGTGAGGTGACTCCAG

TATCTGGACAAGAACGC

TTTGTGTGGAGCTCTCTGGACACCCCATCCCAGAGGAGTTTCTCAGGACCTTG 504821

GCTGGAGGCACAGGAGG

CCCAGCTCCTTTCCCAGCCTTGGCAATGCCAGCTGTACCAGGGGGAGAGGCT

TCTTGGAGCAGCAGTGTA

CTTCACAGAGCTGTCTAGCCCAGGTGCCCAACGCTCTGGGAGAGCCCCAGGT

GCCCTCCCAGCAGGCCAC

CTCCTGCTGTTTCTCATCCTTGGTGTCCTTTCTCTGCTCCTTTTGGTGACTGGA

GCCTTTGGCTTTCACC

TTTGGAGAAGACAGGTGAGCCAGGGACATGGCAACCCCGCCCCCCAGCAGC

TCCCGCTCTTCCATCCTCA

GAGTGCTGATGGCACCCCTTCCTCAGGAAGTGGCGACCAAGACGATTTTCTG

CCTTAGAGCAAGGGATTC

ACCCTCCGCAGGCTCAGAGCAAGATAGAGGAGCTGGAGCAAGAACCGGAGC

CGGAGCCGGAGCCGGAACC

GGAGCCCGAGCCCGAGCCCGAGCCGGAGCAGCTCTGACCTGGAGCTGAGG

CAGCCAGCAGATCTCAGCAG

CCCAGTCCAAATAAACTCCCTGTCAGCAGCAA

An SYBR Green method was used to conduct real-time fluorescent quantitative

PCR. With GAPDH as an internal reference gene, PD-L1, CTLA4, and LAG3-specific

RT-PCR primers were used to conduct real-time fluorescent quantitative PCR to obtain relative expression levels of PD-L1, CTLA4, and LAG3.

PD-L1: RT-PCR forward primer sequence (F: CATTTGCTGAACGCCCCATA) and reverse primer sequence (R: TGTCCAGATGACTTCGGCCT).

CTLA4: RT-PCR forward primer sequence (F: CCATGGACACGGGACTCTAC) and reverse primer sequence (R: GGGCTTCTTTTCTTTAGCAATTACA).

LAG3: RT-PCR forward primer sequence (F: CAGCTCCCGCTCTTCCATC) and reverse primer sequence (R: AATCGTCTTGGTCGCCACTTC).

GAPDH: forward primer sequence (F: GCTAGGGACGGCCTGAAG) and reverse primer sequence (R: GCCCAATACGACCAAATCCGT).

A real-time fluorescent quantitative PCR system was 20 pL in total, including: 10 uL of SYBR Green Realtime PCR Master Mix, 0.5 pL of a forward primer (10 uM), 0.5 uL of a reverse primer (10 uM), 1 uL of cDNA, and the balance of ddH2O.

A reaction system prepared according to the above system was placed in a 7500504821

Fast quantitative PCR System instrument to conduct PCR, and a PCR procedure was shown in Table 3: me ee mem

Cee ee

Table 3

Real time-PCR experimental result analysis: The analysis was conducted with software in the 7500 Fast quantitative PCR System instrument, and analysis results were expressed as mean + standard deviation (SD). The GraphpadPrism software was used to analyze the data differences among different samples and plot a curve (the student T-test was used to statistically analyze a difference between two groups of samples; and P < 0.05, indicating that the difference was statistically significant).

Experimental results were shown in FIG. 5. The results in FIG. 5 showed that a patient with hepatitis B in which an RRM2 level was high was associated with high immune checkpoint levels (PD-L1, CTLA4, and LAG3), that is, a patient with CHB in which an RRM2 level was high may benefit from immunotherapy.

The results in the above examples show that, in the present disclosure, based on bioinformatics analysis, a key diagnostic marker RRM2 for liver fibrosis is found. An expression level of RRM2 in serum of a patient to be tested can be detected to allow diagnosis of a stage of fibrosis in a patient with hepatitis B. Compared with liver fibrosis indexes APRI, FIB-4, and M2BPGi, RRM2 has high diagnostic efficiency, and can allow staging diagnosis of fibrosis in a patient with hepatitis B in which an ALT level is normal or abnormal.

In summary, in the present disclosure, RRM2 is used as a diagnostic marker for staging of liver fibrosis in a patient with hepatitis B, and an expression level of RRM2 in serum of a patient to be tested is detected to allow early diagnosis for a patient with hepatitis B.

The diagnostic marker provided in the present disclosure has excellent specificity, 594821 and can accurately identify a fibrosis stage of a patient with hepatitis B in which an ALT level is normal or abnormal.

The diagnostic marker of the present disclosure has high sensitivity, standards for a detection level of the diagnostic marker are easily formulated, and a detection method of the diagnostic marker is simple, has high diagnostic efficiency and guaranteed reliability, and can allow rapid screening of liver fibrosis in a patient with hepatitis B.

The detection technique established by the present disclosure can be used to determine a course of liver fibrosis in a patient with hepatitis B.

In addition, the diagnostic marker disclosed in the present disclosure may be used to prepare a reagent or a kit for diagnosing liver fibrosis in a patient with hepatitis B or monitoring progression of liver fibrosis in a patient with hepatitis B, and can also be used to prepare an antiviral drug.

SEQUENCE LISTING LU504821 <ST26SequenceListing dtdVersion="V1_3" fleName="DIAGNOSTIC MARKER FOR

LIVER FIBROSIS IN PATIENT WITH HEPATITIS B, AND METHOD AND USE

THEREOF xml" softwareName="WIPO

Sequence" softwareVersion="2.2.0" productionDate="2023-04-18"> <ApplicantFileReference>The First Affiliated Hospital of Wenzhou Medical

University</ApplicantFileReference> <ApplicantName languageCode="zh">The First Affiliated Hospital of Wenzhou Medical

University</ApplicantName> <ApplicantNameLatin>The First Affiliated Hospital of Wenzhou Medical

University</ApplicantNameLatin> <InventionTitle languageCode="zh">DIAGNOSTIC MARKER FOR LIVER FIBROSIS IN

PATIENT WITH HEPATITIS B, AND METHOD AND USE THEREOF</InventionTitle> <SequenceTotalQuantity>11</SequenceTotalQuantity> <SequenceData sequencelDNumber="1"> <INSDSeq> <INSDSeq_length>3292</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..3292</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>mRNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q2"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>agttctgcgcagetteccgagactecgcaccagecgcacttctgtecgccetgcagggcat tccagaaagatgaggatatttgctgtctttatattcatgacctactggcatttgctgaacgccccatacaacaaaatcaacca aagaattttggttgtggatccagtcacctctgaacatgaactgacatgtcaggctgagggctaccccaaggccgaagtcat ctggacaagcagtgaccatcaagtcctgagtggtaagaccaccaccaccaattccaagagagaggagaagcttticaat gtgaccagcacactgagaatcaacacaacaactaatgagatttictactgcacttttaggagattagatcctgaggaaaac catacagctgaattggtcatcccagaactacctctggcacatcctccaaatgaaaggactcacttggtaattictgggagec atcttattatgccttggtgtagcactgacattcatcticcgtttaagaaaagggagaatgatggatgtgaaaaaatgtggcatc caagatacaaactcaaagaagcaaagtgatacacatttggaggagacgtaatccagcattggaacttctgatcttcaage agggattctcaacctgtggtttaggggttcatcggggctgagcgtgacaagaggaaggaatgggeccgtgggatgcagg caatgtgggacttaaaaggcccaagcactgaaaatggaacctggcgaaagcagaggaggagaatgaagaaagatg gagtcaaacagggagcctggagggagaccttgatactttcaaatgcctgaggggctcatcgacgectgtgacagggag 504821 aaaggatacttctgaacaaggagcctccaagcaaatcatccattgctcatcctaggaagacgggttgagaatccctaattt gagggtcagttcctgcagaagtgcecctttgecctccactcaatgectcaatttgttitctgcatgactgagagtctcagtgttgga acgggacagtatttatgtatgagtttttcctatttattttgagtctgtgaggtctictigtcatgtgagtgtggttgtgaatgatticttttg aagatatattgtagtagatgttacaattttgtcgccaaactaaacttgctgcttaatgatttgctcacatctagtaaaacatgga gtatttgtaaggtgcttggtctcctctataactacaagtatacattggaagcataaagatcaaaccgttggtigcataggatgtc acctttatttaacccattaatactctggttgacctaatcttatictcagacctcaagtgtctgtgcagtatctgticcatttaaatatc agctttacaattatgtggtagcctacacacataatctcatttcatcgctgtaaccaccctgttgtgataaccactattattttaccc atcgtacagctgaggaagcaaacagattaagtaacttgcccaaaccagtaaatagcagacctcagactgccacccact gtccttttataatacaatttacagctatattttactttaagcaattcttttattcaaaaaccatttattaagtgcccttgcaatatcaat cgctgtgccaggcattgaatctacagatgtgagcaagacaaagtacctgtcctcaaggagctcatagtataatgaggaga ttaacaagaaaatgtattattacaatttagtccagtgtcatagcataaggatgatgcgaggggaaaacccgagcagtgttg ccaagaggaggaaataggccaatgtggtctgggacggttggatatacttaaacatcttaataatcagagtaattticatttac aaagagaggtcggtacttaaaataaccctgaaaaataacactggaattccttitctagcattatatttattcctgatttgectttg ccatataatctaatgcttgtttatatagtgtctggtattgttitaacagttctgtctttictatttaaatgccactaaattttaaattcatac ctttccatgattcaaaattcaaaagatcccatgggagatggttggaaaatctccacttcatcctccaagecattcaagtttectt tccagaagcaactgctactgcctttcattcatatgtictictaaagatagtctacatttggaaatgtatgttaaaagcacgtatttit aaaatttttttcctaaatagtaacacattgtatgtctgctgtgtactttgctatttttatttattttagtgtticttatatagcagatggaat gaatttgaagttcccagggctgaggatccatgccttctttgttictaagttatctttcccatagcttticattatctttcatatgatcca gtatatgttaaatatgtcctacatatacatttagacaaccaccatttgttaagtatttgctctaggacagagtttggatttgtttatgt ttgctcaaaaggagacccatgggctctccagggtgcactgagtcaatctagtcctaaaaagcaatcttattattaactctgtat gacagaatcatgtctggaacttttgttttctgctttctgtcaagtataaacttcactttgatgctgtactigcaaaatcacattttcttt ctggaaattccggcagtgtaccttgactgctagctaccctgtgccagaaaagcectcattcgttgtgettgaacccttgaatgece accagctgtcatcactacacagccctcctaagaggcttcctggaggtttcgagattcagatgecctgggagatcccagagtt tectttccctcttggccatatictggtgtcaatgacaaggagtacettggctttgccacatgtcaaggctgaagaaacagtatct ccaacagagctccttgtgttatctgtttgtacatgtgcatttgtacagtaattggtgtgacagtgttcttigtgtgaattacaggcaa gaattgtggctgagcaaggcacatagtctactcagtctattcctaagtcctaactcctecttgtggtgttggatttgtaaggcact ttatcccttttgtctcatgtttcatcgtaaatggcataggcagagatgatacctaattctgcatttgattgtcacttttigtacctgcat taatttaataaaatattcttatttattttgttacttggtacaccagcatgtccattttctigtttattttgtgtitaataaaatgttcagtttaa catccca</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="2"> <INSDSeq> <INSDSeq_length>1887</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..1887</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>mRNA</INSDQualifier_value> </INSDQualifier>

<INSDQualifier id="q4"> LU504821 <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>gctttctattcaagtgccttctgtgtgtgcacatgtgtaatacatatctgggatcaaagctatct atataaagtccttgattctgtgtgggttcaaacacatttcaaagcttcaggatcctgaaaggttttgctctacttcctgaagacct gaacaccgctcccataaagccatggcttgecttggatttcagcggcacaaggcetcagetgaacctggcetaccaggacctg gccctgceactctcectgttttttctictcticatcectgtetictgcaaagcaatgcacgtggeccagectgetgtggtactggecag cagccgaggcatcgecagctttgtgtgtgagtatgcatctccaggcaaagecactgaggtccgggtgacagtgcettcgge aggctgacagccaggtgactgaagtctgtgcggcaacctacatgatggggaatgagttgaccttcctagatgattccatct gcacgggcacctccagtggaaatcaagtgaacctcactatccaaggactgagggccatggacacgggactctacatct gcaaggtggagctcatgtacccaccgccatactacctgggcataggcaacggaacccagatttatgtaattgctaaagaa aagaagccctcttacaacaggggtctatgtgaaaatgcccccaacagagccagaatgtgaaaagcaatttcagecttattt tattoccatcaattgagaaaccattatgaagaagagagtccatatttcaatttccaagagctgaggcaattctaacttttttgct atccagctatttttatttgtttgtgcatttggggggaattcatctctctitaatataaagttggatgcggaacccaaattacgtgtact acaatttaaagcaaaggagtagaaagacagagctgggatgtttctgtcacatcagctccactttcagtgaaagcatcactt gggattaatatggggatgcagcattatgatgtgggtcaaggaattaagttagggaatggcacagcccaaagaaggaaa aggcagggagcgagggagaagactatattgtacacaccttatatttacgtatgagacgtttatagccgaaatgatcitttica agttaaattttatgccttttatticttaaacaaatgtatgattacatcaaggcttcaaaaatactcacatggctatgttttagccagt gatgctaaaggttgtattgcatatatacatatatatatatatatatatatatatatatatatatatatatatatatatatatattttaattt gatagtattgtgcatagagccacgtatgtttttgtgtattigttaatggtitgaatataaacactatatggcagtgtctttccaccttg ggtcccagggaagttttgtggaggagctcaggacactaatacaccaggtagaacacaaggtcatttgctaactagcettgg aaactggatgaggtcatagcagtgcttgattgcgtggaattgtgctgagttggtgtigacatgtgcetttggggcttitacaccag ttcctttcaatggtttgcaaggaagccacagcetggtggtatctgagttgacttgacagaacactgtctigaagacaatggctta ctccaggagacccacaggtatgaccttctaggaagctccagttcgatgggcccaattcttacaaacatgtggttaatgccat ggacagaagaaggcagcaggtggcagaatggggtgcatgaaggtttctgaaaattaacactgcttgtgtttttaactcaat attttccatgaaaatgcaacaacatgtataatatttttaattaaataaaaatctgtggtggtcgttttccgga</INSDSeq_s equence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="3"> <INSDSeq> <INSDSeq_length>2062</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..2062</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>mRNA</INSDQualifier_value>

</INSDQualifier> LU504821 <INSDQualifier id="q6"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>agagaccagcagaacggcateccagccacgacggccactttgetctatctgctetccgc cacggacectactctgttecctgggacacececgececcacetectcagactgectgatctgcccagetttccagetttectctg gattecggectctggtcateectececcacecteteteccaaggcectctectggteteccttcttctagaaccecttectecacctc cctetetgcagaacttctcctttaccccccaccecccaccactgcecccctttecttttctgacctecttttggagggcetcagegcet gcccagaccataggagagatgtgggaggctcagttcetgggcettgetgttictgcagecgcetttgggtggetccagtgaage ctctccagecaggggcetgaggteceecggtggtgtgggeccaggagggggctectgeccagcetcecctgcagecccacaat ceccctccaggatctcagecttetgcgaagagcagggatcacttggcagcatcagecagacagtggeccgeecactgec gcceccgaccateccctggececcgacectcacecggegacacectectectgggggeccaggececgecactacacg gtgctgagcatggateccggaggcetgcgcagegggagactgecectgcagecccgegtecagetggatgagegegg ccggcagcgcggggacticicactatggctgcgeccageccggcgcgcggacgecggegagtaccgcgecgegatgc acctcagggacegcacectetectgccgectecgtetgcgccetgggccaggectegatgactgccagececccaggatet ctcagagectccgactggatcattttgaactactcettcagccgecctgaccgcccagectctatgcattggttecggaaccg gggccagggecgagtecctatecgggagtececccatcaccacttageggaaagcticctettectgceccaagtcagec ccatggactctgggcectgggactgcatectcacctacagagatggettcaacgtctccatcatgtataacctcactattctg gatctggageccccaacteccettgacagtgatacgctggagcagaticcaggatggggcetgcectgecacetgectactgat gtggggacecggtetttcctcactgccaagtagactectectgggggaggecctgacctectggtgactggagacaatggce gactttacccttcgactagaggatgtgagccaggeccaggcetgggacctacacctgccatatccatctgcaggaacagea gctcaatgccactgtcacattggcaatcatcacagtgactcccaaatcctttgggtcacctggatccctggggaagcetgcttt gtgaggtgactccagtatctggacaagaacgctttgtgtggagctctctggacaccccatcccagaggagttictcaggac cttggctggaggcacaggaggcccagctcctttcccagecttggcaatgecagcetgtaccagggggagaggcttcttgga gcagcagtgtacttcacagagctgtctagcccaggtgcccaacgcetctgggagagcecccaggtgecctcccagcaggec acctcctgctgtttctcatccttggtgtectttctetgetectttiggtgactggagcectttggcttticacctttggagaagacaggtg agccagggacatggcaaccccgcecccccagcagctcccgctctticcatcctcagagtgetgatggcacccecttcctcagg aagtggcgaccaagacgattttctgccttagagcaagggattcaccctecgcaggctcagagcaagatagaggagetgg agcaagaaccggagecggagecggagecggaaccggageccgageccgageccgagecggagcagetetgacot ggagctgaggcagccagcagatctcagcageccagtccaaataaactecctgtcagcagcaa</INSDSeq_sequ ence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="4"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals> LU504821 <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q8"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>catttgctgaacgcecccata</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="5"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q10"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>tgtccagatgacttcggcct</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="6"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature>

<INSDFeature_key>source</INSDF eature_key> LU504821 <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q12"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>ccatggacacgggactctac</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="7"> <INSDSeq> <INSDSeq_length>25</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..25</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSD Qualifier_value> </INSDQualifier> <INSDQualifier id="q14"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>gggcttctttictttagcaattaca</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="8"> <INSDSeq> <INSDSeq_length>19</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table> LU504821 <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..19</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSD Qualifier_value> </INSDQualifier> <INSDQualifier id="q16"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>cagcteccactcttccatc</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="9"> <INSDSeq> <INSDSeq_length>21</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..21</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q18"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>aatcgtcttggtcgccacttc</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="10"> <INSDSeq> <INSDSeq_length>18</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype> LU504821 <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..18</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q20"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>gctagggacggcctgaag</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="11"> <INSDSeq> <INSDSeq_length>21</INSDSeq_length> <INSDSeq_moltype>RNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..21</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other RNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q22"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>unidentified</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>gcccaatacgaccaaatccgt</INSDSeq_sequence> </INSDSeq> </SequenceData> </ST26SequenceListing>

Claims

CLAIMS LU504821

1. A diagnostic marker for liver fibrosis in a patient with hepatitis B, wherein the diagnostic marker is ribonucleotide reductase M2 (RRM2).

2. A method for diagnosing a stage of liver fibrosis in a patient with hepatitis B using the diagnostic marker for liver fibrosis in a patient with hepatitis B according to claim 1, wherein an expression level of RRM2 in serum of the patient with hepatitis B is detected to identify the stage of liver fibrosis in the patient with hepatitis B.

3. A use of the diagnostic marker for liver fibrosis in a patient with hepatitis B according to claim 1 in preparation of a reagent or a kit for diagnosing a stage of liver fibrosis in a patient with hepatitis B.

4. The use according to claim 3, wherein the reagent or kit comprises RRM2.

5. A use of the diagnostic marker for liver fibrosis in a patient with hepatitis B according to claim 1 in preparation of a drug for preventing or treating liver fibrosis in a patient with chronic hepatitis B (CHB).

6. The use according to claim 5, wherein the drug comprises RRM2.