CN112877440A

CN112877440A - Application of biomarker in prediction of liver cancer recurrence

Info

Publication number: CN112877440A
Application number: CN202110426456.4A
Authority: CN
Inventors: 汪丽燕; 李滨
Original assignee: Affiliated Hospital of Guilin Medical University
Current assignee: Affiliated Hospital of Guilin Medical University
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2021-06-01
Anticipated expiration: 2041-04-20
Also published as: CN112877440B

Abstract

The invention discloses application of biomarkers in predicting liver cancer recurrence, wherein the biomarkers comprise SORBS3, DDX56 and EID 2B. The invention provides application of a reagent for detecting the expression level of the biomarker in preparing a product for predicting liver cancer recurrence. The invention also provides a system for predicting recurrence of liver cancer.

Description

Application of biomarker in prediction of liver cancer recurrence

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a biomarker in prediction of liver cancer recurrence.

Background

The liver is one of the most important organs for maintaining the stable environment and the health of the organism in the organism, and the fatty liver, the hepatitis, the cirrhosis and the liver cancer are the four most common serious diseases in the liver and are also one of the important reasons for harming the health of human beings. In fact, approximately one million people die each year from cirrhosis and liver cancer. Particularly, liver cancer, the most common primary malignancy of the liver, is the leading cause of death among various diseases in humans due to high morbidity, difficulty in finding, few treatment methods, and the like. Currently, liver cancer ranks fifth in terms of tumor fatality rate worldwide, while in some african and asian countries, liver cancer is already the first in neoplastic causes of death.

After decades of efforts, research on liver cancer has been greatly progressed, and the early treatment of small liver cancer and the second resection of narrowed liver cancer are taken as marks in the last century, which respectively contribute 10 percent to the improvement of the survival rate of liver cancer after operation, but the overall curative effect of liver cancer is still poor due to the rapid progression of the disease course of liver cancer and extremely high recurrence rate, and the overall survival rate of liver cancer people in 5 years is still only about 5 percent. In recent years, although some progress has been made in the basic and clinical research of liver cancer, the mechanism of liver cancer recurrence has not been clarified, and effective intervention measures have not been found. The high recurrence rate of liver cancer is the bottleneck affecting the improvement of the curative effect.

The biomarker is an indicator capable of objectively measuring and evaluating normal biological processes, pathological processes or drug intervention reactions, is also an important early warning index when an organism is damaged, and relates to the change of the molecular structure and function of cells, the change of biochemical metabolic processes, abnormal expression of physiological activities, abnormal change of individuals, groups or the whole ecological system and the like. The research of the biomarker is not only an important content of biochemical basic research, but also has important value in the aspects of new drug development, medical diagnosis and clinical research, is helpful for helping researchers to provide more effective diagnosis and treatment means, and particularly has important value in the prevention and control of chronic diseases and complex diseases such as tumors, cardiovascular diseases, diabetes, neurological disorders and the like. Therefore, the method for searching the biomarkers related to the liver cancer recurrence can provide a new method for further reducing the clinical liver cancer recurrence rate and the clinical liver cancer mortality rate.

Disclosure of Invention

One of the objects of the present invention is to provide a biomarker for predicting recurrence of liver cancer.

The second purpose of the invention is to provide a medicine for preventing and/or treating liver cancer recurrence.

The invention also aims to provide a system for predicting recurrence of liver cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a reagent capable of specifically detecting the expression level of a biomarker comprising SORBS3, DDX56 and/or EID 2B.

Further, the biomarkers include EID2B and sobbs 3.

Further, the biomarkers include EID2B and DDX 56.

Further, the biomarkers include a combination of SORBS3, DDX56, and EID 2B.

Further, the reagent comprises a reagent for detecting the expression level of the biomarker by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology and a protein immunization technology.

Further, the reagent comprises a primer, a probe, an antibody and a ligand.

In a second aspect, the invention provides a kit comprising the reagents of the first aspect of the invention.

Further, the kit further comprises one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

In a third aspect, the invention provides a chip comprising a reagent according to the first aspect of the invention.

In a fourth aspect, the present invention provides a composition comprising one or more of the following agents:

(1) an agent that promotes expression of SORBS 3;

(2) an agent that inhibits the expression of DDX 56;

(3) an agent that inhibits expression of EID 2B.

Further, the compositions include an agent that inhibits expression of EID2B and an agent that promotes expression of SORBS 3.

Further, the composition comprises an agent that inhibits expression of EID2B and an agent that inhibits expression of DDX 56.

Further, the composition comprises an agent that promotes the expression of SORBS3, an agent that inhibits the expression of DDX56, and an agent that inhibits the expression of EID 2B.

In a fifth aspect, the present invention provides a system for predicting recurrence of liver cancer, comprising:

(1) a liver cancer recurrence evaluation device including a control unit and a storage unit for evaluating whether a subject is at risk of liver cancer recurrence; and

(2) information communication terminal devices communicatively connected to each other, which provide data on the expression level of the biomarker from the subject;

wherein the control unit of the liver cancer recurrence evaluation device comprises:

1) a data receiving unit that receives data on the expression level of the biomarker transmitted from the information communication terminal device;

2) a discrimination value calculation unit that calculates a discrimination value based on the biomarker expression level received by the data reception unit and a discrimination with the biomarker expression level stored in the storage unit as an explanatory variable;

3) a discrimination value criterion evaluation unit that evaluates the risk of liver cancer recurrence in the subject based on the discrimination value calculated by the discrimination value calculation unit; and

4) an evaluation result transmitting unit that transmits the evaluation result of the subject obtained by the discrimination value reference evaluation unit to the information communication terminal device;

the biomarkers include SORBS3, DDX56, and/or EID 2B.

Further, the biomarkers include EID2B and sobbs 3.

Further, the biomarkers include EID2B and DDX 56.

Further, the biomarkers include a combination of SORBS3, DDX56, and EID 2B.

In a sixth aspect, the invention provides the use of biomarkers comprising SORBS3, DDX56 and/or EID2B in the construction of a computational model or a system incorporating said computational model for the prediction of liver cancer recurrence.

Further, the biomarkers include EID2B and sobbs 3.

Further, the biomarkers include EID2B and DDX 56.

Further, the biomarkers include a combination of SORBS3, DDX56, and EID 2B.

A seventh aspect of the invention provides the use of any one of:

(1) the application of the reagent of the first aspect of the invention in preparing a product for predicting recurrence of liver cancer;

(2) the kit of the second aspect of the invention is applied to the preparation of products for predicting recurrence of liver cancer;

(3) the chip of the third aspect of the invention is applied to the preparation of products for predicting the recurrence of liver cancer;

(4) the composition of the fourth aspect of the invention is applied to the preparation of a medicament for preventing and/or treating liver cancer recurrence.

The medicament also comprises a pharmaceutically acceptable carrier. The active ingredient, the composition according to the fourth aspect of the present invention, is prepared into the medicament of the present invention by a conventional pharmaceutical process together with a pharmaceutically acceptable carrier.

The invention has the advantages and beneficial effects that:

the invention can effectively predict the recurrence of the liver cancer by detecting the biomarkers, and has better clinical application prospect.

The invention also provides a medicine for preventing and/or treating the recurrence of the liver cancer. The invention is reasonably and effectively applied, and can reduce the recurrence rate and the fatality rate of clinical liver cancer.

Drawings

FIG. 1 is a ROC plot of the joint diagnosis in the training set of SORBS3, DDX56, and EID2B for predicting recurrence of liver cancer.

FIG. 2 is a ROC plot of the joint diagnosis prediction of liver cancer recurrence in validation set for SORBS3, DDX56, and EID 2B.

Detailed Description

Nucleic acids and proteins

"SORBS 3" as used herein refers to a nucleic acid encoding all or part of the SORBS3 protein or substantially the same nucleic acid sequence as all or part of the nucleic acid sequence or analog thereof, with the Gene ID of 10174.

"DDX 56" herein refers to a nucleic acid encoding all or part of a DDX56 protein or substantially the same nucleic acid sequence as all or part of a nucleic acid sequence or analog thereof, with the Gene ID of 54606.

"EID 2B" as used herein refers to a nucleic acid encoding all or part of EID2B protein or substantially the same sequence as all or part of the nucleic acid or analog thereof, with the Gene ID of 126272.

By "about the same" is meant that the polypeptide or nucleic acid exhibits at least 75%, 85%, 90%, 95% or even 99% identity to the reference amino acid or nucleic acid sequence. For polypeptides, the aligned sequences are typically at least 35 amino acids, 45 amino acids, 55 amino acids, or even 70 amino acids in length. For nucleic acids, the aligned sequences are typically at least 60 nucleotides, 90 nucleotides, or even 120 nucleotides in length.

Sequence identity is typically determined using publicly available computer programs. Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux et al, nucleic acids Research 12: 387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al, J.mol.biol.215: 403, 1990). The well-known SmithWaterman algorithm can also be used to determine identity. BLAST programs are publicly available from NCBI and other sources (e.g., the BLAST handbook, Altschul et al, NCBI NLM NIH, Bethesda, MD 20894). These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions of amino acid alignments typically include substitutions in the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

In the present disclosure, reagents for detecting the biomarkers include primers, probes, antibodies, ligands.

The term "primer" as used herein is a strand of short nucleic acid sequence that recognizes the target gene sequence, and includes a pair of forward and reverse primers. In particular, the "primers" include a pair of primers that provide an assay result of specificity and sensitivity. The primer is believed to provide a high degree of specificity when used to amplify a target gene sequence, but it does not cause amplification of non-target sequences that are not identical or complementary to the target gene sequence.

The term "probe" as used herein refers to a substance that specifically binds to a target to be detected in a sample. By this binding, the probe can determine the presence of the target in the sample. Any probe can be used in the present disclosure as long as it is generally used in the art. In particular, the probe may be a PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, most preferably a PNA. In particular, the probe is a biological material, which may be derived from an organism or may be synthesized in vitro, or a mimetic thereof. For example, the probe may be an enzyme, protein, antibody, microorganism, animal or plant cell or organ, neuron, DNA or RNA. DNA may include cDNA, genomic DNA, and oligonucleotides. Likewise, genomic RNA, mRNA, and oligonucleotides may fall within the scope of RNA. Examples of proteins include antibodies, antigens, enzymes, and peptides.

The term "antibody" as used herein refers to a substance that specifically binds to an antigen to elicit an antigen-antibody reaction. For the purposes of the present disclosure, the term "antibody" means an antibody that specifically binds to the protein encoded by the biomarker. Falling within the scope of the antibodies of the present disclosure are polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. These antibodies can be readily prepared using techniques well known in the art. Furthermore, antibodies useful in the present disclosure can be whole antibodies consisting of two full-length light chains and two full-length heavy chains, or functional fragments of a whole antibody molecule. The term "functional fragment" of an antibody molecule means a fragment that retains the binding function of the antibody, such as Fab, F (ab')2 and Fv.

Reagent kit

The invention provides a kit comprising reagents for detecting the expression level of the biomarkers. The kit can be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit or an MRM (multiple reaction monitoring) kit.

The kit may further comprise the elements necessary for reverse transcription polymerase chain reaction, RT-PCR kit comprising primers specific for the biomarkers. Each primer is a nucleotide having a nucleic acid sequence specific for the biomarker, which may be about 7 to 50bp, more particularly about 10 to 39bp in length. In addition, the kit may further comprise a primer specific for the nucleic acid sequence of the control gene. In addition, the RT-PCR kit may comprise a test tube or suitable vessel, reaction buffers (different pH values and magnesium concentrations), deoxynucleotides (dntps), enzymes (e.g., Taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water, and sterile water.

In addition, the kit of the present disclosure may contain elements necessary for the operation of the DNA chip. The DNA chip kit may comprise a substrate to which a gene or cDNA or an oligonucleotide corresponding to a fragment thereof is bound, and a reagent, a drug and an enzyme for constructing a fluorescently labeled probe. In addition, the substrate may comprise a control gene or cDNA or an oligonucleotide corresponding to a fragment thereof.

In some embodiments, the kits of the present disclosure may comprise the elements necessary for performing an ELISA. The ELISA kit may comprise antibodies specific for the protein encoded by the biomarker. The antibodies have high selectivity and affinity for the protein encoded by the biomarker, are non-cross-reactive with other proteins, and may be monoclonal, polyclonal or recombinant antibodies. In addition, the ELISA kit may comprise an antibody specific for a control protein. In addition, the ELISA kit may further comprise reagents capable of detecting the bound antibody, e.g., a labeled secondary antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and substrates thereof or substances capable of binding the antibody.

Medicine

The phrase "pharmaceutically acceptable carrier" is art-recognized and includes, for example, pharmaceutically acceptable materials, compositions or excipients, such as liquid or solid fillers, diluents, solvents or encapsulating materials, involved in carrying or transporting any subject composition from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient. In certain embodiments, the pharmaceutically acceptable carrier is pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc powder; (8) cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

The medicaments of the invention are prepared in a manner known to those skilled in the art, for example by means of conventional dissolving, lyophilizing, mixing, granulating or forming processes. Methods well known in the art for making formulations are shown, for example, in Remington: the Science and practice of Pharmacy, 20 th edition, eds A.R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology (Encyclopedia of Pharmaceutical Technology), J.Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York.

The dosage of any agent used in the medicament of the present invention can be readily determined by one skilled in the art. Desirably, the dose of agent in the medicament of the invention will be sufficient to prevent and/or treat liver cancer recurrence.

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Example 1 screening of Gene differentially expressed in recurrence of liver cancer

1. Data source

Chip data and clinical information of the GSE76427 data set were downloaded from GEO as a training set, and the sample size was 63:45 for relapse. And (3) downloading RNA-seq data and clinical information of liver cancer recurrence from a TCGA database as a verification set, wherein the sample size is non-recurrence and recurrence is 203: 168.

2. Data pre-processing

Joint processing, trimming and quality control are carried out on raw data by using fastp software, software default parameters are used for analysis, and high-quality sequencing data are output for subsequent analysis. Linker processing utilizes fastp software to default paired-end sequence automatic detection mode. The analyzed clean data was aligned to the human reference genome, version grch38.d1.vd1, using the Voom method, using ICGC software.

The GEO data was normalized by the RMA method, and annotated by a Platform file, and a plurality of probes corresponded to the same gene, and the average value was taken as the expression level of the gene.

3. Differential expression analysis

Differential expression analysis was performed using the "limma" package in R software, with screening criteria for differential genes being adj. pvalue < 0.05.

4. Results

SORBS3 was significantly reduced in the liver cancer recurrence samples of GEO and TCGA (p is 0.005 in GEO and 0.014 in TCGA) compared to the liver cancer non-recurrence samples; DDX56 was significantly up-regulated in liver cancer relapse samples of GEO and TCGA (p 0.011 in GEO and 0.046 in TCGA); EID2B was significantly up-regulated in liver cancer recurrence samples of GEO and TCGA (p 0.043 in GEO and 0.012 in TCGA).

Example 2 diagnostic Performance validation

1. Experimental methods

Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0), AUC values, sensitivity and specificity were analyzed, and the diagnostic efficacy of the markers alone or in combination was judged. When the diagnosis efficiency of the index combination is judged, logistic regression is carried out on the expression level of each gene, the probability of whether each individual suffers from cancer is calculated through a fitted regression curve, different probability division threshold values are determined, and the sensitivity, specificity, accuracy and the like of the combined detection scheme are calculated according to the determined probability division threshold values.

2. Results of the experiment

The diagnostic potency of SORBS3, DDX56 and EID2B in the training and validation sets, alone or in combination, is shown in tables 1 and 2 and FIGS. 1 and 2. the combination of SORBS3, DDX56 and EID2B showed higher diagnostic potency in both the training and validation sets, with AUC values of 0.736 and 0.739, respectively, and sensitivity and specificity in the training set of 0.711 and 0.683, respectively. The sensitivity and specificity of the validation set were 0.625 and 0.773, respectively.

TABLE 1 AUC values of genes in training set

Gene	AUC
		SORBS3	0.619
DDX56	0.625
		EID2B	0.589
EID2B+SORBS3	0.670
		EID2B+DDX56	0.673
SORBS3+DDX56+EID2B	0.736

TABLE 2 AUC values of genes in the validation set

Gene	AUC
		SORBS3	0.631
DDX56	0.672
		EID2B	0.620
EID2B+SORBS3	0.693
		EID2B+DDX56	0.684
SORBS3+DDX56+EID2B	0.739

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. An agent capable of specifically detecting the level of expression of a biomarker, wherein said biomarker comprises SORBS3, DDX56 and/or EID2B, preferably said biomarker comprises EID2B and SORBS3, preferably said biomarker comprises EID2B and DDX56, preferably said biomarker comprises a combination of SORBS3, DDX56 and EID 2B.

2. The reagent of claim 1, wherein the reagent comprises a reagent for detecting the expression level of the biomarker by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunization technology.

3. The reagent according to claim 1 or 2, wherein the reagent comprises a primer, a probe, an antibody, a ligand.

4. A kit comprising the reagent of any one of claims 1 to 3.

5. The kit of claim 4, further comprising one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

6. A chip comprising the reagent according to any one of claims 1 to 3.

7. A composition comprising one or more of the following agents:

(1) an agent that promotes expression of SORBS 3;

(2) an agent that inhibits the expression of DDX 56;

(3) an agent that inhibits expression of EID 2B;

preferably, the composition comprises an agent that inhibits the expression of EID2B and an agent that promotes the expression of SORBS 3; preferably, the composition comprises an agent that inhibits expression of EID2B and an agent that inhibits expression of DDX 56; preferably, the composition comprises an agent that promotes the expression of SORBS3, an agent that inhibits the expression of DDX56, and an agent that inhibits the expression of EID 2B.

8. A system for predicting recurrence of liver cancer, comprising:

the biomarker comprises SORBS3, DDX56 and/or EID2B, preferably, the biomarker comprises EID2B and SORBS3, preferably, the biomarker comprises EID2B and DDX56, preferably, the biomarker comprises the combination of SORBS3, DDX56 and EID 2B.

9. Use of a biomarker in the construction of a computational model or a system incorporating said computational model for the prediction of liver cancer recurrence, wherein said biomarker comprises SORBS3, DDX56 and/or EID2B, preferably said biomarker comprises EID2B and SORBS3, preferably said biomarker comprises EID2B and DDX56, preferably said biomarker comprises a combination of SORBS3, DDX56 and EID 2B.

10. Use according to any one of the following:

(1) use of an agent according to any one of claims 1 to 3 for the manufacture of a product for predicting recurrence of liver cancer;

(2) use of the kit of claim 4 or 5 for the preparation of a product for predicting recurrence of liver cancer;

(3) the use of the chip of claim 6 for the preparation of a product for predicting recurrence of liver cancer;

(4) the use of the composition of claim 7 in the preparation of a medicament for the prevention and/or treatment of liver cancer recurrence.