CN109633156B

CN109633156B - Application of biomarker in evaluating oral squamous carcinoma risk degree

Info

Publication number: CN109633156B
Application number: CN201811557074.XA
Authority: CN
Inventors: 王月红; 唐瞻贵; 刘欧胜; 邓智元; 潘灏; 方小丹; 全宏志; 王柏胜; 顾立群; 李龙; 戴小寒
Original assignee: XIANGYA STOMATOLOGICAL HOSPITAL CENTRAL SOUTH UNIVERSITY
Current assignee: XIANGYA STOMATOLOGICAL HOSPITAL CENTRAL SOUTH UNIVERSITY
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2022-02-22
Anticipated expiration: 2038-12-19
Also published as: CN109633156A

Abstract

The invention discloses application of a biomarker in evaluating oral squamous cell carcinoma risk degree, wherein the biomarker is GSDMA, samples of oral squamous cell carcinoma with different differentiation grades are detected, the biomarker shows significant difference in cancer samples with different risk degrees, and the GSDMA can be applied to grading judgment of the oral squamous cell carcinoma to prompt doctors to adopt different treatment means for oral squamous cell carcinoma with different processes, so that the treatment effect is improved.

Description

Application of biomarker in evaluating oral squamous carcinoma risk degree

Technical Field

The invention belongs to the field of biological medicines, and relates to application of a biomarker in evaluating the risk degree of oral squamous cell carcinoma, in particular to a biomarker GSDMA (glutathione S-DMA).

Background

Oral Squamous Cell Carcinoma (OSCC) is one of the most malignant malignancies, and likewise the most common type of Oral tumor, and patients with Oral Squamous Cell Carcinoma are often associated with metastasis, high recurrence rate, and poor prognosis. Epidemiological statistical analysis about 10 million people die of oral squamous cell carcinoma and the disease symptoms caused by the oral squamous cell carcinoma every year, so the oral squamous cell carcinoma brings huge challenges and burdens to the medical security system of the whole society. As a disease caused by multiple factors, the current research considers that tobacco and alcohol are important factors causing the occurrence and the development of the disease, chronic inflammation and human herpesvirus infection are also closely related to the generation of the disease, and besides environmental factors, susceptible genetic background is also an important reason causing the generation of oral squamous cell carcinoma.

Although basic tumor research and clinical treatment are rapidly developed in the last decades, the 5-year survival rate of oral squamous cell carcinoma is not remarkably improved, about 50 percent, and great burden is caused to the life quality of patients and social public medical treatment, so that the prevention and treatment research of malignant tumors is strengthened, and the accurate and objective evaluation of tumor biological behaviors and prognosis and treatment scheme establishment are more urgent.

The grade of tumor (grading) is an important index for evaluating the biological behavior and diagnosis of tumor, and malignant tumor generally determines the grade of malignancy according to the degree of differentiation, the size of the abnormal shape and the number of nuclear fission images. More people in recent years tend to use a simple and easy-to-master three-level classification method, i.e. the I level is well differentiated, tumor cells are close to corresponding normal tissues with low malignancy; grade II is moderately differentiated and moderately malignant; grade III is low differentiation, and the tumor cells are highly malignant, with large difference from corresponding normal tissue of origin and poor differentiation. In addition, some researchers refer to a malignant tumor that partially shows no tendency to differentiate as an undifferentiated tumor, and belongs to grade IV (G4), which is highly malignant. Although the grading method has the advantages, the grading method also has certain significance for clinical treatment and prognosis judgment. But the quantitative standard is lacked, and the influence of subjective factors cannot be excluded. With the development of molecular biology, people pay attention to the correlation between genes and tumors, discuss the development process of gene evaluation oral squamous cell carcinoma, and have important significance in realizing accurate treatment of oral squamous cell carcinoma.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a biomarker related to the degree of oral squamous cell carcinoma risk, and by using the biomarker, the development process of patients suffering from oral squamous cell carcinoma can be judged and evaluated.

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

the first aspect of the invention provides application of GSDMA genes in preparing products for evaluating the risk degree of oral squamous cell carcinoma.

Further, the product comprises a reagent for detecting the expression level of the GSDMA in a sample, wherein the sample for detecting the GSDMA comprises cells, tissues, organs, body fluids (blood, lymph fluid and the like), digestive juice, expectoration, alveolar and bronchial lavage fluid, urine, excrement and the like. Preferably, the sample is tissue or blood. In a specific embodiment of the invention, the sample is a tissue.

Further, GSDMA is down-regulated in the high-risk oral squamous cell carcinoma specimens.

Further, the reagent is used for detecting the GSDMA expression level through RT-PCR, real-time quantitative PCR, immunodetection, in-situ hybridization or a chip.

Further, the reagent for detecting the expression level of the GSDMA through RT-PCR at least comprises a pair of primers for specifically amplifying the GSDMA gene; the reagent for detecting the GSDMA expression level by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the GSDMA gene; the reagent for detecting the GSDMA expression level through immunity comprises an antibody and/or a ligand which is specifically combined with GSDMA protein; the reagent for detecting the GSDMA expression level through in situ hybridization comprises a probe hybridized with a nucleic acid sequence of a GSDMA gene; the reagent for detecting the GSDMA expression level through the chip comprises a probe hybridized with a GSDMA gene nucleic acid sequence, or an antibody and/or a ligand specifically bound with a GSDMA protein.

Further, the primer sequence of the specific amplification GSDMA is shown as SEQ ID NO. 3-4.

In a second aspect, the invention provides a product for diagnosing the risk degree of oral squamous cell carcinoma, which comprises a preparation, a nucleic acid membrane strip, a chip or a kit, wherein the preparation, the nucleic acid membrane strip, the chip or the kit comprises a reagent for detecting the GSDMA expression level.

Further, the reagent for detecting the expression level of GSDMA in the chip comprises a probe which specifically recognizes the GSDMA gene, or an antibody or a ligand which specifically binds to a protein encoded by GSDMA.

Further, the reagent for detecting the GSDMA expression level in the kit comprises a primer for specifically amplifying the GSDMA gene; or a probe that specifically recognizes the GSDMA gene; or an antibody or ligand that specifically binds to a protein encoded by GSDMA.

Furthermore, the primer sequence of the specific amplification GSDMA gene is shown as SEQ ID NO.3 and SEQ ID NO. 4.

Further, the kit comprises a SYBR Green polymerase chain reaction system and a primer pair for amplifying housekeeping genes; the SYBR Green polymerase chain reaction system comprises: PCR buffer, dNTPs, SYBR Green fluorescent dye.

The gene chip or the gene detection kit can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to the oral squamous cell carcinoma risk degree) including the GSDMA gene. The protein chip or the protein immunoassay kit can be used for detecting the expression level of a plurality of proteins including GSDMA protein (for example, a plurality of proteins related to the risk degree of oral squamous cell carcinoma). The multiple markers of the oral squamous cell carcinoma risk degree are simultaneously detected, so that the accuracy of diagnosing the oral squamous cell carcinoma risk degree can be greatly improved.

The invention has the advantages and beneficial effects that:

according to the invention, GSDMA is selected as a molecular marker, so that the grading of the risk degree of oral squamous cell carcinoma can be realized, and doctors are guided to adopt different treatment strategies, means and measures for oral squamous cell carcinoma patients with different risk degrees, high, medium and low, not only can the excessive treatment be avoided, but also the insufficient treatment strength can be avoided, so that the treatment effect of the oral squamous cell carcinoma patients is improved, and the medical resources and the cost are saved.

The invention develops GSDMA into detection products, has the advantages of quick and convenient detection, high detection sensitivity and specificity and low cost, can meet the detection requirements of most oral squamous cell carcinoma patients, and has wide application range.

Drawings

Figure 1 is a QPCR assay for GSDMA expression in oral squamous carcinoma tissue, wherein: p <0.01, x: p < 0.001.

Detailed Description

According to the invention, through extensive and intensive research, through high-throughput sequencing and bioinformatics analysis, specific expression of GSDMA in oral squamous cell carcinoma patients with different risk degrees is found for the first time, and a better way and a better method are provided for evaluation of oral squamous cell carcinoma grading.

Biomarkers

A "biomarker," also referred to as a "molecular marker," is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

Any method available in the art for detecting expression of a molecular marker is encompassed herein. Expression of the molecular markers of the invention can be detected at the nucleic acid level (e.g., RNA transcript) or at the protein level. By "detecting expression" is intended the determination of the amount or presence of an expression product of an RNA transcript or its molecular marker gene. Thus, "detecting expression" includes instances where a molecular marker is determined to be not expressed, not to be detected, expressed at a low level, expressed at a normal level, or overexpressed.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. The GSDMA gene has an ID of 284110 in the current International public nucleic acid database GeneBank, and as a non-limiting example, the coding sequence or amino acid sequence of a representative human GSDMA is shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. GSDMA includes polypeptides comprising SEQ ID No.2 and other GSDMA native sequence polypeptides, such as naturally occurring variants and native sequence polypeptides, encoded by the nucleotide sequence set forth in SEQ ID No. 1.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may or may not also include an initial methionine residue.

Polynucleotides encoding mature polypeptides of GSDMA include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The invention also relates to nucleic acid fragments, including sense and antisense nucleic acid fragments, which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments may be used in amplification techniques of nucleic acids (e.g. PCR) to determine and/or isolate polynucleotides encoding GSDMA proteins.

The full-length nucleotide sequence or its fragment of human GSDMA of the present invention can be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Detection method

The present invention can be detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

Generally, PCR uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR Reverse Transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA; TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under substantially constant conditions of temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally including the use of blocking, partial, terminating and other modifying moieties to improve the sensitivity and accuracy of the TMA process; LCR with target nucleic acid adjacent region hybridization of two sets of complementary DNA oligonucleotides. The DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded ligated oligonucleotide product; the SDA uses multiple cycles of the following steps: primer sequence pairs anneal to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded hemiphosphorothioated (phosphorothioated) primer extension products, endonuclease-mediated nicking of the hemimodified restriction enzyme recognition site, and polymerase-mediated extension from the 3' end of the nick to displace the existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, thereby causing geometric amplification of the products.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.

Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

The immunization method according to the present invention may be based on, for example, any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this method measures the amount of precipitate that is formed after the reagent antibody has been incubated with the sample and reacted with the target antigen present therein to form insoluble aggregates. The immunoprecipitation can be either qualitative or quantitative.

In a particle immunoassay, multiple antibodies are attached to the particle and the particle is capable of binding many antigenic molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows for a fast and sensitive detection of the biomarker.

In immunoturbidimetry (immunonephelometry), the interaction of an antibody and a target antigen on a biomarker causes the formation of an immune complex that is too small to precipitate. However, these complexes will scatter incident light, which can be measured using a turbidimeter. The concentration of the antigen (i.e. biomarker) can be determined within a few minutes of the reaction.

Radioimmunoassay (RIA) methods use radioisotopes such as I125 to label antigens or antibodies. The isotope used emits gamma rays, which are usually measured after removal of unbound (free) radiolabel. The main advantages of RIA compared to other immunoassays are higher sensitivity, easy signal detection and confirmation, fast assay. The main disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining the licensed radiation safety and disposal procedures. For this reason, RIA has been largely replaced by enzyme immunoassays in routine clinical laboratory practice.

Enzyme Immunoassays (EIAs) have evolved as alternatives to Radioimmunoassays (RIA). These methods use enzymes to label the antibody or target antigen. The sensitivity of EIA is close to that of RIA and there is no risk caused by radioisotopes. One of the most widely used EIA methods for detection is enzyme-linked immunosorbent assay (ELISA). The ELISA method may use two antibodies, one specific for the target antigen and the other coupled to an enzyme, the addition of an enzyme substrate causing the generation of a chemiluminescent or fluorescent signal.

Fluorescence Immunoassay (FIA) refers to an immunoassay that uses a fluorescent label or an enzyme label that acts on a substrate to form a fluorescent product. Fluorescence measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Thus, the FIA method has higher analytical sensitivity than the EIA method using absorption (optical density) measurement.

Chemiluminescent immunoassays use a chemiluminescent label that produces light when excited by chemical energy; the emission is measured using a photodetector.

Thus, the immunization method according to the present invention can be carried out using well-known methods. Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.

Probe needle

"Probe" refers to a molecule that can be used to measure the expression of a particular gene. Exemplary probes include PCR primers and gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

The term "probe" as used herein refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

As the probe, a labeled probe in which a polynucleotide for cancer detection is labeled, such as a fluorescent label, a radioactive label, or a biotin label, can be used. Methods for labeling polynucleotides are known per se. The presence or absence of the test nucleic acid in the sample can be checked by: immobilizing the test nucleic acid or an amplification product thereof, hybridizing with the labeled probe, washing, and then measuring the label bound to the solid phase. Alternatively, the polynucleotide for cancer detection may be immobilized, a nucleic acid to be tested may be hybridized therewith, and the nucleic acid to be tested bound to the solid phase may be detected using a labeled probe or the like. In this case, the polynucleotide for cancer detection bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The process can be carried out as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within ± 4 ℃) in a buffer for hybridization, washed, and the hybridized labeled probe or template nucleic acid bound to the solid phase probe is then measured.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the coding region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or they may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PN, LNA, ENA, GNA, TNA, etc.

Nucleic acid membrane strip, chip and kit

In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

"chip," also referred to as an "array," refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

Various probe arrays have been described in the literature and can be used in the context of the present invention to detect markers that may be associated with the phenotypes described herein. For example, a DNA probe array chip or a larger DNA probe array wafer (otherwise, individual chips may be obtained by breaking the wafer) is used in one embodiment of the present invention. The DNA probe array wafer generally comprises a glass wafer on which a high-density array of DNA probes (short DNA fragments) is placed. Each of these wafers may hold, for example, about 6000 million DNA probes for identifying longer sample DNA sequences (e.g., from an individual or population, e.g., containing a marker of interest). The identification of sample DNA by the DNA probe set on the glass wafer was performed by DNA hybridization. When a DNA sample is hybridized to an array of DNA probes, the sample binds to those probes whose sample DNA sequences are complementary. By assessing that the individual sample DNA hybridizes more strongly to those probes, it is possible to determine whether a known nucleic acid sequence is present in the sample, and thus whether a marker found in the nucleic acid is present. This approach can also be used to perform ASH by controlling hybridization conditions to allow discrimination of single nucleotides, e.g., for SNP identification and sample genotyping of one or more SNPs. Arrays provide a convenient embodiment for the simultaneous (or tandem) detection of multiple polymorphic markers.

In the present invention, the kit can be used for detecting the expression level of GSDMA genes or proteins, and comprises the ligand and/or the chip of the present invention for GSDMA detection and/or quantification. Optionally together with instructions for the kit.

Kits include one or more sterile containers, which may be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

In the present invention, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to".

Antibodies

In the present invention, the term "antibody" refers to a natural or synthetic antibody that selectively binds to an antigen of interest. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules that selectively bind an antigen of interest, are also included within the scope of the term "antibody" so long as they exhibit the desired biological activity. "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with such variants generally being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences.

Monoclonal antibodies also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric immunoglobulins, immunoglobulin chains or fragments thereof such as Fv, Fab ', F (ab')²Or other antigen binding subsequences of antibodies.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')²And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) are shed from the folded structure of these two domains, contributing to the amino acid residues that bind antigen and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

"functional fragments" of the antibodies of the invention refer to those fragments that retain binding to the polypeptide with substantially the same affinity as the intact full chain molecule from which they are derived and that are active in at least one assay.

Polyclonal antibodies include antibodies derived by immunizing an animal (e.g., a mouse) producing a human antibody with a GSDMA protein. When a chimeric antibody or a humanized antibody is prepared, amino acids in the variable region (e.g., FR) and/or constant region may be replaced with other amino acids, or the like.

Statistical method

In the present invention, the experiment is repeated at least 3 times, the result data are expressed in the form of mean value ± standard deviation, statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is determined by t test, and the statistical significance is considered when P is less than 0.05.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with oral squamous cell carcinoma

1. Sample collection

Each of 68 cases of oral squamous carcinoma tissues and paracarcinoma tissues were collected, including 20 patients histologically graded as grade I (G1), 30 patients histologically graded as grade II (G2), and 18 patients histologically graded as grade III/IV (G3/G4). The patient did not receive any treatment prior to surgery. All the samples were obtained by informed consent, 4 samples per group were taken by the tissue ethics committee for gene expression profiling, screening for differentially expressed genes, and validation experiments were performed on all the samples per group.

2. Preparation of RNA sample (manipulation Using tissue RNA extraction kit of QIAGEN)

Taking out the tissue sample frozen in liquid nitrogen, putting the tissue sample into a precooled mortar for grinding, and extracting and separating RNA according to the instruction in the kit. The method comprises the following specific steps:

1) adding Trizol, and standing at room temperature for 5 min;

2) adding chloroform 0.2ml, shaking the centrifuge tube with force, mixing well, standing at room temperature for 5-10 min;

3) centrifuging at 12000rpm for 15min, transferring the upper water phase into another new centrifuge tube (taking care not to absorb protein substances between the two water phases), adding equal volume of isopropanol precooled at-20 deg.C, fully inverting and mixing, and placing on ice for 10 min;

4) centrifuging at 12000rpm for 15min, carefully removing supernatant, adding 75% DEPC ethanol according to the proportion of 1ml/ml Trizol, washing precipitate (storing at 4 deg.C), shaking, mixing, and centrifuging at 12000rpm for 5min at 4 deg.C;

5) discarding the ethanol liquid, standing at room temperature for 5min, adding DEPC water to dissolve the precipitate;

6) the RNA purity and concentration were measured with a Nanodrop2000 ultraviolet spectrophotometer and frozen in a freezer at-70 ℃.

3. Construction of cDNA library

Ribosomal RNA in total RNA was removed using Ribo-Zero Kit, and cDNA library was constructed using Illumina Truseq RNA sample Prep Kit, according to the instructions.

4. Sequencing on machine

And (3) sequencing the cDNA library by using an Illumina X-Ten sequencing platform, wherein the specific operation is carried out according to the instruction.

5. High throughput transcriptome sequencing data analysis

Bioinformatics analysis was performed on the sequencing results, linear by linear association test was performed using a tool R-3.3.3, each sample was divided into 4 expression level intervals according to the quartile of the expression level of each gene, and then the correlation between the expression level intervals and the tomor grades was examined. Genes were considered significantly differentially expressed when the FDR value was < 0.05.

6. Results

The expression level of the GSDMA gene in oral squamous cell carcinoma tissues with different differentiation degrees shows significant difference, compared with G1, the expression of the GSDMA in G2 and G3/G4 is significantly reduced, compared with G2, the expression of the GSDMA in G3/G4 is significantly reduced, and the GSDMA can effectively distinguish the oral squamous cell carcinoma with different differentiation degrees.

Example 2 QPCR sequencing verification of differential expression of GSDMA genes

1. Large sample QPCR validation was performed on differential GSDMA gene expression.

2. The RNA extraction procedure was as described in example 1.

3. Reverse transcription:

mRNA reverse transcription was performed using FastQ μ ant cDNA first strand synthesis kit (cat # KR106), genomic DNA reaction was first removed, 5 XgDNA B μ ffer 2.0 μ l, total RNA 1 μ g, RNase Free ddH were added to the tube₂O to make the total volume 10. mu.l, heating in a water bath at 42 ℃ for 3min, and adding 10 Xfast RT B. mu.ffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3 min.

4. QPCR amplification assay

QPCR amplification primers were designed based on the sequences of GSDMA and GAPDH and were synthesized by Bomaide Biopsis. The specific primer sequences are as follows:

GSDMA gene:

the forward primer is 5'-CAAAGGCAAAGATGAGTG-3' (SEQ ID NO. 3);

the reverse primer was 5'-CTTCTCCTCTCCTGACTT-3' (SEQ ID NO. 4).

The primer sequence of housekeeping gene GAPDH is as follows:

a forward primer: 5'-CTCTGGTAAAGTGGATATTGT-3' (SEQ ID NO.5)

Reverse primer: 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO.6)

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

A20. mu.l reaction was used: 2 XSuperReal PreMix Plus 10. mu.l, forward and reverse primers (10. mu.M) 0.6. mu.l each, 5 XROX Reference Dye^△2. mu.l, DNA template 2. mu.l, sterilized distilled water 4.8. mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was: 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72)32 s). times.40 cycles, 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15 s). Determination of the band of interest by melting Curve analysis and electrophoresis with SYBR Green as fluorescent marker, 2^-ΔΔCTThe method is used for relative quantification.

6. Results

The results are shown in fig. 1, GSDMA shows significant differences in tissues with different differentiation degrees, and the lower the differentiation degree, the lower the expression level of GSDMA, thus suggesting that GSDMA can be used as a molecular marker for judging the differentiation grade of oral squamous cell carcinoma, so as to guide doctors to take different treatment strategies, means and measures for patients with oral squamous cell carcinoma with different high, medium and low risks, not only can avoid over-treatment, but also can avoid insufficient treatment intensity, thereby improving the treatment effect of the patients with oral squamous cell carcinoma

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.

Sequence listing

<110> Hunan-ya oral Hospital, Zhongnan university in Hunan

Application of <120> biomarker in evaluating oral squamous carcinoma risk degree

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aggaagagga agagcacgct cttctggggg gcccggtacg tccgcaccga ctacacgctg 180

ctggatgtgc ttgagcccgg cagctcacct tcagacccaa cagacactgg gaattttggc 240

tttaagaata tgctggacac ccgagtggag ggagatgtgg atgtaccaaa gacggtgaag 300

gtgaagggaa cggcagggct ctcgcagaac agcactctgg aggtccagac actcagtgtg 360

gctcccaagg ccctggagac cgtgcaggag aggaagctgg cagcagacca cccattcctg 420

aaggagatgc aagatcaagg ggagaacctg tatgtggtga tggaggtggt ggagacggtg 480

caggaggtca cactggagcg agccggcaag gcagaggcct gcttctccct ccccttcttc 540

gccccattgg ggctacaggg atccataaat cacaaggagg ctgtaaccat ccccaagggc 600

tgcgtcctgg cctttcgagt gagacagctg atggtcaaag gcaaagatga gtgggatatt 660

ccacatatct gcaatgataa catgcaaacc ttccctcctg gagaaaagtc aggagaggag 720

aaggtcatcc ttatccaggc atctgatgtt ggggacgtac acgaaggctt caggacacta 780

aaagaagaag ttcagagaga gacccaacaa gtggagaagc tgagccgagt agggcaaagc 840

tccctgctca gctccctcag caaacttcta gggaagaaaa aggagctaca agaccttgag 900

ctcgcacttg aaggggctct agacaaggga catgaagtga ccctggaggc actcccaaaa 960

gatgtcctgc tatcaaagga ggccgtgggc gccatcctct atttcgttgg agccctaaca 1020

gagctaagtg aagcccaaca gaagctgctg gtgaaatcca tggagaaaaa gatcctaccc 1080

gtgcagctaa agctggtgga gagcacgatg gaacagaact tcctgctgga taaagagggt 1140

gttttccccc tgcaacctga gctgctctcc tcccttgggg acgaggagct gaccctcacg 1200

gaggctctag tcgggctgag tggcctggaa gtgcagagat cgggccccca atatatgtgg 1260

gacccagaca ccctccctcg cctctgtgct ctttatgcag gcctctctct ccttcagcag 1320

cttaccaagg cctcctaa 1338

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Phe His Pro Phe Cys Leu Val Leu Arg Lys Arg Lys Ser Thr Leu Phe

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Trp Gly Ala Arg Tyr Val Arg Thr Asp Tyr Thr Leu Leu Asp Val Leu

50 55 60

Glu Pro Gly Ser Ser Pro Ser Asp Pro Thr Asp Thr Gly Asn Phe Gly

65 70 75 80

Phe Lys Asn Met Leu Asp Thr Arg Val Glu Gly Asp Val Asp Val Pro

85 90 95

Lys Thr Val Lys Val Lys Gly Thr Ala Gly Leu Ser Gln Asn Ser Thr

100 105 110

Leu Glu Val Gln Thr Leu Ser Val Ala Pro Lys Ala Leu Glu Thr Val

115 120 125

Gln Glu Arg Lys Leu Ala Ala Asp His Pro Phe Leu Lys Glu Met Gln

130 135 140

Asp Gln Gly Glu Asn Leu Tyr Val Val Met Glu Val Val Glu Thr Val

145 150 155 160

Gln Glu Val Thr Leu Glu Arg Ala Gly Lys Ala Glu Ala Cys Phe Ser

165 170 175

Leu Pro Phe Phe Ala Pro Leu Gly Leu Gln Gly Ser Ile Asn His Lys

180 185 190

Glu Ala Val Thr Ile Pro Lys Gly Cys Val Leu Ala Phe Arg Val Arg

195 200 205

Gln Leu Met Val Lys Gly Lys Asp Glu Trp Asp Ile Pro His Ile Cys

210 215 220

Asn Asp Asn Met Gln Thr Phe Pro Pro Gly Glu Lys Ser Gly Glu Glu

225 230 235 240

Lys Val Ile Leu Ile Gln Ala Ser Asp Val Gly Asp Val His Glu Gly

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Phe Arg Thr Leu Lys Glu Glu Val Gln Arg Glu Thr Gln Gln Val Glu

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Lys Leu Ser Arg Val Gly Gln Ser Ser Leu Leu Ser Ser Leu Ser Lys

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Leu Leu Gly Lys Lys Lys Glu Leu Gln Asp Leu Glu Leu Ala Leu Glu

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Gly Ala Leu Asp Lys Gly His Glu Val Thr Leu Glu Ala Leu Pro Lys

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Asp Val Leu Leu Ser Lys Glu Ala Val Gly Ala Ile Leu Tyr Phe Val

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Gly Ala Leu Thr Glu Leu Ser Glu Ala Gln Gln Lys Leu Leu Val Lys

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Ser Met Glu Lys Lys Ile Leu Pro Val Gln Leu Lys Leu Val Glu Ser

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Thr Met Glu Gln Asn Phe Leu Leu Asp Lys Glu Gly Val Phe Pro Leu

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Gln Pro Glu Leu Leu Ser Ser Leu Gly Asp Glu Glu Leu Thr Leu Thr

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Gln Tyr Met Trp Asp Pro Asp Thr Leu Pro Arg Leu Cys Ala Leu Tyr

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<210> 5

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. The application of the reagent for detecting the GSDMA gene expression level in preparing products for evaluating the oral squamous cell carcinoma risk degree is characterized in that the GSDMA is down-regulated in the oral squamous cell carcinoma with high risk degree.

2. The use of claim 1, wherein the reagents comprise reagents for detecting the level of GSDMA expression by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip.

3. The use of claim 2, wherein the reagent for detecting the expression level of GSDMA by RT-PCR comprises at least one pair of primers for specifically amplifying GSDMA genes; the reagent for detecting the GSDMA expression level by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the GSDMA gene; the reagent for detecting the GSDMA expression level through immunity comprises an antibody and/or a ligand which is specifically combined with GSDMA protein; the reagent for detecting the GSDMA expression level through in situ hybridization comprises a probe hybridized with a nucleic acid sequence of a GSDMA gene; the reagent for detecting the GSDMA expression level through the chip comprises a probe hybridized with a GSDMA gene nucleic acid sequence, or an antibody and/or a ligand specifically bound with a GSDMA protein.

4. The use of claim 1, wherein the product comprises a formulation, nucleic acid membrane strip, chip or kit, wherein the formulation, nucleic acid membrane strip, chip or kit comprises a reagent that detects the expression level of GSDMA.

5. The use of claim 4, wherein the reagent for detecting the expression level of GSDMA in the chip comprises a probe specifically recognizing GSDMA gene, or an antibody or ligand specifically binding to GSDMA-encoded protein.

6. The use of claim 4, wherein the reagent for detecting the expression level of GSDMA in the kit comprises a primer for specifically amplifying the GSDMA gene; or a probe that specifically recognizes the GSDMA gene; or an antibody or ligand that specifically binds to a protein encoded by GSDMA.

7. The use according to claim 6, wherein the primer sequence for specific amplification of the GSDMA gene is shown as SEQ ID No.3 and SEQ ID No. 4.

8. The use of claim 7, wherein the kit comprises a SYBR Green polymerase chain reaction system, a primer pair for amplifying a housekeeping gene; the SYBR Green polymerase chain reaction system comprises: PCR buffer, dNTPs, SYBR Green fluorescent dye.