CN106755372B

CN106755372B - Application of molecular marker in diagnosis and treatment of oral squamous cell carcinoma

Info

Publication number: CN106755372B
Application number: CN201611136262.6A
Authority: CN
Inventors: 杨承刚; 任静; 程城
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2016-12-12
Filing date: 2016-12-12
Publication date: 2020-03-31
Anticipated expiration: 2036-12-12
Also published as: CN106755372A

Abstract

The invention discloses an application of a molecular marker in diagnosis and treatment of oral squamous cell carcinoma, wherein the biomarker is GDPD2, and qPCR experiments show that GDPD2 gene is expressed and down-regulated in oral squamous cell carcinoma tissues and cells, and GDPD2 gene overexpression can inhibit cell proliferation and reduce cancer cell invasion rate. The invention provides application of an accelerant of GDPD2 in diagnosis and treatment of oral squamous cell carcinoma, and also discloses a method for inhibiting cell proliferation.

Description

Application of molecular marker in diagnosis and treatment of oral squamous cell carcinoma

Technical Field

The invention belongs to the field of biological medicines, and relates to application of a molecular marker in diagnosis and treatment of oral squamous cell carcinoma, wherein the molecular marker is GDPD 2.

Background

Oral squamous carcinoma is the most common malignant tumor in the oral cavity and the periphery, the incidence rate of the oral squamous carcinoma accounts for more than 80 percent of the total incidence rate of the tumor in the oral cavity, and the oral squamous carcinoma is one of the malignant diseases which are worthy of being attracted by people. Although the incidence of oral squamous cell carcinoma is reduced in developed countries such as Europe and America, the incidence of oral squamous cell carcinoma is still on the rise worldwide. With the increasing level of medical treatment in recent years, a lot of malignant tumors have been treated with a lot of progress, and the 5-year survival rate of patients is improved by treatment. However, in the treatment of squamous cell carcinoma of the oral cavity, the early stage of the disease is not easy to be detected by patients, so that the treatment of the disease is delayed, and the treatment difficulty of the late stage of the cancer is increased. Therefore, there is a need to increase the attention on oral squamous cell carcinoma therapy and find better treatment methods for oral squamous cell carcinoma.

In the treatment of oral squamous cell carcinoma, conventional treatment methods include surgical treatment, radiotherapy, chemotherapy, immunotherapy and the like. Although the treatment methods can inhibit the occurrence and development of tumors to a certain extent, the treatment methods have great treatment disadvantages. For example, radiotherapy and chemotherapy inevitably cause serious side effects, but the operation is not easy to be carried out, and the tumor can not be radically treated by the simple operation. The efficacy of immunotherapy is not obvious. Gene therapy and molecular targeted therapy are emerging tumor treatment means in recent years, and are favored by researchers due to the advantages of small side effect, definite curative effect and the like.

The molecular targeted therapy is that at the cellular molecular level, tumor-related genes are regulated and controlled, a signal path for interfering tumor abnormality is intervened or an energy path is blocked, and the search for molecular targets which can interfere has important significance for the targeted therapy of oral squamous cell carcinoma.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a biomarker for the diagnosis and treatment of oral squamous cell carcinoma.

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

the invention provides application of a GDPD2 gene or an expression product thereof in preparing a product for diagnosing oral squamous cell carcinoma.

Further, the product comprises detecting changes in the GDPD2 gene and/or its expression product by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, or immunoassays.

Further, the nucleic acid amplification technique is selected from the group consisting of polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification and nucleic acid sequence based amplification.

The invention provides a product for diagnosing oral squamous cell carcinoma, which can diagnose the oral squamous cell carcinoma by detecting the change of GDPD2 gene and/or expression product thereof in a sample.

Further, the product comprises a chip, a kit or a formulation.

Further, the product comprises a chip, a kit or a formulation. Wherein, the chip comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes aiming at GDPD2 gene for detecting the transcription level of GDPD2 gene; the protein chip comprises a solid phase carrier and a specific antibody or ligand of GDPD2 protein fixed on the solid phase carrier; the kit comprises a gene detection kit and a protein immunodetection kit; the gene detection kit comprises a reagent for detecting the transcription level of the GDPD2 gene; the protein immunoassay kit comprises a specific antibody or ligand of GDPD2 protein.

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 oral squamous cell carcinoma) including GDPD2 gene. The protein chip or the protein immunoassay kit can be used for detecting the expression levels of a plurality of proteins (such as a plurality of proteins related to oral squamous cell carcinoma) including GDPD2 protein. The oral squamous cell carcinoma marker can be used for simultaneously detecting a plurality of markers of the oral squamous cell carcinoma, so that the accuracy of oral squamous cell carcinoma diagnosis can be greatly improved.

The invention provides an application of a GDPD2 gene in preparing a pharmaceutical composition for treating oral squamous cell carcinoma.

Further, the pharmaceutical composition comprises an accelerator of the GDPD2 gene and/or its expression product.

Further, the promoter comprises a GDPD2 gene expression product, a promoted miRNA, a promoted transcription regulatory factor, or a promoted targeted small molecule compound

The invention provides a method for inhibiting cell proliferation, which is to add GDPD2 gene, protein or promoter thereof into a culture system.

The invention provides a combined medicament which comprises the medicinal composition and a medicinal composition containing an antitumor agent.

Further, pharmaceutical compositions of antineoplastic agents include, but are not limited to, immunotherapeutic agents such as cetuximab, chemotherapeutic agents or chemoradiotherapeutic agents such as carboplatin or a related class of platinum drugs, taxanes or a class of taxanes, or both.

The invention has the advantages and beneficial effects that:

the invention discovers that the molecular marker GDPD2 is related to the occurrence and development of oral squamous cell carcinoma for the first time, and can be used for diagnosing the oral squamous cell carcinoma by detecting the change of the GDPD2 of a subject.

The invention provides a molecular means for treating oral squamous cell carcinoma, which intervenes or inhibits the oral squamous cell carcinoma by changing the expression of a molecular target of the oral squamous cell carcinoma.

Drawings

FIG. 1 shows the detection of the expression of GDPD2 gene in oral squamous cell carcinoma tissue using QPCR;

FIG. 2 shows the detection of the expression of GDPD2 gene in oral squamous cell carcinoma cells using QPCR;

FIG. 3 shows the detection of the transfection of GDPD2 gene in oral squamous cell carcinoma using QPCR;

FIG. 4 shows MTT assay to determine the effect of GDPD2 on the proliferative activity of oral squamous cell carcinoma cells;

FIG. 5 shows the effect of the GDPD2 gene on the invasion of oral squamous cell carcinoma cells tested using a transwell chamber.

Detailed Description

Through extensive and intensive research, GDPD2 shows specific low expression in oral squamous cell carcinoma is found for the first time through a large amount of screening. Experiments prove that the growth and invasion of oral squamous cell carcinoma can be effectively inhibited by specifically improving the activity of the expression level or the expression product of GDPD2, so that the effect of inhibiting the oral squamous cell carcinoma is achieved. The present invention has been completed based on this finding.

The GDPD2 protein is a diacylglycerol phosphodiesterase enzyme that is used to hydrolyze glycerophosphoinositide to produce inositol 1-phosphate and glycerol. The protein plays an important role in osteogenic differentiation and growth.

GDPD2 polynucleotide or protein (polypeptide) sequences useful in the present invention include GDPD2 of various origins and species, and include wild-type, mutant GDPD2, or active fragments thereof. In some embodiments of the invention, the GDPD2 is derived from human, and the coding sequence or amino acid sequence of a representative human GDPD2 is shown in SEQ ID No.1 and SEQ ID No.2, respectively.

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 the mature polypeptide of GDPD2 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 a polynucleotide encoding the GDPD2 protein.

The full-length nucleotide sequence of human GDPD2 or its fragment can be obtained by PCR amplification, recombination or artificial synthesis. 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.

The gene-carrying vector of the present invention is a variety of vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The term "biomarker" in the present invention is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level in a normal or healthy cell or tissue.

Any method available in the art for detecting biomarker expression is encompassed herein. Expression of the biomarkers 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 to determine the amount or presence of an expression product of an RNA transcript or a biomarker gene thereof. Thus, "detecting expression" includes instances where a biomarker is determined to be not expressed, not to be detected, expressed at a low level, expressed at a normal level, or overexpressed. To determine low expression, the body sample examined can be compared with a corresponding body sample from a healthy person. That is, the "normal" level of expression is the level of expression of a biomarker, for example, in a cervical tissue sample from a human subject or a patient not afflicted with oral squamous cell carcinoma. This sample may be presented in a normalized form. In some embodiments, biomarker overexpression is determined without comparing the body sample to a corresponding body sample from a healthy person.

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 uses Reverse Transcriptase (RT) 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 the target nucleic acid sequence under substantially constant temperature, ionic strength, and pH conditions, wherein the multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally includes the use of a blocker, moiety, terminator, and other modifier to improve the sensitivity and accuracy of the TMA process, LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid, the DNA oligonucleotides are covalently linked by DNA ligase in repeated multiple cycles of thermal denaturation, hybridization, and ligation to produce detectable double-stranded ligated oligonucleotide products, SDA uses multiple cycles of primer sequences annealing to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded half-stranded primer extension (phospho) primer extension, and displacement of the amplified primer sequences to produce a restriction endonuclease displacement product that mediates restriction of the displacement of the phosphomonoesters from the target nucleic acid sequences, and displacement of the amplified products.

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.

The term "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.

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.

The polyclonal antibody includes an antibody obtained by immunizing an animal (e.g., a mouse) producing a human antibody with GDPD2 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.

The promoter of GDPD2 of the present invention, when administered (dosed) therapeutically, promotes the expression or activity of GDPD2 gene and/or protein, thereby inhibiting oral squamous cell carcinoma. In particular embodiments of the invention, the GDPD2 promoter comprises a GDPD2 gene expression product, a promoting miRNA, a promoting transcriptional regulator, or a promoting targeted small molecule compound.

These enhancers are typically formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to about 8, preferably about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical composition may be administered by conventional routes including, but not limited to, oral administration, parenteral administration, administration by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, or administration via an implanted reservoir device. The term parenteral in the context of this invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracoronary, intralesional, and intracranial injection or infusion techniques. As long as the target tissue is achieved.

The pharmaceutical composition of the invention contains a safe and effective amount of the GDPD2 protein or its promoter and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably prepared under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

The pharmaceutical composition can be used for supplementing the deletion or deficiency of endogenous GDPD2 protein, and treating oral squamous cell carcinoma caused by the reduction of GDPD2 protein by improving the expression of GDPD2 protein or enhancing the function of GDPD2 protein.

The medicament of the invention can also be used in combination with other medicaments for treating oral squamous cell carcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

The term "treatment" in the present invention refers to the medical management of a patient for the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapy, i.e., treatment aimed specifically at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition, or disorder. Moreover, the term also includes palliative treatments, i.e., treatments designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, i.e. treatment for supplementing another specific therapy with the purpose of improving the associated disease, pathological state or condition.

The term "sample" as used herein refers to a composition obtained from a target patient that contains cells and/or other molecular entities that are to be characterized and/or identified, for example, according to physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "clinical sample" or "disease sample" and variants thereof, refers to any sample obtained from a subject patient in which it would be expected or known that cellular and/or molecular bodies, such as biomarkers, would be characterized, would be available.

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. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

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

1. Sample collection

6 peripheral normal mucosal tissues and oral squamous cell carcinoma tissues were collected each and all patients were not treated in any form before surgery as confirmed by pathological diagnosis. The surgically excised specimens were cryopreserved in liquid nitrogen and informed consent was obtained from the patients, all of which were obtained with the consent of the tissue ethics committee.

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. Reverse transcription and labelling

mRNA was reverse-transcribed into cDNA using the Low RNA Input Linear Amplification Kit, and the experimental group and the control group were labeled with Cy3, respectively.

4. Hybridization of

The gene chip adopts human whole genome expression profiling chip of Aglient company, and each chip comprises 45015 oligonucleotides, wherein 43376 human gene probes and 1639 experiment control probes are contained in the chip. The steps of the chip use instruction are carried out, the temperature is 65 ℃, rolling hybridization is carried out for 17h 10r/min, and the film is washed at 37 ℃.

5. Data processing

After hybridization, the chip was scanned with an Agilent scanner with a resolution of 5 μm, the scanner automatically scanned 1 time each with 100% and 10% PMT, and the results of 2 Agilent software were automatically merged. And (3) processing and analyzing the scanned image data by adopting Feature Extraction, and performing subsequent data processing on the obtained original data by applying a Bioconductor program package. The final Ratio values are experimental and control. Differential gene screening criteria: the up-regulated gene is the ratio more than or equal to 4, and the down-regulated gene is the ratio less than or equal to 0.25.

6. Results

Compared with normal mucosa tissue, the expression level of the GDPD2 gene in oral squamous cell carcinoma tissue is obviously reduced.

Example 2 QPCR sequencing verification of differential expression of GDPD2 Gene

1. Large sample QPCR validation was performed on differential expression of GDPD2 gene. 80 cases of each of the normal mucosal tissue and the oral squamous cell carcinoma tissue were selected in accordance with the manner of sample collection in example 1.

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

3. Reverse transcription:

1) reaction system:

2) conditions for reverse transcription

The reverse transcription reaction conditions in RNA PCR Kit (AMV) Ver.3.0 were followed.

42℃60min，99℃2min，5℃5min。

3) Polymerase chain reaction

1) Primer design

QPCR amplification primers were designed based on the coding sequences of GDPD2 gene and GAPDH gene in Genebank and synthesized by Bomader Biotech. The specific primer sequences are as follows:

GDPD2 gene:

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

the reverse primer was 5'-CAAGTAGATGACCAGGACAA-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)

2) Prepare 25 μ l PCR reaction as per Table 1:

TABLE 1 PCR reaction System

3) And (3) PCR reaction conditions: 94 ℃ for 4min, (94 ℃ for 20s, 60 ℃ for 30s, 72 ℃ for 30s) x 30 cycles. SYBRGreen is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, relative quantification is carried out by an delta CT method, and each sample is subjected to 3 times of repeated experiments.

5. Statistical method

The experimental results of fluorescent quantitative RT-PCR of oral squamous cell carcinoma tissue and normal mucosa tissue are calculated by taking GAPDH as an internal reference, statistical analysis is carried out by adopting SPSS18.0 statistical software, and the difference between the oral squamous cell carcinoma tissue and the normal mucosa tissue is statistically analyzed by adopting a t test and has statistical difference with P < 0.05.

6. Results

As shown in fig. 1, GDPD2 gene expression was significantly reduced in oral squamous cell carcinoma tissues compared to the control group, with the difference being statistically significant (P <0.05), consistent with the RNA-sep results.

Example 3 differential expression of the GDPD2 Gene in oral squamous cell carcinoma cell lines

1. Cell culture

Oral squamous cell carcinoma cell lines Tca8113, HN13, and the normal mucosal epithelial cell line HIOEC was purchased from the ninth national Hospital affiliated to Shanghai university of transportation. The culture medium of the HIOEC is K-SFM; the culture medium of Tca8113 and HN13 is DMEM; culturing with culture medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. Extraction of Total RNA from cells

1) The culture was terminated when the cells reached 80-90% confluence, and the cells were harvested by 0.25% trypsinization in 1.5m1EP tubes, disrupted by adding lm1Trizol to each tube and left on ice for 10 min.

2) Deproteinization, DNA removal: 0.2ml of chloroform was added to each 1.5m1EP tube, shaken for 15 seconds, and allowed to stand at room temperature for 10 min. Centrifuge at 12000rpm for 15min at 4 ℃.

The remaining procedures were the same as in the extraction of RNA from tissues.

3. Reverse transcription

The specific procedure is the same as in example 2.

4. Statistical method

The experiments were performed in 3 replicates, the results were represented as mean ± sd, and were statistically analyzed using SPSS18.0 statistical software, with the difference between the two using the t-test, and considered statistically significant when P < 0.05.

5. Results

As shown in FIG. 2, compared with normal mucosal epithelial cells, the GDPD2 gene is down-regulated in oral squamous cell carcinoma cells Tca8113 and HN13, and the difference is statistically significant (P <0.05), which is consistent with the result of RNA-sep.

Example 4 overexpression of the GDPD2 Gene

1. Cell culture

Human oral squamous cell carcinoma cell line Tca8113 prepared by culturing 10% fetal calf serum and 1% P/S in DMEM at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. Overexpression of the GDPD2 Gene

Construction of GDPD2 Gene overexpression vector

Designing amplification primers according to the coding sequence (shown as SEQ ID NO. 1) of the GDPD2 gene, wherein the primer sequences are as follows:

a forward primer: 5'-CCGAAGCTTGCCACCATGGACTGGTCCCTGGCATT-3' (SEQ ID NO.7)

Reverse primer: 5'-CGGCTCGAGCTCCATCATGAAATTGTTGATCC-3' (SEQ ID NO.8)

The coding sequence of the full-length GDPD2 gene was amplified from a cDNA library of adult fetal brain (Clontech, cat # 638831), the cDNA sequence was double digested with restriction enzymes HindIII and XhoI and inserted into the eukaryotic cell expression vector pcDNA3.1 double digested with restriction enzymes HindIII and XhoI, and the resulting recombinant vector pcDNA3.1-GDPD2 was ligated for subsequent experiments.

3. Transfection

The oral squamous cell carcinoma cells were divided into three groups, namely a control group (Tca8113), a blank control group (pcDNA3.1-NC transfection), and a GDPD2 overexpression group (pcDNA3.1-GDPD 2 transfection). Transfection of the vector was performed using liposome 2000, and the specific transfection method was performed as indicated in the specification. The transfection concentrations of pcDNA3.1 empty vector and pcDNA3.1-GDPD2 were 0.5. mu.g/ml.

4. QPCR detection of transcription level of GDPD2 gene

4.1 extraction of Total RNA from cells

The specific procedure is the same as in example 3.

4.2 reverse transcription procedure as in example 2.

4.3 QPCR amplification procedure as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± sd, and statistical analysis was performed using SPSS18.0 statistical software, and the differences between the GDPD2 gene overexpression group and the control group were considered statistically significant when P <0.05 using t-test.

6. Results

The results are shown in figure 3, where GDPD2 was overexpressed in the transfected GDPD2 group compared to the non-transfected and transfected null plasmid groups, the difference being statistically significant (P < 0.05).

Example 5 MTT assay for Tca8113 cell proliferation Activity

MTT method is used to detect the influence of GDPD2 gene overexpression on the proliferation activity of Tca8113 cells.

1. Cell culture Tca8113 cells were cultured at 1X 10³Perwell inoculation in 96-well plates, 100. mu.1 per well, 37 ℃ 5% CO₂And (5) incubating and culturing in an incubator.

2. The cell transfection procedure was as in example 3.

3. MTT assay

1) After 1-7 days of transfection, the medium was discarded from each well and 20. mu.l of MTT (5mg/ml) was added. The conventional culture was continued for 4 h.

2) The mixture was aspirated, 200. mu.l of DMSO was added to each well, and the mixture was shaken for 10min to dissolve the crystals sufficiently. The absorbance at 490nm was measured on an enzyme linked immunosorbent and the results recorded.

4. Cell growth curves were plotted with time as the horizontal axis and light absorption (OD) as the vertical axis.

5. As a result:

as a result, as shown in FIG. 4, the proliferation of the cells of the pcDNA3.1-GDPD 2-transfected group was significantly reduced compared to the control.

Example 6 Transwell cell in vitro invasion assay

Tca8113 cells from different groups were collected on day 6 of cell transfection and resuspended in culture medium to a final cell concentration of 2X 10⁶Per ml, aspirate 100. mu.l of cell suspension into a Transwell chamber. The effect of GDPD2 gene overexpression on the invasiveness of Tca8113 cells was observed by using a Transwell chamber method.

1. The Matrigel (4. mu.g/. mu.l) was thawed at 4 ℃ and an ice box (ice bath environment) was prepared. The Matrigel was diluted 8-fold with DMEM and used. Mu.g of human fibronectin was coated on the outer surface of a Transwell cell filter (8 μm pore size) and air-dried in a clean bench.

2. The inner surface of a 6-well Transwell cell filter was coated with 100. mu.1/well Matrigel gel at 37 ℃ with 5% CO₂Incubating for 1h in an incubator to form a matrix barrier layer for later use.

3. DMEM medium containing 20% FBS 2.5m1 was added to each well of the 6-well plate.

4. Cells were collected in logarithmic growth phase and resuspended in culture medium to a final concentration of 2X 10⁶/ml。

5. The cell suspension was added to a Transwell chamber at 100. mu.1 per well, and the chamber was immersed in conditioned medium in 6-well plates at 37 ℃ with 5% CO₂And incubating in an incubator for 24 h.

6. The Transwell chamber was removed and the filter was fixed with methanol for 1 min.

7. HE staining: staining with hematoxylin for 3min, and washing with water; and (5) dyeing with eosin for 10-30 s, and washing with water. And the cells that did not cross the membrane were wiped off with a cotton swab.

8. The number of invaded cells was counted by microscopic observation, photography, and the number of permeated cells in 5 different visual fields, i.e., the upper and lower left and right sides, was counted per membrane, and the average value was calculated. Each group was provided with 3 filters in parallel.

9. Data processing

Statistical analysis of the data was performed using SPSS18.0 software. The metrology data is expressed as mean ± standard deviation. The average number of a plurality of samples is compared by adopting one-factor variance analysis, and the difference with P <0.05 has statistical significance.

10. Results

As shown in FIG. 5, the cell numbers of the cell lower chamber of the polycarbonate membrane of the pcDNA3.1-GDPD2 group were significantly reduced after the cells of the Tca8113, pcDNA3.1-GDPD2 and pcDNA3.1-GDPD2 group were cultured in a transwell chamber for 24 hours.

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> Beijing, the deep biometric information technology GmbH

<120> application of molecular marker in diagnosis and treatment of oral squamous cell carcinoma

<160>8

<170>PatentIn version 3.5

<210>1

<211>1383

<212>DNA

<213> human source

<400>1

atggactggt ccctggcatt cctgctggtc atctctctac tggtcacata tgcatccttg 60

ctattggtcc tggccctgct cctgcggctt tgtagacagc ccctgcatct gcacagcctc 120

cacaaggtgc tgctgctcct cattatgctg cttgtggcgg ctggccttgt gggactggac 180

atccaatggc agcaggagtg gcatagcttg cgtgtgtcac tgcaggccac agccccattc 240

cttcatattg gagcagccgc tggaattgcc ctcctggcct ggcctgtggc tgataccttc 300

taccgtatcc accgaagagg tcccaagatt ctgctactgc tcctattttt tggagttgtc 360

ctggtcatct acttggcccc cctatgcatc tcctcaccct gcatcatgga acccagagac 420

ttaccaccca agcctgggct ggtgggacac cgaggggccc ccatgctggc tcccgagaac 480

accctgatgt ccttgcggaa gacagctgaa tgcggagcta ctgtgtttga gactgatgtg 540

atggtcagct ccgatggggt ccccttcctc atgcatgatg agcacctcag caggaccacg 600

aatgtagcct ctgtattccc aacccgaatc acagcccaca gcagtgactt ctcctggact 660

gaactgaaga gactcaatgc tggatcctgg ttcctagaga ggcgaccctt ctggggggcc 720

aaaccgctgg caggccctga tcagaaagag gctgagagtc agacggtacc agcattagaa 780

gagctattgg aggaagctgc agccctcaac ctttccatca tgttcgactt gcgccgaccc 840

ccacagaacc acacatacta tgacactttt gtgatccaga cattggagac tgtgctgaat 900

gcaagggtgc cccaagccat ggtcttttgg ctaccagatg aagatcgggc taatgtccaa 960

cgacgggcac ctggaatgcg ccagatatat ggacgtcagg gaggcaacag aacggagagg 1020

ccccagtttc ttaacctccc ctatcaagat ctgccactat tggatatcaa ggcattgcat 1080

aaggataatg tctcggtgaa cctatttgta gtgaacaagc cctggctctt ctctctgctt 1140

tggtgtgcag gggtggattc ggtcaccacc aacgactgcc agctgctgca gcagatgcgt 1200

taccctatct ggcttattac ccctcaaacc tacctaatca tatgggtcat taccaattgt 1260

gtttccacca tgctgctttt gtggaccttc ctcctccaaa gaagatttgt taagaagaga 1320

gggaaaactg gcttagaaac agcagtgctg ctgacaagga tcaacaattt catgatggag 1380

tga 1383

<210>2

<211>460

<212>PRT

<213> human source

<400>2

Met Asp Trp Ser Leu Ala Phe Leu Leu Val Ile Ser Leu Leu Val Thr

1 5 10 15

Tyr Ala Ser Leu Leu Leu Val Leu Ala Leu Leu Leu Arg Leu Cys Arg

20 25 30

Gln Pro Leu His Leu His Ser Leu His Lys Val Leu Leu Leu Leu Ile

35 40 45

Met Leu Leu Val Ala Ala Gly Leu Val Gly Leu Asp Ile Gln Trp Gln

50 55 60

Gln Glu Trp His Ser Leu Arg Val Ser Leu Gln Ala Thr Ala Pro Phe

65 70 75 80

Leu His Ile Gly Ala Ala Ala Gly Ile Ala Leu Leu Ala Trp Pro Val

85 90 95

Ala Asp Thr Phe Tyr Arg Ile His Arg Arg Gly Pro Lys Ile Leu Leu

100 105 110

Leu Leu Leu Phe Phe Gly Val Val Leu Val Ile Tyr Leu Ala Pro Leu

115 120 125

Cys Ile Ser Ser Pro Cys Ile Met Glu Pro Arg Asp Leu Pro Pro Lys

130 135 140

Pro Gly Leu Val Gly His Arg Gly Ala Pro Met Leu Ala Pro Glu Asn

145 150 155 160

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

165 170 175

Glu Thr Asp Val Met Val Ser Ser Asp Gly Val Pro Phe Leu Met His

180 185 190

Asp Glu His Leu Ser Arg Thr Thr Asn Val Ala Ser Val Phe Pro Thr

195 200 205

Arg Ile Thr Ala His Ser Ser Asp Phe Ser Trp Thr Glu Leu Lys Arg

210 215 220

Leu Asn Ala Gly Ser Trp Phe Leu Glu Arg Arg Pro Phe Trp Gly Ala

225 230 235 240

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

245 250 255

Pro Ala Leu Glu Glu Leu Leu Glu Glu Ala Ala Ala Leu Asn Leu Ser

260 265 270

Ile Met Phe Asp Leu Arg Arg Pro Pro Gln Asn His Thr Tyr Tyr Asp

275 280 285

Thr Phe Val Ile Gln Thr Leu Glu Thr Val Leu Asn Ala Arg Val Pro

290 295 300

Gln Ala Met Val Phe Trp Leu Pro Asp Glu Asp Arg Ala Asn Val Gln

305 310 315 320

Arg Arg Ala Pro Gly Met Arg Gln Ile Tyr Gly Arg Gln Gly Gly Asn

325 330 335

Arg Thr Glu Arg Pro Gln Phe Leu Asn Leu Pro Tyr Gln Asp Leu Pro

340 345 350

Leu Leu Asp Ile Lys Ala Leu His Lys Asp Asn Val Ser Val Asn Leu

355 360 365

Phe Val Val Asn Lys Pro Trp Leu Phe Ser Leu Leu Trp Cys Ala Gly

370 375 380

Val Asp Ser Val Thr Thr Asn Asp Cys Gln Leu Leu Gln Gln Met Arg

385 390 395 400

Tyr Pro Ile Trp Leu Ile Thr Pro Gln Thr Tyr Leu Ile Ile Trp Val

405 410 415

Ile Thr Asn Cys Val Ser Thr Met Leu Leu Leu Trp Thr Phe Leu Leu

420 425 430

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

435 440 445

Val Leu Leu Thr Arg Ile Asn Asn Phe Met Met Glu

450 455 460

<210>3

<211>18

<212>DNA

<213> Artificial sequence

<400>3

taccgtatcc accgaaga 18

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<400>4

caagtagatg accaggacaa 20

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<400>5

ctctggtaaa gtggatattg t 21

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

ggtggaatca tattggaaca 20

<210>7

<211>35

<212>DNA

<213> Artificial sequence

<400>7

ccgaagcttg ccaccatgga ctggtccctg gcatt 35

<210>8

<211>32

<212>DNA

<213> Artificial sequence

<400>8

cggctcgagc tccatcatga aattgttgat cc 32

Claims

1. Application of reagent for specifically detecting GDPD2 gene or its expression product in preparing products for diagnosing oral squamous cell carcinoma.

2. The use according to claim 1, wherein the product comprises reagents for specifically detecting a change in the GDPD2 gene or its expression product by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, or immunoassays.

3. Use according to claim 2, wherein the nucleic acid amplification technique is selected from the group consisting of polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification and nucleic acid sequence based amplification.

4. The use of claim 1, wherein the product comprises a chip, a kit or a formulation.

5. The use of claim 4, wherein the chip, kit or formulation comprises specific primers, probes for the GDPD2 gene or antibodies or ligands for the protein encoded by GDPD 2.

Use of an enhancer of GDPD2 gene and/or its expression product for the manufacture of a pharmaceutical composition for the treatment of oral squamous cell carcinoma, wherein said enhancer is an over-expression vector of GDPD 2.

7. A method for inhibiting proliferation of oral squamous cell carcinoma cell in vitro is characterized in that GDPD2 gene, protein or its promoter is added into a culture system.