CN109439760B

CN109439760B - Application of ARRDC2 in evaluating development process of oral squamous cell carcinoma

Info

Publication number: CN109439760B
Application number: CN201811566545.3A
Authority: CN
Inventors: 唐瞻贵; 王月红; 潘灏; 邓智元; 刘欧胜; 方小丹; 顾立群; 全宏志; 王柏胜; 戴小寒; 李龙; 陈晓婧
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: 2022-04-08
Anticipated expiration: 2038-12-19
Also published as: CN109439760A

Abstract

The invention discloses application of ARRDC2 in evaluating development process of oral squamous cell carcinoma, and the invention screens gene ARRDC2 with significant change in expression in different development processes of tumor through high throughput sequencing and bioinformatics analysis, and prompts that ARRDC2 can be used as a molecular marker to be applied to judgment of development process of oral squamous cell carcinoma, accurate and objective prediction of tumor prognosis, guidance of treatment scheme formulation and curative effect monitoring.

Description

Application of ARRDC2 in evaluating development process of oral squamous cell carcinoma

Technical Field

The invention belongs to the field of biological medicines, and relates to application of ARRDC2 in evaluating development process of oral squamous cell carcinoma.

Background

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor in the oral cavity, with 500000 new cases occurring worldwide each year, and presents a huge challenge and burden to the medical security system of the whole society. The combined effect of susceptibility to genetic background and environmental factors has led to the development and progression of oral squamous cell carcinoma, with shorter survival of patients with oral squamous cell carcinoma due to frequent distant metastasis, poorer prognosis and high recurrence rate. Currently, most oral squamous cell carcinoma patients are diagnosed with advanced disease, so an appropriate tumor-associated marker will aid in early diagnosis and prognosis of oral squamous cell carcinoma patients.

In the last decades, the research on the mechanism of oral cancer and the treatment method thereof have been remarkably advanced, but the 5-year survival rate of patients still stays at about 50% without remarkable change, so that the prevention and treatment research on malignant tumors is strengthened, and the accurate and objective evaluation on the biological behavior and prognosis of tumors and the establishment of treatment schemes are more urgent. The classification (classification), grading (grading) and staging (staging) of tumors are the three most important indicators for evaluating the biological behavior and diagnosis of tumors at present, wherein the classification and staging are mainly used for the evaluation of the biological behavior and prognosis of malignant tumors. In recent decades, with the breakthrough progress of life science and medical technology, the detection of the target related to the individualized treatment of tumors and the clinical application of individualized treatment medicines including targeted treatment not only greatly improve the detection rate of early tumors, but also obviously improve the prognosis of many tumors. The clinical value and significance of traditional tumor typing, grading and staging have also changed to varying degrees. Provides more accurate molecular biological information for the individual treatment of the tumor, guides the formulation of an individual treatment scheme and monitors the curative effect.

Atypia (atypia) is an important histological feature of malignant tumors, which is essentially a morphological manifestation of the degree of differentiation of the tumor, reflecting the morphological differences of the tumor tissue in terms of tissue structure and cell morphology, to a different extent, between the cells of the normal tissue from which it originates. The magnitude of this tumor tissue heterogeneity can be expressed in the grading (G) of the tumor. Currently, a concise three-level scheme is commonly used for tumor grading: grade I (G1), i.e., well differentiated (referred to as "highly differentiated"), tumor cells are near the corresponding normal tissue of origin, and are less malignant; grade III (G3), a less differentiated cell (termed "poorly differentiated"), a tumor cell that is highly malignant as distinguished from the corresponding normal tissue of origin; grade II (G2), which is intermediate between grade I and grade III in tissue heterogeneity, is of intermediate malignancy.

The grading of the tumor is mainly determined according to parameters such as the size of tumor tissue structure and cell heterogeneity, nuclear fission or proliferation index, necrosis range, invasion condition and the like in an HE stained section under a microscope. However, due to the complexity and heterogeneity of tumor tissue structure, different types of tumors (e.g., adenocarcinoma, squamous carcinoma, renal cell carcinoma, breast cancer, etc.) have different structural features and grading standards, and lack quantitative indicators, and furthermore, due to the influence of the sufficiency of material selection and subjective differences in diagnostic criteria, abnormal interpretation, etc., all affect the objectivity, accuracy and repeatability of tumor grading to different degrees. With the development of molecular biology, people pay attention to the correlation between genes and tumors, discuss the correlation between genes and oral squamous cell carcinoma development process, and actively develop molecular typing diagnosis taking the detection of tumor specific molecular targets as the core is the premise and the basis of accurate and objective prediction of tumor prognosis, guidance of treatment scheme formulation and curative effect monitoring, and has important significance for implementation of tumor individualized treatment.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a biomarker related to the development process of oral squamous cell carcinoma, and by using the biomarker, the cancer grade of an oral squamous cell carcinoma patient can be evaluated, so that the accurate treatment of the oral squamous cell carcinoma is realized.

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

the invention provides application of an agent for detecting ARRDC2 in preparing a product for evaluating the development process of oral squamous cell carcinoma.

Further, the reagent comprises a reagent for detecting the expression level of the ARRDC2 in the sample by RT-PCR, real-time quantitative PCR, immunoassay, in-situ hybridization or a chip. The sample for detecting ARRDC2 includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, expectorations, bronchoalveolar lavage fluids, urine, feces, etc. Preferably, the sample is tissue or blood. In a specific embodiment of the invention, the sample is a tissue.

Further, the agent is selected from:

a probe that specifically recognizes ARRDC 2; or

Primers for specific amplification of ARRDC 2; or

An antibody or ligand that specifically binds to ARRDC 2.

Furthermore, the primer sequence of the specific amplification ARRDC2 is shown in SEQ ID NO. 3-4.

Further, ARRDC2 was up-regulated in patients with highly malignant oral squamous cell carcinoma.

In a second aspect of the present invention there is provided a kit for assessing the progression of oral squamous carcinoma comprising a DNA chip, oligonucleotide chip, protein chip, probe or primer necessary for performing DNA microarray, oligonucleotide microarray, protein array, northern blot, RNase protection assay, western blot and reverse transcriptase polymerase chain reaction to detect the expression of ARRDC 2.

Further, the kit is used for detection by a DNA microarray method, a reverse transcription polymerase chain reaction method or a protein array method.

Further, the reagent for detecting by reverse transcription polymerase chain reaction comprises a primer for specifically amplifying ARRDC2, preferably, the sequence of the primer is shown as SEQ ID NO. 3-4.

Further, the expression level of the ARRDC2 is different in oral squamous cell carcinoma with different development processes, wherein the expression level is relatively lower in oral squamous cell carcinoma with high differentiation degree and low malignancy degree, and the expression level is relatively higher in oral squamous cell carcinoma with low differentiation degree and high malignancy degree.

In a third aspect of the invention, there is provided the use of ARRDC2 in the construction of a computational model for assessing the progression of oral squamous cell carcinoma.

The kit of the invention can be used for detecting the expression levels of a plurality of genes including the ARRDC2 gene (for example, a plurality of genes related to the development process of oral squamous cell carcinoma). The protein chip or the protein immunoassay kit can be used for detecting the expression levels of a plurality of proteins including the ARRDC2 protein (such as a plurality of proteins related to the development process 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, the ARRDC2 is selected as a molecular marker, and the development process of oral squamous cell carcinoma can be judged, so that doctors are guided to adopt different treatment strategies, means and measures for oral squamous cell carcinoma patients in different development processes, further, over-treatment can be avoided, insufficient treatment intensity can be avoided, and individual treatment of the oral squamous cell carcinoma patients is realized.

Drawings

FIG. 1 is a graph showing the detection of the expression of ARRDC2 in oral squamous cell carcinoma tissues by QPCR.

Detailed Description

The present invention has been extensively and intensively studied to screen genes whose expression significantly changes during the transition from G1 to moderately differentiated OSCC (G2) and from G2 to poorly differentiated OSCC (G3) by high throughput sequencing and bioinformatics analysis. The differentiation grade of the tumor tissue can be correctly predicted by selecting genes whose expression is significantly changed in each transition stage. Therefore, these genes can be used to diagnose the differentiation grade of OSCC.

In the present invention, the skilled artisan 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 ID of the ARRDC2 gene in the current International public nucleic acid database GeneBank is 27106.

As a non-limiting example, the coding sequence or amino acid sequence of one representative human ARRDC2 is shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. ARRDC2 includes polypeptides comprising SEQ ID No.2 and other ARRDC2 native sequence polypeptides, such as naturally occurring variants and native sequence polypeptides, which are encoded by the nucleotide sequence shown in SEQ ID No. 1.

The molecular marker of the invention comprises genes and proteins. Such markers include DNA comprising the complete or partial sequence of the nucleic acid sequence encoding the molecular marker or the complement of this sequence. Molecular marker nucleic acids also include RNA containing the entire or partial sequence of any nucleic acid sequence of interest. The molecular marker protein is a protein encoded by or corresponding to the DNA molecular marker of the present invention. The molecular marker protein comprises the complete or partial amino acid sequence of any molecular marker protein or polypeptide. Fragments and variants of molecular marker genes and proteins are also included within the scope of the invention.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional or translational (i.e., protein) level. These techniques include, but are not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

In the present invention, the kit comprises a reagent for detecting ARRDC2, and one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The term "probe" refers to oligonucleotides and their analogs, and relates to a series of chemicals that recognize a polynucleotide target sequence by hydrogen bonding interactions with the nucleotide bases of the target sequence. Is intended to include nucleic acid oligomers or aptamers that specifically hybridize to a target sequence in a nucleic acid or its complement under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection can be direct (i.e., generated by probes that directly hybridize to the target or amplified sequence) or indirect (i.e., generated by probes that hybridize to an intermediate molecular structure linking the probes and the target or amplified sequence). The "target" of a probe generally refers to a sequence of an amplified nucleic acid sequence that specifically hybridizes to at least a portion of the probe sequence through standard hydrogen bonding or "base pairing". Sequences that are "sufficiently complementary" allow for stable hybridization of the probe sequence to the target sequence even if the two sequences are not fully complementary. The probe may be labeled or unlabeled. Probes may be produced by molecular cloning of a particular DNA sequence, or may be synthesized. One skilled in the art to which the invention pertains can readily determine the variety of primers and probes that can be designed and used in the context of the present invention.

The term "primer" means an oligonucleotide, whether naturally occurring or synthetically produced in a purified restriction digest, that serves as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. Factors involved in determining the appropriate length of a primer will be readily known to those skilled in the art.

The term "microarray" is an ordered arrangement 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.

The antibodies against the above-mentioned ARRDC2 protein used in the present invention are used in the broadest sense and specifically encompass, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

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

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 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 (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 ARDDC 2 gene in oral squamous cell carcinoma tissues with different differentiation degrees shows significant difference, compared with G1, the expression of ARDDC 2 in G2 and G3/G4 is significantly up-regulated, compared with G2, the expression of ARDDC 2 in G3/G4 is significantly up-regulated, and the ARDDC 2 can be used as a possible marker for distinguishing oral squamous cell carcinoma with different development processes.

Example 2 QPCR sequencing verification of differential expression of ARRDC2 Gene

1. Large sample QPCR validation was performed on differential ARRDC2 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 ARRDC2 and GAPDH and were synthesized by Bomaide Biopsis. The specific primer sequences are as follows:

ARRDC2 gene:

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

the reverse primer was 5'-ATACAGTAGCGGACACTA-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. DELTA.2. mu.l, DNA template 2. mu.l, and 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 ℃ 15min, (95 ℃ 10s, 55 ℃ 30s, 72 ℃ 32s) x 40 cycles, 95 ℃ 15s, 60 ℃ 60s, 95 ℃ 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, the ARDDC 2 shows significant difference in oral squamous cell carcinoma tissues in different development processes, the lower the differentiation degree, the higher the malignancy degree and the higher the expression level of ARDDC 2, which indicates that the ARDDC 2 can be used as a molecular marker for judging the differentiation grade of oral squamous cell carcinoma, accurately and objectively predicting tumor prognosis, guiding the formulation of a treatment scheme and monitoring the curative effect.

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> ARRDC2 in evaluating development process of oral squamous cell carcinoma

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1209

<212> DNA

<213> Homo sapiens

<400> 1

atgcgctctg ggggcgtgcg cagcttcgcg ctggagctgg cgcggggccc gggcggcgcc 60

taccgcggcg gggagcggct gtgcggccgg gtgctgctgg aggcggcggc gccgctgcgg 120

gtgcgagcgc tcgaggtgaa ggcgcgcggc ggggcggcca cccactggtt ggagggtcgc 180

agcgtgggcg tcaacgccgt atccagcgac tacgcggccg cggagaccta cctgcggcgt 240

cggcagctgc tgctccgaga taccggggag accacgacgc tgcctcctgg gcgccatgag 300

ttcctgttca gcttccagct gcccccgacc ctggtgacat ccttcgaggg caaacacggt 360

agtgtccgct actgtatcaa ggccaccctg caccggccct gggtcccagc acgccgggca 420

aggaaggtgt tcactgtcat cgagcctgtg gacatcaaca cgccagccct gctggcacct 480

caagcggggg ctcgggaaaa ggttgcccga tcctggtact gtaaccgtgg cctagtctcc 540

ctttcggcca agatcgaccg caagggctac accccaggag aggtcatccc tgtctttgcc 600

gagatcgaca acggctccac acgtcctgtg ctgcctcggg cagccgtggt gcagacacag 660

acgttcatgg cccgaggcgc ccgaaagcag aaacgggcag tggtggccag cctcgcgggc 720

gagccggtgg gccccgggca gcgggcgctg tggcagggcc gggcactgcg gatcccccca 780

gtgggtcctt ccatcctgca ctgccgcgtt ctacacgtgg actacgcact caaggtctgt 840

gtggatatcc caggaacgtc caagctgctg ctggagctgc cactggtgat cggcaccatt 900

cccttgcacc cttttggcag ccgttcctcc agcgtgggca gccacgccag cttcctgctg 960

gactggaggc tgggggcctt gccggagcgg cctgaggctc ctcctgagta ctcggaggtg 1020

gtagccgaca ctgaggaggc agccttgggg cagagcccct tcccgcttcc gcaggacccc 1080

gacatgagcc ttgaaggccc gttcttcgcc tacatccaag agttccgcta ccgcccgcca 1140

cccctgtact ctgaggagga tccaaaccca ctcttggggg acatgaggcc gcgctgcatg 1200

acttgctga 1209

<210> 2

<211> 402

<212> PRT

<213> Homo sapiens

<400> 2

Met Arg Ser Gly Gly Val Arg Ser Phe Ala Leu Glu Leu Ala Arg Gly

1 5 10 15

Pro Gly Gly Ala Tyr Arg Gly Gly Glu Arg Leu Cys Gly Arg Val Leu

20 25 30

Leu Glu Ala Ala Ala Pro Leu Arg Val Arg Ala Leu Glu Val Lys Ala

35 40 45

Arg Gly Gly Ala Ala Thr His Trp Leu Glu Gly Arg Ser Val Gly Val

50 55 60

Asn Ala Val Ser Ser Asp Tyr Ala Ala Ala Glu Thr Tyr Leu Arg Arg

65 70 75 80

Arg Gln Leu Leu Leu Arg Asp Thr Gly Glu Thr Thr Thr Leu Pro Pro

85 90 95

Gly Arg His Glu Phe Leu Phe Ser Phe Gln Leu Pro Pro Thr Leu Val

100 105 110

Thr Ser Phe Glu Gly Lys His Gly Ser Val Arg Tyr Cys Ile Lys Ala

115 120 125

Thr Leu His Arg Pro Trp Val Pro Ala Arg Arg Ala Arg Lys Val Phe

130 135 140

Thr Val Ile Glu Pro Val Asp Ile Asn Thr Pro Ala Leu Leu Ala Pro

145 150 155 160

Gln Ala Gly Ala Arg Glu Lys Val Ala Arg Ser Trp Tyr Cys Asn Arg

165 170 175

Gly Leu Val Ser Leu Ser Ala Lys Ile Asp Arg Lys Gly Tyr Thr Pro

180 185 190

Gly Glu Val Ile Pro Val Phe Ala Glu Ile Asp Asn Gly Ser Thr Arg

195 200 205

Pro Val Leu Pro Arg Ala Ala Val Val Gln Thr Gln Thr Phe Met Ala

210 215 220

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

225 230 235 240

Glu Pro Val Gly Pro Gly Gln Arg Ala Leu Trp Gln Gly Arg Ala Leu

245 250 255

Arg Ile Pro Pro Val Gly Pro Ser Ile Leu His Cys Arg Val Leu His

260 265 270

Val Asp Tyr Ala Leu Lys Val Cys Val Asp Ile Pro Gly Thr Ser Lys

275 280 285

Leu Leu Leu Glu Leu Pro Leu Val Ile Gly Thr Ile Pro Leu His Pro

290 295 300

Phe Gly Ser Arg Ser Ser Ser Val Gly Ser His Ala Ser Phe Leu Leu

305 310 315 320

Asp Trp Arg Leu Gly Ala Leu Pro Glu Arg Pro Glu Ala Pro Pro Glu

325 330 335

Tyr Ser Glu Val Val Ala Asp Thr Glu Glu Ala Ala Leu Gly Gln Ser

340 345 350

Pro Phe Pro Leu Pro Gln Asp Pro Asp Met Ser Leu Glu Gly Pro Phe

355 360 365

Phe Ala Tyr Ile Gln Glu Phe Arg Tyr Arg Pro Pro Pro Leu Tyr Ser

370 375 380

Glu Glu Asp Pro Asn Pro Leu Leu Gly Asp Met Arg Pro Arg Cys Met

385 390 395 400

Thr Cys

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccatgagttc ctgttcag 18

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atacagtagc ggacacta 18

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctctggtaaa gtggatattg t 21

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtggaatca tattggaaca 20

Claims

1. Application of reagent for detecting ARRDC2 in preparing products for evaluating development process of oral squamous cell carcinoma.

2. The use of claim 1, wherein the reagents comprise reagents for detecting the expression level of ARRDC2 in a sample by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, or chip assay.

3. Use according to claim 2, wherein said agent is selected from:

a probe that specifically recognizes ARRDC 2; or

Primers for specific amplification of ARRDC 2; or

An antibody or ligand that specifically binds to ARRDC 2.

4. The use according to claim 3, wherein the primer sequence for specific amplification of ARRDC2 is shown in SEQ ID No. 3-4.

5. The use of any one of claims 1-4, wherein ARRDC2 is up-regulated in patients with highly malignant oral squamous cell carcinoma.

6. The use according to claim 1, wherein the product comprises a DNA chip, an oligonucleotide chip, a protein chip, a probe or a primer necessary for performing DNA microarrays, oligonucleotide microarrays, protein arrays, northern blotting, RNase protection assays, western blotting and reverse transcription polymerase chain reactions to detect the expression of ARRDC 2.

7. Use according to claim 6, wherein the product is detected by means of a DNA microarray, reverse transcription polymerase chain reaction or protein array.

8. Use according to claim 7, wherein the reagents for detection by reverse transcription polymerase chain reaction comprise primers for specific amplification of ARRDC 2.

9. Use according to any one of claims 6 to 8, wherein the level of expression of ARRDC2 is different in different developing oral squamous cell carcinomas.

Use of ARRDC2 in the construction of a computational model for assessing the progression of oral squamous carcinoma.