CN112877435A - Oral squamous carcinoma biomarker and application thereof - Google Patents

Abstract

The invention discloses an oral squamous carcinoma biomarker and application thereof, wherein the biomarker is a combination of RP11-733O18.1 or RP11-733O18.1 and AC108142.1 or RP13-297E 16.4; the invention also discloses application of the biomarker in preparing a pharmaceutical composition for treating oral squamous cell carcinoma.

Description

Oral squamous carcinoma biomarker and application thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to oral squamous cell carcinoma biomarkers and application thereof.

Background

According to 2018, global Cancer survey shows that oral Cancer is one of the most common malignant tumors in the world, the number of deaths accounts for 1.9% of all Cancer deaths, the 14 th death is listed, and the male morbidity is significantly higher than that of the female, which is about 2.54 times that of the female (Bray F, Ferlay J, Soerjomataram I, et al. Global Cancer statistics 2018: GLOBOCAN cancers of confidence and birth world wide for 36cancers in 185countries [ J ]. CA: A Cancer Journal for Clinicians,2018,68(6):394 424.). Oral Squamous carcinomas are a common type of cancer (Jan J, Hsu W, Liu S, et al. protective Factors in Patients With dental Square Cell Carcinoma: 10-Yeast Experience [ J ]. Journal of Oral and Maxillofacial Surgery,2011,69(2): 396-. Squamous cell carcinoma of the mouth may be clinically manifested as persistent ulcers, repeatedly erosive lichen planus, white, red plaques, hard and immobile masses, and the like. The treatment is mainly surgical operation. With the continuous development and the advancement of surgical technology and diagnostic technology in recent years, the survival rate of OSCC patients is remarkably improved at present. However, there are still many patients who have poor therapeutic efficacy, manifested by regional local recurrence and distant metastasis after treatment, and the survival rate is still not optimistic. With the increasing research on OSCC, more and more researchers believe that genes play an important role in diagnosis and treatment of OSCC.

With the advance of modern medical technology, omics research brings unprecedented help to the advance of medicine, and genome research reveals molecular mechanisms of many complex human diseases. The development of the second generation sequencing technology provides a research means with high accuracy, high throughput, high sensitivity and low running cost for the research of genomics, and the technology is widely applied to the search of candidate genes of diseases (Shridhar K, Walia GK, Agrarwal A, et al. DNA methylation markers for Oral pre-cancer progression: A clinical review [ J ]. Oral Oncol,2016,53: 1-9.). The search for the gene related to the oral squamous cell carcinoma has important significance for realizing the diagnosis and treatment of the 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 associated with oral squamous cell carcinoma, which marker can be used to diagnose whether a subject has or is at risk of developing oral squamous cell carcinoma.

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

in a first aspect, the invention provides the use of an agent for detecting a biomarker in a sample in the manufacture of a product for diagnosing oral squamous carcinoma, wherein the biomarker comprises RP11-733O 18.1.

Further, the biomarkers also include one or both of AC108142.1 or RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1 and AC 108142.1.

Further, the biomarker is a combination of RP11-733O18.1 and RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1, AC108142.1 and RP13-297E 16.4.

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

Further, the agent is selected from:

a probe that specifically recognizes the biomarker; or

A primer that specifically amplifies the biomarker.

Further, the sample is selected from the group consisting of tissue, blood.

In a second aspect the invention provides a product for the diagnosis of oral squamous cell carcinoma, the product comprising a DNA chip, oligonucleotide chip, probe or primer necessary for performing DNA microarrays, oligonucleotide microarrays, northern blotting, RNase protection assays and reverse transcription polymerase chain reactions to detect the expression of biomarkers including RP11-733O18.1 in a sample.

Further, the biomarkers also include one or both of AC108142.1 or RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1 and AC 108142.1.

Further, the biomarker is a combination of RP11-733O18.1 and RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1, AC108142.1 and RP13-297E 16.4.

Further, the product also includes reagents for processing the sample.

A third aspect of the invention provides the use of a biomarker comprising RP11-733O18.1 in the construction of a computational model or a system incorporating the computational model for predicting oral squamous carcinoma.

Further, the biomarkers also include one or both of AC108142.1 or RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1 and AC 108142.1.

Further, the biomarker is a combination of RP11-733O18.1 and RP13-297E 16.4.

Further, the biomarker is a combination of RP11-733O18.1, AC108142.1 and RP13-297E 16.4.

Further, the calculation model is operated by a bioinformatics method with the level of the biomarker as an input variable.

The fourth aspect of the invention provides an application of RP11-733O18.1 in preparing a pharmaceutical composition for treating oral squamous carcinoma.

Further, the pharmaceutical composition includes an inhibitor of RP11-733O 18.1.

Further, the inhibitor is an agent that specifically reduces expression of RP11-733O 18.1.

Further, the inhibitor is interfering RNA.

In a fifth aspect the invention provides a pharmaceutical composition for the treatment of oral squamous carcinoma, the pharmaceutical composition comprising an inhibitor of RP11-733O 18.1.

Further, the inhibitor is an agent that specifically reduces expression of RP11-733O 18.1.

Further, the inhibitor is interfering RNA.

The product of the invention can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to the development process of oral squamous cell carcinoma) including RP11-733O18.1, AC108142.1 and RP13-297E16.4 genes.

The invention has the advantages and beneficial effects that:

the RP11-733O18.1 is selected as a biomarker, so that whether a subject suffers from oral squamous cell carcinoma or not can be diagnosed or the risk of the subject suffering from the oral squamous cell carcinoma is judged, and doctors are guided to take treatment strategies, means and measures timely.

Drawings

FIG. 1 is a graph of biomarker expression in oral squamous carcinoma tissue, wherein Panel A is a graph of RP11-733O18.1 expression in oral squamous carcinoma tissue; panel B is a graph of AC108142.1 expression in oral squamous carcinoma tissue; panel C is a graph of RP13-297E16.4 expression in oral squamous carcinoma tissue;

FIG. 2 is a ROC plot of biomarkers as the detection variables, wherein plot A is a ROC plot of RP11-733O18.1 as the detection variables; FIG. B is a ROC plot of RP11-733O18.1, AC108142.1, and RP13-297E16.4 in combination as the measured variables.

Detailed Description

In the present invention, the term "biomarker" means a compound, preferably a gene, which is differentially present (i.e. increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g. having a disease) compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g. no disease). The term "biomarker" generally refers to the presence/concentration/amount of one gene or the presence/concentration/amount of two or more genes.

Biomarkers can be differentially present at any level, but are typically present at levels that are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more; or generally at a level that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent).

Preferably, the biomarkers are differentially present at levels of statistical significance (i.e., p-value less than 0.05 and/or q-value less than 0.10, as determined using the Welch's T-Test or the Wilcoxon rank-sum Test).

In a specific embodiment of the invention, the biomarkers include RP11-733O18.1, AC108142.1, and RP13-297E 16.4.

In the present invention, RP11-733O18.1(ensembl gene ID: ENSG00000236120) includes RP11-733O18.1 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed RP11-733O18.1, as well as any form of RP11-733O18.1 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RP11-733O 18.1.

AC108142.1(ensembl gene ID: ENSG00000177822) includes AC108142.1 gene and its homologues, mutations, and isoforms. The term encompasses full-length, unprocessed AC108142.1, as well as any form of AC108142.1 that results from processing in the cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of AC 108142.1.

RP13-297E16.4(ensembl gene ID: ENSG00000223511) includes the RP13-297E16.4 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed RP13-297E16.4, as well as any form of RP13-297E16.4 that results from processing in the cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RP13-297E 16.4.

In the present invention, any suitable method may be used to analyze a biological sample to determine the level of the biomarker in the sample. These methods include, but are not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of the nucleic acid sequencing methods of the present invention include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing method of the present invention includes next generation sequencing (deep sequencing/high throughput sequencing), a high throughput sequencing technique that is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Methods of nucleic acid hybridization in the present 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.

The nucleic acid amplification method of the present 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.

The terms "sample" and "sample" are used interchangeably herein to refer to a composition obtained or derived from a subject (e.g., an individual of interest) that comprises cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluids, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

As a preferred embodiment, the sample is selected from blood, serum, plasma.

In another preferred embodiment, the sample is selected from the group consisting of tissues.

The invention provides a product for diagnosing oral squamous carcinoma, which comprises a reagent for detecting the biomarker in a sample; and instructions for using the product to assess whether the subject is suffering from or susceptible to oral squamous carcinoma.

The most reliable results are possible when processing samples in a laboratory environment. For example, a sample may be taken from a subject in a doctor's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide immediate results at the clinician's office or to allow the subject to perform the test at home. In some cases, the need for testing that is portable, prepackaged, disposable, ready to use by the subject without assistance or guidance, etc., is more important than a high degree of accuracy. In many cases, especially in the case of physician visits, it may be sufficient to perform a preliminary test, even a test with reduced sensitivity and/or specificity. Thus, assays provided in product form can involve detecting and measuring relatively small amounts of biomarkers to reduce the complexity and cost of the assay.

Any form of sample assay capable of detecting a sample biomarker described herein may be used. Typically, the assay will quantify the biomarkers in the sample to an extent, for example whether their concentration or amount is above or below a predetermined threshold. Such kits may take the form of test strips, dipsticks, cartridges, chip-based or bead-based arrays, multi-well plates, or a series of containers, and the like. One or more reagents are provided to detect the presence and/or concentration and/or amount of a selected sample biomarker. The sample from the subject may be dispensed directly into the assay or indirectly from a stored or previously obtained sample.

In the present invention, biomarkers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual retention of each marker, or a combination thereof.

As the skilled artisan will appreciate, the step of associating a marker level with a certain likelihood or risk may be implemented and realized in different ways.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing oral squamous carcinoma is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).

The area under the receiver operating curve (AUC) is an indicator of the performance or accuracy of a diagnostic procedure. The accuracy of a diagnostic method is best described by its Receiver Operating Characteristics (ROC). ROC plots are line graphs of all sensitivity/specificity pairs derived from continuously varying decision thresholds across the entire data range observed.

The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify a subject into a clinically relevant subgroup. Diagnostic accuracy measures the ability to correctly discriminate between two different conditions of the subject under investigation. Such conditions are, for example, health and disease or disease progression versus no disease progression.

One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single numerical value. The most common global metric is the area under the ROC curve (AUC). Conventionally, this area is always ≧ 0.5 (if not, the decision rule can be reversed to do so). The range of values was between 1.0 (test values that perfectly separated the two groups) and 0.5 (no significant distribution difference between the test values of the two groups). The area depends not only on a particular part of the line graph, such as the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire line graph. This is a quantitative, descriptive representation of how the ROC plot is close to perfect (area 1.0).

Overall assay sensitivity will depend on the specificity required to carry out the methods disclosed herein. In certain preferred settings, a specificity of 75% may be sufficient, and statistical methods and resulting algorithms may be based on this specificity requirement. In a preferred embodiment, the method for assessing an individual at risk for oral squamous cell carcinoma is based on specificity of 80%, 85%, or also preferably 90% or 95%.

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 statistical software, and the difference between the two is considered to have statistical significance when P is less than 0.05 by using t test.

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 1QPCR detection of biomarker expression levels

1. Sample collection

15 oral squamous carcinoma tissues and 15 corresponding paracarcinoma tissues were collected, and the patients did not receive any treatment before surgery, with the carcinoma tissues as the experimental group and the paracarcinoma tissues as the normal control group.

2. RNA extraction

RNA in the sample was extracted using a tissue RNA extraction kit of QIAGEN, and detailed procedures were performed according to the instructions.

3. QPCR detection

1) The first strand synthesis kit (cat No.: KR106) to reverse transcription of mRNA;

2) QPCR amplification primers were designed based on the sequences of RP11-733O18.1, AC108142.1, or RP13-297E16.4, synthesized by Bomaide Bio Inc., and GAPDH was used as the reference gene.

Amplification with SuperReal Premix Plus (SYBR Green) (cat # FP205), band of interest by melting curve analysis and electrophoresis, 2^-ΔΔCTThe method is used for relative quantification, and the specific experimental operation is carried out according to the product specification.

4. Results

The results are shown in figure 1, RP11-733O18.1, AC108142.1 or RP13-297E16.4 shows significant difference in the experimental group and the control group, compared with the control group, RP11-733O18.1, AC108142.1 and RP13-297E16.4 show significant up-regulation in the experimental group, and the results suggest that RP11-733O18.1, AC108142.1 and RP13-297E16.4 can be used as biomarkers for diagnosing oral squamous cell carcinoma.

Example 2 diagnostic potency validation of biomarkers

And (3) downloading the sequencing data and clinical information of the pretreated oral squamous cell carcinoma from a TCGA (Chinese character of 'Huanyuan' database, wherein the sample amount is paracarcinoma, namely 44: 331.

Differential lncRNA expression analysis using R software DESeq2 showed that RP11-733O18.1, AC108142.1, or RP13-297E16.4 exhibited significant upregulation in oral squamous carcinoma tissues.

The Receiver Operating Curve (ROC) is drawn by using the R package 'pROC', the AUC value, the sensitivity and the specificity are analyzed, and the diagnosis efficiency of the indexes is judged alone or in combination. When the diagnosis efficiency of the index combination is judged, the expression level of each gene is subjected to logistic regression, the probability of whether each individual suffers from cancer is calculated through a fitted regression curve, different probability division threshold values are determined, and the sensitivity, specificity, accuracy and the like of each combination detection scheme are calculated according to the determined probability division threshold values. The results are shown in Table 1 and FIG. 2, the AUC values of RP11-733O18.1, AC108142.1, RP13-297E16.4 are 0.644, 0.831 and 0.818 respectively; the AUC value of the combination of the three is 0.904, which indicates that the diagnosis of oral squamous cell carcinoma by using RP11-733O18.1, AC108142.1 and/or RP13-297E16.4 has higher diagnosis efficiency.

TABLE 1 area under the curve (AUC)

Gene	AUC
		RP11-733O18.1	0.644
AC108142.1	0.831
		RP13-297E16.4	0.818
RP11-733O18.1+AC108142.1	0.833
		RP11-733O18.1+RP-13.297E16.4	0.856
RP11-733O18.1+AC108142.1+RP13-297E16.4	0.904

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

Claims (10)

1. Use of a reagent for detecting a biomarker in a sample for the manufacture of a product for diagnosing oral squamous carcinoma, wherein the biomarker comprises RP11-733O 18.1; preferably, the biomarkers further comprise one or both of AC108142.1 or RP13-297E 16.4.

2. The use of claim 1, wherein the reagents comprise reagents for detecting biomarker levels by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques.

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

a probe that specifically recognizes the biomarker; or

A primer that specifically amplifies the biomarker.

4. The use according to any one of claims 1 to 3, wherein the sample is selected from the group consisting of tissue, blood.

5. A product for diagnosing oral squamous carcinoma, wherein said product comprises DNA chips, oligonucleotide chips, probes or primers necessary for performing DNA microarrays, oligonucleotide microarrays, northern blotting, RNase protection tests, and reverse transcription polymerase chain reactions to detect the expression of biomarkers in a sample; the biomarker comprises RP11-733O 18.1; preferably, the biomarkers further comprise one or both of AC108142.1 or RP13-297E 16.4.

6. The product of claim 5, further comprising a reagent for processing the sample.

7. Use of a biomarker in the construction of a computational model for predicting oral squamous carcinoma, or a system in which said computational model is embedded, wherein said biomarker comprises RP11-733O 18.1; preferably, the biomarkers further comprise one or both of AC108142.1 or RP13-297E 16.4.

8. Use according to claim 7, wherein the computational model is operated by bioinformatics methods with levels of biomarkers as input variables.

The application of RP11-733O18.1 in preparing a pharmaceutical composition for treating oral squamous carcinoma; preferably, the pharmaceutical composition comprises an inhibitor of RP11-733O 18.1; preferably, the inhibitor is an agent that specifically reduces expression of RP11-733O 18.1; preferably, the inhibitor is interfering RNA.

10. A pharmaceutical composition for treating oral squamous carcinoma, wherein said pharmaceutical composition comprises an inhibitor of RP11-733O 18.1; preferably, the inhibitor is an agent that specifically reduces expression of RP11-733O 18.1; preferably, the inhibitor is interfering RNA.

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