CN112941179B - Application of gene in evaluating oral squamous cell carcinoma - Google Patents

Abstract

The invention discloses application of a gene in evaluating oral squamous cell carcinoma, and particularly relates to a gene AC108142.1 or LINC01160, wherein detection of a cancer sample and a control sample shows that AC108142.1 or LINC01160 shows significant difference in two groups, and the gene has higher sensitivity, specificity and accuracy when applied to diagnosis of oral squamous cell carcinoma.

Description

Application of gene in evaluating oral squamous cell carcinoma

Technical Field

The invention belongs to the field of biological medicine, and relates to application of a gene in evaluating oral squamous cell carcinoma.

Background

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

The pathogenesis of OSCC is not completely understood, which also results in a relatively single, long-term treatment with an effective treatment (Abdalla Z, Walsh, Thakker N, et al. Loss of epithelial markers is an early event in organic dysplassia and is an object with the safety margin [ J ]. PLoS One,2017,12(12): e 0187449.). And the treatment cost of OSCC is high, which is often accompanied with the reduction of the life quality of patients. Therefore, early diagnosis is the key to the treatment of OSCC and is also the basic link for improving the prognosis of patients. For diagnosis of OSCC, the traditional physical examination and auxiliary examination are often difficult to accurately and efficiently judge the tumor type, progress and predict the prognosis. The detection of the biomarker can judge the existence, the progress and the prognosis condition of the tumor to a certain extent, thereby providing possibility for early diagnosis of the tumor and providing guidance and direction for early treatment of the tumor to a certain extent.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a gene marker related to oral squamous cell carcinoma, and by using the gene marker, whether a patient suffers from oral squamous cell carcinoma or not and the risk of suffering from oral squamous cell carcinoma can be judged and evaluated.

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

the invention provides application of a gene in preparing a product for diagnosing oral squamous cell carcinoma, wherein the gene is selected from one or two of AC108142.1 or LINC 01160.

Further, the product comprises a reagent for detecting the expression level of AC108142.1 or LINC01160 in a sample, wherein the sample comprises cells, tissues, organs, body fluids (blood, lymph fluid and the like), digestive juice, expectoration, alveolar and bronchial lavage fluid, urine, excrement and the like. Preferably, the sample is tissue or blood. In a specific embodiment of the invention, the sample is a tissue.

Further, AC108142.1 or LINC01160 is up-regulated in oral squamous carcinoma patients.

Further, the reagents include reagents for detecting the expression level of AC108142.1 or LINC01160 by RT-PCR, real-time quantitative PCR, in situ hybridization or on a chip.

Further, the reagent for detecting the expression level of AC108142.1 or LINC01160 by RT-PCR at least comprises a pair of primers for specifically amplifying the AC108142.1 or LINC01160 gene; the reagent for detecting the expression level of AC108142.1 or LINC01160 by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying AC108142.1 or LINC01160 genes; the reagent for detecting the expression level of AC108142.1 or LINC01160 through in situ hybridization comprises a probe hybridized with a nucleic acid sequence of the AC108142.1 or LINC01160 gene; the reagent for detecting the expression level of AC108142.1 or LINC01160 through the chip comprises a probe hybridized with a nucleic acid sequence of the AC108142.1 or LINC01160 gene.

In a second aspect, the invention provides a product for diagnosing oral squamous carcinoma, the product comprising an agent, a nucleic acid membrane strip, a chip or a kit, wherein the agent, nucleic acid membrane strip, chip or kit comprises an agent for detecting the expression level of AC108142.1 or LINC 01160.

Further, the reagent for detecting the expression level of AC108142.1 or LINC01160 in the chip comprises a probe for specifically recognizing AC108142.1 or LINC01160 genes.

Further, the reagent for detecting the expression level of AC108142.1 or LINC01160 in the kit comprises a primer for specifically amplifying the AC108142.1 or LINC01160 gene; or a probe that specifically recognizes the AC108142.1 or LINC01160 gene.

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 AC108142.1 or LINC 01160. The oral squamous carcinoma marker can be used for simultaneously detecting a plurality of oral squamous carcinoma markers, so that the accuracy of oral squamous carcinoma diagnosis can be greatly improved.

In a third aspect, the invention provides the use of a gene selected from AC108142.1 or LINC01160 in the manufacture of a pharmaceutical composition for the treatment of oral squamous carcinoma.

Further, the pharmaceutical composition comprises an inhibitor of AC108142.1 or LINC 01160.

Further, the inhibitor is an agent that specifically reduces the expression level of AC108142.1 or LINC 01160.

Further, the inhibitor is interfering RNA.

The invention has the advantages and beneficial effects that:

according to the invention, AC108142.1 or LINC01160 is selected as a gene marker, so that auxiliary diagnosis of oral squamous cell carcinoma can be realized, and a new means is provided for personalized treatment of patients.

The invention develops the detection product by using AC108142.1 or LINC01160, has the advantages of quick and convenient detection, high detection sensitivity and specificity and low cost, can meet the detection requirements of most oral squamous cell carcinoma patients, and has wide application range.

Drawings

Fig. 1 is a graph of biomarker expression in oral squamous carcinoma tissue, wherein panel a is a graph of AC108142.1 expression in oral squamous carcinoma tissue; panel B is a graph of LINC01160 expression in oral squamous carcinoma tissue.

Fig. 2 is a ROC plot of biomarkers as detection variables, wherein plot a is a ROC plot of AC108142.1 as detection variables; fig. B is a ROC graph of LINC01160 as a sensed variable, and fig. C is a ROC graph of AC108142.1 in combination with LINC01160 as a sensed variable.

Detailed Description

According to the invention, through extensive and intensive research, AC108142.1 or LINC01160 is found to show significant differential expression in oral squamous cell carcinoma patients for the first time, and the diagnosis efficiency of AC108142.1 or LINC01160 is further evaluated, and the result shows that AC108142.1 and/or LINC01160 has higher efficiency in diagnosing oral squamous cell carcinoma.

Gene marker

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

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

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. The gene ID of the AC108142.1 gene in an ensembl database is ENSG00000177822, and the gene ID of the LINC01160 gene in the ensembl database is ENSG 00000231346.

The present invention also relates to variants of the polynucleotides of the above genes, which may be naturally occurring allelic variants or non-naturally occurring variants. 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, such as PCR, to determine and/or isolate a polynucleotide of AC108142.1 or LINC 01160.

The full-length nucleotide sequence of the human AC108142.1 or LINC01160 or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For the PCR amplification method, the primer can be designed based on the disclosed nucleotide sequence, especially open reading frame sequence, and the sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Detection method

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

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

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

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

Nucleic acid hybridization techniques of the 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.

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

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

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

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

Nucleic acid membrane strip, chip and kit

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

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

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

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

In the invention, the kit can be used for detecting the expression level of the AC108142.1 or LINC01160 gene and comprises the primer, the probe and/or the chip for detecting and/or quantifying the AC108142.1 or LINC 01160. Optionally together with instructions for the kit.

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

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

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 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, for which specific conditions are not noted in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Example 1 QPCR 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 AC108142.1 and LINC01160, synthesized by Bomaide Biopsis, with GAPDH as the reference gene.

Amplification was performed with SuperReal Premix Plus (SYBR Green) (cat # FP205) and the band of interest was determined by melting curve analysis and electrophoresis, 2 ^-ΔΔCT The method is relatively quantitative, and the specific experimental operation is as followsAnd (5) carrying out product instruction.

4. Results

As shown in fig. 1, AC108142.1 and LINC01160 showed significant differences in the experimental group and the control group, and AC108142.1 and LINC01160 showed significant upregulation in the experimental group compared to the control group, suggesting that AC108142.1 and LINC01160 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 AC108142.1 and LINC01160 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 the fitted regression curve, different probability division thresholds are determined, and the sensitivity, specificity, accuracy and the like of each combination detection scheme are calculated according to the determined probability division thresholds. The results are shown in fig. 2, the AUC values of AC108142.1 and LINC01160 are 0.831 and 0.813, respectively; the combination of the two has an AUC value of 0.885, which indicates that the oral squamous carcinoma diagnosis using AC108142.1 and/or LINC01160 has higher diagnosis efficiency.

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 (7)

1. The application of the reagent for detecting the expression levels of AC108142.1 and LINC01160 in preparing a product for diagnosing oral squamous cell carcinoma.

2. The use of claim 1, wherein AC108142.1 and LINC01160 are up-regulated in oral squamous carcinoma patients.

3. The use of claim 1, wherein the reagents comprise reagents for detecting the expression levels of AC108142.1 and LINC01160 by RT-PCR, real-time quantitative PCR, in situ hybridization or on a chip.

4. The use according to claim 3, wherein the reagents for detecting the expression levels of AC108142.1 and LINC01160 by RT-PCR comprise primers for specific amplification of the AC108142.1 and LINC01160 genes; the reagent for detecting the expression levels of AC108142.1 and LINC01160 through real-time quantitative PCR comprises primers for specifically amplifying AC108142.1 and LINC01160 genes; the reagent for detecting the expression level of AC108142.1 and LINC01160 through in situ hybridization comprises a probe hybridized with a nucleic acid sequence of AC108142.1 and LINC01160 genes; the reagent for detecting the expression level of AC108142.1 and LINC01160 through the chip comprises a probe hybridized with the nucleic acid sequence of the AC108142.1 gene and LINC01160 gene.

5. The use of claim 1, wherein the product comprises a formulation, a nucleic acid membrane strip, a chip or a kit.

6. The use of claim 5, wherein the reagents for detecting the expression levels of AC108142.1 and LINC01160 on the chip comprise probes that specifically recognize the AC108142.1 and LINC01160 genes.

7. The use of claim 5, wherein the reagents for detecting the expression levels of AC108142.1 and LINC01160 in the kit comprise primers for specifically amplifying the AC108142.1 and LINC01160 genes; or probes that specifically recognize the AC108142.1 and LINC01160 genes.

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