Oral squamous cell carcinoma related biomarkers and diagnosis and treatment methods

Abstract

The invention discloses a biomarker related to oral squamous cell carcinoma and a diagnosis and treatment method, and the biomarker is lncRNARP11-339B21.10 and / or RP11-426C22.4. The invention discloses application of the biomarker in preparation of a product for diagnosing the oral squamous cell carcinoma and the product for diagnosing the oral squamous cell carcinoma. The invention also discloses application of the biomarker in preparation of a pharmaceutical composition for treating the oral squamous cell carcinoma and the pharmaceutical composition for treating the oral squamous cell carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to oral squamous cell carcinoma related biomarkers and diagnosis and treatment methods. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is an epithelial malignancy that is easily metastatic and is the eleventh most common cancer in the world (Hussein AA, Helder MN, de Visscher JG, et al. Global incidence of oral and oropharynx cancer in patients younger than 45 years versus older patients: A systematic review [J]. Eur J Cancer 2017; 82: 115-127.). There are approximately 600,000 new cases each year, and it is also the fifteenth most common cause of cancer death in the world (Candia J, Fernandez A, Somarriva C, et al. Deaths due to oral cancer in Chile in the period 2002-2012 [J]. Rev Med Chil. 2018; 146 (4): 487-493.). In recent years, the global incidence of oral squamous cell carcinoma has shown a clear upward trend, and the age of onset has gradually become younger, but the reason for the change in the incidence trend is still unclear. Especially in developing countries, the growth rate of its incidence is particularly prominent (Wang F, Zhang H, Wen J, et al. Nomograms forecasting long-term overall and cancer-specific survival of patients with oral squamous cell carcinoma[J]. Cancer Med. 2018; 7(4): 943-952.).

[0003] With the improvement of imaging, surgery, radiotherapy and traditional treatment, the current treatment methods for OSCC are mainly surgical resection, chemotherapy and radiotherapy or a combination of these three methods (Kim SM, Jeong D, Min KK, et al. Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid Chromatography [J]. World Journal of Surgical Oncology. 2017; 15 (1): 151.). Although the treatment methods are constantly improving, oral surgery will be in close contact with important tissues and organs, which seriously restricts the scope of surgery. In addition, the neck and face tissues are rich in blood vessels and nerves, the incidence of cervical lymph node metastasis and invasion is high, and the prognosis is poor. In recent years, the 5-year survival rate has not increased significantly (about 50%-60%). The 5-year survival rate of patients with advanced tumors or tumor recurrence is even lower (Radhika T, Jeddy N, et al. Salivary biomarkers in oral squamous cell carcinoma-An insight[J]. Journal of Oral Biology & Craniofacial Research 2016, 6(Suppl 1): S51-54.). Studies have found that some patients with advanced OSCC who underwent clean surgical resection had a survival time of less than 30 months (Felice FD, Polimeni A, et al. Radiotherapy Controversies and Prospective in Head and Neck Cancer: A Literature-Based Critical Review[J]. Neoplasia 2018; 20(3): 227-232.). In addition, the patient's 5-year survival rate is also related to the location, stage, age and presence of underlying diseases of the tumor. Therefore, for OSCC, finding tumor markers with molecular diagnosis, prognosis prediction and targeted therapy is of great significance for the treatment of tumors and is also the future development direction. In-depth understanding of the occurrence, development, invasion and metastasis mechanisms of OSCC and revealing the oncogenes and tumor suppressor genes of OSCC will help to improve and supplement the treatment of oral squamous cell carcinoma and have important clinical significance. Summary of the invention

[0004] In order to make up for the shortcomings of the existing technology, the present invention studies the genes that are differentially expressed in oral squamous cell carcinoma, and explores the effects of differentially expressed genes on cancer cells through further cell experiments, thereby providing detection and targeting sites for the diagnosis and treatment of oral squamous cell carcinoma, and providing a theory for revealing the pathogenesis of oral squamous cell carcinoma.

[0005] The present invention adopts the following technical solutions:

[0006] One aspect of the present invention provides a biomarker for diagnosing oral squamous cell carcinoma, wherein the biomarker is selected from one or more of LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6.

[0007] Furthermore, compared with normal (adjacent to cancer) samples, the expression of the biomarker was significantly upregulated in oral squamous cell carcinoma.

[0008] The second aspect of the present invention provides the use of the biomarker and / or its expression product described in the first aspect of the present invention or a reagent for specifically detecting the biomarker and / or its expression product described in the first aspect of the present invention, for preparing a product for diagnosing oral squamous cell carcinoma.

[0009] Further, the reagent is selected from: primers that specifically amplify RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and / or AP000695.6 genes; or probes that specifically recognize RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and / or AP000695.6 genes.

[0010] Furthermore, the primer sequences for specifically amplifying RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and / or AP000695.6 genes are shown in SEQ ID NOs. 1 to 14, respectively.

[0011] The third aspect of the present invention provides a product for diagnosing oral squamous cell carcinoma, the product comprising a reagent for detecting the biomarker described in the first aspect of the present invention.

[0012] Furthermore, the product includes a chip, a kit or a test strip. The chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, and the oligonucleotide probe includes an oligonucleotide probe for a biomarker for detecting the expression level of the biomarker; the kit includes a primer, a probe or a chip for detecting the expression level of the biomarker.

[0013] Furthermore, the kit also includes instructions or labels, positive controls, negative controls, buffers, adjuvants or solvents; the instructions or labels indicate that the kit is used to detect oral squamous cell carcinoma.

[0014] Furthermore, the reagents include reagents for detecting the biomarkers of the present invention by reverse transcription PCR, real-time quantitative PCR, in situ hybridization or gene chip.

[0015] Furthermore, the reagents for detecting the biomarkers of the present invention by reverse transcription PCR include at least a pair of primers that specifically amplify the biomarkers; the reagents for detecting the biomarkers of the present invention by real-time quantitative PCR include at least a pair of primers that specifically amplify the biomarkers; the reagents for detecting the biomarkers of the present invention by in situ hybridization include probes that hybridize with the nucleic acid sequences of the biomarkers; the reagents for detecting the biomarkers of the present invention by gene chips include probes that hybridize with the nucleic acid sequences of the biomarkers.

[0016] The fourth aspect of the present invention provides use of the biomarker described in the first aspect of the present invention in preparing a pharmaceutical composition for treating oral squamous cell carcinoma.

[0017] Furthermore, the pharmaceutical composition comprises an inhibitor of the functional expression of the biomarker.

[0018] Further, the inhibitor reduces the expression level of the one or more biomarkers.

[0019] Furthermore, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease.

[0020] Furthermore, the inhibitor is selected from interfering RNA.

[0021] In a specific embodiment of the present invention, the interfering RNA is siRNA. In the present invention, the sequences of siRNA of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and / or AP000695.6 are shown as SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, respectively.

[0022] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises an inhibitor of the functional expression of the biomarker described in the first aspect of the present invention.

[0023] Further, the inhibitor reduces the expression level of the one or more biomarkers.

[0024] Furthermore, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease.

[0025] Furthermore, the inhibitor is selected from interfering RNA.

[0026] In a specific embodiment of the present invention, the interfering RNA is siRNA, and the siRNA has the sequences of SEQ ID NO. 17 to 30 as described above.

[0027] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0028] The sixth aspect of the present invention provides use of the biomarker described in the first aspect of the present invention in screening candidate drugs for treating oral squamous cell carcinoma.

[0029] Further, the steps for screening candidate drugs are as follows:

[0030] (1) treating a system expressing or containing the biomarker described in the first aspect of the present invention with a substance to be screened; and

[0031] (2) detecting the expression level of the biomarker in the system;

[0032] If the substance to be screened can reduce the expression level of the biomarker, it indicates that the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

[0033] Furthermore, the candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, binding molecules, small molecule compounds, etc. targeting the biomarker or its upstream or downstream genes.

[0034] A seventh aspect of the present invention provides a method for screening candidate drugs for preventing or treating oral squamous cell carcinoma, the method comprising:

[0035] (1) treating a system expressing or containing the biomarker described in the first aspect of the present invention with a substance to be screened; and

[0036] (2) detecting the expression level of biomarkers in the system;

[0037] If the substance to be screened can reduce the expression level of the biomarker, it indicates that the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

[0038] The eighth aspect of the present invention provides a method for inhibiting tumor cell proliferation in vitro, wherein the inhibitor of the biomarker described in the first aspect of the present invention is introduced into tumor cells.

[0039] Furthermore, the inhibitor includes siRNA, shRNA, antisense oligonucleotide or loss-of-function gene targeting the biomarker.

[0040] A ninth aspect of the present invention provides a method for diagnosing oral squamous cell carcinoma, the method comprising: detecting the expression level of the biomarker described in the first aspect of the present invention in a sample of a subject.

[0041] If the expression of at least one of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 in the subject's sample is significantly increased compared with that of a normal person, the subject is diagnosed as a patient with oral squamous cell carcinoma.

[0042] Further, the method comprises:

[0043] (1) Collect samples from subjects;

[0044] (2) extracting RNA from the subject's sample and detecting the expression level of the biomarker described in the first aspect of the present invention;

[0045] (3) Compared with normal people, if the expression of at least one of IP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 in the subject's sample is significantly increased, then the subject is diagnosed as a patient with oral squamous cell carcinoma.

[0046] The tenth aspect of the present invention provides a method for preventing or treating oral squamous cell carcinoma, the method comprising: administering to a subject a pharmaceutically effective amount of an inhibitor of the biomarker described in the first aspect of the present invention.

[0047] Further, the inhibitor reduces the expression level of the one or more biomarkers.

[0048] Furthermore, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease.

[0049] Furthermore, the inhibitor is selected from interfering RNA.

[0050] Furthermore, the sequence of the interfering RNA is selected from SEQ ID NOs. 17 to 30.

[0051] Another aspect of the present invention provides a method for inhibiting tumor cell proliferation, wherein the method comprises introducing a down-regulator of the RP11-875O11.3 gene into tumor cells in vitro.

[0052] Furthermore, the down-regulator includes siRNA, shRNA, antisense oligonucleotide or functional loss-type gene targeting the RP11-875O11.3 gene.

[0053] The present invention also provides the use of the biomarker described in the first aspect of the present invention in constructing a computational model for diagnosing oral squamous cell carcinoma and preparing a system / device / equipment / readable storage medium containing the computational model for diagnosing oral squamous cell carcinoma.

[0054] Specific implementation method

[0055] After in-depth research, the present invention first found that the expression of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 genes in oral squamous cell carcinoma tissue was significantly higher than that in normal mucosal tissue, and experiments proved that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 were significantly higher in oral squamous cell carcinoma tissue than in normal mucosal tissue. The results showed that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 were also highly expressed in oral squamous cell carcinoma cells. Down-regulating the expression levels of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 could inhibit the proliferation and invasion of oral squamous cell carcinoma cells, indicating that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 could be used as diagnostic and therapeutic targets in clinical practice.

[0056] The lncRNA in the present invention includes wild type, mutant type or fragments thereof, as long as it can be aligned to the gene when performing sequence alignment. The currently disclosed RP11-875O11.3 has two transcripts, and the sequences are shown in ENST00000520840.1 and ENST00000523806.1, respectively. In a specific embodiment of the present invention, the sequence of the RP11-875O11.3 is shown in ENST00000520840.1. The currently disclosed LINC01679 has one transcript, and the sequence is shown in NR_131902.1. The currently disclosed AP000695.4 has two transcripts, and the sequences are shown in ENST00000428667.1 and ENST00000454980.1, respectively. In a specific embodiment of the present invention, the sequence of the AP000695.4 is shown in ENST00000428667.1. The currently disclosed RP11-339B21.10 has one transcript, and its sequence is shown in ENST00000610052.1. The currently disclosed RP11-426C22.4 has one transcript, and its sequence is shown in ENST00000566070.1. The currently disclosed RP11-426C22.5 has two transcripts, and their sequences are shown in ENST00000562902.1 and ENST00000563477.1, respectively. In a specific embodiment of the present invention, the sequence of the RP11-426C22.5 is shown in ENST00000562902.1. The currently disclosed AP000695.6 has one transcript, and its sequence is shown in ENST00000429588.1.

[0057] As used herein, "marker" and "biomarker" may be used interchangeably to refer to an indication of normal or abnormal progression in an individual or an indication of a disease or other condition in an individual or a target molecule that expresses these. In more detail, a "marker" or "biomarker" is an anatomical, physiological, biochemical or molecular parameter that is normal or abnormal, and if abnormal, is associated with the presence of a specific physiological state or progression, whether chronic or acute. Biomarkers can be detected and measured by a variety of methods including laboratory testing and medical imaging.

[0058] The "biomarker value", "value", "biomarker level" and "level" used in the present invention are measured using any analytical method for detecting biomarkers from a biological sample, and are used interchangeably to refer to measured values ​​representing, used for, or corresponding to the presence or absence of a biomarker, an absolute amount or concentration, a relative amount or concentration, a titer, a level, an expression level, a ratio of measured levels, etc. in the above-mentioned biological sample.

[0059] "Diagnose," "diagnosed," "diagnose," and variations of these terms refer to the discovery, judgment, or knowledge of an individual's health state or condition based on one or more signs, symptoms, data, or other information associated with the individual. An individual's health state may be diagnosed as healthy / normal (i.e., the absence of a disease or condition), or may be diagnosed as unhealthy / abnormal (i.e., the presence of a disease or condition or an assessment of a characteristic). The above terms "diagnose," "diagnosed," "diagnose," etc. include early detection of a disease in relation to a specific disease or condition; the characterization or classification of a disease; the discovery of the progression, cure, or recurrence of a disease; and the discovery of an individual's response to a disease following treatment or therapy. The diagnosis of oral squamous cell carcinoma includes the distinction between individuals who do not have cancer and individuals who do have cancer.

[0060] When a biomarker is a marker of abnormal progression or disease or other conditions in an individual, the biomarker generally indicates the absence of normal progression or disease or other conditions in the individual, or is expressed as one of overexpression or underexpression compared to the expression level or value of the biomarker that is its marker. "Upregulated," "upregulated," "overexpressed," and variations of such expressions are used interchangeably to refer to biomarker values ​​or levels in a biological sample that are higher than the value or level (or range of values ​​or levels) of the biomarker typically detected in a biological sample similar to a healthy or normal individual. A plurality of the above terms may also refer to a biomarker value or level in a biological sample that is higher than the value or level (or range of values ​​or levels) of the biomarker that can be detected in different steps of a particular disease.

[0061] "Downregulated", "underexpressed" and such variations are used interchangeably to refer to a biomarker value or level in a biological sample that is smaller than a value or level (or range of values ​​or levels) of a biomarker typically detected from a similar biological sample of a healthy or normal individual. Multiple of the above terms may also refer to a biomarker value or level in a biological sample that is smaller than a value or level (or range of values ​​or levels) of a biomarker that can be detected from different steps of a particular disease.

[0062] Furthermore, a biomarker that is overexpressed or underexpressed can be said to be indicative of normal progression or the absence of a disease or other condition in an individual, or to have "differential expression" or "differential levels" or "differential values" compared to the "normal" expression level or value of the biomarker that expresses it. Thus, "differential expression" of a biomarker can also be expressed as a change in the "normal" expression level of the biomarker.

[0063] The terms "differential gene expression" and "differential expression" are used interchangeably to refer to genes that are activated at higher or lower levels in subjects with a specific disease compared to expression in normal subjects or control subjects. The above terms also include genes that are activated at high or low levels in different steps of the same disease. Differential gene expression may include comparison of expression between two or more genes or their gene products; or comparison of expression ratios between two or more genes or their gene products; or, conversely, comparison of two products of the same gene that are processed differently between normal subjects and subjects with the disease or between multiple stages of the same disease. Differential expression includes, for example, quantitative differences and qualitative differences in temporal or cellular expression patterns in genes or their expression products between normal and diseased cells, or between multiple cells experiencing different disease events or disease stages.

[0064] The present invention can measure gene expression using any method known in the art. It should be understood by those skilled in the art that the means for measuring gene expression is not an important aspect of the present invention. In order to detect the expression of a gene, a plurality of different detection methods can be used, for example, detection methods such as hybridization assay, mass analysis or real-time fluorescence quantitative nucleic acid amplification detection. In certain embodiments, nucleic acid base sequence analysis methods can be used to detect gene sequences and detect biomarker values. With regard to the "increased" level of the lncRNA gene product mentioned herein, it refers to a higher level than that normally present. Typically, this can be estimated by comparison with a control. According to a specific embodiment, the level of lncRNA increase is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 150%, 200% or even higher than the control. According to another specific embodiment, it means that the lncRNA gene product is expressed or present, while it is missing under normal circumstances (or in the control). In other words, in these embodiments, determining the expression of the increased lncRNA gene product is equivalent to detecting the presence of the lncRNA gene product. Typically, in this case, controls will be included to ensure that the detection reaction is performed correctly. With respect to "functional expression" of lncRNA, it is meant the transcription and / or translation of a functional gene product. For non-protein coding genes like lncRNA, "functional expression" can be dysregulated at least at two levels. First, at the DNA level, for example, by deletion or destruction of the gene, or no transcription occurs (in both cases, the synthesis of the relevant gene product is prevented). The lack of transcription can be caused, for example, by epigenetic changes (such as DNA methylation) or by loss-of-function mutations.

[0065] Second, at the RNA level, for example, by lack of efficient translation - for example because of mRNA instability (e.g., through UTR variants), which can lead to mRNA degradation before the transcript is translated. Or by lack of efficient transcription, for example because mutations induce new splice variants.

[0066] Accordingly, one object of the present invention is to provide an inhibitor of the functional expression of lncRNA genes. Such an inhibitor can act at the DNA level or at the RNA (i.e., gene product) level. Since lncRNA is a non-coding gene, the gene has no protein product.

[0067] If inhibition is achieved at the DNA level, it can be carried out by knocking out or destroying the target gene using gene therapy. As used herein, "knockout" can be gene knockdown, or the gene can be knocked out, by using techniques known in the art, including but not limited to, retroviral gene transfer, causing mutations, such as point mutations, insertions, deletions, frameshifts, or missense mutations. Another way to knock out a gene is to use zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by fusing zinc finger DNA binding domains with DNA cleavage domains. The zinc finger domains can be modified to target the target DNA sequence, which can target the unique sequences in complex genomes for zinc finger nucleases. By utilizing endogenous DNA repair mechanisms, these reagents can be used to accurately change the genomes of higher organisms. Other genome customization technologies that can be used to knock out genes include Meganuclease and TAL effector nucleases (TALENs, Cellectis bioresearch). It is composed of a fusion of the TALE DNA binding domain for sequence-specific recognition and the catalytic domain of the endonuclease that introduces a double-strand break (DSB). Meganuclease is a sequence-specific endonuclease, a naturally occurring "DNA scissors" derived from various unicellular organisms such as bacteria, yeast, algae, and certain plant organelles. Meganuclease has a long recognition site of 12 to 30 base pairs. The recognition site of natural Meganuclease can be altered to target natural genomic DNA sequences (e.g., endogenous genes).

[0068] Another recent genome editing technology is the CRISPR / Cas system, which can be used to achieve RNA-guided genome modification. CRISPR interference is a genetic technology that allows sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) pathway derived from the bacterial immune system.

[0069] Gene inactivation, i.e., inhibition of functional expression of a gene, can also be achieved, for example, by designing a transgenic organism that expresses antisense RNA, or by administering antisense RNA to a subject. Antisense constructs can be delivered, for example, as expression plasmids, wherein when the expression plasmid is expressed in a cell, RNA complementary to at least one unique portion of a cellular lncRNA lncRNA is produced.

[0070] A faster method for inhibiting gene expression is based on the use of shorter antisense oligomers composed of DNA or other synthetic structural types (e.g., phosphorothioate, 2'-0-alkyl ribonucleotide chimera, locked nucleic acid (LNA), peptide nucleic acid (PNA) or morpholino nucleic acid). Except for RNA oligomers, PNA and morpholino nucleic acid, all other antisense oligomers work through the target cleavage mechanism mediated by RNase H in eukaryotic cells. PNA and morpholino nucleic acid highly affinity and specifically bind complementary DNA and RNA targets, thereby acting through simple steric hindrance to the RNA translation machinery, and showing complete resistance to nuclease attack. "Antisense oligomer" refers to an antisense molecule or antigene agent containing an oligomer of at least about 10 nucleotides in length. In an embodiment, the antisense oligomer contains at least 15, 18, 20, 25, 30, 35, 40 or 50 nucleotides. The antisense method includes designing oligonucleotides (DNA or RNA or derivatives thereof) complementary to the RNA encoded by the polynucleotide sequence of lncRNA. Antisense RNA can be introduced into cells to inhibit the translation of complementary mRNA by base pairing with it and physically hindering the translation machinery. The effect is therefore stoichiometric. Although fully complementary is preferred, this is not necessary. As mentioned herein, a sequence is "complementary" to a portion of RNA, meaning that the sequence has sufficient complementarity to hybridize with RNA and form a stable duplex; in the case of a double-stranded antisense polynucleotide sequence, a single strand of duplex DNA can be detected, or the formation of a triplex can be detected. The ability of hybridization will depend on the degree of complementarity and the length of the antisense polynucleotide sequence. Generally, the longer the polynucleotide sequence that is hybridized, the more bases that can be mismatched with RNA can be included and still form a stable duplex (or triplex, as the case may be). The technician can determine the tolerance of mismatches by measuring the melting point of the hybrid complex using standard procedures. The antisense oligomer should be at least 10 nucleotides long, and preferably the oligomer is 15 to about 50 nucleotides long. In certain embodiments, the oligomer is at least 15 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, or at least 50 nucleotides long. A related method uses ribozymes to replace antisense RNA. Ribozymes are catalytic RNA molecules that have the same cleavage properties as enzymes and can be designed to target specific RNA sequences. Successful target gene inactivation using ribozymes has been reported in mice, zebrafish and fruit flies, including time- and tissue-specific gene inactivation. RNA interference (RNAi) is a form of post-transcriptional gene silencing. The RNA interference phenomenon was originally observed and described in Caenorhabditis elegans, where it was shown that exogenous double-stranded RNA (dsRNA) can specifically and powerfully destroy the activity of genes containing homologous sequences by inducing a mechanism of rapid degradation of target RNA.Several reports describe the same catalytic phenomenon in other organisms, including experiments showing controlled gene inactivation in space and / or time, including plants (Arabidopsis thaliana), protozoa (Trypanosoma brucei), invertebrates (Drosophila melanogaster) and vertebrate species (zebrafish and African clawed frogs). The degradation of sequence-specific messenger RNA can be mediated by small interfering RNA (siRNAs), which is produced by ribonuclease III cutting from longer dsRNA. Typically, the length of siRNA is 20-25 nucleotides. siRNA typically comprises a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson Crick base pairing interactions (hereinafter referred to as "base pairing"). The sense strand comprises a nucleic acid sequence consistent with the target sequence in the target mRNA. The sense and antisense strands of the siRNA of the present invention can comprise two complementary single-stranded RNA molecules, or can comprise a single molecule in which two complementary partial bases are paired and covalently linked by a single-stranded "hairpin" region (commonly referred to as shRNA). The term "isolated" means that it is altered or removed from its natural state by human intervention. For example, siRNA naturally present in a living animal is not "isolated", but synthetic siRNA or siRNA partially or completely separated from materials coexisting with its natural state is "isolated". Isolated siRNA may exist in a fairly pure form, or may exist in a non-natural environment, such as a cell into which the siRNA is transferred.

[0071] The siRNA of the present invention can include partially purified RNA, fairly pure RNA, synthetic RNA, or recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by addition, deletion, substitution, and / or change of one or more nucleotides. Such changes can include addition of non-nucleotide material to, for example, the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.

[0072] One or both strands of the siRNA of the present invention may also include a 3' overhang. "3' overhang" refers to at least one unpaired nucleotide extending from the 3' end of the RNA strand. Therefore, in one embodiment, the siRNA of the present invention comprises at least one 3' overhang of 1 to about 6 nucleotides (including RNA or deoxyribonucleic acid), preferably 1 to about 5 nucleotides, more preferably 1 to about 4 nucleotides, and particularly preferably about 1 to about 4 nucleotides.

[0073] In embodiments where both strands of the siRNA molecule comprise a 3' overhang, the length of the overhang may be the same or different for each strand. In a further embodiment, the 3' overhang is present on both strands of the siRNA and is 2 nucleotides long. In order to enhance the stability of the siRNA of the invention, the 3' overhang may also be stabilized to resist degradation. In one embodiment, the overhang is stabilized by including purine nucleotides such as adenosine or guanosine nucleotides.

[0074] Alternatively, replacement of pyrimidine nucleotides with modified analogs, such as replacement of uridine nucleotides in the 3' overhang with 2' deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of the 2' hydroxyl group in 2' deoxythymidine significantly enhances the nuclease resistance of the 3' overhang in tissue culture medium.

[0075] The siRNA of the present invention can target any segment of about 19 to 25 consecutive nucleotides in any target lncRNA RNA sequence ("target sequence"), examples of which are provided in the present application. Techniques for selecting target sequences for siRNA are well known in the art. Therefore, the sense strand of the siRNA of the present invention can comprise a nucleotide sequence consistent with any segment of about 19 to about 25 consecutive nucleotides in the target mRNA.

[0076] Many techniques known to those skilled in the art can be used to obtain siRNA of the present invention. For example, methods known in the art can be used to produce siRNA by chemical synthesis or recombinant production. Preferably, siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

[0077] As used herein, an "effective amount" of siRNA is an amount sufficient to cause RNAi-mediated target mRNA degradation, or an amount sufficient to inhibit metastatic progression in a subject. RNAi-mediated target mRNA degradation can be detected by measuring the level of target mRNA or protein in subject cells using standard techniques for isolating and quantifying mRNA or protein (such as described above).

[0078] One skilled in the art can readily determine the effective amount of an siRNA of the invention to be administered to a given subject by considering, for example, the size and weight of the subject, the extent of disease penetration, the age, health, and sex of the subject, the route of administration, and whether the administration is local or systemic.

[0079] Another special form of the antisense RNA strategy is gapmer. Gapmer is a chimeric antisense oligonucleotide that contains a central segment of deoxynucleotide monomers that is long enough to induce RNase H cleavage. The flanks of the central region of the gapmer are segments of 2'-O modified ribonucleotides or other artificially modified ribonucleotide monomers, such as bridged nucleic acids (BNAs), which protect the internal segments from nuclease degradation. Gapmer has been used to obtain RNase-H-mediated target RNA cleavage while reducing the number of phosphorothioate linkages. Phosphorothioates have increased resistance to nucleases compared to unmodified DNA. However, they have several disadvantages. This includes low binding ability to complementary nucleic acids and nonspecific binding to proteins that leads to toxic side effects, thereby limiting their application. The occurrence of toxic side effects and off-target effects caused by nonspecific binding have inspired people to design new artificial nucleic acids for the development of modified oligonucleotides to provide effective and specific antisense activity in vivo without showing toxic side effects. By recruiting RNaseH, gapmers selectively cut the target oligonucleotide chain. The cutting of this chain triggers the antisense effect. This method has been shown to be a powerful method for inhibiting gene function and has gradually become a popular method for antisense therapy. Gapmer can be provided by business. For example, LNAlongRNA GapmeR provided by Exiqon, or MOE gapmer provided by Isis pharmaceuticals. MOE gapmers or "2'MOE gapmers" are antisense phosphorothioate oligonucleotides of 15-30 nucleotides, in which all backbone connections are modified by adding sulfur (phosphorothioate) to non-bridging oxygens, and a section of at least 10 consecutive nucleotides remains unmodified (deoxy sugar), while the remaining nucleotides contain O'-methyl O'-ethyl substitution (MOE) at the 2' position. For clinical use, the compound according to the present invention or its prodrug form is formulated into a pharmaceutical composition, which is formulated to be compatible with its intended route of administration, such as oral, rectal, parenteral or other modes of administration. Typically, the pharmaceutical formulation is prepared by mixing the active substance with a conventional pharmaceutically acceptable diluent or carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delaying agents, etc. that are compatible with drug administration. Examples of pharmaceutically acceptable diluents or carriers include water, gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is covered.

[0080] The drug of the present invention can also be used in combination with other drugs for treating oral squamous cell carcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, or even administered simultaneously in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment plan.

[0081] The term "sample" as used herein refers to a composition obtained from a target patient, which contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics. For example, the phrases "clinical sample" or "disease sample" and variations thereof refer to any sample obtained from a target patient from which it is expected or known that cells and / or molecular entities, such as biomarkers to be characterized, can be obtained.

[0082] The present invention is further described in detail below in conjunction with the examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

[0083] Example 1 QPCR detection of differential expression of lncRNA

[0084] 1. 33 cases of surrounding normal mucosal tissue and oral squamous cell carcinoma tissue were collected, all of which were confirmed by pathological diagnosis. All patients did not receive any form of treatment before surgery. The samples removed during surgery were frozen in liquid nitrogen.

[0085] 2. RNA Extraction

[0086] Take out the tissue sample frozen in liquid nitrogen, put it into a pre-cooled mortar for grinding, and extract and separate RNA according to the instructions in the kit. The details are as follows:

[0087] 1) Add Trizol and leave at room temperature for 5 minutes;

[0088] 2) Add 0.2 ml of chloroform, shake the centrifuge tube vigorously, mix thoroughly, and leave at room temperature for 5-10 minutes;

[0089] 3) Centrifuge at 12000 rpm for 15 min, transfer the upper aqueous phase to another new centrifuge tube (be careful not to aspirate the protein material between the two aqueous phases), add an equal volume of -20℃ pre-cooled isopropanol, mix thoroughly by inversion, and place on ice for 10 min;

[0090] 4) After centrifugation at 12000 rpm for 15 min, discard the supernatant carefully, add 75% DEPC ethanol at a ratio of 1 ml / ml Trizol to wash the precipitate (stored at 4°C), wash the precipitate, shake and mix, and centrifuge at 4°C, 12000 rpm for 5 min;

[0091] 5) Discard the ethanol liquid, place at room temperature for 5 minutes, and add DEPC water to dissolve the precipitate;

[0092] 6) The RNA purity and concentration were measured using Nanodrop2000 UV spectrophotometer and stored in a -70°C refrigerator.

[0093] 3. Reverse transcription:

[0094] 1) Prepare 10 μl reaction system:

[0095] Mix 2 μl of MgCl2, 1 μl of 10×RT Buffer, 3.75 μl of RNase-free water, 1 μl of dNTP mixture, 0.25 μl of RNase inhibitor, 0.5 μl of AMV reverse transcriptase, 0.5 μl of oligo dT adapter primer, and 1 μl of experimental sample.

[0096] 2) Reverse transcription reaction conditions

[0097] The reverse transcription reaction conditions were followed in RNA PCR Kit (AMV) Ver. 3.0.

[0098] 42℃60min, 99℃2min, 5℃5min.

[0099] 3) Polymerase chain reaction

[0100] 1) Primer design

[0101] QPCR amplification primers were designed based on the coding sequences of RP11-875011.3 gene and GAPDH gene in Genebank and synthesized by Biomed Biotech. The specific primer sequences are shown in Table 1.

[0102] Table 1 Primer sequences

[0103]

[0104]

[0105] 2) Prepare 25 μl PCR reaction system:

[0106] Forward (reverse) primer 1μl, Takara ExTaq HS 12.5μl, template 2μl, deionized water 8.5μl

[0107] 3) PCR reaction conditions: 94°C for 4 min, (94°C for 20 s, 60°C for 30 s, 72°C for 30 s) × 30 cycles.

[0108] SYBR Green was used as a fluorescent marker and PCR reaction was performed on a Light Cycler fluorescent quantitative PCR instrument. The target band was determined by melting curve analysis and electrophoresis. -ΔΔCT Relative quantification was performed by the method, and each sample was repeated 3 times. △△CT method: △CT1 = (target gene, sample to be tested) CT value - (reference gene, sample to be tested) CT value; △CT2 = (target gene, control sample) CT value - (reference gene, control sample) CT value. △△CT = △CT1-△CT2, expression multiple = 2 -ΔΔCT .

[0109] 5. Statistical methods

[0110] Using GAPDH as the internal reference, the experimental results of fluorescence quantitative RT-PCR of oral squamous cell carcinoma tissue and normal mucosal tissue were calculated. The difference between the two was tested by t test, and P < 0.05 was considered statistically significant.

[0111] 6. Results

[0112] The results are shown in Table 2. Compared with the surrounding normal mucosal tissue, the expression of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 genes were upregulated in oral squamous cell carcinoma tissue, and the difference was statistically significant (P<0.05).

[0113] Table 2 Relative expression levels of lncRNA

[0114]

[0115]

[0116] Example 2 Silencing detection and functional verification of lncRNA

[0117] 1. Cell culture

[0118] Human oral squamous cell carcinoma SCC-15 cells stored in liquid nitrogen were taken out and revived and inoculated into DMEM medium in a constant temperature incubator at 37°C and 5% CO2. After 24 hours, the cells were successfully revived if they adhered to the wall and the medium was changed every 1-2 days, and the cells were digested with trypsin and made into cell suspension for experiments.

[0119] 2. Cell transfection

[0120] The cells were divided into 2 × 10 5 / well was inoculated into a six-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. The cells in the logarithmic phase of proliferation (about 80%) were discarded from the culture medium, washed twice with PBS, added with 2ml DMEM and starved for 1h in the incubator, and transfected with liposome transfection reagent 2000 (purchased from Invitrogen), and the specific operation was carried out according to the instructions. The experiment was divided into three groups: a blank control group (SCC-15), a negative control group (siRNA-NC) and an experimental group (siRNA group), wherein the negative control group siRNA had no homology with the sequence of each lncRNA gene.

[0121] Among them, siRNA-NC is a universal negative control provided by Shanghai Jima Pharmaceutical Technology Co., Ltd., and the siRNA sequences for each lncRNA are shown in Table 3.

[0122] Table 3 lncRNA siRNA sequences

[0123]

[0124] 3. QPCR detection of the transcription level of RP11-875O11.3 gene

[0125] After each group of cells were transfected and cultured for 48 h, total cellular RNA was extracted using the Trizol method, and reverse transcription and real-time quantitative PCR were performed according to the method in Example 1.

[0126] 4. CCK-8 cell proliferation assay

[0127] The cells of the negative control group and the experimental group that were transfected for 24 hours were digested and centrifuged by conventional methods, the supernatant was discarded, 1 ml of complete medium was added to resuspend the cells, and the mixture was mixed by pipetting. 3000 cells were inoculated into 96-well plates per well, and the complete medium was supplemented to 100 μl; 100 μl of DEPC water was added to the outermost circle of the well plate, and the 96-well plate was cultured in a constant temperature incubator. After 48 hours of culture, 100 μl of culture medium containing 10% CCK-8 was added, and after continuing to culture in the incubator for 1 hour, the absorbance at 450 nm was measured with an ELISA instrument, and the statistics were calculated.

[0128] 5. Cell migration assay

[0129] The Transwell chamber was placed in a 24-well plate, 200 μl of DMEM solution was added to the upper chamber, and the plate was placed in an incubator for 1 h. 4 The cells were plated, the upper chamber liquid was supplemented to 200μ1, and the mixture was mixed by blowing, and 700μ1 complete culture medium was added to the lower chamber, and the culture was continued in the incubator for 36h; the chamber was taken out, the culture medium in the upper and lower chambers was discarded, and the residual culture medium and cells in the upper chamber were gently wiped with a cotton swab, and the chamber was washed with PBS, shaken for 5min, and PBS was discarded; 500μ14% paraformaldehyde was added to the lower chamber, fixed at room temperature for 30min, and the fixative was discarded, and PBS was washed three times, shaken for 5min, and PBS was discarded; the chamber was placed in a fume hood and air-dried for 30min; 500μ1 prepared 0.1% crystal violet solution was added to the lower chamber, bubbles were removed, and it was left to stand for 30min; the crystal violet solution was discarded, and PBS was washed three times, shaken for 5min, and PBS was discarded, and the excess liquid in the upper chamber was gently wiped with a dry cotton swab, and the chamber was placed under a microscope to count the number of cells.

[0130] 6. Statistical methods

[0131] The experiments were repeated three times and the results were expressed as mean ± standard deviation. The difference between the two was tested by t test and was considered statistically significant when P < 0.05.

[0132] 7. Results

[0133] The silencing effect of siRNA is shown in Table 4. Compared with the blank control group, each siRNA in the experimental group had a better interference effect on the corresponding gene (P<0.05), while siRNA-NC had no significant change (P>0.05).

[0134] Table 4 siRNA transfection effect

[0135]

[0136] Note: P: compared with blank control

[0137] The results of CCK-8 detection are shown in Table 5. The OD value of the experimental group was significantly lower than that of the negative control group, P < 0.05, indicating that the lncRNA in this study plays an important role in the proliferation of oral squamous cell carcinoma cells.

[0138] Table 5 OD values

[0139]

[0140] The results of the migration experiment are shown in Table 6. Compared with the negative control group, the number of migrating cells in the experimental groups RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, and AP000695.6 was significantly decreased (P<0.05), while the number of cells in the RP11-426C22.4 and RP11-426C22.5 groups decreased, but not significantly. Based on the above results, it can be judged that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, and AP000695.6 play an important role in the metastasis of oral squamous cell carcinoma.

[0141] Table 6 Number of migrated cells

[0142]

[0143] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A biomarker for diagnosing oral squamous cell carcinoma, characterized in that: The biomarker is selected from one or more of RP11-339B21.10 and RP11-426C22.4; Preferably, the biomarker is significantly upregulated in oral squamous cell carcinoma.

2. Use of the biomarker and / or its expression product according to claim 1 or a reagent for specifically detecting the biomarker and / or its expression product according to claim 1, characterized in that: For use in the preparation of products for diagnosing oral squamous cell carcinoma; Preferably, the reagent is selected from: primers for specific amplification of RP11-339B21.10 and / or RP11-426C22.4 genes; Or a probe that specifically recognizes RP11-339B21.10 and / or RP11-426C22.4 gene; preferably, the primer sequences that specifically amplify RP11-339B21.10 and / or RP11-426C22.4 gene are shown in SEQ ID NOs.7 to 10, respectively.

3. A product for diagnosing oral squamous cell carcinoma, characterized in that: The product comprises a reagent for detecting the biomarker according to claim 1; preferably, the product comprises a chip, a kit or a test strip. Preferably, the reagents include reagents for detecting the biomarker by reverse transcription PCR, real-time quantitative PCR, in situ hybridization or gene chip; preferably, the reagents for detecting the biomarker gene by reverse transcription PCR include at least one pair of primers for specifically amplifying the biomarker; The reagents for detecting biomarkers by real-time quantitative PCR include at least a pair of primers that specifically amplify the biomarkers; the reagents for detecting biomarkers by in situ hybridization include probes that hybridize with the nucleic acid sequence of the biomarkers; and the reagents for detecting biomarkers by gene chip include probes that hybridize with the nucleic acid sequence of the biomarkers.

4. Use of the biomarker according to claim 1 in preparing a pharmaceutical composition for treating oral squamous cell carcinoma. Preferably, the pharmaceutical composition comprises an inhibitor of the functional expression of the biomarker; preferably, the inhibitor reduces the expression level of the one or more biomarkers; preferably, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease; preferably, the inhibitor is selected from interfering RNA; preferably, the interfering RNA is selected from siRNA, and preferably, the sequence of the siRNA is as shown in SEQ ID NO.23 to 26.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an inhibitor of the functional expression of the biomarker according to claim 1; preferably, the inhibitor reduces the expression level of the one or more biomarkers; preferably, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease; preferably, the inhibitor is selected from interfering RNA; preferably, the sequence of the interfering RNA is selected from SEQ ID NO.23 to 26; preferably, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

6. Use of the biomarker of claim 1 in screening candidate drugs for treating oral squamous cell carcinoma.

7. A method for screening candidate drugs for preventing or treating oral squamous cell carcinoma, characterized in that: The method comprises: (1) treating a system expressing or containing the biomarker of claim 1 with a substance to be screened; and (2) detecting the expression level of biomarkers in the system; If the substance to be screened can reduce the expression level of the biomarker, it indicates that the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

8. A method for inhibiting tumor cell proliferation in vitro, characterized in that: The biomarker inhibitor of claim 1 is introduced into tumor cells.

9. A method for diagnosing oral squamous cell carcinoma, characterized in that: The method comprises: detecting the expression level of the biomarker of claim 1 in a sample of a subject; if the expression of at least one of RP11-339B21.10 and RP11-426C22.4 in the sample of the subject is significantly increased compared with that of a normal person, the subject is diagnosed as a patient with oral squamous cell carcinoma; Preferably, the method specifically comprises: (1) Collect samples from subjects; (2) extracting RNA from a sample of a subject and detecting the expression level of the biomarker described in claim 1; (3) If the expression of at least one of RP11-339B21.10 and RP11-426C22.4 in the subject's sample is significantly increased compared with that of normal subjects, the subject is diagnosed as a patient with oral squamous cell carcinoma.

10. Use of the marker of claim 1 in constructing a computational model for diagnosing oral squamous cell carcinoma, or in preparing a system / device / equipment / readable storage medium comprising a computational model for diagnosing oral squamous cell carcinoma.

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