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, the biomarker is lncRNA RP11-426C22.4, and 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] This invention belongs to the field of biomedicine and relates to biomarkers for oral squamous cell carcinoma and methods for diagnosis and treatment. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is a malignant epithelial tumor that is prone to metastasis.

[0003] With improvements in imaging and surgical techniques, as well as advancements in radiotherapy and traditional treatments, the current treatment methods for OSCC mainly involve 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 treatment methods are constantly being improved, the scope of surgery is severely limited because oral surgery involves close contact with important tissues and organs. In addition, the facial tissues are rich in blood vessels and nerves, with a high incidence of cervical lymph node metastasis and invasion, resulting in a poor prognosis. In recent years, the 5-year survival rate has not increased significantly (around 50%-60%). Patients with advanced or recurrent OSCC have lower 5-year survival rates (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 even some patients with fully surgically removed advanced OSCC have survival times 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.). Furthermore, the 5-year survival rate is also correlated with tumor location, stage, patient age, and the presence of underlying diseases. Therefore, for OSCC, finding tumor markers with molecular diagnostic, prognostic, and targeted therapies is of great significance for tumor treatment and represents a future direction for development. A deeper understanding of the mechanisms of occurrence, development, invasion, and metastasis of OSCC, and the identification of oncogenes and tumor suppressor genes in OSCC, will help improve and supplement treatment methods for oral squamous cell carcinoma, and has important clinical significance. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention studies differentially expressed genes in oral squamous cell carcinoma and explores the effects of differentially expressed genes on cancer cells through further cell experiments. This provides detection and targeting sites for the diagnosis and treatment of oral squamous cell carcinoma, and also provides a theoretical basis for revealing the pathogenesis of oral squamous cell carcinoma.

[0005] The present invention adopts the following technical solution: 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.

[0006] Furthermore, compared to normal (adjacent) samples, the biomarker was significantly upregulated in oral squamous cell carcinoma.

[0007] The second aspect of the present invention provides the use of the biomarkers and / or their expression products described in the first aspect of the present invention, or the reagents for specifically detecting the biomarkers and / or their expression products described in the first aspect of the present invention, for the preparation of products for diagnosing oral squamous cell carcinoma.

[0008] Furthermore, the reagents are selected from: primers that specifically amplify the 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 the RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and / or AP000695.6 genes.

[0009] Furthermore, the primer sequences for specifically amplifying the 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 NO.1~14, respectively.

[0010] A third aspect of the present invention provides a product for diagnosing oral squamous cell carcinoma, the product comprising reagents for detecting the biomarkers described in the first aspect of the present invention.

[0011] Furthermore, the product includes a chip, a kit, or a test strip. The chip includes a solid-phase support and oligonucleotide probes immobilized on the solid-phase support, the oligonucleotide probes including biomarker-specific oligonucleotide probes for detecting biomarker expression levels; the kit includes primers, probes, or a chip for detecting biomarker expression levels.

[0012] Furthermore, the kit also includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent; the instructions for use or the label specify that the kit is for the detection of oral squamous cell carcinoma.

[0013] Furthermore, the reagents include those for detecting the biomarkers described in this invention by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, or gene chip detection.

[0014] Furthermore, the reagent for detecting the biomarker of the present invention by reverse transcription PCR includes at least a pair of primers that specifically amplify the biomarker; the reagent for detecting the biomarker of the present invention by real-time quantitative PCR includes at least a pair of primers that specifically amplify the biomarker; the reagent for detecting the biomarker of the present invention by in situ hybridization includes a probe that hybridizes with the nucleic acid sequence of the biomarker; and the reagent for detecting the biomarker of the present invention by gene chip includes a probe that hybridizes with the nucleic acid sequence of the biomarker.

[0015] A fourth aspect of the invention provides the use of the biomarkers described in the first aspect of the invention in the preparation of pharmaceutical compositions for treating oral squamous cell carcinoma.

[0016] Furthermore, the pharmaceutical composition includes an inhibitor of the functional expression of the biomarker.

[0017] Furthermore, the inhibitor reduces the expression level of the one or more biomarkers.

[0018] Furthermore, the inhibitor is selected from gapmers, interfering RNA, CRISPR, TALEN, or zinc finger nucleases.

[0019] Furthermore, the inhibitor is selected from interfering RNA.

[0020] In a specific embodiment of the present invention, the interfering RNA is siRNA. In the present invention, the sequences of the siRNAs 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, and SEQ ID NO.29-30, respectively.

[0021] A fifth aspect of the present invention provides a pharmaceutical composition comprising an inhibitor of the functional expression of the biomarker described in the first aspect of the present invention.

[0022] Furthermore, the inhibitor reduces the expression level of the one or more biomarkers.

[0023] Furthermore, the inhibitor is selected from gapmers, interfering RNA, CRISPR, TALEN, or zinc finger nucleases.

[0024] Furthermore, the inhibitor is selected from interfering RNA.

[0025] In a specific embodiment of the present invention, the interfering RNA is siRNA, which has the sequences SEQ ID NO. 17~30 as described above.

[0026] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0027] The sixth aspect of the present invention provides the use of the biomarkers described in the first aspect of the present invention in screening candidate drugs for the treatment of oral squamous cell carcinoma.

[0028] Furthermore, the steps for screening candidate drugs are as follows: (1) Treating a system expressing or containing the biomarker described in the first aspect of the present invention with the substance to be screened; and (2) Detect the expression level of the biomarkers in the system; If the substance to be screened can reduce the expression level of the biomarker, then the substance to be screened is a candidate drug for the prevention or treatment of oral squamous cell carcinoma.

[0029] Furthermore, the candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, binding molecules, small molecule compounds, etc., that target the biomarker or its upstream or downstream genes.

[0030] A seventh aspect of the present invention provides a method for screening candidate drugs for the prevention or treatment of oral squamous cell carcinoma, the method comprising: (1) Treating a system expressing or containing the biomarker described in the first aspect of the present invention with the substance to be screened; and (2) Detect the expression levels of biomarkers in the system; If the substance to be screened can reduce the expression level of the biomarker, then the substance to be screened is a candidate drug for the prevention or treatment of oral squamous cell carcinoma.

[0031] The eighth aspect of the present invention provides a method for inhibiting tumor cell proliferation in vitro by introducing an inhibitor of the biomarker described in the first aspect of the present invention into tumor cells.

[0032] Furthermore, the inhibitors include siRNA, shRNA, antisense oligonucleotides, or loss-of-function genes targeting the biomarker.

[0033] 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 subject sample.

[0034] If, compared with normal individuals, the expression of at least one of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, or AP000695.6 is significantly elevated in the subject's sample, then the subject is diagnosed with oral squamous cell carcinoma.

[0035] Furthermore, the method includes: (1) Collecting subject samples; (2) Extract RNA from subject samples and detect the expression level of the biomarkers described in the first aspect of the present invention; (3) If, compared with normal individuals, the expression of at least one of IP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, or AP000695.6 is significantly increased in the subject's sample, then the subject is diagnosed as an oral squamous cell carcinoma patient.

[0036] 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.

[0037] Furthermore, the inhibitor reduces the expression level of the one or more biomarkers.

[0038] Furthermore, the inhibitor is selected from gapmers, interfering RNA, CRISPR, TALEN, or zinc finger nucleases.

[0039] Furthermore, the inhibitor is selected from interfering RNA.

[0040] Furthermore, the sequence of the interfering RNA is selected from SEQ ID NO. 17~30.

[0041] Another aspect of the present invention provides a method for inhibiting tumor cell proliferation, wherein a downregulator of the RP11-875O11.3 gene is introduced into tumor cells in vitro.

[0042] Furthermore, the downregulator includes siRNA, shRNA, antisense oligonucleotide, or loss-of-function gene targeting the RP11-875O11.3 gene.

[0043] The present invention also provides the application of the biomarkers described in the first aspect of the present invention in constructing a computational model for diagnosing oral squamous cell carcinoma and in preparing a system / device / equipment / readable storage medium containing the computational model for diagnosing oral squamous cell carcinoma.

[0044] Specific implementation methods Through in-depth research, this invention has, for the first time, discovered that the expression of the genes RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 in oral squamous cell carcinoma tissue is significantly higher than that in normal mucosal tissue. Furthermore, experiments have demonstrated that these genes play a crucial role in oral squamous cell carcinoma. High expression was also observed in the cells. Downregulation of 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, suggesting that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 could be used as targets for diagnosis and treatment in clinical practice.

[0045] The lncRNAs in this invention include wild-type, mutant, or fragments thereof, as long as they can be aligned to the gene during sequence alignment. Currently, RP11-875O11.3 has two transcripts, with sequences shown as ENST00000520840.1 and ENST00000523806.1, respectively. In a specific embodiment of this invention, the sequence of RP11-875O11.3 is shown as ENST00000520840.1. Currently, LINC01679 has one transcript, with a sequence shown as NR_131902.1. Currently, AP000695.4 has two transcripts, with sequences shown as ENST00000428667.1 and ENST00000454980.1, respectively. In a specific embodiment of this invention, the sequence of AP000695.4 is shown as ENST00000428667.1. The currently disclosed RP11-339B21.10 contains one transcript, with the sequence shown in ENST00000610052.1. The currently disclosed RP11-426C22.4 contains one transcript, with the sequence shown in ENST00000566070.1. The currently disclosed RP11-426C22.5 contains two transcripts, with sequences shown in ENST00000562902.1 and ENST00000563477.1, respectively. In a specific embodiment of this invention, the sequence of RP11-426C22.5 is shown in ENST00000562902.1. The currently disclosed AP000695.6 contains one transcript, with the sequence shown in ENST00000429588.1.

[0046] The terms "marker" and "biomarker" as used in this invention may be used interchangeably to refer to indicators of normal or abnormal progression in an individual, or indicators of disease or other states in an individual, or target molecules expressing these. More specifically, a "marker" or "biomarker" refers to normal or abnormal, and if abnormal, to anatomical, physiological, biochemical, or molecular parameters associated with the presence of a specific physiological state or progression, whether chronic or acute. Biomarkers can be detected and measured using a variety of methods, including laboratory testing and medical imaging.

[0047] In this invention, the terms “biomarker value,” “value,” “biomarker level,” and “level” are used interchangeably to refer to the measured values ​​of a biomarker, used for a biomarker, or corresponding to the presence or absence of a biomarker, including absolute amount or concentration, relative amount or concentration, titration, level, expression level, and ratio of the measured level.

[0048] The terms "diagnostic," "diagnostic," "diagnosis," and variations thereof refer to the discovery, judgment, or recognition of an individual's health status or condition based on one or more signs, symptoms, data, or other information relevant to that individual. An individual's health status may be diagnosed as healthy / normal (i.e., without disease or ailment) or unhealthy / abnormal (i.e., with disease or ailment or an assessment of its characteristics). The terms "diagnostic," "diagnostic," "diagnosis," etc., in relation to a specific disease or ailment include: early detection of the disease; the characteristics or classification of the disease; the discovery of the disease's progression, cure, or recurrence; and the discovery of the individual's response to the disease after treatment or intervention. The diagnosis of oral squamous cell carcinoma includes distinguishing between individuals without cancer and individuals with cancer.

[0049] When a biomarker is a marker of abnormal progression, disease, or other condition in an individual, the biomarker typically indicates the absence of normal progression, disease, or other condition in the individual, or is either overexpressed or underexpressed compared to the expression level or value of the biomarker that marks it. The terms "upregulated," "upregulated," "overexpressed," and variations thereof are used interchangeably to refer to biomarker values ​​or levels in biological samples that are higher than the values ​​or levels (or ranges of values ​​or levels) typically detected in biological samples from individuals similar to healthy or normal individuals. Several of the above terms may also refer to biomarker values ​​or levels in biological samples that are higher than the values ​​or levels (or ranges of values ​​or levels) detectable at different stages of a particular disease.

[0050] "Downregulated," "low-expressed," and this phenotypic variation are used interchangeably to refer to biomarker values ​​or levels in biological samples that are smaller than the values ​​or levels (or ranges of values ​​or levels) typically detected in similar biological samples from healthy or normal individuals. Several of the above terms may also refer to biomarker values ​​or levels in biological samples that are smaller than the values ​​or levels (or ranges of values ​​or levels) of biomarkers that can be detected from different steps in a particular disease.

[0051] Furthermore, biomarkers that are highly or poorly expressed can be described as a representation of the absence of normal progression, disease, or other conditions in an individual, or as having "differential expression," "differential level," or "differential value" compared to the "normal" expression level or value of the biomarker that expresses it. Therefore, the "differential expression" of a biomarker can also be represented by changes in the "normal" expression level of the biomarker.

[0052] The terms "differential gene expression" and "differential expression" are used interchangeably to refer to genes that are expressed at higher or lower levels in subjects with a specific disease compared to their expression in normal or control subjects. These terms also include genes that are expressed at high or low levels at different stages of the same disease. Differential gene expression can include a comparison of the expression of two or more genes or their gene products; or a comparison of the expression ratios of two or more genes or their gene products; or a comparison of two products of the same gene that have been treated differently, rather than between normal subjects and subjects with the disease, or between multiple stages of the same disease. Differential expression includes, for example, quantitative and qualitative differences in the expression patterns of genes or their expression products, based on time or cellular patterns, between normal and diseased cells, or between multiple cells experiencing different disease events or stages.

[0053] This invention can utilize any method known in the art to determine gene expression. Those skilled in the art will understand that the means of determining gene expression is not a key aspect of this invention. Several different detection methods can be used to detect gene expression, such as hybridization assays, quality analysis, or real-time quantitative nucleic acid amplification assays. In some embodiments, nucleic acid base sequence analysis methods can be used to detect gene sequences and biomarker values. Regarding the “increased” level of lncRNA gene product mentioned herein, it refers to a level higher than normally present. Typically, this can be estimated by comparison with a control. According to a specific embodiment, the increased level of lncRNA 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 absent under normal conditions (or in a control). In other words, in these embodiments, determining increased expression of the lncRNA gene product is equivalent to detecting the presence of the lncRNA gene product. Typically, in this case, a control will be included to ensure the detection reaction is performed correctly. The term "functional expression" of lncRNAs refers to the transcription and / or translation of the functional gene product. For non-protein-coding genes like lncRNAs, "functional expression" can be dysregulated at at least two levels. First, at the DNA level, such as through gene deletion or disruption, or the absence of transcription (in both cases, preventing the synthesis of the associated gene product). Loss of transcription can be caused, for example, by epigenetic changes (e.g., DNA methylation) or by loss-of-function mutations.

[0054] Second, at the RNA level, for example, through a lack of efficient translation—e.g., due to mRNA instability (e.g., through UTR variants)—mRNA can be degraded before the transcript is translated. Or through a lack of efficient transcription, for example, because mutations induce new splicing variants.

[0055] Accordingly, one object of the present invention is to provide an inhibitor of functional expression of lncRNA genes. This inhibitor can act at the DNA level or at the RNA (i.e., gene product) level. Since lncRNAs are non-coding genes, they do not produce protein products.

[0056] If inhibition is achieved at the DNA level, it can be done by using gene therapy to knock out or disrupt the target gene. As used herein, “knockout” can mean gene knockdown, or gene knockout can be achieved by using techniques known in the art, including but not limited to, retroviral gene transfer, to induce mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations. Another way to knock out genes is by using zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domain can be modified to target specific DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. By utilizing endogenous DNA repair mechanisms, these agents can be used to precisely alter the genome of higher organisms. Other genome-customized technologies that can be used to knock out genes include meganucleases and TAL effector nucleases (TALENs, Cellectis bioresearch). These consist of a TALE DNA-binding domain for sequence-specific recognition fused with a catalytic domain of a nuclease that introduces double-strand breaks (DSBs). Meganucleases are sequence-specific endonucleases, naturally occurring "DNA scissors" derived from various single-celled organisms such as bacteria, yeast, algae, and certain plant organelles. Meganucleases possess long recognition sites of 12 to 30 base pairs. The recognition sites of natural meganucleases can be altered to target natural genomic DNA sequences (e.g., endogenous genes).

[0057] 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 technique that allows sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on the CRISPR (regularly clustered short palindromic repeats) pathway derived from the bacterial immune system.

[0058] Gene inactivation, i.e., the suppression of functional gene expression, can also be achieved, for example, by designing transgenic organisms that express antisense RNA, or by administering antisense RNA to a subject. The antisense construct can be delivered, for example, as an expression plasmid, wherein when said expression plasmid is expressed in a cell, it produces RNA complementary to at least one unique portion of the cellular lncRNA.

[0059] A faster method for suppressing gene expression is based on the use of shorter antisense oligomers composed of DNA or other synthetic structural types, such as phosphate thioides, 2'-O-alkyl ribonucleotide chimeras, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or morpholine nucleic acids. Except for RNA oligomers, PNAs, and morpholine nucleic acids, all other antisense oligomers function in eukaryotic cells via a target cleavage mechanism mediated by RNase H. PNAs and morpholine nucleic acids bind highly affinity and specifically to complementary DNA and RNA targets, thereby functioning through simple steric hindrance to the RNA translation machine and exhibiting complete resistance to nuclease attack. "Antisense oligomer" refers to an antisense molecule or antigene agent comprising an oligomer of at least about 10 nucleotides in length. In embodiments, the antisense oligomer comprises at least 15, 18, 20, 25, 30, 35, 40, or 50 nucleotides. Antisense methods involve designing oligonucleotides (DNA or RNA or derivatives thereof) complementary to the RNA encoded by the multinucleotide sequence of the lncRNA. Antisense RNA can be introduced into cells to inhibit the translation of complementary mRNA by pairing with its bases and physically disrupting the translation machinery. This effect is therefore stoichiometric. While complete complementarity is preferred, it is not necessary. As mentioned herein, a sequence that is “complementary” to a portion of the RNA means that the sequence has sufficient complementarity to hybridize with the RNA and form a stable double helix; in the case of a double-stranded antisense polynucleotide sequence, the formation of a single strand of the double-stranded DNA or a triple helix can be detected. The ability to hybridize will depend on the degree of complementarity and the length of the antisense polynucleotide sequence. Generally, the longer the polynucleotide sequence that hybridizes, the more bases it can contain that mismatch with the RNA and still form a stable double helix (or triple helix, depending on the case). A technician can determine the degree of mismatch tolerance by measuring the melting point of the hybridization complex using standard procedures. Antisense oligomers should be at least 10 nucleotides long, preferably 15 to about 50 nucleotides long. In some 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. One related approach uses ribozymes instead of antisense RNA. Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties and can be programmed 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 posttranscriptional gene silencing. The RNAi phenomenon was first observed and described in *Caenorhabditis elegans*, where it was shown that exogenous double-stranded RNA (dsRNA) can specifically and powerfully disrupt the activity of genes containing homologous sequences by inducing rapid degradation of target RNA.Several reports describe the same catalytic phenomenon in other organisms, including experiments demonstrating the spatial and / or temporal control of gene inactivation, including plants (Arabidopsis thaliana), protozoa (Trypanosoma bryonis), invertebrates (Drosophila melanogaster), and vertebrate species (zebrafish and Xenopus laevis). The mediator of sequence-specific messenger RNA degradation can be small interfering RNAs (siRNAs), which are produced from longer dsRNAs by cleavage with ribonuclease III. Typically, siRNAs are 20-25 nucleotides in length. siRNAs typically contain 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 contains 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 may comprise two complementary single-stranded RNA molecules, or may comprise a single molecule in which two complementary partial base pairs are covalently linked by a single-stranded "hairpin" region (commonly referred to as shRNA). The term "isolated" means altered or removed from its natural state through human intervention. For example, siRNA naturally present in living animals is not "isolated," but synthetic siRNA or siRNA partially or completely isolated from its natural state is "isolated." Isolated siRNA can exist in fairly pure form or in non-natural environments, such as cells into which the siRNA has been transferred.

[0060] The siRNA of the present invention may include partially purified RNA, relatively pure RNA, synthetic RNA, or recombinant RNA, as well as modified RNA that differs from naturally occurring RNA by adding, deleting, replacing, and / or altering one or more nucleotides. Such alterations may include adding non-nucleotide material to, for example, the ends (one or more) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.

[0061] One or both strands of the siRNA of the present invention may also contain a 3′ overhang. A “3′ overhang” refers to at least one unpaired nucleotide extending from the 3′ end of the RNA strand. Thus, in one embodiment, the siRNA of the present invention contains at least one 3′ overhang of about 1 to about 6 nucleotides (including ribonucleic acid or deoxyribonucleic acid), preferably about 1 to about 5 nucleotides, more preferably about 1 to about 4 nucleotides, and particularly preferably about 1 to about 4 nucleotides.

[0062] In embodiments where both strands of the siRNA molecule contain 3′ overhangs, the length of the overhangs may be the same or different for each strand. In a further embodiment, the 3′ overhangs are present on both strands of the siRNA and are 2 nucleotides long. To enhance the stability of the siRNA of the present invention, the 3′ overhangs may also be stabilized to resist degradation. In one embodiment, the overhangs are stabilized using purine nucleotides such as adenosine or guanosine.

[0063] Alternatively, replacing the pyrimidine nucleotide with a modified analogue, such as replacing the uridine nucleotide at the 3′ overhang with 2′ deoxythymidine, is tolerable and does not affect the efficiency of RNAi degradation. In particular, the deletion of the 2′ hydroxyl group in 2′ deoxythymidine significantly enhances nuclease resistance at the 3′ overhang in tissue culture medium.

[0064] 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 this application. Techniques for selecting the target sequence of siRNA are well known in the art. Therefore, the positive strand of the siRNA of the present invention can contain a nucleotide sequence consistent with any segment of about 19 to about 25 consecutive nucleotides in the target mRNA.

[0065] The siRNA of the present invention can be obtained using many techniques known to those skilled in the art. For example, siRNA can be generated by chemical synthesis or recombination using methods known in the art. Preferably, the siRNA of the present invention is chemically synthesized using a suitably protected ribonucleoside phosphoramide and a conventional DNA / RNA synthesizer. The siRNA can be synthesized as two separate, complementary RNA molecules or as a single RNA molecule having two complementary regions.

[0066] As used herein, the “effective amount” of siRNA is an amount sufficient to cause RNAi-mediated degradation of the target mRNA, or an amount sufficient to inhibit the metastasis process in the subject. RNAi-mediated target mRNA degradation can be detected by measuring the level of the target mRNA or protein in the subject’s cells using standard techniques for isolating and quantifying mRNA or protein (as described above).

[0067] By taking into account factors such as the size and weight of the subject, the extent of disease penetration, the subject's age, health status and sex, the route of administration, and whether the administration is local or systemic, those skilled in the art can readily determine the effective amount of the siRNA of the present invention to be administered to a given subject.

[0068] Another specific form of antisense RNA strategy is the gapmer. A gapmer is a chimeric antisense oligonucleotide containing a central segment of a deoxyribonucleotide monomer long enough to induce RNase H cleavage. The central segment is flanked by segments of 2'-O modified ribonucleotides or other artificially modified ribonucleotide monomers, such as bridged nucleic acids (BNAs), protecting the internal segment from nuclease degradation. Gapmers have been used to achieve RNase-H-mediated target RNA cleavage while reducing the number of phosphate thioester linkages. Phosphothioesters possess increased resistance to nucleases compared to unmodified DNA. However, they have several drawbacks. These include low binding affinity to complementary nucleic acids and non-specific binding to proteins that leads to toxic side effects, limiting their application. The occurrence of toxic side effects and off-target effects caused by non-specific binding have inspired the design of novel artificial nucleic acids for the development of modified oligonucleotides to provide potent and specific antisense activity in vivo without exhibiting toxic side effects. By recruiting RNase H, gapmers selectively cleave target oligonucleotide chains. The cleavage of this chain triggers an antisense effect. This method has proven to be a powerful way to suppress gene function and is gradually becoming a popular approach for antisense therapy. Gapmers are commercially available. For example, LNA longRNA GapmeR is available from Exiqon, or MOE gapmers are available from Isis Pharmaceuticals. MOE gapmers, or “2′MOEgapmers,” are 15-30 nucleotide antisense phosphate-thioester oligonucleotides in which all backbone linkages are modified by adding sulfur (phosphate thioester) to non-bridging oxygens, and at least 10 consecutive nucleotides remain unmodified (deoxy sugar), while the remaining nucleotides contain an O′-methyl O′-ethyl substitution (MOE) at the 2′ position. For clinical use, the compounds of the present invention or their prodrug forms are formulated into pharmaceutical compositions compatible with their intended route of administration, such as oral, rectal, parenteral, or other administration methods. Typically, pharmaceutical formulations are prepared by mixing the active substance with conventional pharmaceutically acceptable diluents or carriers. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delay agents, and the like that compatible with drug administration. Examples of pharmaceutically acceptable diluents or carriers include water, gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium glycolate starch, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silica, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is covered.

[0069] The medicament of this invention can also be used in combination with other medicaments for treating oral squamous cell carcinoma. Other therapeutic compounds can be administered simultaneously with the main active ingredient, or even simultaneously in the same composition. Other therapeutic compounds can also be administered alone as a single composition or in a dosage form different from that of the main active ingredient. A portion of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered alone. During treatment, the dosage of the pharmaceutical composition of this invention can be adjusted according to the severity of symptoms, the frequency of recurrence, and the physiological response to the treatment regimen.

[0070] In this invention, the term "sample" refers to a composition obtained from a target patient that contains cells and / or other molecular bodies—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 in which cells and / or molecular bodies, such as biomarkers to be characterized, are expected or known to be available.

[0071] The present invention will be further described in detail below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0072] Example 1: Detection of differential expression of lncRNA by qPCR 1. Thirty-three cases of surrounding normal mucosal tissue and oral squamous cell carcinoma tissue were collected, all confirmed by pathological diagnosis. None of the patients had received any form of treatment before surgery. The surgically removed samples were cryopreserved in liquid nitrogen.

[0073] 2. RNA extraction Remove the tissue sample from the liquid nitrogen freezer, grind it in a pre-chilled mortar, and extract and isolate RNA according to the kit instructions. Details are as follows: 1) Add Trizol and let stand at room temperature for 5 minutes; 2) Add 0.2 ml of chloroform, shake the centrifuge tube vigorously to mix thoroughly, and let stand at room temperature for 5-10 minutes; 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-chilled isopropanol, mix thoroughly by inverting, and place on ice for 10 min. 4) After centrifuging at 12000 rpm for 15 min, carefully discard the supernatant. Add 75% DEPC ethanol at a ratio of 1 ml / ml Trizol to wash the precipitate (store at 4℃). After washing the precipitate, shake to mix well and centrifuge at 12000 rpm for 5 min at 4℃. 5) Discard the ethanol liquid, let stand at room temperature for 5 minutes, and add DEPC water to dissolve the precipitate; 6) Measure the purity and concentration of RNA using a Nanodrop2000 UV spectrophotometer and freeze at -70°C.

[0074] 3. Reverse transcription: 1) Prepare a 10 μl reaction system: 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 oligomeric dT aptamer primers, and 1 μl of experimental sample. 2) Reverse transcription reaction conditions Perform the reverse transcription reaction according to the conditions in RNA PCR Kit (AMV) Ver. 3.0.

[0075] 42℃ 60min, 99℃ 2min, 5℃ 5min.

[0076] 3) Polymerase chain reaction 1) Primer design qPCR primers were designed based on the coding sequences of the RP11-875O11.3 and GAPDH genes from Genebank and synthesized by Biomed Biotechnology Co., Ltd. The specific primer sequences are shown in Table 1.

[0077] Table 1 Primer Sequences

[0078] 2) Prepare a 25 μl PCR reaction mixture: 1 μl of forward (reverse) primers, 12.5 μl of Takara Ex Taq HS, 2 μl of template, and 8.5 μl of deionized water. 3) PCR reaction conditions: 94℃ for 4 min, (94℃ for 20 s, 60℃ for 30 s, 72℃ for 30 s) × 30 cycles.

[0079] PCR reactions were performed using SYBR Green as a fluorescent label on a Light Cycler real-time PCR instrument. The target band was determined by melting curve analysis and electrophoresis. - CT Relative quantification was performed using the method, with each sample repeated three times. △△CT method: △CT1 = (Target gene, test sample) CT value - (Internal reference gene, test sample) CT value; △CT2 = (Target gene, control sample) CT value - (Internal reference gene, control sample) CT value. △△CT = △CT1 - △CT2, fold change = 2 - CT .

[0080] 5. Statistical methods Using GAPDH as an internal control, the experimental results of quantitative real-time RT-PCR in oral squamous cell carcinoma tissue and normal mucosal tissue were calculated. The differences between the two were analyzed using a t-test. P A value <0.05 indicates a statistically significant difference.

[0081] 6. Results The results are shown in Table 2. Compared with the surrounding normal mucosa, the expression of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5, and AP000695.6 genes was upregulated in oral squamous cell carcinoma tissue, and the difference was statistically significant. P <0.05).

[0082] Table 2. Relative expression levels of lncRNAs

[0083] Example 2: Detection and Functional Verification of lncRNA Silencing 1. Cell Culture Human oral squamous cell carcinoma SCC-15 cells, preserved in liquid nitrogen, were retrieved, revived, and seeded into DMEM medium. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cells adhered to the culture medium, indicating successful revival. The medium was changed every 1-2 days. The cells were digested with trypsin and prepared into a cell suspension for experimental use.

[0084] 2. Cell transfection Cells were divided into 2×10 5Cells were seeded into six-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase (approximately 80% confluency) were discarded from the culture medium, washed twice with PBS, and starved for 1 hour with 2 ml of DMEM. Transfection was then performed using Liposome Transfection Reagent 2000 (purchased from Invitrogen), following the manufacturer's 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). The siRNA in the negative control group showed no homology to any of the lncRNA gene sequences.

[0085] 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.

[0086] Table 3. siRNA sequences of lncRNAs

[0087] 3. qPCR detection of the transcriptional level of the RP11-875O11.3 gene After 48 hours of transfection and culture, total RNA was extracted from the cells using the Trizol method, and reverse transcription and real-time quantitative PCR were performed according to the method in Example 1.

[0088] 4. CCK-8 cell proliferation experiment Cells from the negative control group and experimental group, transfected for 24 hours, were digested and centrifuged using standard methods. The supernatant was discarded, and 1 ml of complete culture medium was added to resuspend the cells. The cells were then mixed thoroughly by pipetting and seeding at 3000 cells per well into a 96-well plate. Complete culture medium was added to a final volume of 100 μL. 100 μL of DEPC water was added around the outermost edge of each well, and the 96-well plate was placed in an incubator. After 48 hours of culture, 100 μL of medium containing 10% CCK-8 was added, and the cells were incubated for another hour. The absorbance at 450 nm was then measured using a microplate reader, and statistical data were collected.

[0089] 5. Cell migration experiment Place the Transwell chamber in a 24-well plate, add 200 μL of DMEM solution to the upper chamber, and incubate. In the incubator, hydrate for 1 hour; according to 2 × 10⁻⁶ per chamber. 4The cells were plated, and the liquid in the upper chamber was added to 200 μL. The mixture was then pipetted and stirred. 700 μL of complete culture medium was added to the lower chamber, and the cells were incubated for 36 h. The chamber was removed, and the culture medium in both the upper and lower chambers was discarded. The remaining culture medium and cells in the upper chamber were gently wiped away with a cotton swab. The chamber was washed with PBS and shaken for 5 min. The PBS was then discarded. 500 μL of 4% paraformaldehyde was added to the lower chamber, and the cells were fixed at room temperature for 30 min. The fixative was discarded. The cells were washed three times with PBS and shaken for 5 min. The PBS was then discarded. The chamber was placed in a fume hood and air-dried for 30 min. 500 μL of 0.1% crystal violet solution was added to the lower chamber, and air bubbles were removed. The chamber was allowed to stand for 30 min. The crystal violet solution was discarded. The cells were washed three times with PBS and shaken for 5 min. The PBS was then discarded. Excess liquid in the upper chamber was gently wiped away with a dry cotton swab. The chamber was placed under a microscope for cell count.

[0090] 6. Statistical methods All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation. Differences between the two sets of data were analyzed using a t-test, which considers the mean to be equal to the standard deviation. P A value <0.05 is statistically significant.

[0091] 7. Results 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 showed no significant change (P>0.05).

[0092] Table 4. Transfection efficiency of siRNA

[0093] Note: P: compared with the blank control The results of CCK-8 testing 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 lncRNA plays an important role in the proliferation of oral squamous cell carcinoma cells in this study.

[0094] Table 5 OD values

[0095] 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 reduced (P<0.05). Although the number of cells in the RP11-426C22.4 and RP11-426C22.5 groups was reduced, it was not significant. Based on the above results, it can be concluded 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.

[0096] Table 6 Number of Migrating Cells

[0097] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope 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 RP11-426C22.4; Preferably, the biomarker is significantly upregulated in oral squamous cell carcinoma.

2. The use of the biomarker and / or its expression product as described in claim 1, or the reagent for specifically detecting the biomarker and / or its expression product as described in claim 1, characterized in that, Used to prepare products for diagnosing oral squamous cell carcinoma; Preferably, the reagent is selected from primers specifically amplifying the RP11-426C22.4 gene; Alternatively, a probe that specifically recognizes the RP11-426C22.4 gene; preferably, the primer sequences for specifically amplifying the RP11-426C22.4 gene are shown in SEQ ID NO. 9~10.

3. A product for diagnosing oral squamous cell carcinoma, characterized in that, The product includes reagents for detecting the biomarkers of claim 1; preferably, the product includes a chip, a reagent 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 a pair of primers that specifically amplify the biomarker; Reagents for detecting biomarkers by real-time quantitative PCR include at least one pair of primers that specifically amplify the biomarker; reagents for detecting biomarkers by in situ hybridization include probes that hybridize with the nucleic acid sequence of the biomarker; reagents for detecting biomarkers by gene chip include probes that hybridize with the nucleic acid sequence of the biomarker.

4. Use of the biomarker of claim 1 in the preparation of 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 one or more biomarkers; preferably, the inhibitor is selected from gapmers, interfering RNA, CRISPR, TALEN, or zinc finger nucleases; preferably, the inhibitor is selected from interfering RNA; preferably, the interfering RNA is selected from siRNA, and preferably, the sequence of the siRNA is shown in SEQ ID NO. 23-26.

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

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

7. A method for screening candidate drugs for the prevention or treatment of oral squamous cell carcinoma, characterized in that, The method includes: (1) Treating a system expressing or containing the biomarker of claim 1 with the substance to be screened; and (2) Detect the expression levels of biomarkers in the system; If the substance to be screened can reduce the expression level of the biomarker, then the substance to be screened is a candidate drug for the prevention or treatment of oral squamous cell carcinoma.

8. A method for inhibiting tumor cell proliferation in vitro, characterized in that, Inhibitors of the biomarkers described in claim 1 are introduced into tumor cells.

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

10. The application of the biomarker of claim 1 in constructing a computational model for diagnosing oral squamous cell carcinoma and in preparing a system / device / equipment / readable storage medium containing the computational model for diagnosing oral squamous cell carcinoma.

Images