CN111575381A - Novel use of biomarkers - Google Patents

Abstract

The invention relates to a new application of a biomarker, in particular to an application of the biomarker AP000695.4 in diagnosing oral squamous cell carcinoma and an application in treating AP000695.4 oral squamous cell carcinoma.

Description

Novel use of biomarkers

Technical Field

The invention belongs to the field of biomedicine, and relates to a new application of a biomarker.

Background

Oral Squamous Cell Carcinoma (OSCC) is an epitheliogenic malignancy that is prone to metastasis and is the most common cancer at the eleventh worldwide (Hussein AA, Helder MN, devisscer JG, et al. Global infection of Oral and oropharmaceutical in patient systems patients: A systematic review [ J ]. EurJcer 2017; 82: 115) cancer 127). About sixty thousand new cases each year are also the fifteenth most common cause of death due to cancer in the world (cancer J, Fernandez A, Somarriva C, et al. In recent years, the global incidence of oral squamous cell carcinoma has been on a markedly increasing trend, and the onset age has become younger, but the cause of the change in the trend has not been understood. The rate of increase in incidence is particularly prominent in developing countries (Wang F, Zhang H, Wen J, et al. nomograms for evaluating long-term overlap and cancer-specific Survival of tissues with organic squamous cell cancer [ J ]. cancer Med.2018; 7(4):943- & 952.).

With the improvement of imaging, surgery, radiotherapy and traditional treatment, the current treatment methods of OSCC are mainly Surgical resection, chemotherapy and radiotherapy or the combination of these three (Kim SM, Jeong D, Min KK, et al. two differential protein expression profiles of organic squamous cell carcinogenized by immunological therapy [ J ] expression Journal of scientific clinical Oncology.2017; 15(1): 151.). Although the treatment methods are continuously improved, the operation range is severely limited because the operation of the oral cavity can be closely contacted with important tissues and organs. In addition, the tissue vessels and nerves of the neck and the face are rich, the metastasis and invasion rate of the neck lymph nodes is high, the prognosis is poor, and the survival rate is not obviously increased (about 50 to 60 percent) in 5years in recent years. The 5-year survival rate of patients with advanced tumors or tumor recurrences is lower (Radihika T, Jeddy N, et al. Salivary biomakers in Oral square cell carcinoma-An insight [ J ]. Journal of Oral Biology & Craniofacial Research 2016,6(SupP 1): S51-54.). It has been found that patients with advanced OSCC who have had a clean partial surgical resection also have survival times of less than 30 months (FeliceFD, Polimeni A, et al. radiotherapeutic controls and Prospectral in Head and tech Cancer: A Literrure-Based clinical Review [ J ]. Neoplasia 2018; 20(3):227 and 232.). In addition, the 5-year survival rate of a patient is also related to the location, stage of the tumor, age of the patient and the presence or absence of underlying disease. Therefore, for OSCC, finding tumor markers with molecular diagnosis, prognosis prediction and targeted therapy is of great significance for tumor treatment and is a future development direction. The occurrence, development, invasion and transfer mechanism of OSCC are deeply understood, the cancer promotion gene and the cancer suppressor gene of OSCC are disclosed, the improvement and the supplement of the treatment means of oral squamous cell carcinoma are facilitated, and the clinical significance is achieved.

Disclosure of Invention

In order to make up the defects of the prior art, the invention researches the gene which shows differential expression in the oral squamous cell carcinoma, and researches the influence of the differential expression gene on cancer cells through further cell experiments, thereby providing detection and target sites for the diagnosis and treatment of the oral squamous cell carcinoma and simultaneously providing a theory for disclosing the pathogenesis of the oral squamous cell carcinoma.

The invention adopts the following technical scheme:

the invention provides an application of an AP000695.4 gene and/or an expression product thereof or a reagent for specifically detecting the AP000695.4 gene and/or the expression product thereof in preparing a product for diagnosing oral squamous cell carcinoma.

Further, the reagent for specifically detecting the AP000695.4 gene and/or the expression product thereof is selected from the group consisting of: primers for specifically amplifying the AP000695.4 gene; or a probe that specifically recognizes the AP000695.4 gene.

Further, the primer sequence of the specific amplification AP000695.4 gene is shown in SEQ ID NO. 5-6.

In another aspect of the invention, there is provided a product for diagnosing oral squamous cell carcinoma, which comprises an agent for detecting the AP000695.4 gene.

Further, the product comprises a chip, a kit or a test strip. Wherein, the chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, and the oligonucleotide probes comprise oligonucleotide probes aiming at the AP000695.4 gene and used for detecting the transcription level of the AP000695.4 gene; the kit comprises a primer, a probe or a chip for detecting the transcription level of the AP000695.4 gene.

Further, the kit may further comprise instructions or labels for use, positive controls, negative controls, buffers, adjuvants or solvents; the instructions or labels indicate that the kit is for detecting oral squamous cell carcinoma.

Further, the reagent comprises a reagent for detecting the AP000695.4 gene by RT-PCR, real-time quantitative PCR, in-situ hybridization or gene chip.

Further, the reagent for detecting the AP000695.4 gene by RT-PCR at least comprises a pair of primers for specifically amplifying the AP000695.4 gene; the reagent for detecting the AP000695.4 gene by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the AP000695.4 gene; reagents for detecting the AP000695.4 gene by in situ hybridization include probes that hybridize to the nucleic acid sequence of the AP000695.4 gene; the reagent for detecting the AP000695.4 gene by the gene chip comprises a probe which is hybridized with the nucleic acid sequence of the AP000695.4 gene.

In another aspect of the invention, there is provided the use of AP000695.4 in the manufacture of a pharmaceutical composition for the treatment of oral squamous carcinoma.

Further, the pharmaceutical composition comprises an inhibitor of functional expression of AP 000695.4.

Further, the inhibitor reduces the expression level of AP 000695.4.

Further, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease.

Further, the inhibitor is selected from interfering RNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 21-22.

Another aspect of the invention provides a pharmaceutical composition comprising an inhibitor of functional expression of AP 000695.4.

Further, the inhibitor reduces the expression level of AP 000695.4.

Further, the inhibitor is selected from gapmer, interfering RNA, CRISPR, TALEN or zinc finger nuclease.

Further, the inhibitor is selected from interfering RNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 21-22.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Another aspect of the invention provides the use of AP000695.4 in screening for a candidate drug for the treatment of oral squamous carcinoma.

Further, the steps of screening candidate drugs are as follows:

(1) treating the system expressing or containing the AP000695.4 gene with a substance to be screened; and

(2) detecting the expression level of the AP000695.4 gene in the system;

if the substance to be screened can reduce the expression level of the AP000695.4 gene, the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

Further, the candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, binding molecules, small molecule compounds, etc., directed against the AP000695.4 gene or its upstream or downstream genes.

Another aspect of the present invention provides a method of screening a candidate drug for preventing or treating oral squamous cell carcinoma, characterized in that the method comprises:

(1) treating the system expressing or containing the AP000695.4 gene with a substance to be screened; and

(2) detecting the expression level of the AP000695.4 gene in the system;

if the substance to be screened can reduce the expression level of the AP000695.4 gene, the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

In another aspect of the present invention, there is provided a method of inhibiting tumor cell proliferation by introducing a down-regulator of the AP000695.4 gene into tumor cells in vitro.

Further, the down-regulator includes siRNA, shRNA, antisense oligonucleotide or loss-of-function type gene to be directed against the AP000695.4 gene.

Detailed Description

Through intensive research, the invention discovers that the expressions of RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 genes in oral squamous carcinoma tissues are obviously higher than those of normal mucosal tissues for the first time, and experiments prove that RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 also show high expression in oral squamous carcinoma cells, and the expression levels of RP down-regulated 11-875O11.3, LINC01679, AP 5, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP 11-426C22.5 and RP 000695.6 can inhibit the proliferation of oral squamous carcinoma cells and the invasion of the oral squamous carcinoma cells and the diagnosis targets of RP 57328-875O 9, RP 869-339B 21.5, RP 863-828653 and RP 3553 can be used as clinical diagnosis targets of the cancer cells.

The lncRNA in the present invention includes a wild type, a mutant type or a fragment thereof, as long as it can be aligned to the gene when sequence alignment is performed. Two transcripts exist for RP11-875O11.3, which have been published so far, and the sequences are shown in ENST00000520840.1 and ENST00000523806.1, respectively. In a specific embodiment of the invention, the sequence of RP11-875O11.3 is shown in ENST 00000520840.1. LINC01679, which has been disclosed so far, has a transcript with a sequence shown in NR _ 131902.1. There are two transcripts of AP000695.4 that have been published so far, the sequences being shown in ENST00000428667.1 and ENST00000454980.1, respectively. In the specific embodiment of the invention, the sequence of the AP000695.4 is shown in ENST 00000428667.1. The currently published RP11-339B21.10 has one transcript with the sequence shown in ENST 00000610052.1. The currently published RP11-426C22.4 has a transcript with the sequence shown in ENST 00000566070.1. The presently disclosed RP11-426C22.5 has two transcripts, the sequences of which are shown in ENST00000562902.1 and ENST00000563477.1, respectively, and in a particular embodiment of the invention, the sequences of RP11-426C22.5 are shown in ENST 00000562902.1. There is a transcript of AP000695.6 which has been previously published, and the sequences are shown in ENST00000429588.1, respectively.

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

The "biomarker value", "biomarker level" and "level (level)" used in the present invention are measured by any analytical method for detecting a biomarker from a biological sample, and are used in combination to refer to a measured value indicating the presence or absence, absolute amount or concentration, relative amount or concentration, titration amount, level, expression level, ratio of measured levels, or the like of the biomarker in the biological sample.

"diagnosing," "diagnosed," "diagnosing," and variations of these terms refer to the discovery, judgment, or cognition of an individual's health state or condition based on one or more signs, symptoms, data, or other information associated with the individual. The health status of an individual may be diagnosed as healthy/normal (i.e., absence of a disease or condition), or may be diagnosed as unhealthy/abnormal (i.e., presence of an assessment of a disease or condition or characteristic). The terms "diagnosis", "diagnosed", "diagnosing" and the like above include early detection of a disease associated with a particular disease or condition; the nature or classification of the disease; discovery of progression, cure or recurrence of disease; discovery of response to disease after treatment or therapy of an individual. The diagnosis of oral squamous carcinoma includes the distinction of individuals who do not have cancer from individuals who do.

Where a biomarker is a biomarker that is indicative of or a marker for abnormal progression or disease or other condition in an individual, the biomarker is typically indicative of normal progression or absence of disease or other condition in the individual, or is one of overexpressed or underexpressed as compared to the level or value of expression of the biomarker as its marker. "Up-regulated", "up-regulated", "over-expression" and variations of this expression are used in a mixture to 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 typically detected from a biological sample that resembles 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 may be detected in mutually different steps of a particular disease.

"Down-regulated", "under-expressed" and the phenotype alterations are used interchangeably to refer to a biomarker value or level in a biological sample that is less than the 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. A plurality of the above terms may also refer to a biomarker value or level in a biological sample that is less than the value or level (or range of values or levels) of the biomarker that can be detected from different steps of a particular disease from each other.

Also, a biomarker that is highly or poorly expressed may be referred to as indicative of normal progression or absence of a disease or other state in an individual, or as having a "differentially expressed" or "differential level" or "differential value" as compared to the "normal" expression level or value of the biomarker for which it is expressed. Thus, a "differential expression" of a biomarker can also be expressed as a change in the "normal" expression level of the biomarker.

The terms "differential gene expression" and "differential expression" are used interchangeably to refer to a gene whose expression is activated at a higher or lower level in a subject with a particular disease than in a normal subject or a control subject. The above term also encompasses genes whose expression is activated at a high level or a low level in mutually different steps of the same disease. Differential gene expression may include a comparison of expression between two or more genes or their gene products; or a comparison of the expression ratio between two or more genes or their gene products; or instead a comparison of two differently processed products of the same gene between a normal subject and a subject with a disease or between various stages of the same disease. Differential expression includes, for example, quantitative and qualitative differences in temporal or cellular expression patterns in genes or their expression products between normal and diseased cells, or between cells undergoing different disease events or disease stages from each other.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is measured is not an important aspect of the present invention. For detecting the expression of the gene, a plurality of detection methods different from each other, for example, a detection method such as hybridization assay, mass analysis, or real-time fluorescence quantitative nucleic acid amplification detection, can be used. In certain embodiments, nucleic acid base sequence analysis methods can be used to detect gene sequences and detect biomarker values. By "increased" level with respect to the incrna gene product referred to herein is meant a higher level than normally present. Typically, this can be estimated by comparison with a control. According to particular embodiments, the increased level of incrna is a level that is 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 150%, 200% or even higher than the control. According to another particular embodiment, it means that the incrna gene product is expressed or present, whereas it is normally (or in a control) absent. In other words, in these embodiments, determining increased expression of the incrna gene product corresponds to detecting the presence of the incrna gene product. Typically, in such a case, a control will be included to ensure that the detection reaction is proceeding correctly. By "functional expression" of lncRNA is meant transcription and/or translation of a functional gene product. For non-protein encoding genes like lncRNA, "functional expression" can be deregulated at least two levels. First, at the DNA level, for example by deletion or disruption of the gene, or no transcription occurs (in both cases preventing synthesis of the relevant gene product). The deletion of transcription can be caused, for example, by an epigenetic change (e.g., DNA methylation) or by a loss-of-function mutation.

Second, at the RNA level, for example by lack of efficient translation-for example because of instability of the mRNA (e.g. by UTR variants), can lead to degradation of the mRNA prior to translation of the transcript. Or by lack of efficient transcription, e.g. because mutations induce new splice variants.

Accordingly, it is an object of the present invention to provide inhibitors of functional expression of lncRNA genes. Such inhibitors may 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.

If inhibition is achieved at the DNA level, it can be done by knocking out or disrupting the target gene using gene therapy. As used herein, a "knockout" can be a gene knock-down, or a gene knock-out can be made by using techniques known in the art, including, but not limited to, retroviral gene transfer, resulting in a mutation, such as a point mutation, insertion, deletion, frameshift, or missense mutation. Another way in which a gene can be knocked out is to use a zinc finger nuclease. Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target a DNA sequence of interest, which can target zinc finger nucleases to unique sequences in a complex genome. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genome of higher organisms. Other genome customization techniques that can be used to knock out a gene are Meganuclease and TAL effector nucleases (TALENs, celectiobioresearch). Consisting of a fusion of a TALE DNA binding domain for sequence specific recognition with the catalytic domain of a Double Strand Break (DSB) introducing endonuclease. Meganuclease is a sequence-specific endonuclease, a naturally occurring "DNA scissors" that is derived from a variety of unicellular organisms such as bacteria, yeast, algae, and certain plant organelles. Meganucleoclean has a long recognition site of 12 to 30 base pairs. The recognition site of native Meganuclease can be altered to target it to a native genomic DNA sequence (e.g., an endogenous gene).

Another recent genome editing technology is the CRISPR/Cas system, which can be used to achieve RNA-guided genome engineering. CRISPR interference is a genetic technique that allows sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on CRISPR (regularly clustered interspaced short palindromic repeats) pathways derived from the bacterial immune system.

Inactivation of a gene, i.e., inhibition of functional expression of the gene, can also be achieved, for example, by designing a transgenic organism that expresses antisense RNA, or by administering antisense RNA to a subject. The antisense construct can be delivered, for example, as an expression plasmid, wherein when the expression plasmid is expressed in a cell, RNA is produced that is complementary to at least a unique portion of the cellular incrna.

A faster method for inhibiting gene expression is based on the use of shorter antisense oligomers composed of DNA or other synthetic structural types such as phosphorothioate, 2' -0-alkylribonucleotide chimeras, Locked Nucleic Acids (LNA), Peptide Nucleic Acids (PNA) or morpholino nucleic acids. With the exception of RNA oligomers, PNA and morpholino nucleic acids, all other antisense oligomers work in eukaryotic cells by the RNase H mediated target cleavage mechanism. PNA and morpholino nucleic acids bind complementary DNA and RNA targets with high affinity and specificity, thereby acting through simple steric hindrance to the RNA translation machinery and appear to be completely resistant to nuclease attack. "anti-sense oligomer" refers to an antisense molecule or antigene agent (anti-gene 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. The antisense approach involves designing oligonucleotides (DNA or RNA or derivatives thereof) that are complementary to the RNA encoded by the polynucleotide sequence of the incrna. Antisense RNA can be introduced into cells to inhibit translation of complementary mRNA by pairing with its base and physically impeding the translation machinery. The effect is therefore stoichiometric. Although complete complementarity is preferred, it is not necessary. As referred to herein, a sequence is "complementary" to a portion of an RNA, meaning that the sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense polynucleotide sequences, single strands of duplex DNA may be detected, or triplex formation may 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 is hybridized, the more mismatched bases can be included with the RNA and still form a stable duplex (or triplex, as the case may be). The skilled artisan can determine the tolerance to mismatches by measuring the melting point of the hybridization complex using standard procedures. The antisense oligomer should be at least 10 nucleotides in length, preferably the oligomer is 15 to about 50 nucleotides in length. 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 in length. A related approach uses ribozymes instead of 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, including time and tissue specific gene inactivation, using ribozymes has been reported in mice, zebrafish, and drosophila. RNA interference (RNAi) is a form of post-transcriptional gene silencing. The phenomenon of RNA interference was originally observed and described in C.elegans, where it was shown that exogenous double-stranded RNA (dsRNA) can specifically and powerfully destroy the activity of genes comprising homologous sequences by inducing a mechanism of rapid degradation of the target RNA. Several reports describe the same catalytic phenomenon in other organisms, including experiments displaying spatially and/or temporally controlled gene inactivation, including plants (arabidopsis thaliana), protozoa (trypanosoma brucei), invertebrates (drosophila melanogaster) and vertebrate species (zebrafish and xenopus laevis). Mediating sequence-specific messenger RNA degradation can be small interfering RNAs (sirnas), which are generated from longer dsrnas by rnase III cleavage. Typically, siRNAs are 20-25 nucleotides in length. sirnas typically comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard watson crick base pairing interactions (hereinafter "base pairing"). The sense strand comprises a nucleic acid sequence identical to a target sequence in a target mRNA. The sense and antisense strands of the siRNA of the invention may comprise two complementary single-stranded RNA molecules, or may comprise a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" region (commonly referred to as shRNA). The term "isolated" means altered or removed from the native state by human intervention. For example, an siRNA that is naturally present in a living animal is not "isolated", but a synthetic siRNA or an siRNA that is partially or completely isolated from material coexisting with its natural state is "isolated". The isolated siRNA can exist in a substantially pure form, or can exist in a non-natural environment, such as a cell into which the siRNA is transferred.

The siRNA of the present invention may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include adding non-nucleotide material, for example, to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.

One or both strands of the siRNA of the invention may also contain 3' overhangs. A "3 'overhang" refers to at least one unpaired nucleotide that extends from the 3' end of an RNA strand. Thus, in one embodiment, the siRNA of the invention comprises at least one 3' overhang of 1 to about 6 nucleotides in length (including ribonucleic or deoxyribonucleic acids), preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 1 to about 4 nucleotides in length.

In embodiments where both strands of the siRNA molecule comprise 3' overhangs, the length of the overhangs may be the same or different for each strand. In the most preferred embodiment, 3' overhang is present on both strands of the siRNA, 2 nucleotides in length. To enhance the stability of the siRNA of the present invention, the 3' overhang may also be stabilized against degradation. In one embodiment, overhangs are stabilized by the inclusion of purine nucleotides, such as adenosine or guanosine nucleotides.

Alternatively, replacement of pyrimidine nucleotides with modified analogs, such as replacement of uridine nucleotides in the 3 'overhang with 2' deoxythymidine, is tolerable without affecting the efficiency of RNAi degradation. In particular, the absence of the 2 ' hydroxyl group in 2 ' deoxythymidine significantly enhances nuclease resistance of the 3 ' overhang in tissue culture media.

The sirnas of the invention can target any segment of about 19 to 25 contiguous nucleotides in any target incrna RNA sequence ("target sequence"), examples of which are provided herein. Techniques for selecting target sequences for siRNA are well known in the art. Thus, the sense strand of the siRNA of the invention can comprise a nucleotide sequence that is identical to any stretch of about 19 to about 25 consecutive nucleotides in the target mRNA.

The siRNA of the present invention can be obtained using a number of techniques known to those skilled in the art. For example, sirnas can be produced by chemical synthesis or recombinantly using methods known in the art. Preferably, the siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.

siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

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

The effective amount of the siRNA of the present invention to be administered to a given subject can be readily determined by one skilled in the art by considering, for example, the size and weight of the subject, the degree of disease infiltration, the age, health and sex of the subject, the route of administration, and whether the administration is local or systemic.

Another special form of antisense RNA strategy is gapmer. Gapmer is a chimeric antisense oligonucleotide comprising a central segment of deoxynucleotide monomers long enough to induce RNase H cleavage. The central region of the Gapmer is flanked by segments of 2' -O modified ribonucleotides or other artificially modified ribonucleotide monomers, such as Bridge Nucleic Acids (BNAs), which protect the inner segments from nuclease degradation. Gapmer has been used to obtain RNase-H mediated cleavage of target RNA while reducing the number of phosphorothioate linkages. Phosphorothioate possesses increased resistance to nucleases compared to unmodified DNA. However, they have several disadvantages. This includes low binding capacity to complementary nucleic acids and non-specific binding to proteins resulting in toxic side effects, limiting their use. The occurrence of toxic side effects and off-target effects due to non-specific binding have motivated the design of new artificial nucleic acids for the development of modified oligonucleotides to provide effective and specific antisense activity in vivo without exhibiting toxic side effects. By recruiting RNaseH, gapmers selectively cleave the target oligonucleotide strand. Cleavage of this strand elicits an antisense effect. This approach has proven to be a powerful approach to inhibiting gene function and is becoming an increasingly popular approach for antisense therapy. Gapmer is commercially available. Such as LNA longRNA GapmeR supplied by Exiqon, or MOEgapmer supplied by Isis pharmaceuticals. MOE gapmers or "2 'MOE gapmers" are anti-sense phosphorothioate oligonucleotides of 15-30 nucleotides, wherein all backbone linkages are modified by addition of sulfur (phosphorothioate) on non-bridging oxygens and a stretch of at least 10 consecutive nucleotides remains unmodified (deoxysugar) while the remaining nucleotides contain an O' -methyl O '-ethyl substitution (MOE) at the 2' position. For clinical use, the compounds according to the invention or their prodrug forms are formulated into pharmaceutical compositions that are compatible with their intended route of administration, e.g., oral, rectal, parenteral or other mode of administration. Typically, pharmaceutical formulations are prepared by mixing the active substance with conventional pharmaceutically acceptable diluents or carriers. 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, and the like, compatible with pharmaceutical administration. Examples of pharmaceutically acceptable diluents or carriers are water, gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated.

The medicament of the invention can also be used in combination with other medicaments for treating oral squamous cell carcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in a separate composition or in a different dosage form than the primary active ingredient. Some doses of the principal component may be administered concurrently with other therapeutic compounds, while other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

The term "sample" as used herein refers to a composition obtained from a subject of interest that contains cells and/or other molecular entities that are to be characterized and/or identified, for example, according to physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "clinical sample" or "disease sample" and variants thereof, refers to any sample obtained from a subject patient in which it would be expected or known that cellular and/or molecular bodies, such as biomarkers, would be characterized, would be available.

The present invention will be described in further detail with reference to examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1QPCR detection of differential expression of lncRNA

1. 33 surrounding normal mucosal tissues and oral squamous cell carcinoma tissues were collected, all confirmed by pathological diagnosis, and all patients did not receive any form of treatment before surgery. The surgically excised specimens were cryopreserved in liquid nitrogen.

2. RNA extraction

Taking out the tissue sample frozen in liquid nitrogen, putting the tissue sample into a precooled mortar for grinding, and extracting and separating RNA according to the instruction in the kit. The method comprises the following specific steps:

1) adding Trizol, and standing at room temperature for 5 min;

2) adding chloroform 0.2ml, shaking the centrifuge tube with force, mixing well, standing at room temperature for 5-10 min;

3) centrifuging at 12000rpm for 15min, transferring the upper water phase into another new centrifuge tube (taking no care to absorb protein substances between the two water phases), adding equal volume of isopropanol precooled at-20 deg.C, fully reversing, mixing, and placing on ice for 10 min;

4) centrifuging at 12000rpm for 15min, carefully removing supernatant, adding 75% DEPC ethanol according to the proportion of 1ml/ml Trizol, washing precipitate (storing at 4 deg.C), shaking, mixing, and centrifuging at 12000rpm for 5min at 4 deg.C;

5) discarding the ethanol liquid, standing at room temperature for 5min, adding DEPC water to dissolve the precipitate;

6) the RNA purity and concentration were measured with a Nanodrop2000 ultraviolet spectrophotometer and frozen at-70 ℃ in a freezer.

3. Reverse transcription:

1) a10. mu.l reaction system was prepared:

taking MgCl₂Mu.l of 2. mu.l, 1. mu.l of 10 × RT Buffer, 3.75. mu.l of RNase-free water, 1. mu.l of dNTP mixture, 0.25. mu.l of RNase inhibitor, 0.5. mu.l of AMV reverse transcriptase, 0.5. mu.l of oligo dT aptamer primer, and 1. mu.l of experimental sample

2) Conditions for reverse transcription

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

42℃60min，99℃2min，5℃5min。

3) Polymerase chain reaction

1) Primer design

QPCR amplification primers were designed based on the coding sequences of RP11-875O11.3 gene and GAPDH gene from Genebank and were synthesized by Bomeide Bio Inc. Specific primer sequences are shown in table 1.

TABLE 1 primer sequences

2) Prepare 25 μ l PCR reaction:

forward (reverse) primer 1. mu.l, Takara Ex Taq HS 12.5. mu.l, template 2. mu.l, deionized water 8.5. mu.l

3) And (3) PCR reaction conditions: 94 ℃ for 4min, (94 ℃ for 20s, 60 ℃ for 30s, 72 ℃ for 30s) x 30 cycles.

SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, and a target band is determined through melting curve analysis and electrophoresis, 2^-ΔΔCTRelative quantification is carried out by the method, and each sample is subjected to 3 repeated experiments, △△ CT method, wherein the CT value of △ CT1 (target gene, sample to be detected) is CT value- (reference gene, sample to be detected), the CT value of △ CT2 (target gene, control sample) is CT value- (reference gene, control sample), the CT value of △△ CT △ CT1- △ CT2, and the expression multiple is 2^-ΔΔCT。

5. Statistical method

The experimental results of fluorescent quantitative RT-PCR of oral squamous cell carcinoma tissue and normal mucosa tissue are calculated by taking GAPDH as an internal reference, and the difference between the two tissues is measured by t test and has statistical difference when P < 0.05.

6. Results

As shown in Table 2, compared with the surrounding normal mucosal tissue, the genes RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 are up-regulated in the oral squamous cell carcinoma tissue, and the difference has statistical significance (P < 0.05).

TABLE 2 relative expression levels of lncRNA

Example 2 silencing detection and functional validation of lncRNA

1. Cell culture

Taking out human oral squamous cell carcinoma SCC-15 cell stored in liquid nitrogen, recovering, inoculating in DMEM culture medium at 37 deg.C and 5% CO₂Cells were cultured in a thermostated incubator. After 24h, cells grow adherently, namely, the cells are recovered successfully and the solution is changed for 1 time every 1-2d, and the cells are digested by trypsin and prepared into cell suspension for experiments.

2. Cell transfection

Pressing the cells according to 2 × 10⁵One well was inoculated into six well cell culture plates at 37 ℃ with 5% CO₂CulturingCulturing in a box. Cells in the logarithmic phase of proliferation (about 80%) were subjected to starvation culture in an incubator for 1 hour by adding 2m 1DMEM, washing with PBS 2 times, and transfection using Lipofectin 2000 (purchased from Invitrogen) 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 siRNA of the negative control group has no homology with the sequence of each lncRNA gene.

Among them, siRNA-NC is a general negative control provided by Shanghai Jima pharmaceutical technology Co., Ltd, and the siRNA sequence for each lncRNA is shown in Table 3.

TABLE 3 siRNA sequences of lncRNA

3. QPCR detection of transcript level of RP11-875O11.3 Gene

After 48h of transfection and culture of each group of cells, total RNA of the cells was extracted by Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method of example 1.

4. CCK-8 cell proliferation assay

Digesting and centrifuging a negative control group transfected for 24 hours and cells of an experimental group by a conventional method, removing supernatant, adding 1ml of complete culture medium for resuspending cells, blowing, uniformly mixing, inoculating 3000 cells per well to a 96-well plate, and supplementing the complete culture medium to 100 mu 1; 100 μ 1DEPC water was added to the outermost periphery of the plate, and the 96-well plate was placed in a incubator for culture. After culturing for 48h, adding 100 mu 1 of culture medium containing 10% of CCK-8, continuing culturing in an incubator for 1h, measuring absorbance at 450nm by using an enzyme-labeling instrument, and counting data.

5. Cell migration assay

Placing Transwell chamber in 24-well plate, adding 200 μ 1DMEM solution into upper chamber, placing in culture box, hydrating for 1 hr, and according to 2 × 10 per chamber⁴Plating the cells, supplementing the liquid in the upper chamber to 200 mu 1, blowing, beating and uniformly mixing, adding a complete culture medium of 700 mu 1 in the lower chamber, and continuously culturing for 36h in an incubator; taking out the small chamber, discarding the culture medium in the upper chamber and the lower chamber, wiping off the residual culture medium and cells in the upper chamber with a cotton swab, washing the small chamber with PBS, shaking for 5min, and discarding the PBS; adding 500 mu 14% paraformaldehyde into the lower chamber, fixing at room temperature for 30min, removing the fixing solution, washing with PBS for 3 times, shaking for 5min, and removing PBS; placing the small chamber in a fume hood, and air-drying for 30 min; adding 500 mu 1 of prepared 0.1% crystal violet solution into the lower chamber, removing bubbles, and standing for 30 min; discard crystal violet solution, wash 3 times with PBS, shake for 5min, discard PBS, wipe off excess liquid in upper chamber with dry cotton swab gently, place chamber under microscope, count cell number.

6. Statistical method

The experiments were performed in 3 replicates, and the results were expressed as mean ± sd, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

7. Results

The silencing effect of siRNA is shown in table 4, compared with the blank control group, each siRNA in the experimental group has better interference effect (P <0.05), while siRNA-NC has no significant change (P > 0.05).

TABLE 4 transfection Effect of siRNA

Note: p compared to blank control

The CCK-8 assay results are shown in table 5, the OD value of the experimental group is significantly reduced compared to the negative control group, and P is less than 0.05, which indicates that lncRNA plays an important role in proliferation of oral squamous cell carcinoma cells.

TABLE 5 OD values

The results of the migration experiment are shown in Table 6, and compared with the negative control group, the number of the migrated cells in the experimental groups RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10 and AP000695.6 is significantly reduced (P <0.05), while the number of the cells in the groups RP11-426C22.4 and RP11-426C22.5 is not significantly reduced, and according to the results, the RP11-875O11.3, LINC01679, AP000695.4, RP11-339B21.10 and AP000695.6 play an important role in the metastasis of oral squamous cell carcinoma.

TABLE 6 number of migrating cells

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

Sequence listing

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Claims (10)

1. Use of the AP000695.4 gene and/or its expression product or a reagent for specifically detecting the AP000695.4 gene and/or its expression product, which is characterized in that the reagent is used for preparing products for diagnosing oral squamous cell carcinoma.

2. Use according to claim 1, characterized in that the agent for the specific detection of the AP000695.4 gene and/or its expression product is selected from: primers for specifically amplifying the AP000695.4 gene; or a probe specifically recognizing the AP000695.4 gene; preferably, the primer sequence for specifically amplifying the AP000695.4 gene is shown in SEQ ID NO. 5-6.

3. A product for diagnosing oral squamous cell carcinoma, comprising an agent for detecting AP000695.4 gene; preferably, the product comprises a chip, a kit or a test strip.

4. The product of claim 3, wherein the reagents comprise reagents for detecting the AP000695.4 gene by RT-PCR, real-time quantitative PCR, in situ hybridization, or gene chip; preferably, the reagent for detecting the AP000695.4 gene by RT-PCR at least comprises a pair of primers for specifically amplifying the AP000695.4 gene; the reagent for detecting the AP000695.4 gene by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the AP000695.4 gene; reagents for detecting the AP000695.4 gene by in situ hybridization include probes that hybridize to the nucleic acid sequence of the AP000695.4 gene; the reagent for detecting the AP000695.4 gene by the gene chip comprises a probe which is hybridized with the nucleic acid sequence of the AP000695.4 gene.

Use of AP000695.4 in the preparation of a pharmaceutical composition for the treatment of oral squamous carcinoma.

6. The use according to claim 5, wherein the pharmaceutical composition comprises an inhibitor of functional expression of AP 000695.4; preferably, the inhibitor reduces the expression level of AP 000695.4; 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 siRNA is shown in SEQ ID NO. 21-22.

7. A pharmaceutical composition comprising an inhibitor of functional expression of AP 000695.4; preferably, the inhibitor reduces the expression level of AP 000695.4; 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 siRNA is shown in SEQ ID NO. 21-22; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Use of AP000695.4 in screening for a candidate drug for the treatment of oral squamous cell carcinoma.

9. A method of screening for a candidate agent for preventing or treating oral squamous cell carcinoma, the method comprising:

(1) treating the system expressing or containing the AP000695.4 gene with a substance to be screened; and

(2) detecting the expression level of the AP000695.4 gene in the system;

if the substance to be screened can reduce the expression level of the AP000695.4 gene, the substance to be screened is a candidate drug for preventing or treating oral squamous cell carcinoma.

10. A method for inhibiting the proliferation of tumor cells, which comprises introducing an inhibitor of AP000695.4 gene into tumor cells in vitro.