CN111455059A

CN111455059A - Application of reagent for detecting and targeting biomarkers in oral squamous cell carcinoma

Info

Publication number: CN111455059A
Application number: CN202010481200.9A
Authority: CN
Inventors: 杨承刚; 肖枫
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2020-05-31
Filing date: 2020-05-31
Publication date: 2020-07-28

Abstract

The invention discloses an application of a reagent for detecting and targeting a biomarker in oral squamous cell carcinoma, wherein the biomarker is lncRNA L INC01679, the invention discloses an application of L INC01679 in preparing a product for diagnosing the oral squamous cell carcinoma and a product for diagnosing the oral squamous cell carcinoma, and simultaneously discloses an application of L INC01679 in preparing a pharmaceutical composition for treating the oral squamous cell carcinoma and a pharmaceutical composition for treating the oral squamous cell carcinoma.

Description

Application of reagent for detecting and targeting biomarkers in oral squamous cell carcinoma

Technical Field

The invention belongs to the field of biomedicine, and relates to application of a reagent for detecting and targeting biomarkers in oral squamous cell carcinoma.

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 methods of OSCC treatment are mainly Surgical resection, chemotherapy and radiotherapy or a combined treatment of the three methods (Kim SM, Jeong D, Min KK, oral.two differential Cancer expression profile of Oral cavity cell Cancer in advanced stage) (clinical diagnosis of Cancer Oncology.2017; 15(1):151.) although the treatment is continuously improved, the Oral cavity surgery closely contacts important tissues and organs, the severe treatment has about the scope of surgery because the Oral cavity surgery closely contacts important tissues and organs, the vessels and nerves of the frontal tissue are abundant, the cervical metastasis and incidence rates are high, the prognosis is poor, the survival rates of 5-60% of tumor metastasis and recurrence rates of Cancer metastasis is not significantly increased in advanced stage [ 7: 7, 15 (7) and the tumor metastasis of Cancer is also more advanced than those of clinical diagnosis of Cancer metastasis of Cancer in advanced stage [ 7: 7, 15: 7. the clinical diagnosis of Cancer metastasis of Cancer is not more advanced stage; 2. A. 7, 7: 7, 7. the clinical diagnosis of Cancer metastasis of Cancer in advanced stage [ 7, 7. A. a. A.

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:

one aspect of the invention provides application of L INC01679 gene and/or expression product thereof or reagent for specifically detecting L INC01679 gene and/or expression product thereof in preparing products for diagnosing oral squamous cell carcinoma.

Furthermore, the reagent for specifically detecting L INC01679 gene and/or expression product thereof is selected from a primer for specifically amplifying L INC01679 gene or a probe for specifically recognizing L INC01679 gene.

Furthermore, the primer sequence of the specific amplification L INC01679 gene is shown in SEQ ID No. 3-4.

Another aspect of the present invention provides a product for diagnosing oral squamous cell carcinoma, which comprises an agent for detecting L INC01679 gene.

The product further 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, the oligonucleotide probes comprise oligonucleotide probes for detecting L INC01679 gene transcription level and aiming at L INC01679 gene, and the kit comprises primers, probes or a chip for detecting L INC01679 gene transcription level.

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 reagents include a reagent for detecting L INC01679 gene by RT-PCR, real-time quantitative PCR, in situ hybridization or gene chip.

Further, the reagent for detecting L INC01679 gene by RT-PCR at least comprises a pair of primers for specifically amplifying L INC01679 gene, the reagent for detecting L INC01679 gene by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying L INC01679 gene, the reagent for detecting L INC01679 gene by in situ hybridization comprises a probe hybridized with the nucleic acid sequence of L INC01679 gene, and the reagent for detecting L INC01679 gene by gene chip comprises a probe hybridized with the nucleic acid sequence of L INC01679 gene.

In another aspect of the invention, L INC01679 is provided for use in preparing a pharmaceutical composition for treating oral squamous carcinoma.

Further, the pharmaceutical composition comprises an inhibitor of L INC01679 functional expression.

Further, the inhibitor reduced the expression level of L INC 01679.

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

Further, the inhibitor is selected from interfering RNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 19-20.

Another aspect of the invention provides a pharmaceutical composition comprising an inhibitor of L INC01679 functional expression.

Further, the inhibitor reduced the expression level of L INC 01679.

Further, the inhibitor is selected from interfering RNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 19-20.

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

According to another aspect of the invention, L INC01679 is provided for use in screening a candidate drug for treating oral squamous cell carcinoma.

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

(1) treating a system expressing or containing the L INC01679 gene with a substance to be screened, and

(2) detecting the expression level of L INC01679 gene in the system;

if the substance to be screened can reduce the expression level of L INC01679 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., against L INC01679 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:

(2) detecting the expression level of L INC01679 gene in the system;

In another aspect of the present invention, there is provided a method for inhibiting tumor cell proliferation by introducing a down-regulator of L INC01679 gene into tumor cells in vitro.

Further, the down-regulator comprises siRNA, shRNA, antisense oligonucleotide or loss-of-function gene against L INC01679 gene.

Detailed Description

Through intensive research, the invention discovers for the first time that the expressions of RP11-875O11.3, L INC01679, 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, and experiments prove that RP11-875O11.3, L INC01679, AP000695.4, RP 53-339B 21.10, RP11-426C22.4, RP11-426C22.5 and AP000695.6 also show high expression in oral squamous carcinoma cells, and the expression levels of RP11-875O11.3, L INC01679, AP L, RP L-339B 21.10, L-426C 22.4, RP L-426C 22.5 and L AP 72 can be reduced to inhibit the proliferation of the oral squamous carcinoma cells and the invasion of the RP L-L, and the RP L, RP 6772, RP L-L, RP 6772, RP 3-L and RP 8472 can be used as clinical diagnosis targets for treating the oral squamous carcinoma cells.

The lncRNA in the invention comprises wild type, mutant or fragment thereof, and can be aligned to the gene only by sequence alignment, the currently disclosed RP11-875O11.3 has two transcripts, the sequences are respectively shown as ENST00000520840.1 and ENST00000523806.1, the sequence of the RP11-875O11.3 is shown as ENST00000520840.1 in the specific embodiment of the invention, the sequence of the currently disclosed L INC01679 has one transcript, the sequence is shown as NR _131902.1, the currently disclosed AP000695.4 has two transcripts, the sequences are respectively shown as ENST00000428667.1 and ENST 6327, the sequence of the AP000695.4 is shown as ENST00000428667.1 in the specific embodiment of the invention, the currently disclosed RP11-339B21.10 has one transcript, the sequence is shown as ENST00000610052.1, the currently disclosed RP 45-46426.4 has one transcript, the sequence is shown as ENST00000566070.1, the sequence of the currently disclosed RP11-339B21.10 has one transcript, the sequence is shown as ENST 6327, the sequences are respectively shown as ENST00000562902.1 6, the sequences are shown as ENST 11 and the sequences are respectively shown as ENST00000562902.1 6.

As used herein, "markers" and "biomarkers" may be used interchangeably to refer to indications of normal or abnormal progression in an individual or indications of a disease or other condition in an individual or target molecules that express 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" 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 biomarker, used for the biomarker, or corresponding to the presence or absence, absolute amount or concentration, relative amount or concentration, titration amount, level, expression level, ratio of measured levels, and the like, 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.

As used herein, "knockout" may be gene knock-down, or may be a gene knock-down, or a gene knock-down, made by using techniques known in the art, including, but not limited to, retroviral gene transfer, causing mutations, such as point mutations, insertions, deletions, frameshifts, or missense mutations another way of gene knock-down is the use of Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing zinc finger DNA binding domains to DNA cleavage domains.

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 more rapid method for inhibiting gene expression is based on the use of a shorter antisense oligomer consisting of DNA or other synthetic structural types (e.g., phosphorothioate, 2' -0-alkylribonucleotide chimeras, locked nucleic acid (L NA), Peptide Nucleic Acid (PNA), or morpholino nucleic acid) except RNA oligomer, PNA, and morpholino nucleic acid, all other antisense oligomers function in eukaryotic cells through the RNase H-mediated target cleavage mechanism, PNA and morpholino nucleic acid have a high affinity and specifically bind to complementary DNA and RNA targets, thereby functioning through simple steric hindrance to the RNA translation machinery, and exhibiting complete resistance to nuclease attack by a sense strand siRNA or antigene agent (anti-gene agent) that comprises an oligomer at least about 10 nucleotides long by an antisense molecule or antigene agent (anti-gene agent) in embodiments, which oligomer comprises at least 15, 18, 20, 25, 30, 35, 40, or 50 nucleotides that is designed to hybridize to a polynucleotide sequence encoding the antisense polynucleotide sequence of a ncncc antisense strand, or antisense RNA sequence thereof, which can be stably transferred to a target RNA, including at least one antisense strand through the sense strand translation of a antisense strand, a antisense strand, or antisense strand, which is capable of interfering RNA, including at least one strand with a naturally occurring in vivo, a antisense strand, a antisense strand, a.

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.

The Gapmer is flanked by 2' -O modified ribonucleotides or other segments of artificially modified ribonucleotide monomers, such as Bridge Nucleic Acids (BNAs), protecting the inner segments from nuclease degradation, Gapmer has been used to obtain RNase-H mediated target RNA cleavage, while reducing the number of phosphorothioate linkages, phosphorothioate possesses increased resistance to nucleases compared to unmodified DNA, however, they have several drawbacks, including low binding capacity to complementary nucleic acids and non-specific binding to proteins that lead to toxic side effects, thereby limiting their use, the occurrence of toxic side effects and off-target effects that result from non-specific binding have motivated the design of new artificial nucleic acids for the development of modified oligonucleotides to provide effective and non-toxic side effects in vivo while exhibiting anti-toxic effects through the use of toxic side effects and anti-target effects that are shown by the use of the anti-toxic side effects and non-specific binding to proteins, which have been formulated with the use of modified oligonucleotides for antisense oligonucleotides, to provide potent antisense activity in vivo by a pharmaceutical formulation, including the use of a pharmaceutically acceptable excipient, or non-toxic substance, such as a pharmaceutically acceptable excipient, or a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, a pharmaceutically acceptable.

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 following examples are intended to illustrate the invention in further detail without limiting the scope of the invention the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L 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) The PCR reaction conditions were × 30 cycles at 94 ℃ for 4min, (94 ℃ for 20s, 60 ℃ for 30s, and 72 ℃ for 30 s).

SYBR Green is used as a fluorescent marker, PCR reaction is carried out on an L 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, L INC01679, 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 is statistically significant (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 plated in six well cell culture plates at 37 deg.C、5％CO₂Culturing in an incubator. 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 the Transwell chamber in a 24-hole plate, adding 200 mu 1DMEM solution into the upper chamber, placing in a culture box, and hydrating for 1 h; push buttonAccording to 2 × 10 per cell⁴Plating 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 migrated cells in the experimental groups RP11-875O11.3, L INC01679, AP000695.4, RP11-339B21.10 and AP000695.6 is significantly reduced (P <0.05), while the number of cells in the groups RP11-426C22.4 and RP11-426C22.5 is not significantly reduced, and according to the results, the results of the migration experiment, RP11-875O11.3, L INC01679, AP000695.4, RP11-339B21.10 and AP000695.6 play an important role in the transfer 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

1. Use of L INC01679 gene and/or its expression product or reagent for specifically detecting L INC01679 gene and/or its expression product, which is used for preparing products for diagnosing oral squamous cell carcinoma.

2. The use of claim 1, wherein the reagent for specifically detecting L INC01679 gene and/or expression product thereof is selected from a primer for specifically amplifying L INC01679 gene or a probe for specifically recognizing L INC01679 gene, and preferably the primer sequence for specifically amplifying L INC01679 gene is shown in SEQ ID No. 3-4.

3. A product for diagnosing oral squamous cell carcinoma is characterized in that the product comprises a reagent for detecting L INC01679 gene, and preferably the product comprises a chip, a kit or a test strip.

4. The product of claim 3, wherein the reagents comprise L INC01679 gene detection reagents by RT-PCR, real-time quantitative PCR, in situ hybridization or gene chip, preferably L INC01679 gene detection reagents by RT-PCR comprise at least one pair of primers for specifically amplifying L INC01679 gene, L INC01679 gene detection reagents by real-time quantitative PCR comprise at least one pair of primers for specifically amplifying L INC01679 gene, L INC01679 gene detection reagents by in situ hybridization comprise probes for hybridizing with the nucleic acid sequence of L INC01679 gene, and L INC01679 gene detection reagents by gene chip comprise probes for hybridizing with the nucleic acid sequence of L INC01679 gene.

Use of L INC01679 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 L INC01679 functional expression inhibitor, preferably the inhibitor reduces L INC01679 expression level, preferably the inhibitor is selected from gapmer, interfering RNA, CRISPR, TA L EN or zinc finger nuclease, preferably the inhibitor is selected from interfering RNA, preferably the siRNA has the sequence shown in SEQ ID NO. 19-20.

7. A pharmaceutical composition is characterized by comprising L INC01679 functional expression inhibitor, preferably, the inhibitor reduces L INC01679 expression level, preferably, the inhibitor is selected from gapmer, interference RNA, CRISPR, TA L EN or zinc finger nuclease, preferably, the inhibitor is selected from interference RNA, preferably, the sequence of the siRNA is shown in SEQ ID NO. 19-20, and preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8, L INC01679 in screening for the use of 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:

(2) detecting the expression level of L INC01679 gene in the system;

10. A method for inhibiting the proliferation of tumor cells, which comprises introducing an inhibitor of L INC01679 gene into tumor cells in vitro.