New uses of LOC285627

Abstract

The application discloses a new use of LOC285627, and particularly discloses a use of LOC285627 in diagnosis and treatment of laryngeal squamous cell carcinoma. The application further discloses a tool for diagnosing laryngeal squamous cell carcinoma, a method for screening a drug for treating laryngeal squamous cell carcinoma, and a drug for treating laryngeal squamous cell carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a new use of LOC285627. Background Technology

[0002] Laryngeal cancer is a common malignant tumor of the head and neck, ranking second among head and neck tumors, with an incidence rate of approximately 5% of all malignant tumors. Squamous cell carcinoma accounts for about 93%-99% of all laryngeal cancers, and most laryngeal squamous cell carcinomas originate in the vocal cords, accounting for about 60%. A retrospective analysis of 306 cases of laryngeal malignant tumors by Huang Xinhui et al. found that squamous cell carcinoma accounted for 99.7% of all malignant tumors, with well- and moderately differentiated squamous cell carcinomas being the most common. The primary site of laryngeal cancer was the glottic type, followed by the supraglottic and subglottic types. Smoking and alcohol consumption were closely related to the occurrence of the tumor. Although surgery, radiotherapy, and chemotherapy can alleviate patients' suffering to varying degrees, they often cause functional impairment, local deformities, and have a high recurrence rate, seriously threatening the physical and mental health of patients. Knudson's two-mutation hypothesis, proposed in 1971, is a scientific prediction about the mechanism of tumorigenesis widely accepted by researchers in tumor genetics, guiding research on the molecular mechanisms of tumorigenesis for over 30 years. Exploring genes associated with different types of tumors is a fundamental prerequisite for understanding the molecular mechanisms of tumorigenesis and for developing gene diagnosis and gene therapy. Cloning and localizing disease-causing genes, including those related to tumors, remains an important component of the Human Genome Project. Studies by Srinivus et al. have found that tumors are accompanied by changes in tumor markers at a very early stage, and detecting these changes can aid in early tumor detection. Research at the gene level will be crucial for the eradication of all tumors, including laryngeal cancer. Therefore, identifying and cloning laryngeal cancer-specific genes will offer broad prospects for research into the molecular mechanisms, gene diagnosis, and gene therapy of laryngeal cancer.

[0003] Although methods for diagnosing and treating tumors are constantly evolving, the overall survival rate of laryngeal cancer worldwide has not improved significantly, especially for patients with advanced-stage disease. This is mainly due to three factors: First, the diverse clinical manifestations of early-stage laryngeal cancer make early diagnosis challenging, and many patients do not receive timely and effective treatment in the early stages. Identifying molecular markers for early diagnosis of laryngeal cancer is crucial to improving the early diagnosis rate. Second, treatment for laryngeal cancer primarily involves surgery, supplemented by radiotherapy and chemotherapy. Although the increased use of partial laryngectomy and CO2 laser therapy for early-stage glottic laryngeal cancer has resulted in complete or partial preservation of laryngeal function and reduced postoperative complications, thus improving patients' quality of life, the recurrence rate remains high. Identifying tumor-promoting genes or tumor suppressor factors in laryngeal cancer and screening for potential drug targets would lay the foundation for developing specific vaccines and / or new anticancer drugs related to laryngeal cancer. Third, different patients with laryngeal cancer have differences in pathological type, degree of tumor differentiation, and severity of lesions at the time of diagnosis. Different laryngeal cancer patients have different clinical manifestations and physical conditions, and their sensitivity to postoperative radiotherapy and chemotherapy varies. Finding molecular markers for monitoring efficacy and predicting prognosis will lay the foundation for personalized treatment of laryngeal cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a long non-coding RNA biomarker for the diagnosis of laryngeal squamous cell carcinoma. This invention utilizes qPCR experiments to demonstrate that the expression level of LOC285627 in the cancerous tissue of laryngeal squamous cell carcinoma patients is significantly lower than that in adjacent normal tissues; therefore, LOC285627 can be used as a molecular biomarker for the diagnosis of laryngeal squamous cell carcinoma.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention provides the application of a reagent for detecting the expression of a long non-coding RNA in the preparation of diagnostic products for laryngeal squamous cell carcinoma. The long non-coding RNA is LOC285627.

[0007] Furthermore, the reagents include those used when detecting the expression level of LOC285627 using reverse transcription PCR, real-time quantitative PCR, microarray detection, DNA blotting, RNA blotting, or in situ hybridization.

[0008] Furthermore, the reagents include amplification primers used to detect the expression level of LOC285627.

[0009] In a specific embodiment of the present invention, the amplification primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0010] This invention provides a product for the diagnosis of laryngeal squamous cell carcinoma, which diagnoses laryngeal squamous cell carcinoma by detecting the expression of the aforementioned long non-coding RNA in a sample.

[0011] Furthermore, the products include, but are not limited to, chips, reagent kits, test strips, or high-throughput sequencing platforms. High-throughput sequencing platforms are specialized diagnostic tools, and with the development of high-throughput sequencing technology, constructing an individual's RNA expression profile will become a very convenient task. By comparing the RNA expression profiles of disease patients and healthy individuals, it is easy to analyze which RNA abnormalities are associated with the disease. Therefore, the discovery in high-throughput sequencing that abnormal LOC285627 expression is associated with laryngeal squamous cell carcinoma is also a use of LOC285627 and falls within the scope of protection of this invention.

[0012] The kit includes reagents for detecting the expression level of LOC285627, the reagents comprising nucleic acids that bind to LOC285627 or its DNA sequence, the nucleic acids comprising primers and / or probes used for detecting the expression level of LOC285627 using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

[0013] The chip includes a reagent for detecting the expression level of LOC285627, the reagent comprising a nucleic acid that binds to LOC285627 or its DNA sequence, the nucleic acid comprising primers and / or probes capable of detecting the expression level of LOC285627.

[0014] The test strip includes a reagent for detecting the expression level of LOC285627, the reagent including a nucleic acid that binds to LOC285627 or its DNA sequence, the nucleic acid including primers and / or probes capable of detecting the expression level of LOC285627.

[0015] The kit may also include: containers, instructions for use, positive controls, negative controls, buffers, auxiliaries, or solvents. Examples include solutions for suspending or fixing cells, detectable tags or labels, solutions for facilitating nucleic acid hybridization, solutions for lysing cells, or solutions for purifying nucleic acids.

[0016] The instruction manual for the kit of the present invention describes how to use the kit for detection and how to use the detection results to judge tumor development and select treatment plans.

[0017] Using the kit of this invention, LOC285627 can be detected by various methods selected from the group consisting of: reverse transcription PCR, real-time quantitative PCR, microarray detection, DNA blotting, RNA blotting, or in situ hybridization. Those skilled in the art can adjust and modify the detection method according to actual conditions and needs.

[0018] Furthermore, the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0019] This invention provides the application of LOC285627 in screening candidate drugs for the prevention or treatment of laryngeal squamous cell carcinoma.

[0020] Furthermore, the steps for screening candidate drugs are as follows:

[0021] Treat systems expressing or containing LOC285627 with candidate substances; and

[0022] The expression of LOC285627 in the system was detected;

[0023] If the candidate substance can increase the expression of LOC285627, preferably significantly, then the candidate substance is a candidate drug for the prevention or treatment of laryngeal squamous cell carcinoma.

[0024] In this invention, the system includes (but is not limited to): a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system. The candidate substances include (but are not limited to): nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins, or carriers.

[0025] In this invention, the steps further include: conducting further cell experiments and / or animal experiments on the obtained candidate drugs to further select and identify drugs useful for the prevention, relief or treatment of laryngeal squamous cell carcinoma from the candidate drugs.

[0026] This invention provides the use of LOC285627 in the preparation of medicaments for the prevention or treatment of laryngeal squamous cell carcinoma.

[0027] The drug includes a substance that promotes the expression of LOC285627.

[0028] The substance that promotes LOC285627 expression includes an overexpression vector containing LOC285627 and a host cell containing the overexpression vector.

[0029] The present invention provides a pharmaceutical composition for treating laryngeal squamous cell carcinoma, the pharmaceutical composition comprising a substance that promotes the expression of LOC285627, and / or other pharmaceutically acceptable carriers compatible with said substance.

[0030] Pharmaceutically acceptable carriers include (but are not limited to) diluents, excipients, binders, wetting agents, absorption enhancers, surfactants, humectants, adsorbents, lubricants, buffers, stabilizers, antibacterial agents, isotonic agents, chelating agents, and pH control agents.

[0031] The pharmaceutical compositions of the present invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted drug delivery device. Oral or injectable administration is preferred. The pharmaceutical compositions of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, excipient, or excipient.

[0032] The pharmaceutical compositions of the present invention can also be used in combination with other drugs for treating laryngeal 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.

[0033] Preferably, gene therapy can be used. For example, substances that promote the expression of LOC285627 (such as overexpression vectors) can be directly administered to the subject by methods such as injection, and such techniques are well known to those skilled in the art.

[0034] The present invention also provides a method for diagnosing laryngeal squamous cell carcinoma, the method comprising the following steps:

[0035] (1) Obtain suspected diseased tissue from the subject's throat;

[0036] (2) Detect the expression level of LOC285627 in the tissue from step 1;

[0037] (3) The measured expression level of LOC285627 was correlated with whether the subject had the disease.

[0038] (4) If the expression level of LOC285627 in the suspected diseased tissue of the larynx of the subject is significantly reduced compared with the normal control, then the subject is judged to have laryngeal squamous cell carcinoma, or the subject is judged to have a high risk of laryngeal squamous cell carcinoma, or the laryngeal squamous cell carcinoma patient is judged to have recurrence, or the laryngeal squamous cell carcinoma patient is judged to have a poor prognosis.

[0039] In the context of this invention, "normal control" can refer to a normal person who does not have laryngeal squamous cell carcinoma, or it can refer to the adjacent tissue of a person who has laryngeal squamous cell carcinoma.

[0040] In the context of this invention, "diagnosis" includes determining whether a subject is already ill, determining whether a subject is at risk of becoming ill, determining whether a patient has relapsed, determining a patient's responsiveness to drug treatment, or determining a patient's prognosis. Attached Figure Description

[0041] Figure 1 This chart displays statistics on LncRNA overexpression.

[0042] Specific implementation methods

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and 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 as recommended by the manufacturer.

[0044] Example 1: Validation of Differentially Expressed Molecules

[0045] I. Research Subjects

[0046] Cancerous tissue and adjacent tissue were collected from 45 patients with laryngeal squamous cell carcinoma. Inclusion and exclusion criteria: ① Patients had no primary tumors in other sites besides the primary tumor, and the primary tumor had not metastasized; ② No history of cardiovascular or cerebrovascular diseases such as diabetes or hypertension; ③ No history of infectious diseases such as hepatitis, syphilis, tuberculosis, or HIV; ④ No family history of hereditary diseases; ⑤ No preoperative radiotherapy, chemotherapy, or biological therapy.

[0047] Sampling precautions: When cutting tumor specimens, pay attention to cutting non-necrotic tumor tissue in the central area of ​​the tumor. When cutting normal tissue adjacent to the cancer, keep it as far away from the tumor as possible, at least 1.5 cm away from the tumor edge. Try to cut mucosal epithelial tissue and avoid deep connective tissue.

[0048] II. Total RNA Extraction from Tissues

[0049] Total RNA was extracted from tissues using the Norgen RNA Extraction Kit:

[0050] tissue lysis

[0051] 1) In a clean area with less RNase interference, weigh about 20 mg of the isolated tissue sample using a mortar containing an appropriate amount of liquid nitrogen, and grind it into powder using a pestle.

[0052] 2) Transfer the sample to a 2 mL centrifuge tube that does not contain RNase;

[0053] 3) Add 300 μL of Lysis solution, place in a homogenizer, and grind thoroughly for 1-5 minutes;

[0054] 4) Centrifuge at 12000g, 4℃ for 10 min, and transfer the supernatant to a new 1.5mL centrifuge tube;

[0055] 5) Add 600ul of RNase-Free Water and mix with a vortex mixer;

[0056] 6) Add 20 μL of proteinase K, incubate at 55°C for 15 minutes, and vortex continuously to mix.

[0057] 7) Centrifuge at 14000g, room temperature, for 1 min to allow cell debris to precipitate at the bottom of the centrifuge tube. Transfer the supernatant to another 1.5 mL centrifuge tube that does not contain RNase.

[0058] 8) Add 450 μL of 95% ethanol and vortex to mix.

[0059] RNA adsorption:

[0060] 9) Add 650 μL of the lysis buffer containing ethanol to a centrifuge column and centrifuge at 14000 g for 1 min;

[0061] 10) Discard the lower layer and reset the collection tube on the column;

[0062] 11) Repeat steps 9) to 10) according to the capacity of the lysis buffer;

[0063] 12) Add 400 μL of Wash solution and centrifuge at 14000 g for 2 minutes;

[0064] 13) Discard the lower layer and place the column on a new collection tube;

[0065] DNase treatment:

[0066] 14) Add 100ul Enzyme Incubation Buffer and 15ul DNase I, centrifuge at 14000g for 1min;

[0067] 15) Transfer the solution from the collection tube back into the column;

[0068] 16) Leave at room temperature for 15 minutes;

[0069] RNA washing:

[0070] 17) Add 400 μL of Wash solution, centrifuge at 14000 g for 1 min, discard the lower layer, and reposition the collection tube onto the column;

[0071] 18) Add 400 μL of Wash solution, centrifuge at 14000 g for 2 min, and discard the collection tube;

[0072] RNA elution:

[0073] 19) Place the column into a 1.7 mL Elution tube;

[0074] 20) Add 30ul of Elution Buffer;

[0075] 21) Centrifuge at 200g for 2 minutes to allow the solution to fully bind with the column, then centrifuge at 14000g for 1 minute.

[0076] III. Reverse Transcription

[0077] The reverse transcription kit (DDR037A) was purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0078] Using the extracted total RNA (1 μg) as a template, the following reaction system was added: Buffer 4μL, 1 μL of RT Enzyme Mix, 1 μL of Oligo dT Primer (50 μM), and 1 μL of Random 6mers (100 μM) were added, and the reaction volume was brought to 20 μL with RNase-free ddH2O. The mixture was incubated at 37°C for 15 min and then at 85°C for 5 s to obtain cDNA. This cDNA can be used for lncRNA real-time PCR detection.

[0079] IV. qPCR

[0080] SYBR Premix Ex Taq is used for real-time quantitative PCR (polymerase chain reaction). TM The (Tli RNaseH Plus) kit was manufactured by Takara Corporation, Japan. Follow the instructions in the manual.

[0081] use The expression level of LncRNA was analyzed using a relative quantitative method. Ct is the intensity value of the fluorescence signal detected by the thermal cycler in the reaction system. The calculation method is: ΔCt = (Ct target gene - Ct internal reference gene) cancer tissue experimental group - (Ct target gene - Ct internal reference gene) control tissue group. This represents the fold change in the expression of the target gene in the experimental group relative to the control group. The experimental data analysis was performed using Bio-RAD analysis software.

[0082] Primer design: Primers were designed based on the lncRNA sequence using NCBI's primer design tool (Primer BLAST). The primer sequences are shown below:

[0083] LOC285627

[0084] Upstream primer: 5'-AATCAAGTGGCTAAGTCA-3' (SEQ ID NO.1); Downstream primer: 5'-GGATTCATCTCATTAACTCTG-3' (SEQ ID NO.2).

[0085] LOC100506860

[0086] Upstream primer: 5'-CCACTGAGAAGTCCTAAG-3' (SEQ ID NO.3); Downstream primer: 5'-TGATGATGAAACTGGCTTA-3' (SEQ ID NO.4).

[0087] LOC388692

[0088] Upstream primer: 5'-CTCACAGTCGGACCTTAG-3' (SEQ ID NO.5); Downstream primer: 5'-ACAGAACGGAAGACAAGA-3' (SEQ ID NO.6).

[0089] Primers were designed based on the GAPDH (internal reference gene) sequence. The upstream primers are: 5'-CTCTGGTAAAGTGGATATTGT-3' (SEQ ID NO.7); 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO.8).

[0090] V. Statistical Analysis

[0091] Data analysis was performed using SPSS 19.0 statistical software. Paired t-tests were applied to determine whether there was a statistically significant difference in LncRNA expression between cancerous and adjacent normal tissue samples. All statistical tests were two-tailed, and P < 0.05 was considered statistically significant.

[0092] VI. Results

[0093] The statistical results are shown in Table 1. Compared with adjacent normal tissue, the expression levels of LOC285627, LOC100506860 and LOC388692 in the cancer tissue of patients with laryngeal squamous cell carcinoma were significantly downregulated, and the differences between groups were statistically significant (P<0.001).

[0094] Table 1. Statistics on LncRNA expression

[0095]

[0096] Example 2: LncRNA Overexpression

[0097] 1. Cell Culture

[0098] Human laryngeal squamous cell carcinoma cell line Hep2 was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days. Cells grew well, forming a monolayer adhering to the culture medium. Cells were routinely passaged using 0.25% trypsin containing EDTA.

[0099] 2. Transfection

[0100] 1) Pre-transfection cell treatment

[0101] The day before transfection, seed 3-5 × 10⁶ cells / wells in a 6-well plate. 5 Cells per well were cultured in antibiotic-free medium for one day. The cell density at transfection was 30-50%. The medium was then replaced with serum-free medium before transfection.

[0102] 2) LncRNA overexpression

[0103] The full-length sequence of the long non-coding RNA was found in NCBI and overexpression plasmid was constructed in pcDNA3.1 by Shanghai Gemma Technology Co., Ltd. E. coli DH5α competent cells were transformed and the plasmid was extracted; Hep2 cells were transfected using the validated overexpression plasmid.

[0104] 3) Transfection

[0105] a. Take 1 μg / μl of overexpression plasmid or pcDNA3.1 empty vector and add it to 49 μl of serum-free medium. Mix gently and incubate at room temperature for 5 min.

[0106] b. Add 2 μl of Lipofectamine 2000 to 48 μl of serum-free culture medium. Mix gently and incubate at room temperature for 5 min;

[0107] c. Mix the two mixtures (total volume 100 μl), mix gently, and incubate at room temperature for 25 min to allow the complex to form;

[0108] d. Add 100 μl of the complex and an appropriate amount of culture medium to each well of a 6-well plate and mix gently.

[0109] e. Observe gene overexpression after incubation for 48–96 hours.

[0110] 3. Cell transfection efficiency detection

[0111] The expression of the target gene was determined using the qPCR method described in Example 1. Figure 1 As shown, the expression levels of LOC285627, LOC100506860, and LOC388692 in cells transfected with the empty vector were set to 100%. In cells transfected with the LncRNA overexpression vector, the expression levels of LOC285627, LOC100506860, and LOC388692 were significantly increased (the differences were statistically significant, *P<0.05; **P<0.01), allowing for further experiments.

[0112] Example 3: Effect of LncRNA Expression on the Proliferation of Laryngeal Squamous Cell Carcinomas

[0113] The effect of LncRNA expression on the proliferation of laryngeal squamous cell carcinoma cells was detected by WST-1 assay.

[0114] On the second day after cell transfection, the cell density was adjusted to 1×10⁶. 2 / μl, seeded into 96-well plates (100μl / well), with 3 replicates per group. After culturing at 37℃ and 5% CO2 for 72 hours, 10μl of WST-1 was added and incubated for 2 hours. The optical density (D) of the cells was then measured at 450nm using a microplate reader.

[0115] The results are shown in Table 2, and the differences between groups were statistically significant (P<0.05). This indicates that promoting the expression of LOC285627, LOC100506860, and LOC388692 can inhibit the proliferation of laryngeal squamous cell carcinoma cells.

[0116] Table 2. Statistics on the proliferation of laryngeal squamous cell carcinoma cells.

[0117] empty carrier 1.302 0.142 LOC285627 overexpression 0.931 0.009 0.041 LOC100506860 overexpression 0.795 0.058 0.034 LOC388692 overexpression 0.668 0.039 0.015

[0118] Example 4: Effect of LncRNA Expression on the Migration of Laryngeal Squamous Cell Carcinomas

[0119] On the second day after cell transfection, cells from the treatment group were collected and counted at 1×10⁻⁶. 5 Cells were resuspended in 100 μl of serum-free medium and added to the upper chamber of a Transwell cell culture plate. 600 μl of complete culture medium was added to the lower chamber. After incubation at 37°C and 5% CO2 for 48 h, the chamber was removed, and the cells in the upper chamber were gently wiped away with a cotton swab. The cells were fixed with 4% paraformaldehyde for 20 min, washed once with PBS, stained with crystal violet for 10 min, washed once with PBS, and the total number of cells in five different fields of view (upper, lower, left, right, and center) was counted under an optical microscope (×200), and the average value was recorded. The experiment was repeated three times.

[0120] The results are shown in Table 3, and the differences between groups were statistically significant (P<0.05). This indicates that promoting the expression of LOC285627, LOC100506860, and LOC388692 can inhibit the migration of laryngeal squamous cell carcinoma cells.

[0121] Table 3. Statistics on migration of laryngeal squamous cell carcinoma cells.

[0122] empty carrier 139.8 15.324 LOC285627 overexpression 115 9.271 0.049 LOC100506860 overexpression 94.2 5.481 0.029 LOC388692 overexpression 74.4 7.233 0.005

[0123] 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. sequence list <110> Beijing Yangshen Biotechnology Co., Ltd. <120> New Uses for LOC285627 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 aatcaagtgg ctaagtca 18 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 ggattcatct cattaactct g 21 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 ccactgagaa gtcctaag 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 tgatgatgaa actggctta 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 ctcacagtcg gaccttag 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 acagaacgga agacaaga 18 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 ctctggtaaa gtggatattg t 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 ggtggaatca tattggaaca 20

Claims

1. Application of an overexpression vector containing LOC285627 in the preparation of a drug for treating laryngeal squamous cell carcinoma.

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