Long-chain non-coding RNA molecule marker combination and application thereof

Abstract

The application relates to the field of tumor biotechnology and discloses a long-chain non-coding RNA molecular marker combination and application thereof. The long-chain non-coding RNA molecular marker combination comprises seven molecules, including ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385 and AL513318.2; the seven long-chain non-coding RNAs are significantly highly expressed in laryngeal squamous cell carcinoma tissues, each molecule can promote the proliferation, migration and invasion ability of laryngeal squamous cell carcinoma cells, and is positively correlated with the poor prognosis of laryngeal squamous cell carcinoma. The long-chain non-coding RNA molecular marker combination is applied to the preparation of a laryngeal squamous cell carcinoma auxiliary prognosis evaluation kit and a preparation for treating laryngeal squamous cell carcinoma; the laryngeal squamous cell carcinoma auxiliary prognosis evaluation kit adopts real-time fluorescent quantitative PCR to detect the expression amount of the seven long-chain non-coding RNAs, and combines the seven expression amount results to conveniently and efficiently judge the disease condition and prognosis evaluation of a laryngeal cancer patient; and the preparation for treating laryngeal squamous cell carcinoma comprises at least one or seven of the seven long-chain non-coding specific interference RNAs.

Description

Technical Field

[0001] This invention relates to the field of tumor biotechnology, specifically to a combination of long non-coding RNA molecular markers and their applications. Background Technology

[0002] Laryngeal squamous cell carcinoma (LSCC) ranks second among head and neck malignancies, with a particularly high incidence in northern China. The annual incidence in my country is approximately 13,000. LSCC has an insidious onset, with early symptoms being subtle or difficult to detect. Furthermore, LSCC is prone to cervical lymph node metastasis, meaning that about 60% of LSCC patients are diagnosed at an advanced stage (clinical stage III or IV), missing the optimal treatment window. Despite continuous advancements in clinical treatment techniques, the 5-year survival rate for LSCC patients worldwide has been declining over the past 40 years (Steuer CE, El-Deiry M, Parks JR, Higgins KA, Saba NF. An update on larynx cancer[J]. CA Cancer J Clin, 2017, 67(1):31-50). Therefore, finding effective and precise clinical diagnosis and prognostic assessment methods is urgently needed for the clinical treatment of LSCC.

[0003] Long non-coding RNAs (LUNs) are a class of gene sequences longer than 200 nt that lack protein-coding potential. They play crucial roles in vital processes such as cell proliferation, metabolism, differentiation, and cell cycle. In recent years, with in-depth research into the mechanisms of malignant tumor development, it has been gradually discovered that LUNs play a significant role in the occurrence and progression of malignant tumors. Furthermore, LUNs are abundant, exist in diverse media, and exhibit tissue and spatiotemporal specificity; therefore, they are considered to have great potential as diagnostic biomarkers and therapeutic targets for malignant tumors.

[0004] With the development of high-throughput sequencing and real-time quantitative PCR technologies, we can comprehensively screen genomes and transcriptomes, and use real-time quantitative PCR for targeted detection and validation of target genes. This opens up possibilities for in-depth research into the mechanisms of malignant tumor development and for using gene transcription products as diagnostic biomarkers.

[0005] Currently, research on single long non-coding RNAs as diagnostic biomarkers is relatively common. However, the specificity and sensitivity of their detection vary depending on the tumor type and the patient's stage. Studies have shown that combinations of different long non-coding RNA biomarkers have been tentatively used for auxiliary diagnosis and prognosis in some malignant tumors, demonstrating good applicability, sensitivity, and specificity. However, research and application in laryngeal squamous cell carcinoma are lacking. The applicant, through in-depth analysis of high-throughput sequencing data from laryngeal squamous cell carcinoma and its corresponding adjacent normal mucosa (ANM), combined with clinical information and validated by real-time quantitative PCR, constructed a biomarker combination for diagnosis and prognostic assessment consisting of seven long non-coding RNAs: ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2. The application of this combination in an auxiliary prognostic assessment kit for laryngeal squamous cell carcinoma is proposed. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a combination of long non-coding RNA molecular markers and their application in an adjuvant prognostic assessment kit for laryngeal squamous cell carcinoma.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A set of long non-coding RNA molecular markers, comprising seven markers: ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2. The nucleotide sequences of the long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0009] Furthermore, any one of the long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 was significantly overexpressed in laryngeal squamous cell carcinoma tissues, promoting the proliferation, metastasis, and invasion of these cells, and showing a positive correlation with poor prognosis. Combining any of these long non-coding RNAs in laryngeal squamous cell carcinoma tissues for biomarker analysis further strengthened this positive correlation. The diagnostic and prognostic assessment of laryngeal squamous cell carcinoma using these seven long non-coding RNAs depends on their expression levels in the cancer, which were detected by real-time quantitative PCR. The combined analysis of the above seven long non-coding RNAs showed that patients with laryngeal squamous cell carcinoma had the worst prognosis.

[0010] Furthermore, the combined analysis involves summing the expression levels of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 in laryngeal squamous cell carcinoma tissue. A comprehensive weighted score is then used to perform prognostic survival analysis and generate a prognostic survival analysis model, based on the R language functions survival::coxph() and glmnet::predict.glmnet.

[0011] The application of the long non-coding RNA molecular marker combination described above is used to prepare a kit for adjuvant prognostic assessment of laryngeal squamous cell carcinoma, and to prepare a formulation or kit for treating laryngeal squamous cell carcinoma; the long non-coding RNA molecular marker combination includes seven types, specifically ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2; the nucleotide sequences of the long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0012] Furthermore, the laryngeal squamous cell carcinoma adjuvant prognostic assessment kit is a kit for real-time quantitative PCR detection of the expression level of the long non-coding RNA using targeted primers for the combination of long non-coding RNA molecular markers.

[0013] Furthermore, the targeted primers are:

[0014] The forward primer sequence of ENSG00000233397 is shown in SEQ ID NO: 8;

[0015] The reverse primer sequence for ENSG00000233397 is shown in SEQ ID NO: 9;

[0016] The BARX1-DT forward primer sequence is shown in SEQ ID NO: 10;

[0017] The BARX1-DT reverse primer sequence is shown in SEQ ID NO: 11;

[0018] The forward primer sequence of LSAMP-AS1 is shown in SEQ ID NO: 12;

[0019] The reverse primer sequence for LSAMP-AS1 is shown in SEQ ID NO: 13;

[0020] The forward primer sequence of HOXB-AS4 is shown in SEQ ID NO: 14;

[0021] The reverse primer sequence of HOXB-AS4 is shown in SEQ ID NO: 15;

[0022] The forward primer sequence of MNX1-AS1 is shown in SEQ ID NO: 16;

[0023] The reverse primer sequence of MNX1-AS1 is shown in SEQ ID NO: 17;

[0024] The forward primer sequence for LINC01385 is shown in SEQ ID NO: 18;

[0025] The reverse primer sequence for LINC01385 is shown in SEQ ID NO: 19;

[0026] The forward primer sequence of AL513318.2 is shown in SEQ ID NO: 20;

[0027] The reverse primer sequence of AL513318.2 is shown in SEQ ID NO: 21.

[0028] Furthermore, the formulation or kit for treating laryngeal squamous cell carcinoma includes one or more specific interfering RNAs of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2, wherein the specific interfering RNAs of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 can respectively knock down the expression levels of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 in laryngeal squamous cell carcinoma cells.

[0029] Furthermore, the specific interfering RNA sequences of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are as follows:

[0030] The positive interfering RNA sequence ENSG00000233397-1 is shown in SEQ ID NO: 22;

[0031] The reverse interference RNA sequence ENSG00000233397-1 is shown in SEQ ID NO: 23;

[0032] The positive interfering RNA sequence ENSG00000233397-2 is shown in SEQ ID NO: 24;

[0033] The reverse interference RNA sequence ENSG00000233397-2 is shown in SEQ ID NO: 25;

[0034] The sequence of BARX1-DT-1 positive interfering RNA is shown in SEQ ID NO: 26;

[0035] The reverse interfering RNA sequence BARX1-DT-1 is shown in SEQ ID NO: 27;

[0036] The sequence of BARX1-DT-2 positive interfering RNA is shown in SEQ ID NO: 28;

[0037] The reverse interfering RNA sequence BARX1-DT-2 is shown in SEQ ID NO: 29;

[0038] The sequence of the positive interfering RNA LSAMP-AS1-1 is shown in SEQ ID NO: 30;

[0039] The reverse interfering RNA sequence of LSAMP-AS1-1 is shown in SEQ ID NO: 31;

[0040] The LSAMP-AS1-2 positive interference RNA sequence is shown in SEQ ID NO: 32;

[0041] The reverse interfering RNA sequence of LSAMP-AS1-2 is shown in SEQ ID NO: 33;

[0042] The sequence of HOXB-AS4-1 positive interfering RNA is shown in SEQ ID NO: 34;

[0043] The reverse interfering RNA sequence HOXB-AS4-1 is shown in SEQ ID NO: 35;

[0044] The sequence of HOXB-AS4-2 positive interfering RNA is shown in SEQ ID NO: 36;

[0045] The reverse interfering RNA sequence HOXB-AS4-2 is shown in SEQ ID NO: 37;

[0046] The MNX1-AS1-1 positive interfering RNA sequence is shown in SEQ ID NO: 38;

[0047] The reverse interfering RNA sequence MNX1-AS1-1 is shown in SEQ ID NO: 39;

[0048] The positive interfering RNA sequence MNX1-AS1-2 is shown in SEQ ID NO: 40;

[0049] The reverse interfering RNA sequence MNX1-AS1-2 is shown in SEQ ID NO: 41;

[0050] The positive interfering RNA sequence LINC01385-1 is shown in SEQ ID NO: 42;

[0051] The reverse interfering RNA sequence LINC01385-1 is shown in SEQ ID NO: 43;

[0052] The positive interfering RNA sequence LINC01385-2 is shown in SEQ ID NO: 44;

[0053] The reverse interfering RNA sequence LINC01385-2 is shown in SEQ ID NO: 45;

[0054] The positive interfering RNA sequence AL513318.2-1 is shown in SEQ ID NO: 46;

[0055] The reverse interfering RNA sequence AL513318.2-1 is shown in SEQ ID NO: 47;

[0056] The positive interfering RNA sequence AL513318.2-2 is shown in SEQ ID NO: 48;

[0057] The reverse interfering RNA sequence AL513318.2-2 is shown in SEQ ID NO: 49.

[0058] We used high-throughput sequencing and real-time quantitative PCR to discover that the aforementioned seven long non-coding RNAs were significantly overexpressed in laryngeal squamous cell carcinoma (LSCC) tissues and were closely related to the prognosis and survival of LSCC patients. Patients with high expression of these seven long non-coding RNAs showed significantly reduced survival time, suggesting that these seven long non-coding RNAs can serve as molecular markers for auxiliary prognostic survival assessment of LSCC. Furthermore, through cell function experiments, we found that knocking down the expression levels of these seven long non-coding RNAs in human LSCC cancer cells inhibited their proliferation, migration, and invasion abilities. This invention provides effective molecular targets for auxiliary prognostic survival assessment of LSCC at the clinical level and for antagonizing precise treatment of LSCC.

[0059] The process of establishing the application of a combination of long non-coding RNA molecular markers in the auxiliary diagnosis, prognosis, and treatment of laryngeal squamous cell carcinoma includes the following steps:

[0060] Step 1: High-throughput sequencing (next-generation sequencing) was used to perform high-throughput sequencing on 107 pairs of laryngeal squamous cell carcinoma tissues and their corresponding adjacent normal mucosal tissues to establish gene transcription expression profiles of laryngeal squamous cell carcinoma tissues and to perform differential analysis.

[0061] Step 2: Through differential expression analysis, we obtained a list of differentially expressed genes in laryngeal squamous cell carcinoma tissue compared to normal tissue. Then, we used the Cox and LASSO algorithms to perform a comprehensive analysis of differential gene expression levels and clinical information. Finally, we obtained seven long non-coding RNAs that were significantly highly expressed in laryngeal squamous cell carcinoma tissue and closely related to the patient's clinical information.

[0062] Step 3: Based on the seven differentially expressed long non-coding RNAs obtained, we established a biomarker combination for the auxiliary diagnosis and prognostic survival prediction of laryngeal squamous cell carcinoma, and used real-time quantitative PCR technology to measure their expression levels.

[0063] verify.

[0064] Step 4: Based on the combination of seven long non-coding RNA biomarkers obtained by high-throughput sequencing technology and the algorithm, we used real-time fluorescence quantitative PCR to measure the expression levels of laryngeal squamous cell carcinoma tissue for auxiliary diagnosis and prognosis.

[0065] The establishment of an application of a combination of long non-coding RNA molecular markers in laryngeal squamous cell carcinoma treatment agents or kits includes the following steps:

[0066] Step 1: Design specific interfering siRNAs for the above seven long non-coding RNA molecules;

[0067] Step 2: Transfect human laryngeal squamous cell carcinoma cell lines with the above-mentioned siRNA interference sequences;

[0068] Step 3: Detect the expression of long non-coding RNA molecules in the transfected cells to verify the effect and level of knockdown;

[0069] Step 4: Detect the knockdown human laryngeal squamous cell carcinoma cell lines using cell function experiments;

[0070] Step 5: Laryngeal squamous cell carcinoma cell lines with knocked-down of the above seven long non-coding RNA molecules showed a decrease in proliferation, migration, and invasion capabilities.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] 1. The combination of long non-coding RNA molecular markers of the present invention are all significantly highly expressed in the tissues of patients with laryngeal squamous cell carcinoma. They can be used as a novel molecular marker for the diagnosis of laryngeal squamous cell carcinoma. They can be applied to the preparation of detection agents or kits for the above seven long non-coding RNA molecules. By measuring the expression levels of the seven molecules, the prognostic assessment of laryngeal squamous cell carcinoma can be carried out efficiently, conveniently and accurately, thereby providing a reference for clinical diagnosis.

[0073] 2. This invention utilizes a combination molecule formed by long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 to further detect the expression levels of these seven long non-coding RNAs in laryngeal squamous cell carcinoma tissues. Combinatorial analysis can then be performed to screen for patients with high expression of all seven molecules, and these patients have the worst prognosis. Therefore, the combination of the seven long non-coding RNA molecules involved in this invention can serve as an auxiliary prognostic biomarker for laryngeal squamous cell carcinoma, possessing advantages such as high detection specificity and sensitivity.

[0074] 3. Using the seven long non-coding RNA molecule siRNA interference sequences described in this invention, preparations or corresponding kits for antagonizing laryngeal squamous cell carcinoma can be prepared. Attached Figure Description

[0075] Figure 1 The expression levels of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 detected by high-throughput sequencing technology in laryngeal squamous cell carcinoma tissue and corresponding adjacent normal mucosa tissue.

[0076] Figure 2 The relationship between seven long non-coding RNAs (ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2) and overall survival in patients with laryngeal squamous cell carcinoma was analyzed using a comprehensive weighted analysis.

[0077] Figure 3 The relationship between seven long non-coding RNAs (ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2) and the total progression-free interval in patients with laryngeal squamous cell carcinoma was analyzed by comprehensive weighted analysis.

[0078] Figure 4 The relative expression levels of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2, detected by real-time quantitative PCR, in laryngeal squamous cell carcinoma tissue and corresponding adjacent normal mucosa tissue.

[0079] Figure 5 The figure shows the effect of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 on the proliferation of laryngeal squamous cell carcinoma cells FD-LSC-1 and TU-177. The detection method was EdU fluorescence assay.

[0080] Figure 6 The graph shows the effect of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 on the migration ability of laryngeal squamous cell carcinoma cells FD-LSC-1 and TU-177. The detection method was Transwell assay.

[0081] Figure 7 The graph shows the effect of long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 on the invasive ability of laryngeal squamous cell carcinoma cells FD-LSC-1 and TU-177. The detection method was Transwell chamber with matrix gel assay. Detailed Implementation

[0082] The specific embodiments and technical solutions of the present invention will be further illustrated with reference to the accompanying drawings and specific examples. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the invention and should not be construed as limiting the invention.

[0083] Example 1:

[0084] A combination of long non-coding RNA molecular markers includes seven long non-coding RNA combinations: ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2. The nucleotide sequences of the long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are shown in SEQ ID NO: 1-7, respectively.

[0085] Example 2:

[0086] A combination of long non-coding RNA molecular markers and its application in the development of a kit for the adjuvant prognostic assessment of laryngeal squamous cell carcinoma.

[0087] 1. Collection of samples and RNA extraction from patients with laryngeal squamous cell carcinoma.

[0088] Total RNA was extracted from 107 cases of laryngeal squamous cell carcinoma tissue and corresponding adjacent normal mucosa tissue using Trizol reagent (Ambion).

[0089] 1) Quickly remove the tissue sample from the liquid nitrogen tank, place it in an RNase-free EP tube, add Trizol and a steel column, and then place the whole sample into a tissue homogenizer that has been pre-cooled to -80 degrees Celsius and homogenize at 75 Hz for 85 seconds.

[0090] 2) Add chloroform at a ratio of 200 μL per 1 mL of Trizol reagent, shake vigorously for 15 seconds, mix thoroughly, and let stand at room temperature for 5 minutes.

[0091] 3) Centrifuge at 14000g for 15 minutes at 4 degrees Celsius, transfer the supernatant to a new RNase-free EP tube, add an equal volume of isopropanol to the supernatant, gently shake well, and let stand at room temperature for 10 minutes or overnight at -20 degrees Celsius.

[0092] 4) Centrifuge at 12000g for 10 minutes at 4 degrees Celsius. The white precipitate at the bottom of the centrifuge tube is RNA. Discard the supernatant.

[0093] 5) Add 75% ethanol to the EP tube to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes at 4 degrees Celsius, discard the supernatant, and after a brief centrifugation, use a pipette to remove any remaining ethanol.

[0094] 6) Place the EP tube containing the precipitate in a clean bench with the fan on and dry for 5 minutes. Finally, dissolve the precipitate in an appropriate amount of RNase-free pure water to dissolve the RNA, and store at -80 degrees Celsius.

[0095] 2. Sequencing and library preparation

[0096] Library preparation for high-throughput sequencing was performed using a standard of 3 micrograms per tissue sample.

[0097] 1) Using Epicentre Ribo-zero TM The rRNA Removal Kit (Epicentre) removes ribosomal RNA (rRNA), and then washes away the remaining RNA with 75% ethanol.

[0098] 2) Use according to the standard operating instructions. Ultra TM Directional RNA LibraryPrep Kit for (NEB) reagents are used to construct libraries for RNA without rRNA.

[0099] 3) Finally, use AMPure XP beads to screen for cDNA of about 200 bp, perform PCR amplification, and purify the PCR product again with AMPure XP beads to obtain the final library.

[0100] 3. Sequencing

[0101] Library quality control is performed before sequencing, and sequencing is performed only after the quality control is passed.

[0102] 1) First, a clustering reaction is performed to fix the library on a flowcell for bridge amplification.

[0103] 2) After bridging amplification, sequencing was performed using the Illumina Hiseq 2000 system.

[0104] 4. Data Analysis

[0105] 1) First, remove the fragment connectors and perform data quality control.

[0106] 2) Use Bowtie (v2.0.6) to build a reference genome and alignment index, and then use TopHat (v2.0) to align the read fragments to the reference genome.

[0107] 3) Use Scripture (beta2) and Cufflinks (v2.1.1) to assemble the aligned fragments.

[0108] 4) Predict the protein coding potential of RNA using CNCI (v2), CPC (0.9-r2), Pfam-scan (v1.3), and PhyloCSF (v20121028).

[0109] 5) Use Cuffdiff for gene differential analysis.

[0110] 6) Clinically relevant long non-coding RNA (LNA) markers were screened using the Cox and LASSO algorithms based on R language. Finally, seven LNA markers—ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2—were selected to prepare diagnostic agents or kits for laryngeal squamous cell carcinoma. The results of these diagnostic agents or kits were used for auxiliary diagnosis and prognostic assessment of LNA. Alternatively, any one or a combination of these LNA markers can be used to prepare diagnostic agents or kits for LNA.

[0111] 7) The expression levels of seven long non-coding RNAs—ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2—were significantly higher in laryngeal squamous cell carcinoma tissues than in the corresponding adjacent normal mucosa tissues (e.g., ...). Figure 1 (As shown).

[0112] 8) Survival analysis comparing the expression levels of seven long non-coding RNAs (LUNs) with overall survival in patients with laryngeal squamous cell carcinoma, after comprehensive weighted analysis, showed that patients with high expression of all seven LUNs had the worst prognosis (e.g., patients with LUNs). Figure 2 (As shown).

[0113] 9) A weighted analysis of the expression levels of seven long non-coding RNAs was performed, and a survival analysis was conducted between this weighted analysis and the total progression-free interval (PFR) of laryngeal squamous cell carcinoma patients. The results showed that laryngeal squamous cell carcinoma patients with high expression of all seven long non-coding RNAs had the worst PFR prognosis (e.g., ...). Figure 3 (As shown).

[0114] Example 3:

[0115] The application process of a combination of long non-coding RNA molecular markers in an adjuvant prognostic assessment kit for laryngeal squamous cell carcinoma.

[0116] 1. Collection of samples and RNA extraction from patients with laryngeal squamous cell carcinoma.

[0117] We collected 47 cases of laryngeal squamous cell carcinoma tissue and corresponding adjacent normal mucosal tissue, and extracted total RNA using Trizol reagent (Ambion).

[0118] 1) Quickly remove the tissue sample from the liquid nitrogen tank, place it in an RNase-free EP tube, add Trizol and a steel column, and then place the whole sample into a tissue homogenizer that has been pre-cooled to -80 degrees Celsius and homogenize at 75 Hz for 85 seconds.

[0119] 2) Add chloroform at a ratio of 200 μL per 1 mL of Trizol reagent, shake vigorously for 15 seconds, mix thoroughly, and let stand at room temperature for 5 minutes.

[0120] 3) Centrifuge at 14000g for 15 minutes at 4 degrees Celsius, transfer the supernatant to a new RNase-free EP tube, add an equal volume of isopropanol to the supernatant, gently shake well, and let stand at room temperature for 10 minutes or overnight at -20 degrees Celsius.

[0121] 4) Centrifuge at 12000g for 10 minutes at 4 degrees Celsius. The white precipitate at the bottom of the centrifuge tube is RNA. Discard the supernatant.

[0122] 5) Add 75% ethanol to the EP tube to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes at 4 degrees Celsius, discard the supernatant, and after a brief centrifugation, use a pipette to remove any remaining ethanol.

[0123] 2. Real-time quantitative PCR detection of expression levels

[0124] 1) Real-time quantitative PCR was used to detect the expression levels of seven long non-coding RNAs: ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2.

[0125] 2) cDNA synthesis: The reaction system consisted of 10 μL of 2×RT Mix, 2 μL of HiScript II Enzyme Mix, 1 μL of Oligo(DT)23VN (50 μmol), 1 μL of Random hexamers (50 ng / μL), 1 μL of RNA, and RNase-free purified water added to a total volume of 20 μL. The reaction conditions were: 25°C for 5 minutes, 50°C for 15 minutes, and 85°C for 2 minutes.

[0126] 3) The primers for real-time quantitative PCR are:

[0127] Forward primer ENSG00000233397 (SEQ ID NO: 8): 5'-CCTCACAGAGAAGAAGGTTGTG-3';

[0128] ENSG00000233397 Reverse primer (SEQ ID NO: 9): 5'-GGGTCATATGGAACCAAGGATAC-3';

[0129] BARX1-DT forward primer (SEQ ID NO: 10): 5'-CTACGCTGAACTCCTCTCTTTG-3';

[0130] BARX1-DT reverse primer (SEQ ID NO: 11): 5'-ACTGAGTTCTCACAGTCTCTCT-3';

[0131] LSAMP-AS1 forward primer (SEQ ID NO: 12): 5'-CAAGAGAGGCCTCAGAAGAATC-3';

[0132] LSAMP-AS1 reverse primer (SEQ ID NO: 13): 5'-TAGTTTGCTAGGGTTGCCATAA-3';

[0133] HOXB-AS4 forward primer (SEQ ID NO: 14): 5'-GCAACAAGAGAGGAGTCAAGAG-3';

[0134] HOXB-AS4 reverse primer (SEQ ID NO: 15): 5'-CAGCAAGTACCCGGCAATAA-3';

[0135] MNX1-AS1 forward primer (SEQ ID NO: 16): 5'-CTCTGCAGGTCGAACCTTATC-3';

[0136] MNX1-AS1 reverse primer (SEQ ID NO: 17): 5'-AGTGTCTATCTGGAGGGTAGTT-3';

[0137] LINC01385 forward primer (SEQ ID NO: 18): 5'-GGAACCTAGGCTATTCCTTGTG-3';

[0138] LINC01385 reverse primer (SEQ ID NO: 19): 5'-CACTATCGCAATACAGCCTTCTA-3';

[0139] AL513318.2 forward primer (SEQ ID NO: 20): 5'-CCTGTTGATAGTGAGGTACCAAG-3';

[0140] AL513318.2 reverse primer (SEQ ID NO: 21): 5'-CGAGACTCTTCGGAGGTTAATG-3';

[0141] Internal reference gene 18S forward primer (SEQ ID NO: 50): 5'-CCTGGATACCGCAGCTAGGA-3';

[0142] Internal reference gene 18S reverse primer (SEQ ID NO: 51): 5'-GCGGCGCAATACGAATGCCCC-3'.

[0143] 4) Add 10 μL of SYBR Green Master Mix solution, 0.4 μL of forward primer (10 μmol), 0.4 μL of reverse primer (10 μmol) and 1 μL of template cDNA to the PCR tube, and finally add RNase-free pure water to a total volume of 20 μL.

[0144] 5) The reaction program is as follows: 95 degrees Celsius for 5 minutes; 95 degrees Celsius for 10 seconds, 60 degrees Celsius for 30 seconds, 40 cycles; 95 degrees Celsius for 15 seconds, 60 degrees Celsius for 60 seconds, 0.5 degrees Celsius / cycle to 95 degrees Celsius, 95 degrees Celsius for 15 seconds.

[0145] 6) After the reaction is complete, follow step 2 -ΔΔt The expression level of the target long non-coding RNA was calculated using this method.

[0146] 7) The high expression of seven long non-coding RNAs—ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2—in laryngeal squamous cell carcinoma tissue was verified by real-time quantitative PCR (e.g., Figure 4 (As shown). By Figure 4 Real-time quantitative PCR confirmed that seven long non-coding RNAs were significantly overexpressed in laryngeal squamous cell carcinoma tissue.

[0147] 8) The expression levels of seven long non-coding RNAs were comprehensively weighted to obtain the expression levels of molecular markers for laryngeal squamous cell carcinoma, and these expression levels were used to assist in the diagnosis and prognosis of laryngeal squamous cell carcinoma.

[0148] Example 4:

[0149] This embodiment verifies the ability of knocking down any one of the long non-coding RNAs in a combination of long non-coding RNA molecular markers to inhibit the proliferation, metastasis, and invasion of laryngeal squamous cell carcinoma cells:

[0150] 1) Transfection: Specific interfering RNA was transfected using Lipofectamine 3000 (Invitrogen). The transfection system was prepared using Opti-MEM (Invitrogen) medium. After incubation for 6 hours, the medium was converted to cell growth medium.

[0151] 2) Cell proliferation detection (EdU method): First, prepare a 1000:1 dilution of EdU solution using culture medium. Add 100 μL of 50 μmol EdU medium to each well of the cell culture plate and incubate for 2 hours. Remove the culture medium, add 100 μL of cell fixation solution (4% paraformaldehyde buffer) to each well, and incubate at room temperature for 15-30 minutes. Next, incubate with glycine for 10 minutes, wash twice with buffer, permeate the cell membrane with 100 μL of permeabilizer, and wash once with buffer. Then, add staining reaction solution to the wells of the culture plate, incubate at room temperature in the dark for 30 minutes, wash once with buffer, precipitate the cells, discard the supernatant, and acquire and analyze images. Ultimately, it was found that the proliferation ability of human laryngeal squamous cell carcinoma cells was weakened when seven long non-coding RNAs were knocked down (e.g., ...). Figure 5 (As shown).

[0152] 3) Cell migration ability assay (Transwell assay): First, the cell culture medium was replaced with serum-free medium and 0.5 μg / mL colchicine was added for 1 hour. The cells were then resuspended in 200 μL of serum-free medium and added to the upper chamber of a Transwell culture plate. 500 μL of complete culture medium was added to the lower chamber. After incubation for 24 hours, the chamber was removed, and residual cells in the upper chamber were gently wiped away with a cotton swab. Cells were fixed with cell fixation solution, washed once with buffer, stained with crystal violet solution for 10 minutes, washed once with buffer, and images were acquired and analyzed. Ultimately, it was found that the migration ability of human laryngeal squamous cell carcinoma cells was weakened when seven long non-coding RNAs were knocked down (e.g., ...). Figure 6 (As shown).

[0153] 4) Cell invasion assay (Transwell assay): Dissolve Matrigel gel overnight at 4°C. Dilute Matrigel gel with serum-free medium at a 1:3 ratio. Add 40 μL of the diluted solution to a pre-chilled Transwell chamber and incubate at 37°C for 2 hours to allow the Matrigel gel to solidify. Next, add 100 μL of serum-free medium to the upper chamber and 600 μL to the lower chamber. Add cells treated with 0.5 μg / mL colchicine to the upper chamber and 500 μL of complete medium to the lower chamber. After incubation for 34 hours, remove the chamber, wipe excess cells from the upper chamber with a cotton swab, wash once with buffer, stain with crystal violet for 10 minutes, wash once with buffer, and acquire and analyze images. Ultimately, it was found that the invasive ability of human laryngeal squamous cell carcinoma cells with knockdown of seven long non-coding RNAs was reduced (e.g., ...). Figure 7 (As shown).

[0154] 5) According to the seven long non-coding RNAs described in this invention, knocking down the expression level of any one of them with siRNA can inhibit the proliferation, migration and invasion of human laryngeal squamous cell carcinoma cells, indicating that any one of the seven long non-coding RNAs has the potential to treat laryngeal squamous cell carcinoma.

[0155] Therefore, as can be seen from the experiments in Examples 4(1)-4), the specific interfering RNA designed for one or seven of the long non-coding RNAs included in the long non-coding RNA molecular markers of the present invention, such as ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385 and AL513318.2, can be used to prepare preparations or kits for treating laryngeal squamous cell carcinoma.

[0156] The specific interfering RNA sequences of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are as follows:

[0157] ENSG00000233397-1 positive interfering RNA sequence (SEQ ID NO: 22): 5'-GCACAUAUGGAUAGAACAATT-3';

[0158] The reverse interfering RNA sequence ENSG00000233397-1 (SEQ ID NO: 23): 5'-UUGUUCUAUCCAUAUGUGCTT-3';

[0159] ENSG00000233397-2 positive interfering RNA sequence (SEQ ID NO: 24): 5'-GCAAAUUAUGGCAUCCCUUTT-3';

[0160] ENSG00000233397-2 reverse interference RNA sequence (SEQ ID NO: 25): 5'-AAGGGAUGCCAUAAUUUGCTT-3';

[0161] BARX1-DT-1 positive interfering RNA sequence (SEQ ID NO: 26): 5'-GGAUUCGAGGAGAAGGGAATT-3';

[0162] BARX1-DT-1 reverse interfering RNA sequence (SEQ ID NO: 27): 5'-UUCCCUUCUCCUCGAAUCCTT-3';

[0163] BARX1-DT-2 positive interfering RNA sequence (SEQ ID NO: 28): 5'-GGCAGUACCUUCUCUAGAUTT-3';

[0164] BARX1-DT-2 reverse interfering RNA sequence (SEQ ID NO: 29): 5'-AUCUAGAGAAGGUACUGCCTT-3';

[0165] LSAMP-AS1-1 positive interfering RNA sequence (SEQ ID NO: 30): 5'-GAGCAAACUAAUACAUUAAACAGAA-3';

[0166] LSAMP-AS1-1 reverse interfering RNA sequence (SEQ ID NO: 31): 5'-UUCUGUUUAAUGUAUUAGUUUGCUCAG-3';

[0167] LSAMP-AS1-2 positive interfering RNA sequence (SEQ ID NO: 32): 5'-GCAUUUCCAUCUGAGGUGGAAUGAC-3';

[0168] LSAMP-AS1-2 reverse interfering RNA sequence (SEQ ID NO: 33): 5'-GUCAUUCCACCUCAGAUGGAAAUGCAG-3';

[0169] HOXB-AS4-1 positive interfering RNA sequence (SEQ ID NO: 34): 5'-UCAGUUUAGGGAAGAAGUUTT-3';

[0170] HOXB-AS4-1 reverse interfering RNA sequence (SEQ ID NO: 35): 5'-AACUUCUUCCCUAAACUGATT-3';

[0171] HOXB-AS4-2 positive interfering RNA sequence (SEQ ID NO: 36): 5'-UCCACCACCGUCUCCUUAUTT-3';

[0172] HOXB-AS4-2 reverse interfering RNA sequence (SEQ ID NO: 37): 5'-AUAAGGAGACGGUGGUGGATT-3';

[0173] MNX1-AS1-1 positive interfering RNA sequence (SEQ ID NO: 38): 5'-GGUCGAACCUUAUCUGCUATT-3';

[0174] MNX1-AS1-1 reverse interfering RNA sequence (SEQ ID NO: 39): 5'-UAGCAGAUAAGGUUCGACCTT-3';

[0175] MNX1-AS1-2 positive interfering RNA sequence (SEQ ID NO: 40): 5'-GCUACGUGAGUCUUGCAAATT-3';

[0176] MNX1-AS1-2 reverse interfering RNA sequence (SEQ ID NO: 41): 5'-UUUGCAAGACUCACGUAGCTT-3';

[0177] LINC01385-1 positive interfering RNA sequence (SEQ ID NO: 42): 5'-CCAUGAACGUGAACAUGAACGUGUU-3';

[0178] LINC01385-1 reverse interfering RNA sequence (SEQ ID NO: 43): 5'-AACACGUUCAUGUUCACGUUCAUGG-3';

[0179] LINC01385-2 positive interfering RNA sequence (SEQ ID NO: 44): 5'-GGUGAAGUGGGUAUCUCCAUGUGAU-3';

[0180] LINC01385-2 reverse interfering RNA sequence (SEQ ID NO: 45): 5'-AUCACAUGGAGAUACCCACUUCACC-3';

[0181] AL513318.2-1 positive interfering RNA sequence (SEQ ID NO: 46): 5'-CCUUCUCACUUUCUGCCUUTT-3';

[0182] The reverse interfering RNA sequence AL513318.2-1 (SEQ ID NO: 47) is: 5'-AAGGCAGAAAGUGAGAAGGTT-3'.

[0183] AL513318.2-2 positive interfering RNA sequence (SEQ ID NO: 48): 5'-CCGAGCCUGUUCAGUCUUUTT-3';

[0184] The reverse interfering RNA sequence AL513318.2-2 (SEQ ID NO: 49) is: 5'-AAAGACUGAACAGGCUCGGTT-3'.

[0185] The specific interfering RNA of ENSG00000233397 can knock down the expression level of ENSG00000233397 in laryngeal squamous cell carcinoma cells; the specific interfering RNA of BARX1-DT can knock down the expression level of BARX1-DT in laryngeal squamous cell carcinoma cells; the specific interfering RNA of LSAMP-AS can knock down the expression level of LSAMP-AS1 in laryngeal squamous cell carcinoma cells; the specific interfering RNA of HOXB-AS4 can knock down the expression level of HOXB-AS4 in laryngeal squamous cell carcinoma cells; the specific interfering RNA of MNX1-AS1 can knock down the expression level of MNX1-AS1 in laryngeal squamous cell carcinoma cells; the specific interfering RNA of LINC01385 can knock down the expression level of LINC01385 in laryngeal squamous cell carcinoma cells; and the specific interfering RNA of AL513318.2 can knock down the expression level of AL513318.2 in laryngeal squamous cell carcinoma cells.

Claims

1. A combination of long non-coding RNA molecular markers, characterized in that: The long non-coding RNA molecular marker combination includes seven markers: ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.

2. The nucleotide sequences of the long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

2. The RNA molecular marker combination according to claim 1, characterized in that: The seven long non-coding RNAs ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 were all significantly highly expressed in laryngeal squamous cell carcinoma tissues and were positively correlated with poor prognosis of laryngeal squamous cell carcinoma. Combined analysis of the above seven long non-coding RNAs showed that patients with laryngeal squamous cell carcinoma had the worst prognosis.

3. The application of the RNA molecular marker combination as described in any one of claims 1-2, characterized in that: The targeted primers for detecting the combination of said RNA molecular markers are used to prepare a kit for adjuvant prognostic assessment of laryngeal squamous cell carcinoma, or the specific interfering RNA sequences of said RNA molecular markers are used to prepare a formulation or kit for treating laryngeal squamous cell carcinoma.

4. The application of the RNA molecular marker combination according to claim 3, characterized in that: The laryngeal squamous cell carcinoma adjuvant prognostic assessment kit is a kit for detecting the expression level of the long non-coding RNA using targeted primers for the combination of RNA molecular markers in real-time quantitative PCR.

5. The application of the RNA molecular marker combination according to claim 4, characterized in that: The targeted primers are: The forward primer sequence of ENSG00000233397 is shown in SEQ ID NO: 8; The reverse primer sequence for ENSG00000233397 is shown in SEQ ID NO: 9; The BARX1-DT forward primer sequence is shown in SEQ ID NO: 10; The BARX1-DT reverse primer sequence is shown in SEQ ID NO: 11; The forward primer sequence of LSAMP-AS1 is shown in SEQ ID NO: 12; The reverse primer sequence for LSAMP-AS1 is shown in SEQ ID NO: 13; The forward primer sequence of HOXB-AS4 is shown in SEQ ID NO: 14; The reverse primer sequence of HOXB-AS4 is shown in SEQ ID NO: 15; The forward primer sequence of MNX1-AS1 is shown in SEQ ID NO: 16; The reverse primer sequence of MNX1-AS1 is shown in SEQ ID NO: 17; The forward primer sequence for LINC01385 is shown in SEQ ID NO: 18; The reverse primer sequence for LINC01385 is shown in SEQ ID NO: 19; The forward primer sequence of AL513318.2 is shown in SEQ ID NO: 20; The reverse primer sequence of AL513318.2 is shown in SEQ ID NO:

21.

6. The application of the long non-coding RNA molecular marker combination according to claim 3, characterized in that: The formulation or kit for treating laryngeal squamous cell carcinoma includes one or more specific interfering RNAs of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.

2. The specific interfering RNA sequences of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 can respectively knock down the expression levels of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 in laryngeal squamous cell carcinoma. The specific interfering RNA sequences of ENSG00000233397, BARX1-DT, LSAMP-AS1, HOXB-AS4, MNX1-AS1, LINC01385, and AL513318.2 are as follows: The positive interfering RNA sequence ENSG00000233397-1 is shown in SEQ ID NO: 22; The reverse interference RNA sequence ENSG00000233397-1 is shown in SEQ ID NO: 23; The positive interfering RNA sequence ENSG00000233397-2 is shown in SEQ ID NO: 24; The reverse interference RNA sequence ENSG00000233397-2 is shown in SEQ ID NO: 25; The sequence of BARX1-DT-1 positive interfering RNA is shown in SEQ ID NO: 26; The reverse interfering RNA sequence BARX1-DT-1 is shown in SEQ ID NO: 27; The sequence of BARX1-DT-2 positive interfering RNA is shown in SEQ ID NO: 28; The reverse interfering RNA sequence BARX1-DT-2 is shown in SEQ ID NO: 29; The sequence of the positive interfering RNA LSAMP-AS1-1 is shown in SEQ ID NO: 30; The reverse interfering RNA sequence of LSAMP-AS1-1 is shown in SEQ ID NO: 31; The LSAMP-AS1-2 positive interference RNA sequence is shown in SEQ ID NO: 32; The reverse interfering RNA sequence of LSAMP-AS1-2 is shown in SEQ ID NO: 33; The sequence of HOXB-AS4-1 positive interfering RNA is shown in SEQ ID NO: 34; The reverse interfering RNA sequence HOXB-AS4-1 is shown in SEQ ID NO: 35; The sequence of HOXB-AS4-2 positive interfering RNA is shown in SEQ ID NO: 36; The reverse interfering RNA sequence HOXB-AS4-2 is shown in SEQ ID NO: 37; The MNX1-AS1-1 positive interfering RNA sequence is shown in SEQ ID NO: 38; The reverse interfering RNA sequence MNX1-AS1-1 is shown in SEQ ID NO: 39; The positive interfering RNA sequence MNX1-AS1-2 is shown in SEQ ID NO: 40; The reverse interfering RNA sequence MNX1-AS1-2 is shown in SEQ ID NO: 41; The positive interfering RNA sequence LINC01385-1 is shown in SEQ ID NO: 42; The reverse interfering RNA sequence LINC01385-1 is shown in SEQ ID NO: 43; The positive interfering RNA sequence LINC01385-2 is shown in SEQ ID NO: 44; The reverse interfering RNA sequence LINC01385-2 is shown in SEQ ID NO: 45; The positive interfering RNA sequence AL513318.2-1 is shown in SEQ ID NO: 46; The reverse interfering RNA sequence AL513318.2-1 is shown in SEQ ID NO: 47; The positive interfering RNA sequence AL513318.2-2 is shown in SEQ ID NO: 48; The reverse interfering RNA sequence AL513318.2-2 is shown in SEQ ID NO: 49.

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