Application of DDX39B protein or gene as target spot in treatment and detection of head and neck squamous cell carcinoma

Abstract

The invention relates to the field of biological medicine, in particular to application of DDX39B protein or gene as a target spot in treatment and detection of head and neck squamous cell carcinoma, the DDX39B gene is highly expressed in HNSCC tissue, and the DDX39B protein can be used as a kit or a detection marker according to the discovery, namely, the content of the DDX39B protein in a patient can be detected, and the content of the DDX39B protein in the patient can be detected. The expression level can be used as a powerful prediction index independent of the traditional clinical staging, which is beneficial for clinicians to formulate a more targeted follow-up plan and a more positive adjuvant therapy strategy; besides, the DDX39B gene can also be used as a potential treatment target of the head and neck squamous cell carcinoma, expression of the DDX39B gene is reduced by constructing siRNA, the purposes of inhibiting migration and growth of cancer cells and reducing tumor cells are achieved through cell and animal experiments, and the discovery can be used for purposefully treating the head and neck squamous cell carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of DDX39B protein or gene as a target point in treatment and detection of head and neck squamous cell carcinoma. BACKGROUND

[0002] Head and neck malignant tumor ranks one of the seven most common malignant tumors in the world, and its histological types are diverse. The most important pathological subtype is head and neck squamous cell carcinoma (HNSCC), with a high incidence of about 890,000 new cases and 450,000 deaths per year. Although the comprehensive sequential treatment mode for HNSCC is becoming more and more mature, from single treatment mode such as surgery, chemotherapy to multi-means combined treatment such as concurrent chemoradiotherapy, electrochemical therapy and neoadjuvant therapy combined with immunotherapy, the 5-year survival rate of HNSCC patients is still less than 50%, especially for patients in stage Ⅲ and Ⅳ. In addition, the biological behavior of HNSCC shows high heterogeneity, resulting in large differences in treatment efficacy and prognosis among different patients. At present, the main factors used to predict the prognosis of tumor patients in clinic are tumor stage, tumor marker and patient age, but these clinical characteristics have great limitations in predicting patient efficacy and prognosis, and cannot realize individualized treatment guidance. In the past research, great progress has been made in the study of pathogenesis, prognostic markers and therapeutic targets of HNSCC. Zhang Guosen et al. also developed an online survival analysis tool for head and neck squamous cell carcinoma. Although these have great hope in predicting the prognosis of HNSCC patients, most of them are only focused on the changes in the overall transcription level of genes, and the differences in post-transcriptional modification of genes are not analyzed.

[0003] Alternative splicing (AS) is an important process of post-transcriptional processing of mRNA, which modifies more than 90% of human genes. It generates mature mRNAs with different structures and functions by selectively retaining or removing exons, fundamentally causing protein diversity. AS plays an important role in controlling human growth and development. Genome-wide studies have shown that tumorigenesis often involves large-scale AS changes, and AS disorders promote tumor occurrence, invasion, metastasis, drug resistance, immune escape, and are closely related to patient prognosis. Splicing factors are protein factors involved in the splicing process of mRNA precursors. In the process of alternative splicing, RNA splicing factors can act as proto-oncogenes and tumor suppressor genes. Studies have shown that the expression disorder of splicing factors (SF) can promote or inhibit the progression of diseases, and alternative splicing may be complexly regulated by SF. Serine and arginine rich splicing factor 2 (SRSF2) is involved in the alternative splicing of various pre-mRNAs associated with human diseases. The up-regulation of BUD31, a key oncogenic splicing factor, is associated with poor prognosis of ovarian cancer, and the down-regulation is associated with poor prognosis of prostate cancer. Abnormal expression of specific SFs is closely related to the progression, treatment resistance and poor prognosis of cancer. For example: serine / arginine-rich splicing factor 2 (SRSF2) is involved in the regulation of the splicing of precursor mRNAs of various genes associated with human diseases (including cancer). BUD31, as a key oncogenic splicing factor, is up-regulated in ovarian cancer, indicating poor prognosis; on the contrary, low expression in prostate cancer is associated with poor prognosis, highlighting that different target sites show different prognostic effects for different diseases.

[0004] DDX39B is a member of the DEAD-box RNA helicase family, mainly responsible for post-transcriptional modification, almost involved in the regulation of all processes of RNA metabolism, and maintaining the stability of the genome. In colorectal cancer, it increases the proliferation, migration and invasion of colorectal cancer cells by promoting PMK2 and nuclear translocation, and in hepatocellular carcinoma, it promotes cancer malignant progression by activating SREBP1-mediated lipid synthesis process. There is no report on the correlation between DDX39B gene and HNSCC. Therefore, it is necessary to conduct in-depth research on HNSCC-related genes in order to find new treatment and detection strategies for specific treatment and detection of HNSCC. SUMMARY

[0005] In view of the above, it is necessary to conduct in-depth research on HNSCC-related genes in order to find new treatment and detection strategies for specific treatment and detection of HNSCC.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is:

[0007] The application of the reagent for inhibiting the expression of DDX39B gene or reducing the activity of DDX39B protein with DDX39B protein or DDX39B gene as a target in the treatment of head and neck squamous cell carcinoma, wherein the nucleic acid sequence of the DDX39B gene is shown as SEQ ID NO. 1, and the amino acid sequence of the DDX39B protein is shown as SEQ ID NO. 2.

[0008] Further, the reagent is selected from a DDX39B antibody, an siRNA capable of inhibiting the expression of the DDX39B gene, or a gene editing vector capable of inhibiting the expression of the DDX39B gene.

[0009] Further, the siRNA is double-stranded, and is any one of siRNA1, siRNA2, siRNA3 and / or siRNA4; the sense strand of the siRNA1 is shown as SEQ ID NO. 3, and the antisense strand is shown as SEQ ID NO. 4; the sense strand of the siRNA2 is shown as SEQ ID NO. 5, and the antisense strand is shown as SEQ ID NO. 6; the sense strand of the siRNA3 is shown as SEQ ID NO. 7, and the antisense strand is shown as SEQ ID NO. 8; and the sense strand of the siRNA4 is shown as SEQ ID NO. 9, and the antisense strand is shown as SEQ ID NO. 10.

[0010] The present application also includes the application of the siRNA for down-regulating the expression of the DDX39B gene in the preparation of a drug for treating head and neck squamous cell carcinoma, inhibiting the migration, invasion and / or growth of head and neck squamous cell carcinoma cells, wherein the siRNA is double-stranded, and is any one of siRNA1, siRNA2, siRNA3 and / or siRNA4; the sense strand of the siRNA1 is shown as SEQ ID NO. 3, and the antisense strand is shown as SEQ ID NO. 4; the sense strand of the siRNA2 is shown as SEQ ID NO. 5, and the antisense strand is shown as SEQ ID NO. 6; the sense strand of the siRNA3 is shown as SEQ ID NO. 7, and the antisense strand is shown as SEQ ID NO. 8; and the sense strand of the siRNA4 is shown as SEQ ID NO. 9, and the antisense strand is shown as SEQ ID NO. 10.

[0011] The present invention also includes a protein biomarker for diagnosing or predicting head and neck squamous cell carcinoma. The protein biomarker is DDX39B protein. The nucleic acid sequence of the DDX39B protein is shown in SEQ ID NO.1, and the amino acid sequence of the DDX39B protein is shown in SEQ ID NO.2.

[0012] The present invention also includes a kit for diagnosing head and neck squamous cell carcinoma, which comprises a reagent for the protein biomarker. The expression level of the protein biomarker is determined using one or more of fluorescence method or Western blot.

[0013] The present invention also includes the use of the kit in preparing products for diagnosing head and neck squamous cell carcinoma.

[0014] The present invention also includes the use of the kit, which comprises the following steps:

[0015] (1) extracting extracellular vesicles from the plasma or serum of the subject to be tested;

[0016] (2) Determine the content of DDX39B protein;

[0017] (3) The results are determined based on the content of the above-mentioned protein biomarkers.

[0018] The present invention also includes the use of the protein biomarker in screening therapeutic drugs for head and neck squamous cell carcinoma.

[0019] The present invention has the following beneficial effects:

[0020] Through in-depth research, the present invention discovered that the DDX39B gene is highly expressed in HNSCC tissues. This discovery can use the DDX39B protein as a kit or detection marker, that is, the DDX39B protein content in the patient's body can be detected, and its expression level can serve as a powerful predictive indicator independent of traditional clinical staging, which helps clinicians develop more targeted follow-up plans and more proactive adjuvant treatment strategies; in addition, the DDX39B gene can also be used as a potential therapeutic target for head and neck squamous cell carcinoma. The present application also constructs siRNA to downregulate DDX39B gene expression, and achieves the purpose of inhibiting cancer cell migration and growth and shrinking tumor cells through cell and animal experiments. This discovery can be used for the targeted treatment of head and neck squamous cell carcinoma, and is expected to become a new treatment strategy for improving HNSCC treatment outcomes and overcoming drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the SF-AS regulatory network.

[0022] Figure 2These are the RT-qPCR experimental results of the DDX39B gene in different HNSCC cell lines (NP69, CAL27, SAS, HONE1, and HN4).

[0023] Figure 3 The Western blot experimental results of DDX39B gene in different HNSCC cell lines (NP69, CAL27, SAS, HONE1, HN4); in the figure, A is the electrophoresis result and B is the statistical result.

[0024] Figure 4 The figure shows the RT-qPCR experimental results of different siRNAs of the DDX39B gene in different HNSCC cell lines; in the figure, A is the HONE1 cell line and B is the HN4 cell line.

[0025] Figure 5 Figure 2 is a graph showing the results of Western blot experiments of different siRNAs for the DDX39B gene in different HNSCC cell lines; in the figure, A is the electrophoresis diagram of the HONE1 cell line, B is the electrophoresis diagram of the HN4 cell line, C is the statistical results of the HONE1 cell line, and D is the statistical results of the HN4 cell line.

[0026] Figure 6 This is a graph of cck8 experimental results; in the figure, A is the OD value of the HONE1 cell line, and B is the OD value of the HN4 cell line.

[0027] Figure 7 These are photos of the well plates used in the cloning experiment. In the photos, the upper row shows the HN4 cell line and the lower row shows the HONE1 cell line.

[0028] Figure 8 It is a statistical chart of the cloning experiment; in the figure, A is the HN4 cell line and B is the HONE1 cell line.

[0029] Figure 9 These are photos of the scratch test on cell lines; in the figure, A is the HN4 cell line and B is the HONE1 cell line.

[0030] Figure 10 Statistical chart of scratch test of cell lines; in the figure, A is HN4 cell line, and B is HONE1 cell line.

[0031] Figure 11 The results of the Transwell experiment are shown in Figure 1. In the figure, A is the HN4 cell line and B is the HONE1 cell line.

[0032] Figure 12 This is a statistical diagram of the Transwell experiment. In the figure, A is the HN4 cell line and B is the HONE1 cell line.

[0033] Figure 13 This is a flow cytometry apoptosis experiment result diagram of HN4 cell line. In the figure, A is the NC group, B is the si-RNA group, and C is the flow cytometry apoptosis experiment statistical diagram of the cell line.

[0034] Figure 14 This is the result of the subcutaneous tumor formation experiment in nude mice. DETAILED DESCRIPTION

[0035] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Example 1:

[0037] This example shows the process of obtaining markers for predicting the prognosis of patients with head and neck squamous cell carcinoma:

[0038] (1) Construction of splicing factor-variable splicing (SF-AS) regulatory network: RNA-seq data of HNSCC patients were obtained using the TCGA database (https: / / portal.gdc.cancer.gov / ) and variable splicing data of HNSCC were obtained using the TCGA SpliceSeq database (https: / / bioinformatics.mdanderson.org / TCGASpliceSeq / ). The expression levels of SF-related genes were extracted using RNA-seq data. The Pearson test was used to analyze the expression levels of SF genes related to the prognosis of HNSCC patients. At the same time, the correlation between them and the variable splicing events PSI related to the prognosis of HNSCC was evaluated. Cytoscape (version 3.9.1) was used to construct the SF-AS regulatory network, as shown in Figure 3. Figure 1 As shown in the figure, red circles represent AS events with favorable prognosis, green circles represent AS events with unfavorable prognosis, triangles represent key splicing factors associated with prognosis, red lines indicate positive regulation, and green lines indicate negative regulation. The figure shows that the SF-AS regulatory network reveals that DDX39B is a key splicing factor associated with prognosis in HNSCC, negatively regulating both favorable and unfavorable AS events. The key splicing factor DDX39B has been discovered.

[0039] (2) Detection of DDX39B expression in HNSCC cell lines: Western blot and RT-qPCR were used to detect the expression level of DDX39B in HNSCC cell lines (HONE1, SAS, HN4, CAL27) and normal head and neck epithelial cells (NP69). DDX39B primer sequences: F: 5'-GAGCAGAACTTCCCAGCCAT-3', R: 5'-AATGTTCACCCGCTCGATGT-3'; β-actin primer sequences: F: 5'-GCACTCTTCCAGCCTTC CTTCC-3', R: 5'-GCGGATGTCCACGTCACACTTC-3'.

[0040] ① Western blot experiment: Extract total cell protein and prepare a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis. Remove the protein from the -80°C freezer and heat at 100°C for 5 minutes to fully denature it. Then remove and place on ice. Place the gel in an electrophoresis chuck and check for leaks. Slowly pour the electrophoresis buffer into the electrophoresis tank. Slowly remove the comb and add 3 μl of a two-color prestained protein marker or 30 μg of total protein to each well. Electrophoresis parameters: 90V for 30 minutes, then 150V for 30 minutes. Transfer: Confirm the location of the target protein. Cut the PVDF membrane to the appropriate size and soak it in anhydrous ethanol for 2 minutes. Then remove and soak it in transfer buffer. Soak a filter paper-free sponge in transfer buffer and place it on both sides of the transfer chuck. Cut the gel and transfer it to the sponge. Cover the PVDF membrane with the electrophoresis gel to avoid air bubbles. Close the transfer chuck and place it in the electrophoresis tank. Add transfer buffer and run at 400mA for 30 minutes. After transfer, remove the PVDF membrane and wash three times in TBST solution on a horizontal shaker for 5 minutes each. Then, transfer the PVDF membrane to 5% skim milk powder and block on a horizontal shaker for 1 hour at room temperature. Prepare the primary antibody at a 1:1500 ratio using the primary antibody diluent. Place the PVDF membrane in the prepared primary antibody solution, allowing it to fully soak, and incubate on a shaker at 4°C overnight. The next day, remove the PVDF membrane and wash three times in TBST for 5 minutes each. After washing, place the membrane in the secondary antibody solution and incubate at room temperature for 50 minutes. Wash three times in TBST for 5 minutes each. Then, perform chemical development.

[0041] ②RT-qPCR experiment: Use Mona Bio's reverse transcription kit (MonScriptTM RTIII All-in-One Mix with dsDNase) and operate on ice. Add the extracted RNA to a 200ul enzyme-free sterile centrifuge tube according to the following scheme to synthesize the first-strand template cDNA: template RNA 500ng, MonScriptTM 5×RTIII All-in-One Mix 2ul, MonScriptTM dsDNase 0.5μL, and Nuclease-Free Water to make up the volume to 10μL. After vortexing and instant centrifugation, place it in a reverse transcription instrument for reverse transcription to obtain cDNA. Reaction procedure: 37℃ reaction for 2min, 55℃ reaction for 15min, and 85℃ reaction for 5min. Depending on the amount of template cDNA required for the experiment, the various components in the reverse transcription system can be added in appropriate proportions. After the obtained cDNA is removed, place it on ice and add 40ul pre-cooled enzyme-free water for dilution. The cDNA can be temporarily stored at -20℃ or stored at -80℃ for a long time. Use Mona Bio's MonAmp TM PCR amplification was performed using PCR Green qPCR Mix (MQ00501S, Monad, China).

[0042] The experimental results of Western blot and RT-qPCR are as follows Figure 2-Figure 3 As shown, Figure 2 The experimental results of RT-qPCR are shown in Figure 2. Figure 3 A is the experimental result of Western bLot, A is the electrophoresis result, and B is the statistical result. Both Western bLot and RT-qPCR experiments indicate that DDX39B is highly expressed in HNSCC.

[0043] Example 2:

[0044] This example uses the DDX39B gene as a protein biomarker for the diagnosis or prediction of head and neck squamous cell carcinoma, as follows:

[0045] 1. The nucleic acid sequence of the DDX39B protein in this example is shown in SEQ ID NO. 1, and the amino acid sequence of the DDX39B protein is shown in SEQ ID NO. 2.

[0046] 2. From Example 1, we know that DDX39B is highly expressed in HNSCC. We can prepare the DDX39B protein into a kit for diagnosing head and neck squamous cell carcinoma. The kit contains the DDX39B protein. The specific detection method is as follows:

[0047] (1) extracting extracellular vesicles from the plasma or serum of the subject to be tested;

[0048] (2) measuring the content of DDX39B protein therein;

[0049] (3) diagnosing the subject as suffering from head and neck squamous cell carcinoma or predicting the subject as likely to suffer from head and neck squamous cell carcinoma if the content of DDX39B protein is measured to be higher than the blank.

[0050] Example 3:

[0051] This example is to use DDX39B gene for screening of head and neck squamous cell carcinoma treatment drugs, as follows:

[0052] Screening of drugs capable of down-regulating DDX39B gene, such as DDX39B antibody, siRNA capable of inhibiting DDX39B gene expression or gene editing vector capable of inhibiting DDX39B gene expression; if the expression amount of DDX39B gene can be down-regulated, it indicates that the drug can treat head and neck squamous cell carcinoma.

[0053] Example 4:

[0054] This example is the screening of DDX39B gene down-regulation siRNA and the verification of tumor cell treatment, as follows:

[0055] 1. Constructing DDX39B down-regulation cell strain in HNSCC cell line: siRNA and its negative control are purchased from Shanghai Jimma Pharmaceutical Technology Co., Ltd. HONE1 and HN4 cells are plated in a 6-well plate, and when the cell density is 50%-60%, Lipofectamine 3000 is used for transfection. Collect cells to extract RNA and protein 48h after transfection, and detect the expression level of DDX39B by Western bLot and RT-qPCR experiment to verify the transfection efficiency; the sequence of siRNA1 is: S: 5'-GCUCUGGCUUUCGUGACUUTT-3', AS: 5'-AAGUCACGAAACCAGAGCTT-3'(SEQ ID NO. 3-NO. 4) The sequence of siRNA2 is: S: 5'-GCAGCAGUACUACGUGAAATT-3', AS: 5'-UUUCACGUAGUACUGCUGCTT-3'(SEQ ID NO. 5-NO. 6) The sequence of siRNA3 is: S: 5'-CUCGGUAUCAGCAGUUUAATT-3', AS: 5'-UUAAACUGCUGAUACCGAGTT-3'(SEQ ID NO. 7-NO. 8) The sequence of siRNA4 is: S: 5'-GCCUCAACCUCAAACACAUTT-3', AS: 5'-AUGUGUUUGAGGUUGAGGCTT-3'(SEQ ID NO. 9-NO. 10).

[0056] (1) Cell transfection: inoculate cells: prepare cell suspension (HONE1: 1 x 10 5 / ml; HN4: 1 x 10 5 / ml) with complete medium, 2 ml per well in a 6-well plate, and transfect when the cell confluence is 50%-60%. Dilute 3.75 ul Lipofectamine 3000 with 125 ul Opti-MEM medium, dilute 10 ul P3000 and 1 ul siRNA with 125 ul Opti-MEM medium, mix them after 5 min respectively, incubate at room temperature for 15 min. Add the compound to the cells, mix well and put into the incubator, change the liquid after 4-6 h. As Figure 4-Figure 5 shown: Figure 4 RT-qPCR experiment results of different siRNAs of DDX39B gene in different HNSCC cell lines; in the figure, A is HONE1 cell line, B is HN4 cell line; Figure 5 Western blot experiment results of different siRNAs of DDX39B gene in different HNSCC cell lines; in the figure, A is the electrophoresis of HONE1 cell line, B is the electrophoresis of HN4 cell line, C is the statistical result of HONE1 cell line, D is the statistical result of HN4 cell line; from the figure, from the Western blot expression and RT-qPCR detection results, compared with the blank, the down-regulation level of siRNA1, siRNA2, siRNA3 and siRNA4 experimental groups is significantly different. It shows that siRNA1, siRNA2, siRNA3 and siRNA4 can construct DDX39B gene down-regulation cell lines in HONE1 and HN4 cell lines.

[0057] 2, select siRNA1, siRNA4 cell lines for cell proliferation experiment: inoculate cells into 96-well plates (1.5 x 10 3 / well), use cck8 experiment to calculate the relative proliferation activity of DDX39B knockdown cell lines and negative control cell lines. Inoculate cells into 6-well plates (600 / well), use colony formation experiment to calculate the number of colonies of DDX39B knockdown cell lines and negative control cell lines, the specific method is:

[0058] (1) cck8 experiment: collect cells by centrifugation after trypsin digestion. Resuspend the collected cells with serum-containing medium, count, and dilute to 1.5 x 10 4A single cell suspension of 100 μl / ml was inoculated into a 96-well plate. The cell suspension was inoculated into each well with 100 μl. The culture plate was placed in an incubator for pre-culture for 24 h, 48 h, and 72 h. The culture medium was discarded and 100 μl of culture medium containing 10 μl CCK8 solution was added to each well. After adding the reagent, the culture plate was gently shaken to help mix (to prevent errors caused by the CCK8 reagent sticking to the well wall). During the addition process, try not to generate bubbles to avoid affecting the OD value reading. The culture plate was placed in an incubator and incubated for 1-2 h. The absorbance (OD) at 450 nm was measured with an enzyme-linked microplate reader. The results are as follows: Figure 6 As shown in the figure, A is the OD value of HONE1 cell line, B is the OD value of HN4 cell line; Figure 6 It can be seen that the proliferation activity of si-DDX39B cell line was significantly lower than that of the blank (NC) group.

[0059] (2) Clone formation experiment: Take cells in the logarithmic growth phase, digest them with trypsin, and then completely resuspend the cells in complete culture medium. Count them, dilute them into a single cell suspension of 300 cells / ml, and inoculate them into a 6-well plate with 2 ml per well. Continue to culture until the number of cells in most single clones exceeds 50. During the culture process, the culture medium was changed every 3 days, and the cell status was observed. After the clone formation was completed, 4% paraformaldehyde was used to fix the cells for 15 minutes. After washing with PBS, 1 mL of crystal violet stain was added to each well and stained for 20 minutes. The cells were washed several times with PBS, and photographed after drying (the entire six-well plate and each well were photographed separately); the results were as follows: Figure 7-Figure 8 As shown: Figure 7 This is a photo of the well plate used in the cloning experiment. In the picture, the upper row is the HN4 cell line and the lower row is the HONE1 cell line; Figure 8 This is a statistical chart of the cloning experiment; in the figure, A is the HN4 cell line and B is the HONE1 cell line; as can be seen from the figure, in the photographs, the density of cells is NC cell line>siRNA1 cell line>siRNA4 cell line; from the statistical results Figure 8 From the results, the cell proliferation activity of the blank group (CN) was significantly greater than that of the siRNA1 cell line, and the cell proliferation activity of the siRNA1 cell line was significantly greater than that of the siRNA4 cell line.

[0060] 3. Scratch test to detect the proliferation ability of cells with different expression levels of DDX39B: On the bottom of the six-well plate, use a marker to draw three horizontal lines along the ruler as marking lines. Plant the cells according to the grouping, and after the cells are fully grown, use a ruler to compare, and use a 200ul gun tip to draw two vertical lines perpendicular to the well plate and the marked line, so that the scratches intersect with the marked lines, and several intersections can be formed as fixed detection points. Discard the old culture medium and gently rinse with PBS two to three times until the scratched cells are rinsed clean. Add serum-free culture medium, observe the width of the scratch at the same position under a microscope at 0h, 24h, and 48h after the scratch, and take pictures. Count the relative migration area; the results are as follows. Figure 9-10 As shown, Figure 9 The following are photos of the scratch test of cell lines; in the figure, A is the HN4 cell line and B is the HONE1 cell line; it can be seen from the figure that at 24 hours, the scratches of the high-expressing cell line (NC) were significantly less than those of the down-regulated cell line si-DDX39B, indicating that high expression of DDX39B promotes the invasion and migration of tumor cells; the statistical results of the relative migration area of ​​the scratch test are shown in Figure 2. Figure 10 Statistical graph of scratch test of cell lines; in the figure, A is HN4 cell line, B is HONE1 cell line, and the figure shows that the relative migration area of ​​blank cells (NC) is significantly higher than that of down-regulated cells si-DDX39B.

[0061] 4. Transwell assay to detect cell proliferation at different DDX39B expression levels: Select a Transwell chamber with an appropriate pore size (usually 8 μm). Dilute the Matrigel to culture medium at a ratio of 1:8, then add 100 μl of the culture medium to the chamber for pre-coating. Place the chamber in an incubator for 30 minutes to allow the Matrigel to solidify. Digest the cells with trypsin, terminate the digestion, resuspend in a serum-free incubator, count, and adjust the cell suspension to an appropriate concentration (2.5×10 5 Add 200ul of cell suspension to the upper chamber of Transwell and 800ul of complete culture medium to the lower chamber. Place the well plate in a cell culture incubator and incubate for 24 hours. Remove the chamber and gently wipe off the upper layer of cells with a cotton swab. Fix with 4% paraformaldehyde solution for 15 minutes, stain with crystal violet solution for 20 minutes, wash, dry and take pictures. Summarize the number of invading cells, perform statistical analysis and draw a cell invasion map. Summarize the number of invading cells, perform statistical analysis and draw a cell invasion map. The results are as follows Figure 11-12 As shown, Figure 11 The results of the TransweLL experiment are shown in Figure 1. A is the HN4 cell line and B is the HONE1 cell line. As can be seen from the pictures, the number of blank cells (NC) is significantly greater than that of the siRNA1 cell line and the siRNA2 cell line. The statistical analysis results are shown in Figure 1. Figure 12As shown in the figure: A is the statistical result of HN4 cell line, B is the statistical result of HONE1 cell line. From the statistical results, it can be seen that the number of invasion of blank cells (NC) is significantly higher than that of siRNA1 cell line and siRNA4 cell line.

[0062] 5. Flow cytometry was used to detect apoptosis of cells with different DDX39B expression levels: cells were digested with trypsin and washed with pre-cooled PBS, and 3×10 5 Cells were diluted with double distilled water to 1× working solution of 5× Binding Buffer, and 300ul of 1× Binding Buffer was used to resuspend the cells. The apoptosis rate of HN4 cells with different DDX39B expression levels was detected by the instrument. The results are as follows: Figure 13 As shown in the figure, A is the result of the NC group, B is the result of the si-RNA group, and C is the flow cytometry apoptosis experiment statistical graph of the HN4 cell line. It can be seen from the figure that the apoptosis level of low-expressing cells (si-DDX39B) is higher than that of DDX39B high-expressing cells (NC).

[0063] 6. Nude mouse subcutaneous tumor formation experiment: Twelve 6-week-old SPF-grade BALB / c male nude mice were randomly divided into NC group and DDX39B knockdown group. After trypsinization, the cells were resuspended in PBS, counted, and the cell suspension was adjusted to an appropriate concentration (3×10 7 Each mouse was injected subcutaneously with 100 μl of the drug. Tumor volume and mouse weight were measured every 3 days. When the tumor diameter was ≥1.5 cm, the animal was sacrificed by excessive anesthesia, the subcutaneous tumor was removed, and the size was measured. Figure 14 As shown: After DDX39B gene downregulation (si-DDX39B), the size of tumor cells was significantly reduced, indicating that si-DDX39B gene downregulation can inhibit the growth of head and neck squamous cell carcinoma tumor cells in mice.

[0064] In summary, it is shown that the DDX39B gene is negatively correlated with head and neck squamous cell carcinoma cells. The siRNA of the present application has the effect of inhibiting the expression of the DDX39B gene in head and neck squamous cell carcinoma cells, that is, upregulating the expression of the DDX39B gene will promote the migration and growth of head and neck squamous cell carcinoma cells, and downregulating the expression of the DDX39B gene will inhibit the migration and growth of head and neck squamous cell carcinoma cells. Moreover, the present application achieves the purpose of inhibiting the growth of head and neck squamous cell carcinoma by constructing siRNA to interfere with the expression of the DDX39B gene, thereby providing a new therapeutic basis for the treatment of head and neck squamous cell carcinoma.

[0065] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of an agent that inhibits DDX39B gene expression or reduces DDX39B protein activity by targeting DDX39B protein or DDX39B gene in the treatment of head and neck squamous cell carcinoma, characterized in that: The nucleic acid sequence of the DDX39B gene is shown in SEQ ID NO.1; the amino acid sequence of the DDX39B protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The reagent is selected from a DDX39B antibody, an siRNA capable of inhibiting DDX39B gene expression, or a gene editing vector capable of inhibiting DDX39B gene expression.

3. The use according to claim 1, characterized in that The siRNA is double-stranded, and is any one of siRNA1, siRNA2, siRNA3 and / or siRNA4; the sense chain of the siRNA1 is shown in SEQ ID NO.3 in the sequence listing, and the antisense chain is shown in SEQ ID NO.4 in the sequence listing; the sense chain of the siRNA2 is shown in SEQ ID NO.5 in the sequence listing, and the antisense chain is shown in SEQ ID NO.6 in the sequence listing; the sense chain of the siRNA3 is shown in SEQ ID NO.7 in the sequence listing, and the antisense chain is shown in SEQ ID NO.8 in the sequence listing; the sense chain of the siRNA4 is shown in SEQ ID NO.9 in the sequence listing, and the antisense chain is shown in SEQ ID NO.10 in the sequence listing.

4. Use of siRNA that downregulates DDX39B gene expression in the preparation of drugs for treating head and neck squamous cell carcinoma and inhibiting the migration, invasion and / or growth of head and neck squamous cell carcinoma cells, characterized in that: The siRNA is double-stranded, and is any one of siRNA1, siRNA2, siRNA3 and / or siRNA4; the sense chain of the siRNA1 is shown in the sequence listing SEQ ID NO.3, and the antisense chain is shown in the sequence listing SEQ ID NO.4; the sense chain of the siRNA2 is shown in the sequence listing SEQ ID NO.5, and the antisense chain is shown in the sequence listing SEQ ID NO.6; the sense chain of the siRNA3 is shown in the sequence listing SEQ ID NO.7, and the antisense chain is shown in the sequence listing SEQ ID NO.8; the sense chain of the siRNA4 is shown in the sequence listing SEQ ID NO.9, and the antisense chain is shown in the sequence listing SEQ ID NO.

10.

5. A protein biomarker for the diagnosis or prediction of head and neck squamous cell carcinoma, characterized in that: The protein biomarker is DDX39B protein, the nucleic acid sequence of the DDX39B protein is shown in SEQ ID NO.1, and the amino acid sequence of the DDX39B protein is shown in SEQ ID NO.

2.

6. A kit for diagnosing head and neck squamous cell carcinoma, comprising a reagent for detecting the protein biomarker of claim 5, wherein the expression level of the protein biomarker is determined using one or more of fluorescence or Western blot.

7. Use of the kit according to claim 6 in preparing a product for diagnosing head and neck squamous cell carcinoma.

8. The method according to claim 7, comprising the steps of: (1) extracting extracellular vesicles from the plasma or serum of the subject to be tested; (2) Determine the content of DDX39B protein; (3) The results are determined based on the content of the above-mentioned protein biomarkers.

9. Use of the protein biomarker according to claim 5 in screening therapeutic drugs for head and neck squamous cell carcinoma.

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