Application of PRPF39 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 PRPF39 protein or gene as a target spot in treatment and detection of head and neck squamous cell carcinoma, research finds that the PRPF39 gene has remarkable expression up-regulation in HNSCC tissue, and the PRPF39 protein can be used as a kit or a detection marker to be applied to treatment and detection of head and neck squamous cell carcinoma. The expression level can be used as a powerful prediction index independent of traditional clinical staging; besides, the PRPF39 gene can also be used as a potential treatment target of the head and neck squamous cell carcinoma, expression of the PRPF39 gene is lowered by constructing siRNA, the purposes of inhibiting migration and growth of cancer cells and reducing tumor cells are achieved through cell and animal experiments, the head and neck squamous cell carcinoma can be treated purposefully, and the method is expected to become a brand new treatment strategy for improving the treatment outcome of HNSCC and overcoming drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of PRPF39 protein or gene as a target in the treatment and detection of head and neck squamous cell carcinoma. Background Art

[0002] Head and neck malignancies are among the seven most common malignant tumors worldwide, with significant clinical and biological heterogeneity. Among them, head and neck squamous cell carcinoma (HNSCC) is its most common pathological subtype, accounting for the vast majority of cases. Its global burden is heavy, with approximately 890,000 new cases each year and approximately 450,000 deaths, highlighting its severity as a major public health issue. HNSCC often appears pathologically as a poorly differentiated or undifferentiated state, which makes the tumor cells highly invasive and metastatic. Unfortunately, about two-thirds of patients are already in the late stage (stage III or IV) at the time of their first visit, missing the best window for early intervention. This late discovery feature, coupled with the malignant biological behavior of the tumor itself, leads to a generally poor prognosis for HNSCC. Despite significant progress in treatment strategies, evolving from traditional single modalities (surgery, radiotherapy, or chemotherapy) to multidisciplinary, personalized comprehensive treatment approaches (e.g., surgery combined with chemoradiotherapy, targeted therapy, and immunotherapy), improvements in overall survival (OS) remain limited, with 5-year survival rates hovering at a low 40%-50%, significantly lower than those of many other common solid tumors. One of the core factors contributing to the treatment dilemma for HNSCC is its significant intra- and inter-tumor heterogeneity. This complex heterogeneity is not only reflected in histological morphology and genomic profiles but also profoundly influences tumor biological behavior, leading to significant variability in sensitivity and response rates to the same treatment regimen among different patients, ultimately resulting in diverse clinical outcomes and prognosis. Currently, clinical practice primarily relies on traditional indicators such as the anatomical TNM staging system, limited tumor markers, and patient age to predict prognosis and guide treatment selection. However, the inherent limitations of these conventional clinical features are becoming increasingly prominent - they fail to fully capture the molecular differences in the intrinsic driving mechanisms of tumors and the potential for personalized treatment, and are insufficiently accurate in precisely dividing prognostic risk groups and predicting the efficacy of specific therapies, making it difficult to truly achieve the goal of "personalized medicine."

[0003] In previous studies, much progress has been made in exploring the pathogenesis of HNSCC, screening of prognostic biomarkers and discovery of therapeutic targets. A large number of works have focused on genomic variations (such as TP53 mutation, PIK3CA amplification, etc.) and abnormal overall expression profiles of transcriptome, and the online survival analysis tool developed by Zhang Guosen team is a representative of using such macroscopic transcriptome data to predict prognosis. However, the importance of a key regulatory level: post-transcriptional processing modification of genes, especially the role of pre-mRNA splicing in HNSCC has not been fully explored and analyzed.

[0004] More and more studies have proved that the expression disorder of splicing factors (SFs) is one of the key drivers of the occurrence and development of malignant tumors. SFs can generate protein subtypes with different structures and functions by accurately regulating the process of alternative splicing, thereby deeply affecting various cancer-related pathways (such as cell cycle regulation, apoptosis resistance, DNA repair, invasion and metastasis, etc.). 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): involved in the regulation of pre-mRNA splicing of various genes related to human diseases (including cancer). BUD31: as a key oncogenic splicing factor, its up-regulated expression in ovarian cancer indicates 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.

[0005] PRPF39 gene as an important protein-coding gene and splicing factor, studies have suggested that its expression level may be related to tumor cells, but there is no report on PRPF39 gene related to HNSCC. Therefore, it is necessary to conduct in-depth research on HNSCC related genes in order to find new treatment strategies for specifically treating HNSCC. SUMMARY

[0006] In view of the above, it is necessary to conduct in-depth research on HNSCC related genes in order to find new treatment strategies for specifically treating HNSCC.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is:

[0008] The invention relates to the use of an agent that inhibits PRPF39 gene expression or reduces PRPF39 protein activity by targeting PRPF39 protein or PRPF39 gene in the treatment of head and neck squamous cell carcinoma, wherein the PRPF39 gene nucleic acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of PRPF39 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the reagent is selected from a PRPF39 antibody, an siRNA capable of inhibiting PRPF39 gene expression, or a gene editing vector capable of inhibiting PRPF39 gene expression.

[0010] Furthermore, the siRNA is double-stranded, which 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.

[0011] The present invention also includes the use of siRNA that downregulates PRPF39 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. 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.

[0012] The present invention also includes a protein biomarker for diagnosing or predicting head and neck squamous cell carcinoma. The protein biomarker is PRPF39 protein. The nucleic acid sequence of the PRPF39 protein is shown in SEQ ID NO.1, and the amino acid sequence of the PRPF39 protein is shown in SEQ ID NO.2.

[0013] 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.

[0014] The present invention also includes the use of the kit in preparing products for diagnosing head and neck squamous cell carcinoma.

[0015] The present invention also includes the use of the kit, which comprises the following steps:

[0016] (1) extracting extracellular vesicles from the plasma or serum of the subject to be tested;

[0017] (2) determining the content of PRPF39 protein;

[0018] (3) The results are determined based on the content of the above-mentioned protein biomarkers.

[0019] The present invention also includes the use of the protein biomarker in screening therapeutic drugs for head and neck squamous cell carcinoma.

[0020] The present invention has the following beneficial effects:

[0021] Through in-depth research, the present invention discovered that the PRPF39 gene is significantly upregulated in HNSCC tissues. This discovery can use the PRPF39 protein as a kit or detection marker, and its expression level can be used as a powerful predictive indicator independent of traditional clinical staging to more accurately identify patient subgroups with high recurrence risk and low survival rate, which helps clinicians to formulate more targeted follow-up plans and more active auxiliary treatment strategies; in addition, the PRPF39 gene can also be used as a potential therapeutic target for head and neck squamous cell carcinoma. This application also constructs siRNA to downregulate PRPF39 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 to improve the treatment outcome of HNSCC and overcome drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the SF-AS regulatory network.

[0023] Figure 2 These are the RT-qPCR experimental results of the PRPF39 gene in different HNSCC cell lines (NP69, HONE1, SAS, HN4, CAL27).

[0024] Figure 3The Western blot experimental results of PRPF39 gene in different HNSCC cell lines (NP69, HONE1, SAS, HN4, CAL27); in the figure, A is the electrophoresis result and B is the statistical result.

[0025] Figure 4 The figures show the RT-qPCR experimental results of different siRNAs of the PRPF39 gene in different HNSCC cell lines; in the figure, A is the SAS cell line and B is the HN4 cell line.

[0026] Figure 5 Figure 2 is a graph showing the results of Western blot experiments of different siRNAs for the PRPF39 gene in different HNSCC cell lines; in the figure, A is the electrophoresis diagram of the SAS cell line, B is the electrophoresis diagram of the HN4 cell line, C is the statistical result of the SAS cell line, and D is the statistical result of the HN4 cell line.

[0027] Figure 6 This is a graph of cck8 experimental results; in the figure, A is the OD value of the SAS cell line, and B is the OD value of the HN4 cell line.

[0028] 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 SAS cell line.

[0029] Figure 8 It is a statistical chart of the cloning experiment; in the figure, A is the HN4 cell line and B is the SAS cell line.

[0030] 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 SAS cell line.

[0031] Figure 10 Statistical chart of scratch test of cell lines; in the figure, A is HN4 cell line, and B is SAS cell line.

[0032] Figure 11 These are the results of the TransweLL experiment. In the figure, A is the HN4 cell line and B is the SAS cell line.

[0033] Figure 12 This is a statistical chart of the TransweLL experiment. In the figure, A is the HN4 cell line and B is the SAS cell line.

[0034] Figure 13 Figure 2 is a flow cytometry apoptosis experiment result diagram, in which A is the NC group, B is the si-RNA group, and C is the flow cytometry apoptosis experiment statistical diagram of the HN4 cell line.

[0035] Figure 14 This figure shows the results of the subcutaneous tumor formation experiment in nude mice. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1:

[0038] This example shows the process of obtaining markers for predicting the prognosis of patients with head and neck squamous cell carcinoma:

[0039] (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. As can be seen from the figure: the SF-AS regulatory network shows that PRPF39 is a key splicing factor associated with prognosis in HNSCC, and it negatively regulates favorable prognosis-related AS events and positively regulates unfavorable prognosis-related AS events. The key splicing factor PRPF39 was discovered.

[0040] (2) Detection of PRPF39 expression in HNSCC cell lines: Western blot and RT-qPCR experiments were used to detect the expression level of PRPF39 in HNSCC cell lines (HONE1, SAS, HN4, CAL27) and normal head and neck epithelial cells (NP69). PRPF39 primer sequence: F: 5'-GGTGCATATGCTTTGGGCAG-3', R: 5'-AGATGCCGGGCTAGTTTGAC-3'; β-actin primer sequence: F: 5'-GCACTCTTCCAGCCTTCCTTCC-3', R: 5'-GCGGATGTCCACGTCACACTTC-3'.

[0041] 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 solution 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.

[0042] ② RT-qPCR: The MonaBio Reverse Transcription Kit (MonScript™ RTIII ALL-in-One Mix with dsDNase) was used on ice. First-strand template cDNA was synthesized by adding the extracted RNA to a 200 µL sterile, enzyme-free centrifuge tube according to the following protocol: 500 ng template RNA, 2 µL MonScript™ 5× RTIII ALL-in-One Mix, 0.5 µL MonScript™ dsDNase, and NucLease-Free Water to a volume of 10 µL. After vortexing and brief centrifugation, the tube was placed in a reverse transcription instrument for reverse transcription to obtain cDNA. The reaction protocol was: 37°C for 2 min, 55°C for 15 min, and 85°C for 5 min. Depending on the amount of template cDNA required, the components of the reverse transcription system were added in appropriate proportions. The resulting cDNA was removed and placed on ice. Diluted with 40 µL of pre-chilled enzyme-free water. The cDNA can be temporarily stored at -20°C or long-term stored at -80°C. Using MonAmp TM PCR amplification was performed using PCR Green qPCR Mix (MQ00501S, Monad, China).

[0043] 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 PRPF39 is highly expressed in HNSCC.

[0044] Example 2:

[0045] This example uses the PRPF39 gene as a protein biomarker for the diagnosis or prediction of head and neck squamous cell carcinoma, as follows:

[0046] 1. The nucleic acid sequence of the PRPF39 protein in this example is shown in SEQ ID NO.1, and the amino acid sequence of the PRPF39 protein is shown in SEQ ID NO.2.

[0047] 2. From Example 1, we know that PRPF39 is highly expressed in HNSCC. We can prepare the PRPF39 protein into a kit for diagnosing head and neck squamous cell carcinoma. The kit contains the PRPF39 protein. The specific detection method is as follows:

[0048] (1) extracting extracellular vesicles from the plasma or serum of the subject to be tested;

[0049] (2) determining the content of PRPF39 protein;

[0050] (3) If the measured PRPF39 protein content is higher than the blank, the subject is diagnosed as having head and neck squamous cell carcinoma or predicted to be likely to have head and neck squamous cell carcinoma.

[0051] Example 3:

[0052] This example uses the PRPF39 gene to screen for drugs for treating head and neck squamous cell carcinoma, as follows:

[0053] Screen related drugs that can downregulate the PRPF39 gene, such as PRPF39 antibodies, siRNA that can inhibit the expression of the PRPF39 gene, or gene editing vectors that inhibit the expression of the PRPF39 gene; if the expression of the PRPF39 gene can be downregulated, it means that the drug can treat head and neck squamous cell carcinoma.

[0054] Example 4:

[0055] This example is about screening siRNA for down-regulating the PRPF39 gene and verifying its therapeutic effect on tumor cells. The specific method is as follows:

[0056] 1. Construction of PRPF39 downregulation cell lines in HNSCC cell lines: siRNA and negative control were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. SAS and HN4 cells were plated in 6-well plates and transfected using Lipofectamine 3000 when the cells grew to a cell density of 50%-60%. 48 h after transfection, cells were collected to extract RNA and protein, and the expression level of PRPF39 was detected by Western blot and RT-qPCR to verify the transfection efficiency. The sequence of siRNA1 was: S: 5'-GGAUUGGCAAUGGUUCGUUTT-3', AS: 5'-AACGAACCAUUGCCAAUCCTT-3' (SEQ ID NO.3-NO.4) The sequence of siRNA2 was: S: 5'-GGCAAUACCUCUUAGUGUUTT-3', AS: 5'-AACACUAAGAGGUAUUGCCTT-3' (SEQ ID NO.5-NO.6) The sequence of siRNA3 was: S: 5'-GCGGCACGACAACAUUAAATT-3', AS: 5'-UUUAAUGUUGUCGUGCCGCTT-3' (SEQ ID No. 7-No. 8) The sequence of siRNA4 is: S: 5'-GGGAAUUAGCUUCUGUAAATT-3', AS: 5'-UUUACAGAAGCUAAUUCCCTT-3' (SEQ ID No. 9-No. 10).

[0057] (1) Cell transfection: Inoculate cells: Prepare cell suspension with complete culture medium (SAS: 1×10 5 / mL; HN4:1×10 5 / mL), 2mL per well was inoculated into a 6-well plate, and transfection was performed when the cell confluence reached 50%-60%. 125uL Opti-MEM medium was used to dilute 3.75uL Lipofectamine 3000, 10uL P3000 and 1uL siRNA were diluted in 125uL Opti-MEM medium, and the two were mixed after standing for 5 minutes, and incubated at room temperature for 15 minutes. The complex was added to the cells, mixed and placed in the incubator, and the medium was changed after 4-6 hours. Figure 4-Figure 5 As shown: Figure 4 Figure 2 is the RT-qPCR experimental results of different siRNAs of the PRPF39 gene in different HNSCC cell lines; in the figure, A is the SAS cell line and B is the HN4 cell line; Figure 5 Figure 2 shows the Western blot results of different siRNAs targeting the PRPF39 gene in different HNSCC cell lines. Figure A shows the electrophoresis of the SAS cell line, B shows the electrophoresis of the HN4 cell line, C shows the statistical results of the SAS cell line, and D shows the statistical results of the HN4 cell line. As can be seen from the figure, based on the RT-qPCR expression results, the downregulation levels of siRNA1 and the blank were not significantly different, while the downregulation levels of siRNA2, siRNA3, and siRNA4 were significantly different. Western blot results show that the downregulation levels of siRNA1, siRNA2, siRNA3, and siRNA4 were significantly different. This indicates that we have successfully established PRPF39 downregulation cell lines in the SAS and HN4 cell lines.

[0058] 2. Select siRNA2 and siRNA3 cell lines for cell proliferation ability experiment: seed cells into 96-well plates (1.5×10 3 / well), and the relative proliferation activity of the PRPF39 knockdown cell line and the negative control cell line was calculated using the CCK8 assay. Cells were seeded into 6-well plates (600 / well), and the number of clones of the PRPF39 knockdown cell line and the negative control cell line was calculated using a colony formation assay. The specific method is as follows:

[0059] (1) CCK8 experiment: After trypsin digestion, cells were collected by centrifugation. The collected cells were resuspended in serum-containing medium, counted, and diluted to 1.5×10 4Cell suspension of 100 μL / mL. Inoculate the cell suspension in a 96-well plate, 100 μL per well, place the culture plate in an incubator for pre-culture for 24h, 48h, and 72h, then discard the culture medium and add 100uL of culture medium containing 10μL cck8 solution to each well. After adding the reagent, gently shake the culture plate to help mix (to prevent errors caused by CCK8 reagent sticking to the well wall). Try not to generate bubbles during the addition process to avoid affecting the OD value reading. Place the culture plate in an incubator and incubate for 1-2h. Measure the absorbance (OD) at 450nm with an enzyme reader. The results are as follows. Figure 6 As shown in the figure, A is the OD value of SAS cell line, B is the OD value of HN4 cell line; Figure 6 It can be seen that the proliferation activity of si-PRPF39 cell line was significantly lower than that of the blank (NC) group.

[0060] (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 SAS 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 SAS cell line; it can be seen from the figure that in the photographs, the density of cells is NC cell line>siRNA2 cell line>siRNA3 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 siRNA2 cell line, and the cell proliferation activity of the siRNA2 cell line was significantly greater than that of the siRNA3 cell line.

[0061] 3. Scratch test to detect the proliferation ability of cells with different expression levels of PRPF39: 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 SAS 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 cells si-PRPF39, indicating that high expression of PRPF39 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 SAS cell line, and the figure shows that the relative migration area of ​​blank cells (NC) is significantly higher than the relative migration area of ​​cells down-regulated with si-PRPF39.

[0062] 4. Transwell assay to detect cell proliferation at different PRPF39 expression levels: Dilute Matrigel to culture medium at a ratio of 1:8, then take 100 μL and add it to the chamber for pre-coating. Place the chamber in an incubator for 30 minutes to allow the Matrigel to solidify. Add 800 μL of complete culture medium to the bottom of the chamber, trypsinize the cells, 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 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, rinse, dry and take pictures. Summarize the number of invading cells, perform statistical analysis and draw a cell invasion map. The results are shown in Figure 2. 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 SAS cell line. It can be seen from the pictures that the number of blank cells (NC) is significantly greater than that of the siRNA2 cell line and the siRNA3 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 strain, B is the statistical result of SAS cell strain, from the statistical result, it can be seen that the invasion number of blank cells (NC) is significantly higher than that of siRNA2 cell strain and siRNA3 cell strain.

[0063] 5. Flow cytometry experiment detects the apoptosis of cells with different expression levels of PRPF39: after trypsin digestion, the cells are washed with pre-cooled PBS, centrifuged to collect 3x10 5 cells, according to the instructions of the kit, dilute 5xBindding Buffer with double distilled water to 1xworking solution, take 300uL of 1xBindding Buffer to resuspend the cells, and detect the apoptosis rate of cells with different PRPF39 expression levels on the machine, and the results are shown in Figure 13 the figure: A is the flow cytometry apoptosis experiment statistical result graph of NC group, B is si-RNA group, and C is HN4 cell strain, from the figure, it can be seen that the apoptosis level of low expression cells (si-PRPF39) is higher than that of PRPF39 high expression cells (NC).

[0064] 6. Subcutaneous tumor experiment of nude mice: 12 SPF level BALB / c male nude mice of 6 weeks old are selected, the mice are randomly divided into NC group and PRPF39 knockdown group, after trypsin digestion, the cells are resuspended with PBS, counted, and the cell suspension is adjusted to an appropriate concentration (3x10 7 cells / mL), and each is subcutaneously injected with 100uL. The tumor volume and mouse weight are measured every 3 days. When the tumor diameter is ≥1.5cm, the animals are sacrificed after excessive anesthesia, and the subcutaneous tumor is peeled off, and the size is measured, and the results are shown in Figure 14 : After down-regulation of PRPF39 gene (si-PRPF39), the size of tumor cells is significantly reduced, which shows that down-regulation of si-PRPF39 gene can inhibit the growth of head and neck squamous cell carcinoma tumor cells in mice.

[0065] In summary, it is shown that PRPF39 gene is negatively correlated with head and neck squamous cell carcinoma cells, the siRNA of the application has the effect of inhibiting the expression of PRPF39 gene of head and neck squamous cell carcinoma, that is, up-regulation of PRPF39 gene expression can promote the migration and growth of head and neck squamous cell carcinoma cells, and down-regulation of PRPF39 gene expression can inhibit the migration and growth of head and neck squamous cell carcinoma cells, and the application can inhibit the growth of head and neck squamous cell carcinoma by constructing siRNA to interfere with the expression of PRPF39 gene, which provides a new treatment basis for the treatment of head and neck squamous cell carcinoma.

[0066] 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 targets PRPF39 protein or PRPF39 gene to inhibit PRPF39 gene expression or reduce PRPF39 protein activity in the treatment of head and neck squamous cell carcinoma, characterized in that: The nucleic acid sequence of the PRPF39 gene is shown in SEQ ID NO.1; the amino acid sequence of the PRPF39 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The reagent is selected from PRPF39 antibodies, siRNA capable of inhibiting PRPF39 gene expression, or a gene editing vector capable of inhibiting PRPF39 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 PRPF39 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 PRPF39 protein, the nucleic acid sequence of the PRPF39 protein is shown in SEQ ID NO.1, and the amino acid sequence of the PRPF39 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) determining the content of PRPF39 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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