Application of LncRNA GSEC in preparation of medicine for treating oral squamous cell carcinoma

Abstract

The invention discloses application of LncRNA GSEC in preparation of a medicine for treating oral squamous cell carcinoma, and relates to the technical field of biological medicine. The nucleotide sequence of the LncRNA GSEC is shown as SEQ ID NO: 1, the medicine contains an active component capable of inhibiting expression or functions of the LncRNA GSEC, the active component is one or more of siRNA, shRNA or a small-molecule inhibitor, and the medicine is used for inhibiting proliferation, migration and / or invasion of OSCC cells and / or inducing cell apoptosis. The specific high expression of the LncRNA GSEC in oral squamous cell carcinoma tissues and cells is disclosed for the first time, and the expression level of the LncRNA GSEC is remarkably and positively correlated with adverse clinical parameters such as tumor T staging and lymph node metastasis, so that the LncRNA GSEC is proved to serve as a novel molecular target with clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of LncRNA GSEC as a target in the preparation of drugs for treating oral squamous cell carcinoma. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, characterized by local invasiveness and high recurrence rate. Despite significant advancements in treatment, the 5-year survival rate for OSCC remains largely unchanged. The poor prognosis of OSCC is primarily due to the often subtle early symptoms, frequently confused with oral ulcers, leading to diagnosis typically at an intermediate or advanced stage. Furthermore, it is associated with a high rate of local recurrence or distant metastasis after initial treatment. Therefore, the development of reliable biomarkers and the discovery of therapeutic targets are crucial for the early diagnosis and effective prognosis assessment of OSCC.

[0003] Long non-coding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides that lack protein-coding capabilities. As important competing endogenous RNAs (ceRNAs), they act as miRNA sponges in vivo. LncRNAs can exert various biological functions by regulating gene expression and function at the transcriptional, translational, and post-translational levels. In the past decade, numerous studies both domestically and internationally have confirmed that lncRNAs participate in the development and progression of various diseases, including malignant tumors, metabolic diseases, and cardiovascular diseases, through intercellular communication and regulation of gene expression within recipient cells. Previous studies have shown that G-quadruplexes (G4S) in DNA and RNA are related to transcription, polyadenylation, splicing, RNA turnover, and stability. It has been reported that GSEC is abnormally expressed in colorectal cancer and promotes colorectal cancer migration by antagonizing DHX36. Researchers have found that the expression level of LncRNA GSEC is upregulated in osteosarcoma cell lines. Overexpression of LncRNA GSEC promotes the proliferation and migration of osteosarcoma cells and inhibits their apoptosis (Liu R, Ju C, Zhang F, et al. LncRNA GSEC promotes the proliferation, migration and invasion by sponging miR-588 / EIF5A2 axis in osteosarcoma. Biochem Biophys Res Commun. 2020 Nov 5;532(2):300-307.). Other researchers have demonstrated the functional role of the lncRNA GSEC / miR-101-3p axis in the lung adenocarcinoma (LUAD) environment and proposed potential biomarkers for the clinical diagnosis and treatment of this disease (Jiang X, Yuan Y, Tang L, et al. Systematic Analysis and Validation of the Prognosis, Immunological Role and Biology Function of the Ferroptosis-Related lncRNAGSEC / miRNA-101-3p / CISD1 Axis in Lung Adenocarcinoma. Front Mol Biosci. 2022 Mar 7; 8: 793732.). However, the function of lncRNA GSEC in OSCC and its potential as a therapeutic target have not yet been revealed in current technologies. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of LncRNA GSEC in the preparation of drugs for treating oral squamous cell carcinoma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of LncRNA GSEC in the preparation of a drug for treating oral squamous cell carcinoma.

[0006] Furthermore, the nucleotide sequence of the LncRNA GSEC is shown in SEQ ID NO:1.

[0007] Furthermore, the drug contains an active ingredient capable of inhibiting the expression or function of LncRNA GSEC.

[0008] Furthermore, the active ingredient is one or more of siRNA, shRNA, or small molecule inhibitors.

[0009] Furthermore, the drug is used to inhibit the proliferation, migration, and / or invasion of OSCC cells, and / or induce apoptosis.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals for the first time that lncRNA GSEC is specifically and highly expressed in oral squamous cell carcinoma (OSCC) tissues and cells, and its expression level is significantly positively correlated with adverse clinical parameters such as tumor T stage and lymph node metastasis, confirming it as a novel molecular target with clinical application potential. This provides a new direction and approach to overcome the current limitations of OSCC treatment and its poor prognosis. Cellular-level experiments demonstrated that knocking down lncRNA GSEC expression using technologies such as siRNA significantly inhibits the proliferation, migration, and invasion of OSCC cells and effectively induces apoptosis. This indicates that intervention targeting this point can directly reverse the malignant phenotype of the tumor. Animal-level experiments confirmed that in a nude mouse xenograft model, knocking down lncRNA GSEC significantly inhibits tumor growth, manifested as a significant reduction in tumor volume and weight, powerfully validating its therapeutic potential in vivo. Based on this, developing highly effective OSCC therapeutic drugs targeting lncRNA GSEC can provide candidate strategies for targeted OSCC treatment. Attached Figure Description

[0011] Figure 1 The expression of GSEC in OSCC cancer and adjacent normal tissues is shown in Figure 1. A: GSEC expression in OSCC cancer and adjacent normal tissues, and B: GSEC expression in OSCC cancer and adjacent normal tissues. Figure 2The correlation between GSEC and clinical parameters of OSCC is as follows: A: Relationship between GSEC expression in OSCC tissue and T stage; B: Relationship between GSEC expression in OSCC tissue and lymph node metastasis; C: Relationship between GSEC expression in OSCC tissue and age; D: Relationship between GSEC expression in OSCC tissue and sex; E: Relationship between GSEC expression in OSCC tissue and high / low differentiation. Figure 3 The expression of GSEC in NHOK and OSCC cell lines is shown in Figure 1. A: GSEC expression in NHOK and OSCC cell lines, and B: transfection efficiency of si-GSEC. Figure 4 The results of the CCK8 cell proliferation experiment are shown. A: Bar graph of OSCC cell proliferation at 24h, 48h, and 72h after transfection with si-GSEC-1 / si-NC; B: Line graph of OSCC cell proliferation at 24h, 48h, and 72h after transfection with si-GSEC-1 / si-NC. Figure 5 The results are from flow cytometry experiments. Among them, A: apoptosis results of OSCC cells after transfection with si-NC, B: apoptosis results of OSCC cells after transfection with si-GSEC-1, and C: a comparison of apoptosis results of OSCC cells after transfection with si-NC / si-GSEC-1. Figure 6 The results of the Transwell experiment are shown in Figure 1. A: Comparison of Transwell experiment results of OSCC cells after transfection with si-NC / si-GSEC-1; B: Figure of Transwell experiment results of OSCC cells after transfection with si-NC / si-GSEC-1. Figure 7 The results are shown in the scratch assay. A: Scratch assay results of OSCC cells transfected with si-NC / si-GSEC-1 at 0h, 24h, and 48h. B: Comparison of scratch assay results of OSCC cells transfected with si-NC / si-GSEC-1 at 0h, 24h, and 48h. Figure 8 To detect the expression levels of apoptosis-related markers BAX, caspase-9, and BCL-2, as well as proliferation-related markers Ki67 and PCNA, at the mRNA level using qRT-PCR after transfection with si-NC / si-GSEC-1; Figure 9The results of Western blot detection of Bax, Bcl-2 and caspase-9 are shown. A: Expression level of Bax, B: Expression level of caspase-9, C: Expression level of Bcl-2, D: Western blot results of Bax, Bcl-2 and caspase-9. Figure 10 The images show the results of immunofluorescence assays for Ki67 and PCNA. A: PCNA results in OSCC cells transfected with si-NC / si-GSEC-1; B: PCNA results in OSCC cells transfected with si-NC / si-GSEC-1; C: Ki67 results in OSCC cells transfected with si-NC / si-GSEC-1; D: Ki67 results in OSCC cells transfected with si-NC / si-GSEC-1. Figure 11 The expression of GSEC in NHOK and OSCC cell lines during the tumor-bearing experiment in nude mice is shown. A: GSEC expression in NHOK and OSCC cell lines, B: transfection efficiency of si-GSEC. Figure 12 The graph shows the results of body weight and tumor volume in nude mice bearing tumors. In the graph, A represents the tumor volume at different time points, and B represents the body weight of nude mice at different time points. Figure 13 The results show the comparison of tumors in different groups of nude mice. In this figure, A is a comparison of the body weight of tumor-bearing nude mice in different groups, and B is the tumor formation in different groups of nude mice. Figure 14 HE staining results of tumors in different groups of nude mice; Figure 15 The results of Western blot analysis of Bax, Bcl-2 and caspase-9 in tumors of different groups of nude mice were obtained. A: expression level of Bax, B: expression level of Bcl-2, and C: expression level of caspase-9. Figure 16 Immunohistochemical results of Ki67 and PCNA in tumors from different groups of nude mice. A: Ki67 immunohistochemical result, B: PCNA immunohistochemical result.

[0012] In the figure, ns P >0.05, * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Detailed Implementation

[0013] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0014] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0015] This invention elucidates the molecular mechanism by which LncRNA GSEC affects cell proliferation and migration in the OSCC process through tissue, cell, and animal level experiments in the following embodiments. LncRNA GSEC may be a new molecular marker for OSCC, providing a basis for finding drug targets for OSCC in the future, and providing a reference for early diagnosis, prognosis assessment and treatment of OSCC.

[0016] Example 1: Tissue Experiment 1. qRT-PCR detection of the expression of target lncRNA GSEC in OSCC tissue specimens Tissue was cut into small pieces, and 10 mg was weighed out. Total RNA was extracted using TriQuick Reagent reagent (Solepro, Beijing) according to the manufacturer's instructions. Then, the total RNA was reverse transcribed using the SureScript™ First-Strand cDNA Synthesis Kit Cat. (GeneCopeia, USA) to obtain cDNA according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using 2×SYBR Green qPCR Master Mix (None ROX) reagent (Servicebio, Wuhan) according to the manufacturer's instructions, with actin as a standardized control. The relative expression level of the target gene was determined using the threshold cycle 2 DDCt method. Primer sequences: The primers used in this example are shown in Table 1.

[0017] Table 1 Primer Design

[0018] The results showed that the expression level of GSEC in OSCC cancer tissue was significantly increased compared with that in adjacent normal tissue. P <0.05)( Figure 1 AB).

[0019] 2. Correlation between the expression of target lncRNA GSEC in OSCC tissue specimens and clinical parameters This study included 30 patients with OSCC who visited the Affiliated Hospital of Inner Mongolia Medical University in China from July 2024 to February 2025. All patients were pathologically diagnosed with primary OSCC, without other malignant tumors, and had not received radiotherapy, chemotherapy, or other treatments before surgery. Cancerous tissue and adjacent normal tissue specimens, as well as complete clinical medical records, were collected. This study will be conducted after being discussed and approved by the ethics committee, and all patients signed informed consent forms. The expression level of the target lncRNA GSEC in cancerous tissue and adjacent normal tissue of OSCC patients was detected using qRT-PCR, and its correlation with clinical parameters of OSCC was analyzed.

[0020] The results show that: In OSCC, GSEC in the TMN period, T Ⅰ-Ⅱ With T Ⅲ-Ⅳ The expression in them is different ( P <0.05)(Table 2, Figure 2 A), GSEC expression is differential in lymph node metastasis ( P <0.05)( Figure 2 B), GSEC expression showed no significant differences in age, sex, or high / low differentiation. P >0.05)( Figure 2 CE).

[0021] Table 2. Correlation between GSEC expression and clinical parameters in OSCC tissues

[0022] Example 2 Cell Experiment 1. Experimental Methods (1) Cell culture Oral epithelial keratinocytes (NHOK) and oral squamous cell carcinoma cell lines (SCC-25, CAL-27, and SCC-9) (purchased from Procyno) were used. All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco). Cultured further at 37°C with 5% carbon dioxide. The expression of lncRNA GSEC was detected by qRT-PCR. Cell lines with relatively high expression were selected for further research.

[0023] (2) Plasmid transfection All plasmids were constructed by Shanghai Hanheng Biotechnology Co., Ltd. GSEC siRNA and negative controls were purchased from Suzhou Hongxun Biotechnology Co., Ltd. OS cells were transfected using Lipofectamine™ 2000 transfection reagent (Thermo Fisher) according to the manufacturer's instructions. Sequences are shown in Table 3.

[0024] Table 3 Sequence List

[0025] (3) RNA isolation and qRT-PCR Total RNA was extracted using TriQuick Reagent reagent (Solepro, Beijing) according to the manufacturer's instructions. Then, the total RNA was reverse transcribed using the SureScript™ First-Strand cDNA Synthesis Kit Cat. (GeneCopeia, USA) to obtain cDNA according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using 2×SYBR Green qPCRMaster Mix (None ROX) reagent (Servicebio, Wuhan) according to the manufacturer's instructions, with actin as a standardized control. The relative expression level of the target gene was determined using the threshold cycle 2 ΔΔCt method. The primers used in this example are shown in Table 4.

[0026] Table 4 Primer Design

[0027] (4) Western Blotting experiment Total protein was extracted from cultured cells using RIPA cell lysis buffer (Bai Rui Ji). An appropriate amount of protein sample was loaded and subjected to SDS-PAGE (10% polyacrylamide gel electrophoresis) to separate the proteins. The proteins in the gel were transferred to a PVDF membrane to form a blot. The membrane was incubated overnight at 4°C with the following antibodies: Anti-Caspase9 Rabbit mAb (A18676) (Wuhan Sanying, China; 1:1000), Anti-Bcl2 Polyclonal antibody (12789-1-AP) (Wuhan Sanying, China; 1:1000), Anti-Bax Polyclonal antibody (50599-2-Ig) (Wuhan Sanying, China; 1:1000), and Anti-β-Actin antibody (ab8227) (abcam, UK; 1:10000). The membrane was then placed in an incubator containing the corresponding secondary antibody (1:8000) (goat anti-rabbit) and incubated at room temperature for 1.5 hours. Protein bands on PVDF membranes were visualized using an ECL detection kit (Wanleibio). The membranes were photographed using a chemiluminescence imaging and analysis system. The brightness values ​​of each protein band were analyzed, and bar charts were plotted.

[0028] (5) Cell counting kit-8 (CCK-8) detection Cell proliferation was detected using CCK-8 (Beyotime, Shanghai) according to the manufacturer's instructions. Transfection was performed according to experimental groups. After 24 hours, cells were digested and counted, and then seeded into 96-well plates at 1000 cells per well. CCK-8 assays were performed after 24, 48, and 72 hours by adding 10 μL of CCK-8 solution to each well and incubating for another 1 hour in a cell culture incubator. Finally, absorbance at 450 nm was measured using a microplate reader.

[0029] (6) Flow cytometry analysis Cells were seeded into 6-well plates until the cell density reached approximately 60% after 24 hours. Transfection was performed according to the assigned groups, and cells were collected for apoptosis assays after 48 hours. The supernatant was discarded, and the cells were washed once with PBS. Cells were digested with trypsin without EDTA, and 1 ml of complete culture medium was used to collect the cells. The cells were centrifuged at 1500 rpm for 5 minutes, resuspended in pre-chilled PBS, centrifuged again at 1500 rpm for 5 minutes, and the supernatant was carefully aspirated. The cells were resuspended in 1× Binding Buffer to achieve a cell density of 1×10⁶ cells / well. 6 Cells / ml. Take 100µl of cell suspension (1×10⁻⁶ cells / ml). 5 Add 5µl of Annexin V-FITC and 5µl of PI Solution to each EP tube. Mix gently. Incubate at room temperature (25°C) in the dark for 15 min. Add 400µl of 1×Binding Buffer to each tube and mix gently. Perform instrumental analysis within 1 hour.

[0030] (7) Transwell experiment Invasion assays were performed using a Nunc 24-well 8.0 μm permeable plate (Thermo Fisher Scientific). Matrigengel was polymerized into a film within the chamber. 500 µL of medium containing 20% ​​FBS was added to the lower chamber of the 24-well plate. Then, using forceps, the Transwell chamber was placed into the 24-well plate, and 200 µL of medium at a density of 3 × 10⁻⁶ ppm was added to each well. 5 / ml of cell suspension was transferred to the upper chamber of the Transwell incubator and incubated at 37°C for 48 hours. After incubation, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 10 minutes. The invasive cells were counted at 100x magnification. Three fields of view were randomly selected from each sample for counting, and the average value was taken.

[0031] (8) Wound healing experiment Transfect the cells according to their groups. When the cell density reaches about 90%, use the tip of a 200 μl pipette to make a scratch perpendicular to the cell plane. Discard the supernatant, rinse once with PBS to remove cell debris, and observe under a microscope. Take pictures of each group and mark them as 0 hours. Add fresh serum-free culture medium and take pictures under a microscope at 24 h and 48 h respectively. Use Photoshop software to count the migration distance of each experimental group.

[0032] (9) Immunofluorescence detection Cells transfected for 48 hours were removed, fixed with 4% paraformaldehyde at room temperature for 20 minutes, permeabilized with 0.2% Triton X-100, and incubated at room temperature for 10 minutes. Cells were then blocked with 5% BSA and incubated at 37°C for 30 minutes. Primary antibodies were added; the optimal concentrations for Ki67 and PCNA monoclonal antibodies were 1:200, and incubation was performed overnight at 4°C. The next day, secondary antibodies (1:200) (goat anti-mouse) were added, and the cells were incubated at room temperature in the dark for 2 hours. DAPI working solution was then added to counterstain the cell nuclei, and the cells were incubated at room temperature in the dark for 10 minutes. After drying, the cells were observed and photographed under a fluorescence microscope. ImageJ software was used for semi-quantitative analysis of the images to calculate the average fluorescence intensity.

[0033] (10) Statistical analyses were performed using GraphPad Prism 10.0 (GraphPad, San Diego, CA, California, USA). All experiments were performed at least three times for each treatment. Two-tailed t-tests were used to compare differences between two groups, and one-way ANOVA was used to compare differences among three or more groups. A p-value ≤ 0.05 was considered statistically significant.

[0034] 2. Experimental Results (1) Expression of LncRNA GSEC in OSCC cell line GSEC expression was detected by qRT-PCR ( Figure 3 A) The results showed that GSEC expression in OSCC cell lines (SCC-25, CAL-27, SCC-9) was significantly higher than that in NHOK ( P <0.05), the expression of GSEC in the CAL-27 cell line was significantly higher than that in the other two OSCC cell lines, so the CAL-27 cell line with relatively high expression was selected for further research.

[0035] (2) Detection of LncRNA GSEC knockdown efficiency after transfection with si-LncRNA GSEC To determine the biological function of GSEC in OSCC, si-GSEC plasmids si-GSEC-1 and si-GSEC-2 were constructed and transfected into CAL-27 cells using si-GSEC-1, si-GSEC-2, and si-NC, respectively. Transfection efficiency was assessed by qRT-PCR. The results showed that si-GSEC-1 had a significantly higher transfection efficiency. P <0.05, Figure 3 B), si-GSEC-1 was selected for subsequent experiments.

[0036] (3) Effects of GSEC on the biological behavior of OSCC cells ①GSEC promotes the proliferation of CAL27 cells and inhibits their early apoptosis. CCK-8 results showed that, compared with si-NC, si-GSEC-1 significantly inhibited cell proliferation in the CAL27 cell line. P <0.05, Figure 4 A) The proliferation of CAL27 cells was significantly inhibited at 24h, 48h, and 72h, with the most significant difference observed at 72h, which was statistically significant. P <0.05, Figure 4 B).

[0037] Flow cytometry experiments showed that si-GSEC-1 significantly promoted apoptosis in CAL27 cells compared to the control group. Cell density reached approximately 60% after 24 hours. Cells were transfected according to groupings, and apoptosis experiments were performed after 48 hours. This demonstrated that si-GSEC-1 significantly promoted early apoptosis. P <0.05, Figure 5 AC).

[0038] ②GSEC promotes the invasion and migration of OSCC cells Transwell assays showed that si-GSEC-1 effectively inhibited the cell invasion ability of CAL27 cells compared to si-NC. P <0.05%. After 48 hours of incubation, the number of cells in the si-GSEC-1 group that invaded the chamber matrix gel was significantly reduced ( P <0.05, Figure 6 (AB). The experimental results suggest that GSEC can significantly enhance the invasive ability of CAL27 cells.

[0039] Scratch assay results showed that the percentage of wound healing distance between scratches in the si-GSEC-1 group was significantly lower than that in the si-NC group, and the cell migration rate was also lower in the si-NC group at both 24h and 48h. P <0.05, Figure 7(AB). This experimental result suggests that GSEC can significantly improve the migration ability of CAL27 cells.

[0040] ③ mRNA level qRT-PCR detection of LncRNA GSEC and proliferation and apoptosis proteins Ki67, PCNA, BCL-2, BAX, and caspase-9 qRT-PCR results showed successful cell transfection. Compared with the si-NC group, the expression levels of apoptosis-related marker proteins BAX and caspase-9 were significantly upregulated in the si-GSEC-1 group, while the expression level of BCL-2 was significantly inhibited. The expression levels of proliferation-related marker proteins Ki67 and PCNA were significantly low, and the differences were statistically significant. P <0.05. This further confirms that GSEC affects the biological behaviors of OSCC cells, such as proliferation and apoptosis, at the mRNA level. Figure 8 ).

[0041] ④ Protein level detection: LncRNA GSEC and proliferation and apoptosis proteins Ki67, PCNA, BCL-2, BAX, and caspase-9 Western blot analysis showed that three proteins, Bax, Bcl-2, and caspase-9, were detected. Compared with the control group, the expression levels of apoptosis-related marker proteins BAX and caspase-9 were significantly upregulated in the si-GSEC-1 group, while the expression level of BCL-2 was significantly inhibited, and the differences were statistically significant. P <0.05, Figure 9 (AD). Protein levels demonstrated that GSEC inhibits apoptosis in OSCC tumors.

[0042] Immunofluorescence was used to detect the proteins Ki67 and PCNA. The results showed that, compared with the control group, the proliferation-related marker proteins Ki67 and PCNA in the si-GSEC-1 group were significantly underexpressed. P <0.05, Figure 10 (AD). Protein levels demonstrated that GSEC promotes tumor growth.

[0043] In summary, this embodiment reveals for the first time the high expression of GSEC in OSCC and its oncogenic function, confirming that it affects tumor progression by regulating the biological behaviors of OSCC cells, such as proliferation, apoptosis, invasion and migration.

[0044] Example 3 Animal Experiment 1. Experimental Methods (1) Cell culture Oral epithelial keratinocytes (NHOK) and oral squamous cell carcinoma lines (SCC-25, CAL-27, SCC-9) (purchased from Procyno). All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco). Cultured further at 37°C in a 5% CO2 incubator.

[0045] (2) Plasmid transfection All plasmids were constructed by Shanghai Hanheng Biotechnology Co., Ltd. GSEC siRNA and negative controls were purchased from Suzhou Hongxun Biotechnology Co., Ltd. OS cells were transfected using Lipofectamine™ 2000 transfection reagent (Thermo Fisher) according to the manufacturer's instructions. Sequences are shown in Table 5.

[0046] Table 5 Sequence List

[0047] (4) HE staining (Hematoxylin and Eosin staining) Fresh tissue was rapidly fixed in 10% neutral buffered formaldehyde for 24 hours. The tissue was then dehydrated stepwise with ethanol solutions of varying concentrations (75%, 85%, 95%, 100%, 100%), each for 1 hour. The tissue was then sequentially immersed in two xylene solutions for clearing (Cavity I: 20 min; Cavity II: 30 min). The tissue was then sequentially immersed in three paraffin baths (Cavity I: 1 hour; Cavity II: 1.5 hours; Cavity III: 2 hours). The tissue was then further immersed in molten paraffin, and after cooling, formed a wax block. The pre-cooled wax block was fixed on a paraffin microtome and sliced ​​into 4 μm thick sections. After attaching the sections to glass slides, they were allowed to air dry slightly, then baked in a 60°C slide oven for 1 hour, followed by baking in an oven for another 1 hour. The slides were immersed in xylene three times, 15-20 minutes each time, to remove paraffin. Then, they were immersed in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes, finally rehydrating with distilled water for 1 minute. The slides were then immersed in hematoxylin staining solution for 5-10 minutes, rinsed with running tap water for 1-2 minutes to remove excess dye. Next, the slides were immersed in eosin staining solution for 1-3 minutes, monitoring the staining intensity under a microscope. The slides were then rinsed with distilled water to remove excess dye. Further dehydration with a gradient of alcohols was performed, followed by xylene clearing. The slides were then mounted with a neutral resin (such as DPX), ensuring the coverslip completely covered the slide without air bubbles. The results were observed under a microscope.

[0048] (5) Western Blotting experiment Total protein was extracted from cultured cells using RIPA cell lysis buffer (Bai Rui Ji). The appropriate concentration of polyacrylamide gel was selected based on the molecular weight of the target protein; in this experiment, the stacking gel concentration was 5%, and the separating gel concentration was 10%. An appropriate amount of protein sample was loaded, and proteins were separated by SDS-PAGE with 10% polyacrylamide gel electrophoresis. The proteins in the gel were then transferred to a PVDF membrane to form a blot. The membrane was incubated overnight at 4°C with the following primary antibodies: Anti-Caspase9Rabbit mAb (A18676) (Wuhan Sanying, China; 1:1000), Anti-Bcl2 Polyclonal antibody (12789-1-AP) (Wuhan Sanying, China; 1:1000), Anti-Bax Polyclonal antibody (50599-2-Ig) (Wuhan Sanying, China; 1:1000), and Anti-β-Actin antibody (ab8227) (abcam, UK; 1:10000). The membrane was then placed in an incubator containing the corresponding secondary antibody (1:8000) (goat anti-rabbit) and incubated at room temperature for 1.5 hours. The protein bands of the PVDF membrane were visualized using an ECL detection kit (Wanleibio). The membrane was photographed using a chemiluminescence imaging and analysis system, and the brightness values ​​of each protein band were analyzed to create a bar chart.

[0049] (6) Immunohistochemical detection Tumor tissues excised from mice in different groups were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and 4μm thick tissue sections were cut using a microtome. The baked sections were dewaxed in xylene, then hydrated to inactivate endogenous peroxidase. Antigen retrieval was performed, followed by inactivation with 3% hydrogen peroxide. Non-specific binding sites were blocked with goat serum. PCNA monoclonal antibody (60097-1-Ig, Wuhan Sanying, China) and Ki67 monoclonal antibody (60097-1-Ig, Wuhan Sanying, China) were used as markers at a concentration of 1:200, and the sections were incubated overnight at 4°C. The next day, the sections were incubated with HRP-labeled goat anti-mouse IgG (BN20602, Bairuiji, China) at 37°C for 30 min to detect the binding of primary antibodies. Slides were washed three times with PBS for 5 min each time. For immunohistochemistry, freshly prepared DAB chromogenic reagent was used for the colorimetric reaction, and the sections were rinsed with distilled water to terminate the reaction. Subsequently, hematoxylin was used for nuclear counterstaining, followed by blue staining in TBS buffer, and then dehydration, clearing, and mounting. Finally, the color development effect was observed under a fluorescence microscope (200x magnification).

[0050] (7) Xenotransplantation experiment Healthy BALB / c-nu 5-week-old mice (purchased from Beijing SPAF Biotechnology Co., Ltd.) were selected and randomly divided into 3 groups of 5 mice each. CAL27, si-NCCAL27, and si-GSECCAL27 cells in the logarithmic growth phase were collected and adjusted to a density of 1.0 × 10⁻⁶ cells using serum-free medium. 7 / ml, 0.2ml of tumor cells were subcutaneously injected into the axilla of each group of mice. From the date of inoculation, the mice's condition and tumor growth were monitored daily. Tumor volume was calculated using the formula V(mm²). 3 )=(LxS 2 (L, longest diameter; S, shortest diameter). Mice were sacrificed on day 13, and subcutaneous tumor tissue was excised, weighed, and photographed. The tumor was then divided into two parts: one part was fixed in 4% formaldehyde solution for HE staining and immunohistochemistry, and the other part was flash-frozen in liquid nitrogen and stored at -80°C for subsequent experiments.

[0051] (8) Statistical analysis Data analysis was performed using Graphpad Prism 10.0 (GraphPad, San Diego, CA, USA). Quantitative data are expressed as mean ± SD. All experiments were performed at least three times in each treatment. T-tests were used for comparisons between two groups, and ANOVA was used for comparisons among multiple groups. Chi-square tests were used for comparisons of categorical data. P A difference of <0.05 is statistically significant.

[0052] 2. Experimental Results (1) Expression of LncRNA GSEC in OSCC cell line GSEC expression was detected by qRT-PCR ( Figure 11 A), the results showed that GSEC expression in OSCC cell lines (SCC25, CAL27, SCC9) was significantly higher than that in NHOK ( P (CAL27 <0.05), CAL27 was significantly higher than the other two OSCC cell lines, so the relatively high-expressing CAL27 cell line was selected for further research.

[0053] (2) Detection of LncRNA GSEC knockdown efficiency after transfection with si-LncRNA GSEC To determine the biological function of GSEC in OSCC, we transfected CAL27 cells with siGSEC-1, siGSEC-2, and si-NC, respectively, to downregulate its expression level, and confirmed the transfection efficiency by qRT-PCR. P <0.01, Figure 11B). It is evident that the si-GSEC-1 transfection cell line has a more significant transfection rate, and this cell line was selected for subsequent experiments.

[0054] (3) Results of animal experiments ① Changes in body weight and tumor volume in nude mice After plasmid transfection, xenograft tumor models were established in nude mice according to the groups. We found that knockdown of GSEC expression significantly inhibited tumor volume and weight. The results showed that all mice inoculated with CAL27, si-NC CAL27, and si-GSEC CAL27 developed tumors after 3 days. After 5 days, the tumor growth rate in the si-GSEC CAL27 group was significantly lower than that in the control group (Figures 12A and 12B). Mice were sacrificed after 13 days, and the tumors were removed and their weight measured. The results showed that the tumor sizes in the CAL27 and si-NC CAL27 groups were relatively similar, while the tumor size in the si-GSEC CAL27 group was significantly smaller than that in the control group. Figure 13 A, B). The above results indicate that GSEC significantly promotes OSCC cell proliferation and tumorigenesis in the in vivo environment. Representative tumor images show that the xenograft tumors in the si-GSEC group were significantly smaller than those in the si-NC group. Furthermore, compared to the si-NC group, siGSEC significantly reduced tumor weight. P <0.05).

[0055] ② HE staining HE staining (hematoxylin and eosin staining) is the most commonly used staining method in histopathology. It selectively stains the nucleus and cytoplasm with two dyes, making the morphology and structure of tissue cells clearly visible. Under a microscope, HE staining results show that in moderately differentiated squamous cell carcinoma, the nucleus appears blue and the cytoplasm red, the tissue structure is disordered, there is increased mitotic figures, and a small number of keratin beads and intercellular bridges are visible. In contrast, the si-GSEC group shows a relatively more orderly tissue structure, fewer cancer cells, better cell differentiation, fewer mitotic figures, and less malignant transformation. Figure 14 ).

[0056] ③Western blot experiment Western blot analysis showed that three proteins, Bax, Bcl-2, and caspase-9, were detected. Compared with the control group, the expression levels of apoptosis-related marker proteins BAX and caspase-9 were significantly upregulated in the si-GSEC-1 group, while the expression level of BCL-2 was significantly inhibited, and the differences were statistically significant. Figure 15 (AC). Protein levels demonstrated that GSEC has an inhibitory effect on tumor apoptosis.

[0057] ④ Immunohistochemical results showed Immunohistochemical analysis showed that the expression of proliferation-related marker proteins Ki67 and PCNA in the transplanted tumor tissue was significantly lower than that in the control group. Figure 16 (A, B) Ki67 antibody specifically recognizes proliferation-related proteins in the cell nucleus. Immunohistochemical staining showed that Ki67-positive expression in tumor cells was localized in the nucleus, appearing as brownish-yellow granules. Positive cells showed heterogeneous distribution within the cancer nest, with higher density in the peripheral and poorly differentiated areas, and fewer in the central keratinized and necrotic areas. PCNA antibody specifically recognizes PCNA protein involved in DNA replication in the cell nucleus, with positive signals appearing as brownish-yellow granules distributed in the nucleus. These were mainly concentrated in the proliferative areas at the periphery of the cancer nest, while fewer or no positive cells were found in the central, mature differentiation area. The expression of Ki67 and PCNA was significantly reduced in the si-GSEC group compared to the control group, indicating that si-GSEC reduced tumor cell proliferation activity, thus suggesting that GSEC has a function of promoting tumor growth in vivo.

[0058] This study focuses on the role of lncRNA GSEC in OSCC, finding that it is significantly overexpressed in OSCC cell lines, a result consistent with the aberrant expression patterns of many tumor-associated lncRNAs. These findings suggest that GSEC plays a crucial role in the development and progression of OSCC. In functional validation experiments, successful knockdown of GSEC using siRNA technology resulted in significant inhibition of tumor growth in a nude mouse xenograft model, directly indicating that GSEC promotes OSCC tumor growth. From a mechanistic perspective, analysis of proliferation and apoptosis-related proteins revealed that knockdown of GSEC upregulated the expression of pro-apoptotic proteins BAX and Caspase-9, while inhibiting the expression of the anti-apoptotic protein Bcl-2, suggesting that GSEC may regulate cell fate through the mitochondrial apoptosis pathway. Caspase-9 activation further confirms the key role of this pathway, consistent with previous reports of lncRNAs influencing apoptosis through regulation of mitochondrial membrane potential. Simultaneously, the expression of proliferation-related proteins Ki67 and PCNA was significantly reduced, revealing the regulatory role of GSEC in cell cycle progression, potentially affecting tumor cell proliferation by influencing the formation of the DNA replication-related complex. These changes strongly suggest that GSEC may influence the biological behavior of OSCC by regulating apoptosis and proliferation-related pathways, thereby promoting tumor development.

[0059] In summary, this invention provides a new perspective for a deeper understanding of the pathogenesis of OSCC and confirms the potential of lncRNA GSEC as a therapeutic target. This invention is the first to reveal the association between GSEC and the Bcl-2 family regulatory network, providing a new perspective for targeted therapy of OSCC—developing small molecule inhibitors targeting the GSEC-Bcl-2 axis may break down tumor cell apoptosis resistance. This study found a positive correlation between GSEC expression levels and the Ki67 index, suggesting that it can serve as a biomarker for prognostic assessment. Future research should explore its mechanism of action more deeply and actively develop GSEC-targeted therapies to open new avenues for precision medicine in OSCC, ultimately improving patient prognosis.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of LncRNA GSEC in the preparation of drugs for the treatment of oral squamous cell carcinoma.

2. The application according to claim 1, characterized in that: The nucleotide sequence of the LncRNA GSEC is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that: The drug contains an active ingredient that can inhibit the expression or function of LncRNA GSEC.

4. The application according to claim 3, characterized in that: The active ingredient is one or more of siRNA, shRNA, or small molecule inhibitors.

5. The application according to any one of claims 1-4, characterized in that: The drug is used to inhibit the proliferation, migration and / or invasion of OSCC cells, and / or induce apoptosis.

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