Use of lncrna gsec in preparation of a drug for treating oral squamous cell carcinoma

Abstract

The application 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 comprises an active ingredient capable of inhibiting expression or function of the LncRNA GSEC, the active ingredient 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 application firstly discloses that the LncRNA GSEC is specifically highly expressed in oral squamous cell carcinoma tissues and cells, and the expression level of the LncRNA GSEC is significantly positively correlated with adverse clinical parameters such as tumor T stage and lymph node metastasis, and it is confirmed that the LncRNA GSEC is a novel molecular target with clinical application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of LncRNA GSEC as a target point in preparation of a drug for treating oral squamous cell carcinoma. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of head and neck with local invasion and high recurrence. Although great progress has been made in the treatment, the 5-year survival rate of OSCC has not been significantly improved. The main reason for the poor prognosis of OSCC is that the early symptoms of most patients are not obvious, which is often confused with oral ulcer, so that the patients are usually diagnosed in the middle and late stages, and the local recurrence or distant metastasis rate after the initial treatment is high. Therefore, it is of great significance to develop reliable biomarkers and find drug treatment targets for early diagnosis and effective evaluation of prognosis of OSCC.

[0003] Long non-coding RNA (LncRNA) is a class of RNA with no protein-coding ability and more than 200 nucleotides in length. As an important competing endogenous RNA (ceRNA), it plays a role as a miRNA sponge in the body. LncRNA can regulate gene expression and function at the transcriptional, translational, and post-translational levels to exert various biological effects. In the past decade, many studies have confirmed that LncRNA can participate in the development of various diseases such as malignant tumors, metabolic diseases, and cardiovascular diseases by regulating gene expression in recipient cells through cell-to-cell communication. 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 colon cancer and promotes the migration of colon cancer by antagonizing DHX36. Some researchers have found that the expression level of LncRNA GSEC is up-regulated in osteosarcoma cell lines, and 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 shown that the LncRNA GSEC / miR-101-3p axis has a related functional role in the environment of lung adenocarcinoma (LUAD) and has proposed a possible biomarker for the clinical diagnosis and treatment of the 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 lncRNA GSEC / 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 been disclosed in the prior art. SUMMARY

[0004] The application provides application of LncRNA GSEC in preparation of a drug for treating oral squamous cell carcinoma.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0006] The application provides application of LncRNA GSEC in preparation of a drug for treating oral squamous cell carcinoma.

[0007] Further, the nucleotide sequence of the LncRNA GSEC is shown as SEQ ID NO: 1.

[0008] Further, the drug comprises an active ingredient capable of inhibiting expression or function of the LncRNA GSEC.

[0009] Further, the active ingredient is one or more of siRNA, shRNA or a small molecule inhibitor.

[0010] Further, the drug is used for inhibiting proliferation, migration and / or invasion of the OSCC cells, and / or inducing apoptosis of the cells.

[0011] Compared with the prior art, the application has the following beneficial effects:

[0012] The application first discloses that LncRNA GSEC is specifically highly expressed in oral squamous cell carcinoma tissues and cells, and the expression level is significantly positively correlated with tumor T stage, lymph node metastasis and other adverse clinical parameters, which confirms that it is a new molecular target with clinical application potential. This provides a new direction and idea for overcoming the current difficulties of limited treatment methods and poor prognosis of OSCC. The cell level experiment proves that the expression of LncRNA GSEC can be significantly inhibited by siRNA and other technologies, which can significantly inhibit the proliferation, migration and invasion ability of the OSCC cells, and effectively induce apoptosis of the cells. This shows that the intervention of the target can directly reverse the malignant phenotype of the tumor. The animal level experiment proves that in the nude mouse tumor model, the expression of LncRNA GSEC can significantly inhibit the growth of the tumor, which is manifested as significant reduction of the tumor volume and weight, and the treatment potential is verified in vivo. Based on this, the LncRNA GSEC is taken as a target to develop an efficient OSCC treatment drug, which can provide a candidate strategy for the OSCC targeted treatment scheme. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 GSEC expression in OSCC cancer and paracancer tissues, wherein A: GSEC expression in OSCC cancer tissues and paracancer tissues, B: GSEC expression in OSCC cancer tissues and paracancer matched tissues;

[0014] Figure 2 Correlation of GSEC with clinical parameters of OSCC, wherein, A: the expression of GSEC in OSCC tissue and the relationship with T stage, B: the expression of GSEC in OSCC tissue and the relationship with lymph node metastasis, C: the expression of GSEC in OSCC tissue and the relationship with age, D: the expression of GSEC in OSCC tissue and the relationship with gender, E: the expression of GSEC in OSCC tissue and the relationship with high / low differentiation;

[0015] Figure 3 Expression of GSEC in NHOK and OSCC cell lines, wherein, A: expression of GSEC in NHOK and OSCC cell lines, B: transfection efficiency of si-GSEC;

[0016] Figure 4 CCK8 cell proliferation experiment results, wherein, A: column chart of proliferation results of OSCC cells after transfection of si-GSEC-1 / si-NC at 24h, 48h, 72h, B: line chart of proliferation results of OSCC cells after transfection of si-GSEC-1 / si-NC at 24h, 48h, 72h;

[0017] Figure 5 Flow cytometry experiment results, wherein, A: apoptosis experiment results of OSCC cells after transfection of si-NC, B: apoptosis experiment results of OSCC cells after transfection of si-GSEC-1, C: comparison of apoptosis experiment results of OSCC cells after transfection of si-NC / si-GSEC-1;

[0018] Figure 6 Transwell experiment results, wherein, A: comparison of Transwell experiment results of OSCC cells after transfection of si-NC / si-GSEC-1, B: Transwell experiment results chart of OSCC cells after transfection of si-NC / si-GSEC-1;

[0019] Figure 7 Scratch experiment results, wherein, A: scratch experiment results chart of OSCC cells after transfection of si-NC / si-GSEC-1 at 0h, 24h, 48h, B: comparison of scratch experiment results of OSCC cells after transfection of si-NC / si-GSEC-1 at 0h, 24h, 48h;

[0020] Figure 8 mRNA level qRT-PCR detection of expression levels of apoptosis-related marker proteins BAX, caspase-9, BCL-2 and proliferation-related marker proteins ki67, pcna after transfection of si-NC / si-GSEC-1;

[0021] Figure 9Results of Western blot detection of Bax, Bcl-2 and caspase-9, wherein 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;

[0022] Figure 10 Results of immunofluorescence experiment detection of ki67 and pcna, wherein A: results of immunofluorescence experiment detection of pcna after transfection of si-NC / si-GSEC-1 in OSCC cells, B: results of immunofluorescence experiment detection of pcna after transfection of si-NC / si-GSEC-1 in OSCC cells, C: results of immunofluorescence experiment detection of ki67 after transfection of si-NC / si-GSEC-1 in OSCC cells, D: results of immunofluorescence experiment detection of ki67 after transfection of si-NC / si-GSEC-1 in OSCC cells;

[0023] Figure 11 Expression of GSEC in NHOK and OSCC cell lines in tumor-bearing nude mice experiment, wherein A: expression of GSEC in NHOK and OSCC cell lines, B: transfection efficiency of si-GSEC;

[0024] Figure 12 Results of tumor volume and body weight of tumor-bearing nude mice, wherein A: results of tumor volume at different time points, B: results of body weight of nude mice at different time points;

[0025] Figure 13 Comparison results of tumors of nude mice in different groups, wherein A: comparison chart of results of body weight of tumor-bearing nude mice in different groups, B: tumorigenesis of nude mice in different groups;

[0026] Figure 14 HE staining results of tumors of nude mice in different groups;

[0027] Figure 15 Results of Western blot detection of Bax, Bcl-2 and caspase-9 in tumors of nude mice in different groups, wherein A: expression level of Bax, B: expression level of Bcl-2, C: expression level of caspase-9;

[0028] Figure 16 Results of ki67 and pcna immunohistochemistry of tumors of nude mice in different groups, wherein A: results of ki67 immunohistochemistry, B: results of pcna immunohistochemistry.

[0029] In the figure, ns P >0.05, P <0.05, P<0.01, *** P <0.001, **** P <0.0001. DETAILED DESCRIPTION

[0030] In order to make the objectives and advantages of the present application clearer and more apparent, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are intended to explain the present application and are not intended to limit the present application.

[0031] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are all conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are all conventional experimental methods, detection methods, etc. already existing in the prior art.

[0032] The present application will be illustrated by the following examples of tissue, cell and animal level experiments to clarify the molecular mechanism of LncRNA GSEC affecting cell proliferation, migration, etc. in the process of OSCC, and LncRNA GSEC may be a new molecular marker of OSCC, which provides a basis for finding drug treatment targets of OSCC in the future, and provides a reference for early diagnosis, prognosis evaluation and treatment of OSCC.

[0033] Example 1 Tissue Experiment

[0034] 1. qRT-PCR detection of the expression of target lncRNA GSEC in OSCC tissue specimens

[0035] The tissue was cut into small pieces, and 10 mg was weighed. The total RNA was extracted using TriQuick Reagent reagent (Solabio, Beijing) according to the manufacturer's instructions. Then, the total RNA was reverse transcribed using SureScriptTM First-Strand cDNA Synthesis Kit Cat. (GeneCopeia, USA) according to the manufacturer's instructions to obtain cDNA. We used 2xSYBR Green qPCR Master Mix (None ROX) reagent (Servicebio, Wuhan) to perform quantitative real-time PCR (qRT-PCR) according to the manufacturer's instructions, and used actin (Actin) as a standard control. The threshold cycle 2 DDCt method was used to determine the relative expression level of the target gene. The primer sequences used in this example are shown in Table 1.

[0036] Table 1 Primer design

[0037]

[0038] 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).

[0039] 2. Correlation between the expression of target lncRNA GSEC in OSCC tissue specimens and clinical parameters

[0040] 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.

[0041] 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).

[0042] Table 2. Correlation between GSEC expression and clinical parameters in OSCC tissues

[0043]

[0044] Example 2 Cell Experiment

[0045] 1. Experimental Methods

[0046] (1) Cell culture

[0047] 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.

[0048] (2) Plasmid transfection

[0049] All plasmids were constructed by Shanghai Hanheng Biotechnology Co., Ltd. GSEC siRNA and negative control were purchased from Suzhou Hongxin Biotechnology Co., Ltd. Transfection of OS cells was performed using Lipofectamine™ 2000 transfection reagent (Thermo Fisher) according to the manufacturer's instructions. The sequences are shown in Table 3.

[0050] Table 3 Sequence table

[0051]

[0052] (3) RNA isolation and qRT-PCR

[0053] Total RNA was extracted using TriQuick Reagent reagent (Solebao, Beijing) according to the manufacturer's instructions. Then, total RNA was reverse transcribed using 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 2xSYBR Green qPCR Master Mix (None ROX) reagent (Servicebio, Wuhan) according to the manufacturer's instructions, and actin (Actin) was used as a standard control. The threshold cycle 2 ΔΔCt method was used to determine the relative expression level of the target gene. The primers used in this example are shown in Table 4.

[0054] Table 4 Primer design

[0055]

[0056] (4) Western Blotting experiment

[0057] Total protein was extracted from cultured cells using RIPA cell lysis buffer (Beyotime). The appropriate amount of protein sample was loaded for SDS denaturation 10% polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins. The proteins in the gel were transferred to the PVDF membrane to form a blot. Place in the incubation box containing the first antibody Anti-Caspase9 Rabbit mAb (A18676) (Sanying, China; 1:1000), Anti-Bcl2 Polyclonal antibody (12789-1-AP) (Sanying, China; 1:1000), Anti-Bax Polyclonal antibody (50599-2-Ig) (Sanying, China; 1:1000), Anti-β-Actin antibody (ab8227) (abcam, UK; 1:10000), 4°C shaking overnight. Then place the membrane in the incubation box containing the corresponding secondary antibody (1:8000) (goat anti-rabbit), room temperature for 1.5 h. The PVDF membrane was visualized with the ECL detection kit (Wanleibio), and the membrane was photographed with the chemiluminescence imaging and analysis system. The brightness values of the protein bands in each group were analyzed, and a column chart was drawn.

[0058] (5) Cell counting kit-8 (CCK-8) detection

[0059] CCK-8 (Bi Yun Tian, Shanghai) was used to detect cell proliferation, and the manufacturer's instructions were followed. According to the experimental grouping, transfection was performed, and 24 h later, digestion and counting were performed, and 1000 cells were inoculated into each well of a 96-well plate. After 24 / 48 / 72 h, CCK8 experiment was performed, and 10 μL CCK-8 solution was added to each well, and incubated in the cell incubator for 1 h. Finally, the absorbance at 450 nm was detected using a microplate reader.

[0060] (6) Flow cytometry analysis

[0061] Cells were inoculated into a 6-well plate so that the cell density reached about 60% after 24 h, and transfection was performed according to the grouping. After 48 h, the cells were collected for apoptosis experiment. Discard the supernatant, PBS rinse once, use trypsin without EDTA to digest the cells, collect the cells with 1 ml complete medium, centrifuge at 1500 rpm for 5 min, resuspend the cell pellet with pre-cooled PBS, centrifuge at 1500 rpm for 5 min, carefully aspirate the supernatant, resuspend the cells with 1×Binding Buffer to make the cell density reach 1×10 6 5 ​Each tube was added with 5 μΐ Annexin V-FITC and 5 μΐ PI Solution, mixed gently. Incubated at room temperature (25℃) for 15 min in the dark. Added 400 μΐ 1x Binding Buffer to each tube, mixed gently, and completed the machine detection within 1 h.

[0062] (7) Transwell experiment

[0063] Invasion assay was performed using Nunc 24-well 8.0 μm pore transwell plates (Thermo Fisher Scientific). Matrigengel was polymerized into a thin film in the chamber. 500 μΐ of 20% FBS medium was added to the lower chamber of the 24-well plate. Then the transwell chamber was placed in the 24-well plate with forceps, 200 μΐ of cell suspension with a density of 3 x 10 5 / ml was added to the upper chamber of the transwell, and after incubation in a 37℃ incubator for 48 h, 4% paraformaldehyde was used for fixation, and 0.1% crystal violet was used for staining for 10 min. The invasive cells were counted under 100x magnification, and 3 fields of view were randomly selected for each sample, and the average value was taken.

[0064] (8) Wound healing experiment

[0065] According to the cell grouping, the cells were transfected, and when the cell density reached about 90%, a 200 μΐ pipette tip was used to vertically scratch the cell plane, the supernatant was discarded, and PBS was rinsed once to remove cell debris. The cells were observed under a microscope, photographed, and marked as 0 h. Fresh serum-free medium was added, and the cells were taken out at 24 h and 48 h for microscopic photography and recording. The migration distance of each experimental group was counted by Photoshop software.

[0066] (9) Immunofluorescence detection

[0067] After 48 h of transfection, the cells were taken out, 4% paraformaldehyde was added for room temperature fixation for 20 min, 0.2% Triton X-100 was added for cell permeation, and the cells were incubated at room temperature for 10 min. 5% BSA was used for blocking treatment, and the cells were incubated at 37℃ for 30 min. The primary antibody was added, and the optimal antibody concentration of Ki67 monoclonal antibody was 1:200, and the optimal concentration of PCNA monoclonal antibody was 1:200. Incubate overnight at 4℃. The next day, the secondary antibody (1:200) (goat anti-mouse) was added, and the cells were incubated at room temperature in the dark for 2 h. DAPI working solution was added to restain the cell nucleus, and the cells were incubated at room temperature in the dark for 10 min. After air drying, the cells were observed and photographed under a fluorescence microscope, and the average fluorescence intensity was calculated by semi-quantitative analysis of the images using ImageJ software.

[0068] (10) Statistical analysis was performed using GraphPad prism 10.0 (GraphPad, San Diego, CA, USA). All experiments were performed at least 3 times in each treatment. Differences between two groups were compared using a two-tailed t-test, and differences between three or more groups were compared using one-way ANOVA. P≦0.05 was considered statistically significant.

[0069] 2. Experimental results

[0070] (1) Expression of LncRNA GSEC in OSCC cell lines

[0071] The expression of GSEC was detected by qRT-PCR Figure 3 A), and the results showed that the expression of GSEC in OSCC cell lines (SCC-25, CAL-27, SCC-9) was significantly higher than that in NHOK P <0.05), and the expression of GSEC in 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 study.

[0072] (2) Detection of LncRNA GSEC knockdown efficiency after transfection of si-LncRNA GSEC

[0073] To determine the biological function of GSEC in OSCC, si-GSEC plasmids si-GSEC-1 and si-GSEC-2 were constructed, and CAL-27 cells were transfected with si-GSEC-1, si-GSEC-2 and si-NC, respectively, and the transfection efficiency was detected by qRT-PCR. The results showed that the transfection efficiency of si-GSEC-1 was more significant P <0.05, Figure 3 B), and si-GSEC-1 was selected for later experiments.

[0074] (3) Effect of GSEC on biological behavior of OSCC cells

[0075] ①GSEC promotes CAL27 cell proliferation and inhibits early apoptosis

[0076] CCK-8 results showed that compared with si-NC, si-GSEC-1 significantly inhibited cell proliferation in CAL27 cell line P <0.05, Figure 4 A), and the proliferation ability of CAL27 cells was significantly inhibited at 24h, 48h and 72h, and the difference at 72h was the most obvious, which was statistically significant P <0.05, Figure 4 B).

[0077] Flow cytometry showed that si-GSEC-1 can significantly promote the apoptosis of CAL27 cells compared with the control group. The cell density reached about 60% after 24h, according to the grouping, the cells were collected for apoptosis experiment after 48h, and it was found that si-GSEC-1 had a significant effect on promoting early apoptosis (Fig. 4A). P <0.05, Figure 5 A-C).

[0078] ②GSEC promotes the invasion and migration of OSCC cells

[0079] Transwell experiment showed that si-GSEC-1 can effectively inhibit the invasion ability of CAL27 cells compared with si-NC (Fig. 5A). P <0.05). After 48h of incubation, the number of cells invading the Matrigel of the si-GSEC-1 group was significantly reduced (Fig. 5B). P <0.05, Figure 6 A-B). The experimental results suggest that GSEC can significantly enhance the invasion ability of CAL27 cells.

[0080] The results of scratch test showed that the percentage of wound healing distance of si-GSEC-1 group was significantly lower than that of si-NC group, and the cell migration rate was lower than that of si-NC group at 24h and 48h (Fig. 6A-B). P <0.05, Figure 7 A-B). The experimental results suggest that GSEC can significantly enhance the invasion ability of CAL27 cells.

[0081] ③mRNA level qRT-PCR detection of LncRNA GSEC and proliferation, apoptosis proteins ki67, pcna, BCL-2, BAX, caspase-9

[0082] The results of qRT-PCR detection showed that the cell transfection was successful, compared with si-NC group, the expression level of apoptosis related marker protein BAX, caspase-9 in si-GSEC-1 group was significantly up-regulated, while the expression level of BCL-2 was significantly inhibited, the expression of proliferation related marker protein ki67, pcna was significantly lower, the difference was statistically significant (Fig. 7A-B). P <0.05). Further confirmed that GSEC affects the biological behavior of OSCC cells such as proliferation and apoptosis at mRNA level (Fig. 7C). Figure 8 .

[0083] ④ Protein level detection of LncRNA GSEC and proliferation, apoptosis proteins ki67, pcna, BCL-2, BAX, caspase-9

[0084] Western blot experiment 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 in the si-GSEC-1 group were significantly up-regulated, while the expression level of BCL-2 was significantly inhibited, and the difference was statistically significant (P < 0.05). P <0.05, Figure 9 A-D). Protein levels prove that GSEC has an inhibitory effect on OSCC tumor apoptosis.

[0085] Immunofluorescence detected two proteins, ki67 and pcna. The experimental results showed that, compared with the control group, the proliferation-related marker proteins ki67 and pcna in the si-GSEC-1 group were significantly low expressed (P < 0.05). P <0.05, Figure 10 A-D). Protein levels prove that GSEC has an inhibitory effect on OSCC tumor apoptosis.

[0086] In summary, this embodiment first discloses the high expression of GSEC in OSCC and its pro-cancer function, and confirms that it affects tumor progression by regulating the biological behaviors of OSCC cell proliferation, apoptosis, invasion and migration, etc.

[0087] Example 3 Animal experiment

[0088] 1. Experimental method

[0089] (1) Cell culture

[0090] Oral epithelial keratinocytes NHOK, oral squamous cell carcinoma cell lines (SCC-25, CAL-27, SCC-9) (purchased from Promocell). All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco). Continue to be cultured in a 37°C, 5% carbon dioxide concentration incubator.

[0091] (2) Plasmid transfection

[0092] All plasmids were constructed by Shanghai Hanheng Biotechnology Co., Ltd. GSEC siRNA and negative control were purchased from Suzhou Hongxin Biotechnology Co., Ltd. Transfection of OS cells used Lipofectamine™ 2000 transfection reagent (Thermo Fisher) and followed the manufacturer's instructions. The sequence is shown in Table 5.

[0093] Table 5 Sequence list

[0094]

[0095] (4) HE staining (Hematoxylin and Eosin Staining)

[0096] Fresh tissue was quickly placed in 10% neutral buffered formalin for 24 hours. Tissue was dehydrated through graded ethanol solutions (75%, 85%, 95%, 100%, 100%) for 1 h each; tissue was cleared in 2 xylene baths (I bath for 20 min; II bath for 30 min). Tissue was infiltrated with paraffin in 3 paraffin baths (1 h in first bath, 1.5 h in second bath, 2 h in third bath); tissue was then placed in molten paraffin and allowed to cool to form a paraffin block. The pre-cooled paraffin block was mounted on a microtome and sections were cut at 4 μm thickness. Sections were attached to glass slides and allowed to air dry for a short period of time, then placed in a 60 °C slide warmer for 1 h and then placed in an oven for 1 h. Sections were deparaffmized by placing in xylene for 3 times for 15-20 min each, then placed in absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and finally distilled water for 1 min. Sections were stained in hematoxylin for 5-10 min, rinsed in running tap water for 1-2 min to remove excess stain. Sections were stained in eosin for 1-3 min, monitored under a microscope for intensity of staining, and rinsed in distilled water to remove excess stain. Sections were dehydrated through graded alcohols, cleared in xylene, and mounted with a neutral resin (e.g., DPX) to ensure that the coverslip covers the entire section without air bubbles. Sections were observed under a microscope.

[0097] (5) Western Blotting experiments

[0098] Total protein was extracted from cultured cells using RIPA cell lysis buffer (Beyotime). According to the molecular weight of the target protein, the corresponding concentration of polyacrylamide gel was selected, and the concentration of the concentrated gel in this experiment was 5%, and the concentration of the separation gel was 10%. Take the appropriate amount of protein sample and load it into the 10% polyacrylamide gel for SDS denaturation. The proteins in the gel were transferred to the PVDF membrane to form a print. Place it in an incubation box containing the first antibody Anti-Caspase9 Rabbit mAb (A18676) (Sanying, China; 1:1000), Anti-Bcl2 Polyclonal antibody (12789-1-AP) (Sanying, China; 1:1000), Anti-Bax Polyclonal antibody (50599-2-Ig) (Sanying, China; 1:1000), Anti-β-Actin antibody (ab8227) (abcam, UK; 1:10000), 4°C shaking overnight. Then put the membrane in the incubation box containing the corresponding secondary antibody (1:8000) (goat anti-rabbit), and incubate at room temperature for 1.5 h. The PVDF membrane was visualized with the ECL detection kit (Wanleibio), and the membrane was photographed with the chemiluminescence imaging and analysis system. The brightness values of the protein bands in each group were analyzed, and a column chart was drawn.

[0099] (6) Immunohistochemical detection

[0100] Tumor tissues removed from different groups of mice were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and cut into 4 μm thick tissue sections with a microtome. The baked sections were deparaffinized in xylene, then hydrated and endogenous peroxidase was inactivated. Antigen retrieval, 3% hydrogen peroxide inactivation, goat serum blocking of non-specific binding sites, PCNA monoclonal antibody (60097-1-Ig, Sanying, China) and Ki67 monoclonal antibody (60097-1-Ig, Sanying, China) were used as markers, with a concentration of 1:200, and the sections were incubated at 4°C overnight. The next day, the sections were incubated with HRP-labeled goat anti-mouse IgG (BN20602, Beyotime, China) at 37°C for 30 min to detect the bound primary antibody. The glass slides were washed with PBS three times, each for 5 min. For immunohistochemistry, freshly prepared DAB color developing agent was used for color development, the sections were rinsed with distilled water to stop the reaction. Then, the cell nuclei were counterstained with hematoxylin, and then dehydrated, transparent, and mounted in TBS buffer, and finally observed under a fluorescence microscope (200x magnification) for color development.

[0101] (7) Xenotransplantation experiment

[0102] 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.

[0103] (8) Statistical analysis

[0104] 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.

[0105] 2. Experimental Results

[0106] (1) Expression of LncRNA GSEC in OSCC cell line

[0107] 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 CAL27 cell line with relatively high expression was selected for further research.

[0108] (2) Detection of LncRNA GSEC knockdown efficiency after transfection with si-LncRNA GSEC

[0109] 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 11 B). 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.

[0110] (3) Results of animal experiments

[0111] ① Changes in body weight and tumor volume in nude mice

[0112] 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).

[0113] ② HE staining

[0114] 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 ).

[0115] ③Western blot experiment

[0116] 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 15A-C). Protein level proved that GSEC had the effect of inhibiting apoptosis on tumor.

[0117] IV. Immunohistochemical results showed

[0118] Immunohistochemical analysis showed that the expression of proliferation-related marker proteins ki67 and PCNA in transplanted tumor tissues was significantly lower than that in the control group Figure 16 A, B). Ki67 antibody specifically recognizes proliferation-related proteins in the nucleus. Immunohistochemical staining showed that tumor cell Ki67 positive expression was located in the nucleus and showed brownish yellow granules. Positive cells were heterogeneously distributed in cancer nests, with higher density in the edge area and poorly differentiated area, and fewer in the central keratinization area and necrotic area. PCNA antibody specifically recognizes PCNA protein involved in DNA replication in the nucleus, and the positive signal is brownish yellow granules distributed in the nucleus. Mainly concentrated in the edge of the cancer nest proliferation area, and fewer positive cells or negative in the central differentiated mature area. The expression of Ki67 and PCNA in the si-GSEC group was significantly reduced compared with the control group, indicating that si-GSEC reduced the proliferation activity of tumor cells, and further indicating that GSEC had the function of promoting tumor growth in vivo.

[0119] This example focuses on the role of LncRNAGSEC in OSCC, and finds that it is significantly highly expressed in OSCC cell lines, which is consistent with the abnormal expression pattern of many tumor-related LncRNAs. The above research results suggest that GSEC plays an important role in the occurrence and development of OSCC. In the functional verification experiment, after successfully knocking down GSEC using si-RNA technology, the nude mouse transplanted tumor model showed a significant inhibition of tumor growth, which directly indicates that GSEC has a promoting effect on OSCC tumor growth. From the mechanism exploration level, through the detection of proliferation and apoptosis-related proteins, it was found that after knocking down GSEC, the expression of pro-apoptotic proteins BAX and Caspase-9 was up-regulated, and the expression of anti-apoptotic protein Bcl-2 was inhibited, suggesting that it may regulate cell fate through the mitochondrial apoptosis pathway. The activation of Caspase-9 further confirms the key role of this pathway, which is consistent with the mechanism of lncRNA affecting cell apoptosis by regulating mitochondrial membrane potential reported in previous studies. At the same time, the expression of proliferation-related proteins Ki67 and PCNA was significantly reduced, revealing the regulatory effect of GSEC on cell cycle progression, which may affect tumor cell proliferation by affecting the formation of DNA replication-related complexes. This series of changes fully demonstrate that GSEC may affect the biological behavior of OSCC by regulating apoptosis and proliferation-related pathways, and further promote the development of tumors.

[0120] In summary, the present application provides a new perspective for understanding the pathogenesis of OSCC, and confirms the possibility of LncRNA GSEC as a potential diagnosis and treatment target. The present application first reveals the association of GSEC with the Bcl-2 family regulatory network, which provides a new perspective for the targeted therapy of OSCC - developing small molecule inhibitors targeting the GSEC-Bcl-2 axis may break the apoptosis resistance of tumor cells. The positive correlation between the expression level of GSEC and the Ki67 index found in this study suggests that it can be used as a biomarker for prognosis evaluation. Further research should focus on the mechanism of action and actively develop treatment methods targeting GSEC to open up new ways for the precision medicine of OSCC and ultimately improve the prognosis of patients.

[0121] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of an active ingredient for inhibiting the expression of LncRNA GSEC in the preparation of a drug for treating oral squamous cell carcinoma, characterized in that: The nucleotide sequence of the LncRNA GSEC is shown as SEQ ID NO:1; the active ingredient is siRNA specifically targeting the LncRNA GSEC; the sense strand of the siRNA is shown as SEQ ID NO:6, and the antisense strand of the siRNA is shown as SEQ ID NO:7; the drug is used for inhibiting the proliferation, migration and / or invasion of OSCC cells, and / or inducing cell apoptosis.

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