CN119818681A - Application of PAX 9 agonist in preparation of medicines for preventing or treating head and neck squamous cell carcinoma - Google Patents

Abstract

The present invention provides the use of a PAX 9 agonist in the preparation of a method for preventing or treating head and neck squamous cell carcinoma. The present invention establishes stable overexpression of PAX 9 in SNU 1076 cells through cell experiments, and uses CCK-8 to detect the proliferation ability of cells in the PAX 9 overexpression group and the control group, and the proliferation ability of cells in the overexpression group is significantly weakened. Animal experiments show that the significant deceleration of tumor growth in the PAX 9 overexpression group is demonstrated by measuring tumor volume; and the HE, Ki 67, Bax and Bcl-2 staining results in tumor tissues show that the expression level of ki-67 in the overexpression group is significantly reduced, Bax expression is increased, and Bcl-2 expression is reduced, indicating that PAX 9 has an inhibitory effect on tumor cell proliferation and promotes the induction of tumor cell apoptosis.

Description

Application of PAX 9 agonist in preparation of medicines for preventing or treating head and neck squamous cell carcinoma

Technical Field

The invention relates to the field of biological medicine, in particular to application of PAX 9 agonist in preparation of a medicine for preventing or treating head and neck squamous cell carcinoma.

Background

Head and neck squamous cell carcinoma includes squamous cell carcinoma occurring in the mouth, oropharynx, nasopharynx, larynx and hypopharynx. The epidemiological data issued by national cancer centers of about 93 ten thousand new cases and 46.7 ten thousand (Sung H,Ferlay J,Siegel RL,et al.Global cancer statistics 2020:GLOBOCAN estimates ofincidence and mortality worldwide for 36cancers in 185countries[J].CACancer JClin,2021,71(3):209-249.).2022 years of death cases are shown, wherein 13 ten thousand new cases are issued each year in China, the death rate is about 7 ten thousand, the overall incidence rate of lips, oral cavities and pharynx is highest, the incidence rate of men is higher than that of women (Zheng RS,Zhang SW,Zeng HM,et al.Cancer incidence and mortality in China,2016[J].JNatl Cancer Cent,2022,2(1):1-9.)., head and neck squamous cell carcinoma (HEAD ANDNECK squamous cell carcinoma, HNSCC) accounts for about 90% of head and neck malignant tumors, and 70% -80% of head and neck squamous cell carcinoma patients are locally advanced at the time of initial diagnosis. The recurrence rate of the patients with local advanced stage is up to 40% -60% within 2 years of comprehensive treatment

(Lorch JH,Goloubeva O,HaddadRI,et al.Induction chemotherapy with cisplatin and fluorouracil alone or in combination with docetaxel in locally advanced squamous-cell cancer ofthe head andneck:long-term results ofthe TAX 324randomisedphase 3trial[J].Lancet Oncol,2011,12(2):153-159.). For patients with recurrent or metastatic head and neck squamous cell carcinoma, median survival time after traditional chemotherapy or targeted therapy is about (Winquist E,Agbassi C,Meyers BM,et al.Systemic therapy in the curative treatment ofhead and neck squamous cell cancer:a systematic review[J].J Otolaryngol HeadNeck Surg,2017,46(1):29.). years

Biological behavior, clinical manifestations, responses to treatment and prognosis of head and neck squamous cell carcinoma show significant heterogeneity, whereas the high invasiveness of head and neck squamous cell carcinoma hampers improvement of overall survival of patients. Thus, an insight into its etiology and pathogenesis, as well as the identification of novel diagnostic and prognostic biomarkers and molecular targets for therapeutic intervention, is of great clinical significance in improving its prognosis.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the use of a PAX 9 agonist in the preparation of a medicament for the prevention or treatment of squamous cell carcinoma of the head and neck.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

use of a PAX 9 agonist in the preparation of a medicament for the prevention or treatment of head and neck squamous cell carcinoma.

PAX 9 gene coding is a transcription factor, in embryonic development and organogenesis. PAX 9 has been found to play a very important role in the initiation and differentiation of teeth (Chu KY,WangYL,Chen JT,Lin CH,Yao CJ,Chen YJ,Chen HW,Simmer JP,Hu JC,Wang SK.PAX9 mutations and genetic synergismin familial tooth agenesis.AnnNYAcad Sci.2023Jun;1524(1):87-96.;Bhol CS,Mishra SR,Patil S,Sahu SK,Kirtana R,Manna S,Shanmugam MK,Sethi G,Patra SK,Bhutia SK.PAX9 reactivationby inhibiting DNAmethyltransferase triggers antitumor effect in oral squamous cell carcinoma.Biochim Biophys ActaMol Basis Dis.2022Sep

1;1868 (9): 166428.). The PAX 9 sequence is as follows:

ATGGAGCCAGCCTTCGGGGAGGTGAACCAGCTGGGAGGAGTGTTCGTGAACGGG

AGGCCGCTGCCCAACGCCATCCGGCTTCGCATCGTGGAACTGGCCCAACTGGGCA

TCCGACCGTGTGACATCAGCCGCCAGCTACGGGTCTCGCACGGCTGCGTCAGCAA

GATCCTGGCGCGATACAACGAGACGGGCTCGATCTTGCCAGGAGCCATCGGGGGC

AGCAAGCCCCGGGTCACTACCCCCACCGTGGTGAAACACATCCGGACCTACAAGC

AGAGAGACCCCGGCATCTTCGCCTGGGAGATCCGGGACCGCCTGCTGGCGGACG

GCGTGTGCGACAAGTACAATGTGCCCTCCGTGAGCTCCATCAGCCGCATTCTGCG

CAACAAGATCGGCAACTTGGCCCAGCAGGGTCATTACGACTCATACAAGCAGCAC

CAGCCGACGCCGCAGCCAGCGCTGCCCTACAACCACATCTACTCGTACCCCAGCC

CTATCACGGCGGCGGCCGCCAAGGTGCCCACGCCACCCGGGGTGCCTGCCATCCC

CGGTTCGGTGGCCATGCCGCGCACCTGGCCCTCCTCGCACTCCGTCACCGACATC

CTGGGCATCCGCTCCATCACCGACCAAGTGAGCGACAGCTCCCCCTACCACAGCC

CCAAGGTGGAGGAGTGGAGCAGCCTGGGCCGCAACAACTTCCCCGCCGCCGCCC

CGCACGCGGTGAACGGGTTGGAGAAGGGAGCCCTGGAGCAGGAAGCCAAGTAC

GGTCAGGCACCAAATGGTCTCCCAGCTGTGGGCAGTTTTGTGTCAGCATCCAGCA

TGGCTCCTTACCCTACCCCAGCCCAAGTGTCGCCTTACATGACCTACAGTGCTGCT

CCTTCTGGTTATGTTGCTGGACATGGGTGGCAACATGCTGGGGGCACCTCATTGTC

TCCCCACAACTGTGACATTCCGGCATCGCTGGCGTTCAAGGGAATGCAGGCAGCC

AGAGAAGGTAGTCATTCTGTCACGGCTTCCGCGCTCTGA。

According to one embodiment of the invention, the use is to inhibit head and neck squamous cell carcinoma cell proliferation, invasion, migration and tumorigenesis.

The beneficial effects are that:

The invention provides the use of a PAX9 agonist in the manufacture of a medicament for the prevention or treatment of squamous cell carcinoma of the head and neck. According to the invention, through a cell experiment, stable over-expression of PAX9 is established in SNU1076 cells, and the proliferation capacities of cells of a PAX9 over-expression group and a control group are detected by using CCK-8, so that the proliferation capacities of cells of the over-expression group are obviously reduced. The cell experiments of the invention show that the clone forming capacity of the PAX9 over-expressed SNU1076 cells is obviously reduced, the migration capacity of the PAX9 over-expressed cells is obviously reduced within 24 hours, and meanwhile, the migration capacity and invasion of the PAX9 over-expressed cells are obviously reduced. The inventors also revealed by western blot and qPCR analysis studies that up-regulation of pro-apoptotic protein Bax and relative down-regulation of anti-apoptotic protein Bcl-2 in PAX9 groups overexpressed by SNU1076 cells, suggesting that PAX9 may promote the apoptotic process in SNU 1076. The inventors also established a subcutaneous xenograft model of PAX 9-overexpressing SNU1076 cells, demonstrated a significant reduction in tumor growth in the PAX9 over-expression group by measuring tumor volume, whereas HE, ki 67, bax and Bcl-2 staining results in tumor tissue showed a significant reduction in the level of expression of Ki-67 in the over-expression group, increased Bax expression, decreased Bcl-2 expression, demonstrating that PAX9 has an inhibitory effect on tumor cell proliferation and promotes induction of tumor cell apoptosis.

It was found that PAX 9 overexpression has an important impact on head and neck squamous cell carcinoma cell proliferation, invasion, migration and tumor formation, and PAX 9 agonists have the potential to be prepared for preventing or treating head and neck squamous cell carcinoma.

Drawings

FIG. 1 is a graph of the results of stable overexpression of PAX 9 in SNU 1076 cells;

FIG. 2 is a graph showing the results of the detection of proliferation potency of cells of PAX 9 over-expression group and control group using CCK-8;

FIG. 3 is a graph of the clonogenic potential of PAX9 overexpressed SNU1076 cells;

FIG. 4 is a graph of migration capacity results of PAX 9 overexpressing cells over 24 hours;

FIG. 5 is a graph of the results of cell migration and invasion capacity over-expressing PAX 9;

FIG. 6 is a graph of the relative down-regulation results of up-regulation of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in the PAX9 group;

FIG. 7 is a graph of the results of a subcutaneous xenograft model of PAX 9-overexpressing SNU 1076 cells;

FIG. 8 is a representative graph of HE, ki 67, bax and Bcl-2 staining patterns in tumor tissue.

Detailed Description

The present invention is described in detail below by way of specific examples, which are given herein for the purpose of further illustration only and are not to be construed as limiting the scope of the present invention, as many insubstantial modifications and variations of the present invention will become apparent to those skilled in the art in light of the foregoing disclosure. The raw materials and the reagents used in the invention are all commercial products.

Example 1 cell experiment

1. Human laryngeal squamous cell carcinoma cell lines (SNU 1076) were divided into two groups, a control group (transfected with control lentiviral vector) and a PAX 9 over-expression group (transfected with lentiviral vector-mediated PAX 9 over-expression). The SNU 1076 cells under optimal growth conditions were seeded in 6-well plates at a density of 7.5×10 ⁴ cells/well. 24 hours after inoculation, when cell confluence reached about 30%, lentiviruses were transfected using moi=50. To increase transfection efficiency, a concentration of 5ug/ml of polybrene was added. 24 hours after lentivirus transfection, the medium was replaced with complete medium, and thereafter daily. After 72 hours of transfection, about 70% transfection efficiency was achieved under fluorescent microscopy, puromycin was introduced at a concentration of 5ug/ml to select resistant cells. After two rounds of screening, puromycin concentration was halved while continuing to select to obtain stable cell lines for subsequent experiments.

2. Total protein was extracted from the laryngeal squamous cell carcinoma cell line and its concentration was determined using the BCA protein concentration determination kit according to the manufacturer's instructions. Subsequently, an equal volume of protein (30. Mu.g) was separated by electrophoresis on a 12% SDS-PAGE gel and then transferred to a 0.22 μm PVDF membrane. The film was sealed in a quick seal solution for 15 minutes at room temperature. Then, it was combined with an anti-PAX 9 antibody (1:500; # AG2804, beyotime), a BAX antibody (1:500; # WL01637, wan Lei Biotech), a bcl-2 antibody (1:500; # WL01556, wan Lei Biotech) and a β -actin antibody (1:2000; # R23613 Zenbio). After 10min each, the membrane was incubated with a second antibody (#a0208, beyotime) for 2 hours at room temperature and then washed 3 more times with TBST for 10min each. Finally, enhanced chemiluminescence (ECL, P0018 AS, beyotime) was used to detect protein signals. Relative protein expression levels were normalized to the level of β -actin and quantified automatically using ImageJ software.

3. The over-expression and vector groups were divided into 5 experimental groups, 24-hour, 48-hour, 72-hour, 96-hour and 120-hour groups, depending on the culture time. Cells (8×10 ³/well) were inoculated into 96-well plates for incubation. Subsequently, at each respective time point of 24h, 48h, 72h, 96h and 120h, the respective group was added with CCK-8 (C0041; beyotime; china), in a volume of 10. Mu.l/well. After incubation in an incubator at 37 ℃ for 4 hours, absorbance values at 450nm were measured using SpectraMax iD 5 (Molecular Devices).

4. The ability of laryngeal squamous cell carcinoma to migrate horizontally was assessed by a wound healing experiment. Initially, cells were plated uniformly onto 6-well plates at a density of 1 x 10 ⁶ cells/well. After 24 hours, the scratched areas were observed and compared by culturing the cells in serum-free medium for an additional 24 hours. To determine vertical migration and intrusion capability, transwell analysis was used. For migration experiments, a total of 2×10 ⁵ cells were seeded into the upper chamber of a Transwell, which had an 8 μm pore membrane, a volume of 200 μl, and the lower chamber contained 600 μl of medium supplemented with 20% fbs. After incubation for 24 hours, cells migrating to the lower surface were fixed, stained with a solution containing 0.5% crystal violet, photographed and counted. To assess invasive capacity, a gel matrix (C0372; beyotime; china) was applied to the Transwell membrane prior to performing a procedure similar to that used in the migration test.

Experimental results:

To investigate the specific role of PAX 9 in laryngeal squamous cell carcinoma, the inventors selected the SNU 1076 cell line, which had relatively low expression levels of PAX 9 compared to other HNSCC cell lines. First, the inventors successfully established stable overexpression of PAX 9 in SNU 1076 cells, as demonstrated by western blot analysis (fig. 1). In addition, the proliferation capacity of cells in the PAX 9 overexpressing group was significantly reduced (fig. 2), and their clonogenic capacity was significantly reduced (fig. 3). In addition, a significant decrease in both migration capacity (fig. 4) and invasion capacity (fig. 5) was observed in cells overexpressing PAX 9. These findings suggest that PAX 9 may exert tumor-inhibiting functions in HNSCC by participating in processes such as cell proliferation, migration, and invasion. Further studies by western blot analysis revealed an up-regulation of pro-apoptotic protein Bax and a relative down-regulation of anti-apoptotic protein Bcl-2 in the overexpressed group of SNU 1076 cells (fig. 6), indicating that PAX 9 can promote the apoptotic process in SNU 1076. The results of this study demonstrate that PAX 9 has an inhibitory effect on proliferation, migration and invasion of SNU 1076 cells while promoting apoptosis of these cells.

FIG. 1 is the establishment of stable overexpression of PAX 9 in SNU 1076 cells, confirmed by Western blot and qPCR analysis.

FIG. 2 shows that the proliferation capacity of cells of the PAX 9 over-expression group and the control group is detected by CCK-8, and the proliferation capacity of cells of the over-expression group is obviously reduced.

Figure 3 illustrates that PAX9 overexpressed SNU1076 cells had significantly reduced clonogenic capacity.

Figure 4 illustrates that PAX 9 overexpressing cells have significantly reduced migration capacity within 24 hours.

Figure 5 illustrates a significant reduction in cell migration capacity and invasion of PAX 9 over-expressed cells.

Fig. 6 reveals an up-regulation of pro-apoptotic protein Bax and a relative down-regulation of anti-apoptotic protein Bcl-2 in PAX9 groups overexpressed by SNU 1076 cells by western blot and qPCR analysis studies, suggesting that PAX9 can promote the apoptotic process in SNU 1076.

Example 2 animal experiments

Establishment of nude mice xenograft tumor model A control group (SNU 1076 cells transfected with control vector) was inoculated subcutaneously with nude mice at a concentration of 1X 10 ⁷ cells/cell (150. Mu.l cell suspension) to establish a xenograft tumor mouse subcutaneous tumor model. Tumor size (length L and width W) was measured weekly and tumor volume V was calculated using the formula v=l×w2/2 to obtain a tumor survival curve. Four weeks later, mice were euthanized by anesthesia, followed by choking with high concentration CO 2. Tumor tissue was excised for immunohistochemical analysis to determine Ki-67, bax and Bcl-2 expression levels.

The data were processed using SPSS21.0 software. Measurement data are expressed as mean ± Standard Deviation (SD) and are statistically analyzed using t-test. A p-value of less than 0.05 is considered to be statistically significant.

Experimental results:

Examining the effect of PAX9 on subcutaneous tumor formation in vivo, the inventors established a subcutaneous xenograft model of SNU 1076 cells overexpressing PAX9, demonstrating a significant reduction in tumor growth in the PAX9 overexpressing group, exhibiting statistically significant differences. In addition, a significant reduction in ki-67 expression levels was observed in tumor tissue from the over-expression group, consistent with the results obtained from in vitro experiments. In addition, bax expression was increased and Bcl-2 expression was decreased in tumor tissues derived from the over-expression group. In vivo experiments also prove that PAX9 has an inhibiting effect on tumor cell proliferation and promotes the induction of tumor cell apoptosis.

Fig. 7 establishes a subcutaneous xenograft model of PAX 9-overexpressing SNU 1076 cells. Significant reduction in tumor growth in PAX 9 over-expressed groups was demonstrated by measuring tumor volume. FIG. 8 is a representative graph of HE, ki 67, bax and Bcl-2 staining patterns in tumor tissue with significantly reduced levels of expression of the overexpression group Ki-67, increased Bax expression, reduced Bcl-2 expression, demonstrating that PAX 9 has an inhibitory effect on tumor cell proliferation and promotes induction of tumor cell apoptosis.

Claims (5)

1. Use of a PAX 9 agonist in the preparation of a drug for preventing or treating head and neck squamous cell carcinoma; the PAX 9 sequence is as follows: ATGGAGCCAGCCTTCGGGGAGGTGAACCAGCTGGGAGGAGTGTTCGTGAACGGGAGGCCGCTGCCCAACGCCATCCGGCTTCGCATCGTGGAACTGGCCCAACTGGGCATCCGACCGTGTGACATCAG CCGCCAGCTACGGGTCTCGCACGGCTGCGTCAGCAAGATCCTGGCGCGATACAACGAGACGGGCTCGATCTTGCCAGGAGCCATCGGGGGCAGCAAGCCCCGGGTCACTACCCCCACCGTGGTGAAAC ACATCCGGACCTACAAGCAGAGAGACCCCGGCATCTTCGCCTGGGAGATCCGGGACCGCCTGCTGGCGGACGGCGTGTGCGACAAGTACAATGTGCCCTCCGTGAGCTCCATCAGCCGCATTCTGCGC AACAAGATCGGCAACTTGGCCCAGCAGGGTCATTACGACTCATACAAGCAGCACCAGCCGACGCCGCAGCCAGCGCTGCCCTACAACCACATCTACTCGTACCCCAGCCCTATCACGGCGGCGGCCGCC AAGGTGCCCACGCCACCCGGGGTGCCTGCCATCCCCGGTTCGGTGGCCATGCCGCGCACCTGGCCCTCCTCGCACTCCGTCACCGACATCCTGGGCATCCGCTCCATCACCGACCAAGTGAGCGACAG CTCCCCCTACCACAGCCCCAAGGTGGAGGAGTGGAGCAGCCTGGGCCGCAACAACTTCCCCGCCGCCGCCCCGCACGCGGTGAACGGGTTGGAGAAGGGAGCCCTGGAGCAGGAAGCCAAGTACGGTCA GGCACCAAATGGTCTCCCAGCTGTGGGCAGTTTTGTGTCAGCATCCAGCATGGCTCCTTACCCTACCCCAGCCCAAGTGTCGCCTTACATGACCTACAGTGCTGCTCCTTCTGGTTATGTTGCTGGAC ATGGGTGGCAACATGCTGGGGGCACCTCATTGTCTCCCCACAACTGTGACATTCCGGCATCGCTGGCGTTCAAGGGAATGCAGGCAGCCAGAGAAGGTAGTCATTCTGTCACGGCTTCCGCGCTCTGA. 2. The use according to claim 1, characterized in that: the use is to inhibit the proliferation ability of head and neck squamous cell carcinoma cells. 3. The use according to claim 1, characterized in that: the use is to inhibit the invasive ability of head and neck squamous cell carcinoma cells. 4. The use according to claim 1, characterized in that: the use is to inhibit the migration ability of head and neck squamous cell carcinoma cells. 5. The use according to any one of claims 1 to 4, characterized in that the use is to inhibit the tumor formation ability of head and neck squamous cell carcinoma.