Hypopharyngeal cancer is a subtype of the head and neck malignancies. We aimed to explore the role of lysine-specific demethylase 1 (LSD1/KDM1A) in the progression of hypopharyngeal cancer and to identify the potential mechanisms. First, LSD1 expression in head and neck squamous cell carcinoma (HNSCC) tissues and the correlation between LSD1 and the stage of HNSC were analyzed by the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Following LSD1 silencing, proliferation of pharyngeal cancer cell line FaDu cells was evaluated by cell counting kit-8 and colony formation assays. Wounding healing and transwell assays were used to measure the capacities of migration and invasion. In addition, expression of proteins related to epithelial-to-mesenchymal transition (EMT), autophagy, and pyroptosis was tested by Western blot analysis or immunofluorescence. After treatment with autophagy inhibitor 3-methyladenine (3-MA) or NLR family pyrin domain containing 3 (NLRP3) inhibitor MCC950, the malignant biological properties were measured again. High LSD1 expression was observed in HNSC tissues, which was correlated with stage. LSD1 knockdown significantly suppressed the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. Moreover, autophagy and pyroptosis were induced by LSD1 depletion, observed by the enhanced fluorescence intensity of LC3, gasdermin-D (GSDMD)-N, and apoptosis-associated speck-like protein containing a CARD (ASC), accompanied by upregulated expression of LC3II/LC3I, Beclin-1, NLRP3, cleaved-caspase 1, ASC, interleukin (IL)-1β, and IL-18 and downregulated expression of p62. Importantly, 3-MA or MCC950 addition obviously reversed the inhibitory effects of LSD1 silencing on the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. To sum up, LSD1 silencing could restrain the progression of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.
Keywords: Autophagy, epithelial-to-mesenchymal transition, hypopharyngeal cancer, invasion, lysine-specific demethylase 1, pyroptosis
|How to cite this URL:|
Wang H, Liu F. LSD1 silencing inhibits the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of hypopharyngeal cancer cells by inducing autophagy and pyroptosis. Chin J Physiol [Epub ahead of print] [cited 2023 May 29]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=377442
| Introduction|| |
Hypopharyngeal cancer is a subtype of the head and neck malignancies, which arises from the mucosa of the upper aerodigestive tract. Among head and neck carcinomas, such as oropharyngeal cancer, oral cancer, and laryngeal cancer, hypopharyngeal cancer which accounts for approximately 3% of all head and neck malignancies is less common than other cancers. Hypopharyngeal cancer is often difficult to diagnose in its early stage because of its insidious location. More than 75% of patients diagnosed with hypopharyngeal cancer are in advanced stages and often develop late regional lymph node metastases or distant metastases, which directly affects the prognosis of the disease., Therefore, understanding the underlying oncogenic mechanisms of hypopharyngeal cancer is crucial to advancing therapeutic strategies for hypopharyngeal cancer.
Lysine-specific demethylase 1 (LSD1, also known as KDM1A) was characterized in 2004 as the first histone demethylase. Substantial evidence exists to suggest that LSD1 is significantly upregulated in various cancer types, such as esophageal cancer, non-small cell lung cancer, and gastric cancer.,, Within these cancers, LSD1 is often associated with advanced cancer stage and poor prognosis. Importantly, as a flavin adenine dinucleotide-dependent demethylase, LSD1 is highly expressed in head and neck squamous cell carcinomas (HNSCC) originating in the hypopharynx and elevated LSD1 expression correlated with advanced tumor stage and poor progression-free survival. Compelling evidence indicate that inducing autophagy contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma (HSCC). LC3-II and Beclin-1, two important marker proteins of autophagy, are downregulated in HSCC, and their abnormal expression is associated with poor prognosis of this disease. A growing body of literature has shown that LSD1 could negatively regulate autophagy to affect cancer progression in multiple cancers, such as ovarian cancer and prostate cancer., It is worthy of note that activating Gasdermin-E (GSDME)-dependent pyroptosis contributes to the treatment of HNSCC. However, the effects of LSD1 on the malignant tumor progression of hypopharyngeal cancer and the potential mechanisms related to autophagy and pyroptosis remain to be elucidated.
In the present study, it was hypothesized that LSD1 was overexpressed in laryngeal cancer, and LSD1 depletion might suppress the malignant progression of this disease. The mechanisms of LSD1 related to autophagy and pyroptosis were explored in the subsequent experiments.
| Materials and Methods|| |
Online database analysis
LSD1 expression in HNSC tissues (Primary tumor (n = 520)) and normal tissues (Normal (n = 44)) as well as the correlation between LSD1 and the stages of HNSC were analyzed by The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN; http://ualcan.path.uab.edu/).
Cell culture and treatment
The pharyngeal cancer cell line FaDu provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Rockford, IL, USA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
Two small hairpin RNAs (sh-RNA) specific to LSD1 (sh-LSD1-1 and sh-LSD1-2) and the negative control (sh-NC) were constructed by GenePharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was applied for the transfection of these plasmids into FaDu cells. After 24 h, reverse transcription-quantitative polymerase chain reaction (qPCR) and Western blot analysis were used to detect the transfection efficacy.
Cell viability assay
The viability of FaDu cells was evaluated by the Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China). A total of 5×103 FaDu cells were seeded in a 96-well plate. After the indicated treatment, 10 μL CCK-8 reagent was put into each well for further cultivation. 4 h later, a microplate reader (Molecular Devices, USA) was adopted for the assessment of absorbance at 450 nm.
Colony formation assay
The proliferation ability of FaDu cells was determined by means of colony formation assay. Briefly, FaDu cells (500 cells/well) were seeded in 6-well plates. After incubation in DMEM for two weeks, cells were fixed by methanol and stained with 0.5% crystal violet solution for 30 min. Colonies (>50 cells) were counted manually using an inverted light microscope (Olympus, Japan).
Wound healing assay
The migrated capability of FaDu cells was detected using of wound healing assay. Briefly, cells at the logarithmic phase were seeded in 6-well plates, following which was the cultivation until 95% cell fusion was achieved. Then, the cell monolayer was gently scratched in a straight line with a disposable pipette tip. After washed three times with phosphate buffer solution (PBS), FaDu cells were cultivated in serum-free DMEM at 37°C. At 0 and 24 h, images of the wound areas were pictured using a light microscope (Olympus, Japan) and the width of the open area was immediately measured using ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA) to estimate motility.
Using the Matrigel invasion assay, the capacity of FaDu cell invasion was evaluated. FaDu cells resuspended in 200 μL serum-free medium were inoculated in the upper chamber which coated with Matrigel (8 μm pore size). 600 μL of DMEM containing 10% FBS was added to the lower chamber to act as the chemoattractant. After 24 h incubation, the cells in the bottom of the invasion chamber were fixed in 4% paraformaldehyde, stained with crystal violet and then photographed by a light microscope (Olympus, Japan).
GFP-LC3 plasmid was obtained from GENOMEDITECH Co., LTD, (Shanghai, China). FaDu cells were seeded in glass bottom cell culture dishes. After 24 h, Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, California, USA) was used for transient transfection in the light of the manufacturer's protocol for 24 h. FaDu cells were subjected to 4% paraformaldehyde fixation at 4°C for 15min and then permeabilized with 0.2% Triton X-100 at 37°C for 30 min. Subsequently, the PBS-rinsed cells were impeded with 3% bovine serum albumin (BSA). The cells were incubated with primary antibody against GSDMD-N at 4°C overnight, after which was the probe with 100μl/well working solution containing Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody. Thereafter, cell nuclear were counterstained applying 4',6-diamidino-2-phenylindole. With the adoption of a confocal microscope (Olympus, Japan), the stained slides were photographed.
Immunofluorescent staining for ASC
To visualize ASC speck formation in response to different treatments, FaDu cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton X-100 for 30 min at 37°C. Then, cells were blocked with 3% BSA and incubated with primary antibody against ASC at 4°C overnight. Afterward, Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody was used to incubate FaDu cells. The slides were monitored using a confocal microscope (Olympus, Japan).
Enzyme-linked immunosorbent assay
The levels of interleukin (IL)-1β (Cat. No. F01220) and IL-18 (Cat. No. F01460) were examined by specific enzyme-linked immunosorbent assay kits (Shanghai WestTang Bio-Tech Co., LTD.) according to the manufacturer's instructions. The optical density values were determined at 450 nm by a microplate reader (Molecular Devices, USA).
Reverse transcription-quantitative polymerase chain reaction
With the application of TRIzol reagent (Guangzhou Saiyan Biotechnology Co., Ltd.), the extraction of total RNA from FaDu cells was carried out. Reverse transcription was performed to generate complementary DNA by means of PrimeScript RT reagent kit (TaKaRa, Dalian, China). Afterward, qPCR was implemented with iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Inc.) in light of recommended specifications in an ABI 7500 system (Applied Biosystems, Carlsbad, CA, USA). Primers pairs used in this study were as follows: LSD1 forward, 5'-GCTTGGCCAACCTCTCAGAA-3' and reverse, 5'-GACAGTGTCAGCTTGTCCGTTG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-GGAGCGAGATCCCTCCAAAAT-3' and reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'. Relative gene expression was calculated using the 2−ΔΔCT method. GAPDH served as an internal reference.
Western blot analysis
For the extraction of total protein, cells were homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). And then, the concentration of protein samples was detected by the Bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific). The separation of equal amounts of 40 μg protein samples in each group was carried out adopting 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were then separated and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking in 5% nonfat milk for 2 h at room temperature, PVDF membranes were incubated with primary antibodies at 4°C overnight and then incubated with secondary antibody at room temperature for 1 h. An enhanced chemiluminescence kit (Beyotime) was used to show the protein bands, which were quantified by ImageJ 1.8.0 software. GAPDH was used as an internal control.
Data from three independent replicates were presented as mean ± standard deviation (SD). GraphPad Prism 8.0.1 (San Diego, CA, USA) was used for the statistical analysis. The difference between the two groups was estimated with the Student's t-test. Comparisons among multiple groups were conducted by one-way ANOVA followed by Tukey's post hoc test. Statistical significance was achieved when P < 0.05.
| Results|| |
LSD1 expression is notably upregulated in HNSC tissues
First, LSD1 expression in HNSC tissues was analyzed using the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) database. As shown in [Figure 1]a, LSD1 level was significantly enhanced in the HNSC tissues (primary tumor (n = 520)) when compared to the normal group (n = 44). It was also observed in [Figure 1]b that LSD1 expression was associated with stage of HNSC. These findings indicate that LSD1 expression was notably upregulated in HNSC tissues.
|Figure 1: LSD1 expression was notably upregulated in HNSC tissues. (a) LSD1 expression in HNSC tissues and (b) the connection between LSD1 expression and stages were analyzed using UALCAN database. ***P < 0.001 versus normal. LSD1: Lysine-specific demethylase 1.|
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LSD1 silencing inhibits the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of FaDu cells
To explore the role of LSD1 in the progression of hypopharyngeal cancer, LSD1 was silenced by transfection with LSD1-1/2. As displayed in [Figure 2]a and [Figure 2]b, LSD1 level was noticeably downregulated after transfection as comparison to the sh-NC group. Cells transfected in sh-LSD1-2 presented the lower LSD1 expression, which was selected to perform the subsequent experiments. LSD1 depletion significantly inhibited the proliferation of FaDu cells, as exhibited in [Figure 2]c and [Figure 2]d. In [Figure 3]a and [Figure 3]b, the capacities of migration and invasion were reduced in the sh-LSD1 group when compared to the sh-NC group. Meantime, as what is observable from [Figure 3]c, LSD1 knockdown decreased matrix metalloproteinase (MMP) 2, MMP9, N-cadherin, Vimentin, and Snail expression and increased E-cadherin expression. These results demonstrate that LSD1 silencing inhibits the malignant biological behaviors of FaDu cells.
|Figure 2: LSD1 silencing inhibits the proliferation of FaDu cells. (a) LSD1 mRNA expression was evaluated by means of RT-qPCR. (b) LSD1 protein expression was tested by Western blot analysis. (c) Cell viability was determined by CCK-8 assay. (d) Cell proliferation was analyzed using colony formation assay. Data from three independent replicates were presented as mean ± SD. ***P < 0.001 versus sh-NC. LSD1: Lysine-specific demethylase 1, SD: Standard deviation, sh-NC: Small hairpin-negative control, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.|
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|Figure 3: LSD1 depletion suppresses the migration, invasion, and EMT of FaDu cells. (a) Cell migration was assessed by wound healing assay. (b) Cell invasion was tested by transwell assay. (c) Western blotting was used to determine the expression of MMP2, MMP9, E-cadherin, N-cadherin, Vimentin and Snail. Data from three independent replicates were presented as mean ± standard deviation (SD). ***P < 0.001 versus sh-NC. LSD1: Lysine-specific demethylase 1, EMT: Epithelial-to-mesenchymal transition, sh-NC: Small hairpin-negative control, MMP: Matrix metalloproteinase.|
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LSD1 depletion induces autophagy and pyroptosis of FaDu cells
It has been reported that inducing autophagy and pyroptosis are closely related to the process of HSCC., Inducing GSDME-dependent pyroptosis is considered a new therapeutic idea to treat in HNSCC., First, the effects of LSD1 knockdown on the autophagy were measured with the help of immunofluorescent staining. As exhibited in [Figure 4]a, cells in the sh-LSD1 group presented the stronger fluorescence intensity than that in the sh-NC group. Consistently, LC3II/LC3I and Beclin-1 protein expression in the silenced group was obviously upregulated, accompanied by downregulated expression of p62 [Figure 4]b and [Figure 4]c. Furthermore, results of [Figure 5]a, [Figure 5]b, [Figure 5]c showed that LSD1 depletion enhanced GSDMD-N fluorescence intensity and elevated NLR family pyrin domain containing 3 (NLRP3), cleaved-caspase 1, and apoptosis-associated speck-like protein containing a CARD (ASC) expression when compared to the sh-NC group. In addition, as shown in [Figure 5]d, the aggregation of ASC was substantially increased after LSD1 depletion when compared to the sh-NC group. Consistently, LSD1 silencing significantly elevated the levels of IL-1β and IL-18 relative to the sh-NC group [Figure 5]e and [Figure 5]f. These observations reveal that LSD1 deletion induces autophagy and pyroptosis of FaDu cells.
|Figure 4: LSD1 depletion induces the autophagy of FaDu cells. (a) The expression of GFP-LC3 was examined with immunofluorescent staining. (b and c) Western blotting was adopted for the measurement of LC3 II/LC3 I, Beclin-1 and p62 expression. Data from three independent replicates were presented as mean ± SD. ***P < 0.001 versus sh-NC. LSD1: Lysine-specific demethylase 1, sh-NC: Small hairpin-negative control, SD: Standard deviation.|
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|Figure 5: LSD1 silencing induces the pyroptosis of FaDu cells. (a) GSDMD-N fluorescence intensity was evaluated by immunofluorescent staining. (b and c) The expression of proteins related to pyroptosis was determined by Western blot analysis. (d) ASC expression was determined by immunofluorescent staining. The levels of (e) IL-1β and (f) IL-18 were measured using ELISA. Data from three independent replicates were presented as mean ± SD. ***P < 0.001 versus sh-NC. LSD1: Lysine-specific demethylase 1, sh-NC: Small hairpin-negative control, SD: Standard deviation, ELISA: Enzyme-linked immunosorbent assay.|
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LSD1 silencing suppresses the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of FaDu cells by inducing autophagy and pyroptosis
By using autophagy inhibitor 3-methyladenine (3-MA) or NLRP3 inhibitor MCC950, whether LSD1 silencing could regulate proliferation of FaDu cells by inducing autophagy and pyroptosis was investigated in the following experiments. Results from CCK-8 and colony formation assays suggested that 3-MA or MCC950 treatment markedly reversed the impact of LSD1 knockdown on the proliferation of FaDu cells [Figure 6]a and [Figure 6]b. Simultaneously, migration and invasion of FaDu cells were also promoted by 3-MA or MCC950 addition as comparison to the sh-LSD1 group [Figure 7]a and [Figure 7]b. In addition, 3-MA or MCC950 intervention upregulated MMP2, MMP9, N-cadherin, Vimentin, and Snail expression and downregulated E-cadherin expression in FaDu cells transfected with sh-LSD1 [Figure 7]c. Overall, these data suggest that LSD1 silencing suppresses the progression of FaDu cells by inducing autophagy and pyroptosis.
|Figure 6: LSD1 silencing suppresses the proliferation of FaDu cells by inducing autophagy and pyroptosis. (a) Cell viability was tested using CCK-8 assay. (b) Colony formation assay was used to detect the ability of cell proliferation. Data from three independent replicates were presented as mean ± SD. ***P < 0.001 versus sh-NC; ##P < 0.01, ###P < 0.001 versus sh-LSD1. LSD1: Lysine-specific demethylase 1, sh-NC: Small hairpin-negative control, SD: Standard deviation.|
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|Figure 7: LSD1 silencing suppresses the migration, invasion, and EMT of FaDu cells by inducing autophagy and pyroptosis. (a) The capacity of cell migration was assessed by wound healing assay. (b) Cell invasion was measured using transwell assay. (c) Western blotting was used to determine the expression of MMP2, MMP9, E-cadherin, N-cadherin, Vimentin and Snail. Data from three independent replicates were presented as mean ± SD. ***P < 0.001 versus sh-NC; #P < 0.05, ##P < 0.01, ###P < 0.001 versus sh-LSD1. LSD1: Lysine-specific demethylase 1, EMT: Epithelial-to-mesenchymal transition, sh-NC: Small hairpin-negative control, MMP: Matrix metalloproteinase, SD: Standard deviation.|
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| Discussion|| |
Hypopharyngeal cancer is a malignant tumor originating from the mucosal epithelial tissue of the larynx, accounting for about 3% of head and neck malignant tumors. The anatomy of the inferior pharynx is hidden and the early clinical symptoms of hypopharyngeal cancer are not obvious, which lead to misdiagnosis of this disease. In recent years, the incidence of hypopharyngeal cancer is on the rise, and there is a trend of younger age. Despite great advances in diagnosis and treatment, the 5-year overall survival rate of hypopharyngeal cancer is only 30%–35%. Therefore, it is of great importance to investigate the potential mechanism of hypopharyngeal carcinoma. The important role of LSD1 in the progression of hypopharyngeal cancer has been explored in this study. We found that LSD1 was highly expressed in HNSC tissues. The further mechanism exploration experiments showed that LSD1 silencing could inhibit the malignant biological properties of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.
In recent years, an increasing number of researches have paid attention to the role of invasion, distant metastasis, and EMT in malignant tumor and tumor metastasis remains the leading cause of cancer-related death in hypopharyngeal cancer., EMT is a complex reprogramming process of epithelial cells, in which epithelial tumor cells lose epithelial polarity, adhesion, and motility and translate to a mesenchymal phenotype. This process includes the loss of epithelial cell markers, such as E-cadherin and the increase of mesenchymal components, such as N-cadherin, Vimentin, and Snail., As an important early event of tumor metastasis, EMT is the main mode of metastasis and invasion of most malignant tumors., MMP2 and MMP9 are expressed in cancer cells during malignant invasion and migration, which are proteolytic enzymes that degrade extracellular matrix and can induce cancer cells to permeate the basement membrane. Therefore, suppression of tumor cell invasion and metastasis might become a new treatment for hypopharyngeal cancer. As the first histone demethylase reported in 2004, LSD1 has been demonstrated to highly express in hypopharyngeal cancer and elevated LSD1 expression indicated poor prognosis in this disease. Consistently, in this study, from the analysis of UALCAN database, LSD1 level was significantly enhanced in the HNSC tissues and was associated with stage of HNSC. Another previous study has demonstrated that LSD1 ablation could repress cancer stem cell-like characteristics in HNSCC in vitro and suppress tumorigenicity in vivo in immune-deficient xenografts. By facilitating EMT, LSD1 has been reported to promote migration and invasion in the progression of bladder cancer, gastric cancer, and cervical cancer.,, In the present study, LSD1 silencing inhibited the migration, invasion, and EMT of hypopharyngeal cancer cells, reflected by the decrease in MMP2, MMP9, N-cadherin, Vimentin, and Snail expression and the increase in E-cadherin.
It has been reported that inducing autophagy contributes to the increase of cisplatin sensitivity in HSCC cells., LC3-II and Beclin-1, two important marker proteins of autophagy, are downregulated in HSCC, and their abnormal expression is associated with poor prognosis of this disease. A growing body of literature has shown that LSD1 could negatively regulate autophagy to affect cancer progression in ovarian cancer and prostate cancer., By regulating autophagy network, LSD1 plays an important role in the treatment of various diseases. In addition, the novel LSD1 inhibitor ZY0511 inhibits the proliferation of diffuse large B-cell lymphoma by inducing apoptosis and autophagy. Pyroptosis is a newly discovered type of programmed cell death initiated by inflammasomes, accompanied by inflammatory and immune responses. Upon activation, NLRP3 oligomerizes and binds to adaptor protein ASC, which then recruits caspase-1 to form NLRP3 inflammasome. Caspase-1 is well known as a protease that can be activated by NLRP3 inflammasomes. Accumulating study confirms that inflammasomes break down caspase 1 to cleaved-caspase 1 and cells can not complete the process of pyroptosis without the activation of caspase-1. Pyroptosis is executed by the GSDM family and the linker of GSDM is specifically cleaved by caspase-1, generating a GSDM-N fragment to form membrane pore, thereby promoting pyroptosis. Furthermore, the activated caspase-1 leads to the maturation of IL-1β and IL-18, which can further induce pyroptosis. An increasing number of recent studies have demonstrated that pyroptosis can influence the proliferation, invasion, and metastasis of tumors., Inducing GSDME-dependent pyroptosis is considered as a new therapeutic idea to treat in HNSCC., In this study, autophagy and pyroptosis were activated in hypopharyngeal cancer cells after LSD1 silencing, coupled with elevated LC3, Beclin-1, NLRP3, cleaved-caspase-1, ASC, IL-1β, and IL-18 levels and reduced p62 level. On the contrary, 3-MA or MCC950 intervention alleviated the inhibitory effects of LSD1 depletion on the malignant biological behaviors of FaDu cells.
The present study has a limitation. In this study, we only discussed the regulatory effect of LSD1 on the progression of hypopharyngeal cancer cells. The further in vivo experiments involved in transgenic animals will be performed in the future investigation to support the conclusion obtained in this study.
| Conclusion|| |
Taken together, we demonstrated for the first time that LSD1 silencing could restrain the progression of hypopharyngeal cancer cells by inducing autophagy and pyroptosis. This study provides a good theoretical and molecular basis for LSD1 as a new targeted therapy against hypopharyngeal cancer.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Hao Wang and Fang Liu designed, conceived the study, and conducted the experiments. Hao Wang analyzed the data and drafted the manuscript. Fang Liu revised the manuscript. All authors have read and approved the final manuscript.
Financial support and sponsorship
This work was supported by the Scientific Research Project of Health and Family Planning Commission of Huangpu District, Shanghai, China (HLQ202001).
Conflicts of interest
There are no conflicts of interest.
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Department of Otolaryngology, Shanghai Ruijin Rehabilitation Hospital, 1100 Quxi Road, Huangpu District, Shanghai 200020
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]