Hypopharyngeal Squamous Cell Carcinoma Cells Research Articles

ETV4/NSUN2 Axis modulates aerobic glycolysis and malignancy in HSCC.

This study delves into the molecular intricacies of hypopharyngeal squamous cell carcinoma (HSCC), specifically focusing on the pivotal role played by ETS translocation variant 4 (ETV4) in aerobic glycolysis. The objective is to uncover new targets for early diagnosis and treatment of HSCC. ETV4 expression in HSCC tissues was rigorously examined, revealing its association with patient survival. Through comprehensive experimentation, we demonstrated that ETV4 activation promotes HSCC cell proliferation and invasion while inhibiting apoptosis. Furthermore, in vivo experiments confirmed the tumor-promoting effect of ETV4 activation. The study elucidated the binding of ETV4 to the NSUN2 promoter and its influence on PKM2 expression, thereby regulating glycolysis and cellular functions in HSCC.

MiR-107 Targets NSG1 to Regulate Hypopharyngeal Squamous Cell Carcinoma Progression through ERK Pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of malignant tumor with a poor prognosis and low quality of life in the otolaryngology department. It has been found that microRNA (miRNA) plays an important role in the occurrence and development of various tumors. This study found that the expression level of miRNA-107 (miR-107) in HSCC was significantly reduced. Subsequently, we screened out the downstream direct target gene Neuronal Vesicle Trafficking Associated 1 (NSG1) related to miR-107 through bioinformatics analysis and found that the expression of NSG1 was increased in HSCC tissues. Following the overexpression of miR-107 in HSCC cells, it was observed that miR-107 directly suppressed NSG1 expression, leading to increased apoptosis, decreased proliferation, and reduced invasion capabilities of HSCC cells. Subsequent experiments involving the overexpression and knockdown of NSG1 in HSCC cells demonstrated that elevated NSG1 levels enhanced cell proliferation, migration, and invasion, while the opposite effect was observed upon NSG1 knockdown. Further investigations revealed that changes in NSG1 levels in the HSCC cells were accompanied by alterations in ERK signaling pathway proteins, suggesting a potential regulatory role of NSG1 in HSCC cell proliferation, migration, and invasion through the ERK pathway. These findings highlight the significance of miR-107 and NSG1 in hypopharyngeal cancer metastasis, offering promising targets for therapeutic interventions and prognostic evaluations for HSCC.

TMEM16A inhibits autophagy and promotes the invasion of hypopharyngeal squamous cell carcinoma through mTOR pathway.

Previous studies have indicated that transmembrane protein 16A (TMEM16A) plays a crucial role in the pathogenesis and progression of various tumors by influencing multiple signaling pathways. However, the role of TMEM16A in regulating autophagy via the mammalian target of rapamycin (mTOR) pathway and its impact on the development of hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. Immunohistochemistry and western blotting were used to assess the expression of TMEM16A in HSCC tissues and metastatic lymph nodes. Manipulation of TMEM16A expression levels was achieved in the FaDu cell line through overexpression or knockdown, followed by assessment of its biological effects using cell colony formation, wound healing, transwell and invasion assays. Additionally, apoptosis and autophagy-related proteins, as well as autophagosome formation, were evaluated through western blotting, transmission electron microscopy and immunofluorescence following TMEM16A knockdown or overexpression in FaDu cells. Our study revealed significantly elevated levels of TMEM16A in both HSCC tissues and metastatic lymph nodes compared with normal tissues. In vitro experiments demonstrated that silencing TMEM16A led to a notable suppression of HSCC cell proliferation, invasion and migration. Furthermore, TMEM16A silencing effectively inhibited tumor growth in xenografted mice. Subsequent investigations indicated that knockdown of TMEM16A in HSCC cells could suppress mTOR activation, thereby triggering autophagic cell death by upregulating sequestosome-1 (SQSTM1/P62) and microtubule-associated protein light chain 3 II (LC3II). This study highlights the crucial role of TMEM16A in modulating autophagy in HSCC, suggesting its potential as a therapeutic target for the treatment of this malignancy.

Linc00662 sponges miR-15b-5p to promote hypopharyngeal squamous cell carcinoma progression by facilitating cancer stem cell-like phenotypes.

Background: Long non-coding RNAs (lncRNAs) are associated with multiple head and neck tumors and play important roles in cancer. This study explored the molecular mechanism of Linc00662 in hypopharyngeal squamous cell carcinoma (HSCC). Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression in HSCC tissues. The viability and proliferation of tumor cells were measured using CCK-8 assays. HSCC cell apoptosis was measured using flow cytometry and western blotting. Cell stemness was examined using the sphere formation assay. A xenograft tumor model was established to investigate the role of Linc00662 in vivo. Results: The expression level of Linc00662 in HSCC tissues was significantly higher than that in adjacent normal tissues. The expression of Linc00662 had no significant relationship with the tumor stage. Patients with high Linc00662 expression were found to have shorter overall survival than those with low Linc00662 expression. Linc00662 over-expression promoted cell viability and inhibited apoptosis. Using online databases and a dual luciferase reporter, miR-15b-5p was confirmed as a potential downstream sponge of Linc00662. Moreover, Linc00662 was negatively associated with miR-15b-5p in HSCC cells. Depletion of miR-15b-5p can reverse the function of Linc00662 in vivo and in vitro. Furthermore, Linc00662 promotes tumor growth, which was abolished by miR-15b-5p mimics. Importantly, the stemness of cancer stem cells was mediated by the Linc00662/miR-15b-5p axis. Conclusion: Patients with HSCC with high Linc00662 showed poor prognosis and high Linc00662 induced stemness of tumor cells by targeting miR-15b-5p. Linc00662 may serve as a novel diagnostic and target marker for head and neck squamous cell carcinoma.

Caffeic acid phenethyl ester: Unveiling its potential as a potent apoptosis inducer for combating hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare form of head and neck cancer that is notorious for its poor prognosis and low overall survival rate. This highlights the need for new therapeutic options for this malignancy. The objective of the present study was to examine the ability of caffeic acid phenethyl ester (CAPE), which is an active compound found in propolis, to combat HSCC tumor growth. CAPE exerted its tumor‑suppressive activity in HSCC cell lines through the induction of apoptosis. Mechanistically, the CAPE‑mediated apoptotic process was attributed to the perturbation of the mitochondrial membrane potential and the activation of caspase‑9. CAPE also modulated survivin and X‑linked inhibitor of apoptosis, which are potent members of the inhibitors of apoptosis protein family, either through transcriptional or post‑translational regulation, leading to HSCC cell line death. Therefore, the findings of the present study suggested that CAPE is an effective treatment alternative for HSCC via the stimulation of mitochondria‑dependent apoptosis.

CPT1A mediates chemoresistance in human hypopharyngeal squamous cell carcinoma via ATG16L1-dependent cellular autophagy

Hypopharyngeal squamous cell carcinoma (HSCC) is a highly aggressive malignancy that constitutes approximately 95% of all hypopharyngeal carcinomas, and it carries a poor prognosis. The primary factor influencing the efficacy of anti-cancer drugs for this type of carcinoma is chemoresistance. Carnitine palmitoyltransferase 1A (CPT1A) has been associated with tumor progression in various cancers, including breast, gastric, lung, and prostate cancer. The inhibition or depletion of CPT1A can lead to apoptosis, curbing cancer cell proliferation and chemoresistance. However, the role of CPT1A in HSCC is not yet fully understood. In this study, we discovered that CPT1A is highly expressed in HSCC and is associated with an advanced T-stage and a poor 5-year survival rate among patients. Furthermore, the overexpression of CPT1A contributes to HSCC chemoresistance. Mechanistically, CPT1A can interact with the autophagy-related protein ATG16L1 and stimulate the succinylation of ATG16L1, which in turn drives autophagosome formation and autophagy. We also found that treatment with 3-methyladenine (3-MA) can reduce cisplatin resistance in HSCC cells that overexpress CPT1A. Our findings also showed that a CPT1A inhibitor significantly enhances cisplatin sensitivity both in vitro and in vivo. This study is the first to suggest that CPT1A has a regulatory role in autophagy and is linked to poor prognosis in HSCC patients. It presents novel insights into the roles of CPT1A in tumorigenesis and proposes that CPT1A could be a potential therapeutic target for HSCC treatment.

ALKBH5 promotes hypopharyngeal squamous cell carcinoma apoptosis by targeting TLR2 in a YTHDF1/IGF2BP2-mediated manner

Hypopharyngeal squamous cell carcinoma (HPSCC) is one of the most aggressive cancers and is notorious for its extremely poor prognosis. However, very few molecular biological studies have been performed. As a novel method of epigenetic gene modulation, N6-methyladenosine (m6A) RNA modification occurs in HPSCC. The expression of the m6A demethylase AlkB homolog 5 (ALKBH5) is frequently downregulated in human HPSCC. Furthermore, we found that ALKBH5 impaired cell proliferation by regulating human Toll-like receptor 2 (TLR2) in an m6A-dependent manner in HPSCC cells. ALKBH5 decreased TLR2 m6A modification, which could be recognized by the m6A readers IGF2BP2 and YTHDF1. IGF2BP2 facilitates TLR2 mRNA stability, whereas YTHDF1 promotes TLR2 mRNA translation. The current work uncovered a critical function of ALKBH5 in TLR2 regulation and provides a novel role for m6A demethylation of mRNA in HPSCC. The inhibition of m6A modification of ALKBH5 in HPSCC deserves further clinical investigation.

Long Non-coding RNA Linc01224 Regulates Hypopharyngeal Squamous Cell Carcinoma Growth Through Interactions with miR-485-5p and IGF2BP3.

Increasing evidence illustrates that long non-coding RNAs (lncRNAs) play significant oncogenic roles, including hypopharyngeal squamous cell carcinoma (HSCC). The function and mechanism of long non-coding RNAs (lncRNAs) in hypopharyngeal squamous cell carcinoma (HSCC) have not been fully elucidated. Therefore, this study aimed to investigate the role of a specific lncRNA, linc01224, in regulating the miR-485-5p/IGF2BP3 axis in HSCC. We confirmed the lncRNA expression profiles in 5 pairs of HSCC and normal tissues by lncRNA sequencing. Another 28 HSCC tissues were further validated by quantitative real-time PCR (qRT-PCR). qRT-PCR was also used to detect the expression levels of linc01224, miR-485-5p and IGF2BP3 in HSCC cell lines. Next, functional experiments in vitro and in vivo were applied to determine the effects of linc01224 silencing on tumor proliferation, migration, apoptosis and progression in HSCC. Linc01224 expression was significantly higher in HSCC tissues than in adjacent normal tissues. In addition, HSCC patients with low IGF2BP3 expression had good survival. In vitro assays were mechanistically performed to explore whether linc01224 positively regulates IGF2BP3 expression via its competitive inhibition of miR-485-5p. An in vivo animal model also confirmed that linc01224 could promote the occurrence and development of HSCC. Our study first identified that linc01224 plays an oncogenic role in HSCC. It suggests that linc01224 may act as a prognostic biomarker and potential therapeutic target for HSCC.

HOXA11-AS1 Promotes PD-L1-Mediated Immune Escape and Metastasis of Hypopharyngeal Carcinoma by Facilitating PTBP1 and FOSL1 Association.

Simple SummaryThe metastasis of hypopharyngeal squamous cell carcinoma (HSCC) is the main reason for the poor prognosis of patients. Increasing studies have shown that abnormally expressed lncRNAs play crucial roles in HSCC, providing new perspectives for exploring cancer pathogenesis and matastasis. The expressions of HOXA11-AS1 and PD-L1 were found to be closely related to the overall survival of HSCC patients. Subsequently, the potential target genes, namely PBTP1 and FOSL1, were identified by expression correlation analysis. Finally, HOXA11-AS1/FOSL1/PTBP1/PD-L1 axis was identified to be a novel pathway provided a feasible preliminary basis for the future application of immunotherapy or targeted therapies in HSCC. Background: The metastatic characteristics of hypopharyngeal squamous cell carcinoma (HSCC) lead to many diagnostic and therapeutic challenges, while functional long non-coding RNAs (lncRNAs) can provide effective strategies for its diagnosis and treatment. Methods: RT-qPCR, Western blot, immunohistochemistry, and an immunofluorescence assay were used to detect the related gene expression. Flow cytometry was used to measure the percentage of CD8+ and CD4+ T cells. CCK-8 and transwell assays were performed to analyze the role of HOXA11-AS1. The targeted relationship of the FOSL1/PD-L1 promoter was measured by ChIP and dual-luciferase reporter assays. RNA pulldown and RIP assays were used to measure the interaction between HOXA11-AS1, FOSL1, and PTBP1. A tumor xenograft study was used to analyze HOXA11-AS1 function in vivo. Results: HOXA11-AS1, PD-L1, and FOSL1 were upregulated in HSCC, and HOXA11-AS1 positively correlated with PD-L1. HOXA11-AS1 knockdown upregulated CD8+ T cells through an increase in IFN-γ concentration while decreasing the proliferation, migration, and invasion of HSCC cells. FOSL1 bound the PD-L1 promoter, increasing gene expression. HOXA11-AS1 enhanced the stability of FOSL1 mRNA by binding to PTBP1. HOXA11-AS1 or PTBP1 overexpression increased FOSL1 and PD-L1 expression. PD-L1 knockdown arrested the inhibiting function of HOXA11-AS1 overexpression on CD8+ T cell content. HOXA11-AS1 knockdown inhibited immune escape and metastasis through PD-L1 regulation by downregulating FOSL1 in vivo. Conclusion: HOXA11-AS1 promoted PD-L1 expression by upregulating FOSL1 levels through PTBP1, thereby facilitating immune escape, proliferation, and metastasis of HSCC cells.

CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells.

Simple SummarySquamous cell carcinoma is a type of cancer resulting from cancer of the squamous cells that line some organs of the body such as the skin, lungs, and vulva. It is one of the leading causes of cancer-related deaths worldwide. In the current study, we show that a protein called CD109 plays an important role in the progression of this type of cancer. We also show that CD109 acts by associating with another protein called the epidermal growth factor receptor (EGFR), stabilizing its levels and promoting its action, leading to increased progression of squamous cell carcinoma. Together, these findings highlight a potential clinical utility for targeting CD109 in squamous cell carcinoma.(1) Background: Squamous cell carcinoma (SCC) is one of the leading causes of cancer-related deaths worldwide. CD109 is overexpressed in many cancers including SCC. Although a pro-tumorigenic role for CD109 has been shown in non-SCC cancers, and in one type of SCC, the mechanisms and signaling pathways reported are discrepant. (2) Methods: The CD109-EGFR interaction and CD109-mediated regulation of EGFR expression, signaling, and stemness were studied using microarray, immunoblot, immunoprecipitation, qPCR, immunofluorescence, and/or spheroid formation assays. The role of CD109 in tumor progression and metastasis was studied using xenograft tumor growth and metastatic models. (3) Results: We establish the in vivo tumorigenicity of CD109 in vulvar SCC cells and demonstrate that CD109 is an essential regulator of EGFR expression at the mRNA and protein levels and of EGFR/AKT signaling in vulvar and hypopharyngeal SCC cells. Furthermore, we show that the mechanism involves EGFR-CD109 heteromerization and colocalization, leading to the stabilization of EGFR levels. Additionally, we demonstrate that the maintenance of epithelial morphology and in vitro tumorigenicity of SCC cells require CD109 localization to the cell surface. (4) Conclusions: Our study identifies an essential role for CD109 in vulvar SCC progression. We demonstrate that CD109 regulates SCC cellular stemness and epithelial morphology via a cell-surface CD109-EGFR interaction, stabilization of EGFR levels and EGFR/AKT signaling.

Suppression of Long Noncoding RNA SNHG1 Inhibits the Development of Hypopharyngeal Squamous Cell Carcinoma via Increasing PARP6 Expression.

Purpose This study aimed to explore the function and molecular mechanism of long noncoding RNA Small Nucleolar RNA Host Gene 1 (SNHG1) in the development of hypopharyngeal squamous cell carcinoma (HSCC). Methods Human HSCC cell line FaDu was used in this study. Cell viability and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. Cell migration and invasion were measured by Transwell assay. The expression of PARP6, XRCC6, β-catenin, and EMT-related proteins (E-cadherin and N-cadherin) were determined using western blotting. Moreover, the regulatory relationship between SNHG1 and PARP6 was investigated. Furthermore, the effects of the SNHG1/PARP6 axis on tumorigenicity were explored in vivo. Results Suppression of SNHG1 suppressed the viability, migration, and invasion but promoted apoptosis of FaDu cells in vitro (P < 0.01). PARP6 is a target of SNHG1, which was upregulated by SNHG1 knockdown in FaDu cells (P < 0.01). SNHG1 suppression and RARP6 overexpression inhibited FaDu cell proliferation, migration, and invasion (P < 0.05). SNHG1 suppression and RARP6 overexpression also inhibited tumorigenicity of HSCC in vivo. Furthermore, the protein expression of E-cadherin was significantly increased and that of N-cadherin, β-catenin, and XRCC6 was dramatically decreased in HSCC after SNHG1 suppression or/and RARP6 overexpression both in vitro and in vivo (P < 0.01). Conclusions SNHG1 silencing inhibits HSCC malignant progression via upregulating PARP6. XRCC6/β-catenin/EMT axis may be a possible downstream mechanism of the SNHG1/PARP6 axis in HSCC. SNHG1/PARP6 can be used as a promising target for the treatment of HSCC.

LncRNA LEF1-AS1 Acts as a Novel Biomarker and Promotes Hypopharyngeal Squamous Cell Carcinoma Progression and Metastasis by Targeting the miR-221-5p/GJA1 Axis.

Hypopharyngeal squamous cell carcinoma (HSCC) is highly malignant and extremely aggressive, making it one of the worst prognoses among all kinds of head and neck squamous cell carcinoma (HNSCC); therefore, gaining insight into molecular mechanisms of HSCC is of profound significance. In the current manuscript, we revealed the elevated expression of long noncoding RNA (lncRNA) LEF1-AS1 in HNSCC which was associated with the poor prognosis by bioinformatic analysis. Moreover, we noticed that LEF1-AS1 dramatically accelerated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process in HSCC cell line FaDu. Most importantly, we illustrated that LEF1-AS1 played as a competitive endogenous RNA (ceRNA) via sponging miR-221-5p and thereby positively regulated gap junction protein alpha 1 (GJA1) expression, thus aggravated tumor progression and EMT. In conclusion, for the first time, we demonstrated lncRNA LEF1-AS1 as a novel biomarker for HNSCC and suggested LEF1-AS1/miR-221-5p/GJA1 axis as promising diagnostic and therapeutic target for HSCC treatment.

UBE2L3 promotes squamous cell carcinoma progression in the oral cavity and hypopharynx via activating the NF-κB signaling by increasing IκBα degradation.

Oral squamous cell carcinoma (OSCC) and hypopharyngeal squamous cell carcinoma (HSCC) are representative of head and neck squamous cell carcinoma (HNSCC) and the molecular pathogenesis has not been completely clarified. Ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is the key member of the E2 family that encodes 153 amino acid residues. Previous studies demonstrate that UBE2L3 is aberrantly overexpressed in various types of human cancers, suggesting that UBE2L3 may function as an oncogene. However, its functional role and the potential mechanisms in the OSCC and HSCC remain unclear. In the present study, we found that UBE2L3 was significantly upregulated in clinical HNSCC samples and HNSCC cell lines, and patients with lower UBE2L3 expression have a higher survival rate. Two HNSCC cell lines FaDu (HSCC cells) and CAL-27 (OSCC cells) with moderate expression of UBE2L3 were selected for in vitro experiments. We proved that UBE2L3 overexpression was positively associated with cellular malignant phenotypes in vitro, including proliferation, invasion, migration, and tumor growth in vivo. Conversely, UBE2L3 suppression diametrically yielded opposing results. Our further study demonstrated that overexpression of UBE2L3 significantly activated the nuclear factor kappa B (NF-κB) signaling pathway through promoting NF-κB p65 nuclear translocation and the ubiquitination and degradation of IκBα protein. Additionally, UBE2L3 was proved to be targeted and negatively regulated by miR-378a-5p, and UBE2L3 overexpression reversed the effects of miR-378a-5p upregulation. Collectively, the present study indicates that UBE2L3 may promote OSCC and HSCC progression via activating the NF-κB signaling by increasing IκBα degradation, indicating that UBE2L3 may be a potential therapeutic target for the treatment of HNSCC.

Encoding gene RAB3B exists in linear chromosomal and circular extrachromosomal DNA and contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma via inducing autophagy

Cisplatin (DDP) resistance is an important factor that decreases the effect of chemotherapy, thus leading to local recurrence and lymph node metastasis of hypopharyngeal squamous cell carcinoma (HSCC). We aimed to explore the role and mechanism of extrachromosomal circular DNA (eccDNA) in the DDP resistance of HSCC. In our research, the HSCC cell line FaDu and the DDP-resistant cell line FaDu/DDP were used as subjects. eccDNA sequencing and whole transcriptome sequencing were conducted, followed by a combined analysis of the two sequencing profiles. Outward PCR, inward PCR and Sanger sequencing were used to verify sequences of the eccDNAs. Bioinformatics analysis based on TCGA/GEO was performed in addition to plasmid transfection, RNA interference, qRT-PCR and Western blot experiments to verify the expression level of RAB3B amplified from eccDNA. mRFP-GFP-LC3 adenoviral particle transfection and transmission electron microscopy were used to detect autophagic flux. Finally, we evaluated the role of RAB3B in FaDu/DDP cells and patient-derived organoids. Our results showed that we purified and sequenced more than 10 thousand eccDNAs from the two cell lines, and the size of the eccDNAs was distributed from 0.01 kb to 1000 kb. The combined analysis between eccDNA and transcript sequencing indicated that there were some highly expressed genes that were completely or partially transcribed from related sequences of eccDNAs and not from genome linear DNA. We further screened and verified the encoding gene RAB3B using full-length sequences that might be amplified from eccDNA [chr1circle 46219-52682 kb]. Finally, we confirmed that RAB3B could promote DDP resistance in HSCC by inducing autophagy. The eccDNA might play significant roles in DDP resistance in HSCC by amplifying related functional genes. Further study is needed to explore the novel mechanisms of eccDNA in the drug resistance of HSCC.

Circ_0058106 promotes proliferation, metastasis and EMT process by regulating Wnt2b/\u03b2-catenin/c-Myc pathway through miR-185-3p in hypopharyngeal squamous cell carcinoma

Hypopharyngeal squamous cell carcinoma (HSCC) accounts 95% of hypopharyngeal cancer, which is characterized by high early metastasis rate and poor prognosis. It is reported that circular RNA is involved in the occurrence and development of cancer; however, the role of circRNA in hypopharyngeal cancer has little been investigated. We performed hypopharyngeal carcinoma circRNA microarray and qRT-PCR verification. The results showed circ_0058106 expression level was significantly upregulated in tumor tissues than in corresponding normal tissues. We found that circ_0058106 upregulation promoted proliferation, migration and invasion of HSCC cells, while knockdown of circ_0058106 inhibited proliferation, migration and invasion of HSCC cells both in vitro and in vivo. Bioinformatics predicted circ_0058106 may interact with miR-185-3p. We verified circ_0058106 directly bound miR-185-3p and downregulated miR-185-3p expression by using dual-luciferase reporter assay and qRT-PCR. Moreover, we proved circ_0058106 promoted HSCC cells tumorigenesis and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway via miR-185-3p. In conclusion, our findings firstly confirmed the carcinogenic effect of circ_0058106 in promoting HSCC cells tumorigenesis, metastasis, invasion and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway through sponging miR-185-3p, indicating that circ_0058106 may be a new therapeutic target and prognostic marker for HSCC.

Metformin Inhibits the Development of Hypopharyngeal Squamous Cell Carcinoma through Circ_0003214-Mediated MiR-489-3p-ADAM10 Pathway.

Purpose This study aims to explore the function of metformin in hypopharyngeal squamous cell carcinoma (HSCC) and the underlying mechanism. Methods Cell viability, colony formation, cell apoptosis, and cell cycle were investigated using cell counting kit-8 assay, colony formation, and flow cytometry assay. Gene expression was detected by quantitative real-time polymerase chain reaction and western blot. The target relationship was validated by dual-luciferase reporter assay or RNA immunoprecipitation assay. An animal study was implemented to clarify the effect of metformin in vivo. Results Metformin suppressed HSCC cell viability and colony formation ability and induced cell cycle arrest and apoptosis, and circ_0003214 overexpression weakened these effects. Circ_0003214 regulated A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) expression via targeting miR-489-3p. Besides, miR-489-3p restoration reversed the role of circ_0003214, and ADAM10 knockdown reversed miR-489-3p inhibition-mediated effect. Moreover, metformin blocked tumor growth via the circ_0003214-miR-489-3p-ADAM10 axis in vivo. Conclusion Metformin inhibits HSCC progression through the circ_0003214/miR-489-3p/ADAM10 pathway.

PHF20 inhibition promotes apoptosis and cisplatin chemosensitivity via the OCT4‑p‑STAT3‑MCL1 signaling pathway in hypopharyngeal squamous cell carcinoma.

Cisplatin is a widely used platinum-based chemotherapeutic agent for hypopharyngeal squamous cell carcinoma (HSCC). However, resistance to cisplatin limits its use for the treatment of HSCC, and the underlying molecular mechanism requires further investigation. The present study performed functional assays to determine whether the expression of plant homeodomain finger protein 20 (PHF20) may be involved in the apoptosis and cisplatin resistance of HSCC. The expression levels of PHF20 were higher in cisplatin-resistant HSCC cells compared with those in cisplatin-sensitive cells. The inhibition of PHF20 suppressed cell viability but did not affect the migratory and invasive abilities of HSCC cells compared with those of negative control-transfected cells. Furthermore, PHF20 inhibition reduced cell viability by enhancing apoptosis compared with those in the control cells in vitro. Notably, the inhibition of PHF20 sensitized HSCC cells to cisplatin, thus increasing apoptosis via the signal transducer and activator of transcription 3 (STAT3)-myeloid cell leukemia-1 (MCL1) pathway. Octamer-binding transcription factor 4 (OCT4) overexpression restored phosphorylated STAT3-MCL1-mediated apoptosis induced by PHF20 inhibition. In vivo experiments confirmed that PHF20 silencing induced tumor growth and increased apoptosis in HSCC cells compared with those in the control cells. Thus, PHF20 inhibition may promote apoptosis and improve cisplatin chemosensitivity via the OCT4-p-STAT3-MCL1 signaling pathway in HSCC.

The miR‑136‑5p/ROCK1 axis suppresses invasion and migration, and enhances cisplatin sensitivity in head and neck cancer cells

Laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) are two types of head and neck cancers with high incidence rates and relatively poor prognoses. The aim of the present study was to determine the effects of microRNA (miR/miRNA)-136-5p and its downstream target, Rho-associated coiled-coil containing protein kinase 1 (ROCK1), on LSCC and HPSCC progression and cisplatin sensitivity. The miRNA and protein expression levels in head and neck cancer cell lines were evaluated using reverse transcription-quantitative PCR and western blotting, respectively. MTT, wound healing assays, transwell assays and flow cytometry analysis were performed to measure cell properties. The binding between miR-136-5p and ROCK1 was detected using a dual-luciferase reporter assay. Autophagy double-labeled adenoviral infection assays were used to assess cell autophagy. The results showed that miR-136-5p was expressed in LSCC and HPSCC cells. Functional experiments showed that the expression of miR-136-5p in LSCC and HPSCC cells was negatively correlated with cell viability, invasion and migration. Additionally, miR-136-5p overexpression inhibited epithelial-mesenchymal transition, whereas miR-136-5p knockdown had the opposite effect. Dual-luciferase reporter assays confirmed the targeting relationship between miR-136-5p and ROCK1. miR-136-5p overexpression increased the cisplatin sensitivity of LSCC and HPSCC cells by reducing cell viability, as well as promoting cell apoptosis and autophagy. miR-136-5p overexpression decreased the expression levels of its downstream target ROCK1 and attenuated activity of the Akt/mTOR signaling pathway in cisplatin-treated LSCC and HPSCC cells. Conversely, miR-136-5p knockdown increased ROCK1 levels and decreased cisplatin sensitivity of the LSCC and HPSCC cells by increasing cell viability and inhibiting cell apoptosis, which was reversed by ROCK1 inhibition using the ROCK1 inhibitor, Y27632. Taken together, the results showed that the miR-136-5p/ROCK1 axis inhibits cell invasion and migration, and increases the sensitivity of LSCC and HPSCC cells to cisplatin.

Elevated O-GlcNAcylation Promotes Malignant Phenotypes of Hypopharyngeal Squamous Cell Carcinoma by Stabilizing Nrf2 through Regulation of the PI3K/Akt Pathway.

O-GlcNAcylation is a significant protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc) for intracellular signaling. Elevated O-GlcNAcylation contributes to cell proliferation, cell migration, cell apoptosis and signal transduction in various cancers. However, the expression level and functional role of O-GlcNAcylation in Hypopharyngeal Squamous Cell Carcinoma (HSCC) is not clearly elucidated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a master transcriptional factor that has been found to be aberrantly activated in HSCC. Here, we provide a molecular rationale between O-GlcNAcylation and Nrf2 in HSCC patients. The protein levels of O-GlcNAcylation and Nrf2 in HSCC tissues were detected by immunohistochemistry technique and western blot analysis. Then, O-GlcNAcylation knockdown HSCC cells were applied in this study. Cell proliferation was detected by CCK8, colony-forming analysis, and cell cycle assays. Cell migration and invasion ability was evaluated by transwell assays. Cell apoptosis was measured by TUNEL analysis. O-GlcNAcylation was obviously up-regulated in HSCC tissues, which correlated with tumor size and lymph node metastasis. In addition, the protein level of Nrf2 was found to positively correlate with the expression of O-GlcNAcylation both in vivo and in vitro. Knockdown of O-GlcNAcylation significantly inhibited HSCC cell growth, suppressed cell migration, and promoted cell apoptosis, whereas overexpression of Nrf2 reversed these phenotypes. Mechanismly, the upregulation of O-GlcNAcylation promoted the phosphorylation of Akt, leading to the stabilization of Nrf2; this could be attenuated by inhibition of the PI3K/Akt signaling pathway. Here, we provide a molecular association between O-GlcNAcylation and Nrf2 in HSCC patients, thus providing valuable therapeutic targets for the disease.

RP11‑156L14.1 regulates SSR1 expression by competitively binding to miR‑548ao‑3p in hypopharyngeal squamous cell carcinoma.

Emerging studies have demonstrated that long non-coding RNAs (lncRNAs) play essential roles in tumorigenesis. However, the role and function of lncRNAs in hypopharyngeal squamous cell carcinoma (HSCC) have not been completely elucidated. The present study explored the function of a novel lncRNA, RP11-156L14.1, in HSCC. RP11-156L14.1 was revealed to be highly expressed in HSCC tissues and cell lines. Knockdown of RP11-156L14.1 inhibited proliferation, migration, and invasion in HSCC cells. Furthermore, RP11-156L14.1 regulated epithelial-mesenchymal transition (EMT) by controlling EMT-related protein expression. Mechanistically, RP11-156L14.1 exerted its function as a competing endogenous RNA (ceRNA) and directly interacted with miR-548ao-3p. The present study also demonstrated that miR-548ao-3p regulated signal sequence receptor subunit 1 (SSR1) expression by targeting SSR1 3′-UTR. Moreover, the xenograft HSCC tumor model revealed that knockdown of RP11-156L14.1 markedly suppressed HSCC tumor growth in vivo. In summary, these findings indicated that the lncRNA RP11-156L14.1 functions as an oncogene in HSCC by competing with miR-548ao-3p in regulating SSR1 expression. The RP11-156L14.1/miR-548ao-3p/SSR1 axis could be utilized as a potential novel biomarker and therapeutic target for HSCC.