STYK1 promotes the malignant progression of laryngeal squamous cell carcinoma through targeting TGF-β1.

This study aims to investigate the expression level of long non-coding RNA (lncRNA) SNHG20 in laryngeal squamous cell carcinoma (LSCC), and to explore further whether it can promote the development of LSCC by regulating microRNA-140 (miR-140). Expression levels of SNHG20 in 56 pairs of LSCC tissues and adjacent normal tissues were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between SNHG20 expression with pathological parameters and the prognosis of LSCC was analyzed. Besides, the SNHG20 expression in LSCC cells was also analyzed by qRT-PCR. The SNHG20 knockdown and overexpression model were constructed by lentivirus transfection in AMC-HN-8 and Hep-2 cells. Cell counting kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay were used to analyze the effect of SNHG20 on the biological function of LSCC cells. Finally, the dual-luciferase reporter gene assay was performed to explore the potentials of SNHG20 and miR-140 in LSCC. The SNHG20 expression in LSCC tissues or cells remarkably increased than controls, and the difference was statistically significant. The LSCC patients with the high expression level of SNHG20 were more likely to develop advanced tumor compared with patients with low expression of SNHG20. Moreover, the LSCC patients with the high expression level of SNHG20 had a shorter overall survival than those with low level. The cell proliferation ability significantly decreased in the SNHG20 knockdown group, while notably increased in SNHG20 overexpression group. MiR-140 was negatively correlated with SNHG20 in LSCC tissues and cells. Dual-luciferase reporter gene assay showed that SNHG20 could be targeted by miR-140 through a certain binding site. The cell rescue experiment also indicated that there was a mutual regulation between SNHG20 and miR-140, which could together affect the malignant progression of LSCC. We showed that the expression levels of SNHG20 in LSCC tissues or cell lines significantly increased and was associated with advanced tumor staging and undesirable prognosis of LSCC. In addition, SNHG20 could promote the malignant progression of LSCC.

To evaluate the expression of EphA2 protein in tissue specimens and cell lines of laryngeal squamous cell carcinoma (LSCC), and to further study the correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in LSCC. Western blot was applied to assess the EphA2 protein expression in LSCC cell line Hep-2 cells and the head and neck immortalized epithelial cell line NP-69 cells. Immunohistochemical staining was performed on paraffin sections of 88 cases of LSCC specimens and 16 cases of adjcent normal tissue samples to investigate the EphA2 protein expression, and to futher elucidate its correlation with clinicopathological characteristics. Compared with the NP-69 cells, EphA2 expression in LSCC cell line Hep-2 cells was upregulated. The positive rates of EphA2 expression in LSCC and adjcent normal tissues samples were 80.7% and 43.8%, respectively, with a significant difference between the two groups (P < 0.001). EphA2 overexpresion was closely correlated with clinical stage (I + II/III + IV, P = 0.005), metastasis (P = 0.025) and recurrence (P = 0.021) in LSCC. Furthermore, patients with EphA2 overexpression had poorer tumor-free survival and 5-year overall survival compared with that in patients with low EphA2 expression (33.3% vs. 63.2%, P = 0.003; 46.7% vs. 81.6%, P = 0.002). EphA2 expression combined with clinical stage provided a better predictive value in prognosis. Univariate and multivariate Cox regression analysis revealed that EphA2 expression is an independent prognostic factor for patients with LSCC (P = 0.019). The results of this study demonstrate that EphA2 protein expression is significantly increased in LSCC tissues and cell lines, and EphA2 protein overexpression is associated with tumor recurrence, metastasis and poorer prognosis in LSCC patients. These results suggest that EphA2 may play a critical role in the initiation and progression of LSCC, implicating EphA2 as a valuable marker for the prediction of recurrence, metastasis and prognosis in LSCC.

BackgroundAlthough great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure.MethodsWe collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1.ResultsBased on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine.ConclusionsTPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.

Recent research has explored the potential use of serum-derived biomarkers in cancer screening, and mounting evidence has illustrated the pivotal roles of long noncoding RNAs (lncRNAs) in regulating laryngeal squamous cell carcinoma (LSCC) progression. LINC02191 is a newly identified lncRNA and no studies have investigated its role in malignant tumors. This study aims to explore the functions and mechanisms of lncRNA LINC02191 in LSCC. LINC02191 was knocked down in LSCC cells using shRNAs for loss-of-function experiments. RT-qPCR revealed that LINC02191was upregulated in LSCC patients' serum exosomes, tissues and cells. Western blotting and RT-qPCR were implemented for detecting molecular protein and RNA levels. Colony formation, CCK-8, wound healing and Transwell assays were employed for examining LSCC cell malignant behaviors in vitro. A tumor-bearing mouse model (n = 4/group) was established for examining LINC02191 role in vivo.The results showed that LINC02191 silencing hindered LSCC cell proliferation, invasiveness, migration as well as EMT in vitro and impeded tumorigenesis in xenograft mouse model. Luciferase reporter assay was utilized for verifying the interaction between LINC02191, miR-204-5p and RAB22A. Pearson correlation analysis was employed to evaluate their expression correlation in LSCC tissue specimens (N = 30). Mechanistically, LINC02191 upregulated RAB22A by binding to miR-204-5p, and knocking down LINC02191 inhibited PI3K/Akt/mTOR signaling transduction in LSCC cells and tumor-bearing mice. Moreover, RAB22A overexpression reversed LINC02191 depletion-triggered suppression of LSCC cell aggressiveness and inactivation of PI3K/Akt/mTOR signaling. In conclusion,LINC02191 aggravates LSCC by targeting miR-204-5p/RAB22A/PI3K/Akt/mTOR signaling pathway, which indicates that LINC02191 may serve as a promising target for LSCC treatment.

In this study, we investigated the role of Y-box binding protein-1 (YB-1), c-Myb and miR-155 in human laryngeal squamous cell carcinoma (LSCC) progression. Quantitative real-time PCR, western blot, MTT and Transwell were conducted to determine the expression and function of YB-1/miR-155 pathway. Univariate and multivariate analyses were used to determine the prognostic factors. Expression of YB-1, c-Myb and miR-155 was higher in LSCC tissues. YB-1 promoted proliferation, invasiveness and migration of Hep-2 cells in vitro. Patients with higher YB-1 correlated with advanced T stage, poor differentiation and cervical metastasis. LSCC patients with high YB-1 expression showed poor overall survival. YB-1 promotes LSCC progression by increasing miR-155 levels via c-Myb and acts as a prognostic factor.

PurposeLong noncoding RNAs (lncRNAs) have been identified as an important class of noncoding RNAs that are deeply involved in multiple biological processes in tumorigenesis. This study is to investigate the critical roles and biological function of lncRNA growth arrest-specific 5 (GAS5) in tumorigenesis of laryngeal squamous cell carcinoma (LSCC).Patients and methodsA total of 59 samples of LSCC and paired adjacent tissue, as well as corresponding clinicopathological information were collected. GAS5 expression in both LSCC tissues and human SUN1076 and SNU899 cell lines were analyzed by Real-time quantitative RT-PCR method. Ectopic expression of GAS5 by vector transfection in LSCC cell lines and followed by in vitro experiments was to investigate the critical roles and function of GAS5 in LSCC. Cell Counting Kit 8 (CCK8) assay and PE/7AAD Annexin V Apoptosis analysis was to evaluate cell proliferation ability and cell apoptosis. Co-transfection of GAS5 and miR-21 was to explore the interaction between GAS5 and miR-21 in LSCC. BAX and CDK6 protein level were analyzed by western blot method.ResultsThis study demonstrated that GAS5 was significantly downregulated in LSCC tissue and human LSCC cell lines. GAS5 levels were correlated with the clinicopathological features of LSCC patients. In addition, the ectopic expression of GAS5 significantly inhibited cell proliferation and promoted apoptosis. Co-expression analyses indicated that GAS5 is negatively correlated with miR-21 in LSCC tissues. Overexpression of miR-21 eliminated GAS5-mediated cell apoptosis and proliferation suppression. Furthermore, GAS5, which upregulated BAX mRNA expression and downregulated CDK6 mRNA expression, was reversed by ectopic expression of miR-21.ConclusionGAS5 suppresses LSCC progression through the negative regulation of miR-21 and its targets involved in cell proliferation and apoptosis, indicating that GAS5 may serve as a biomarker and potential target for LSCC therapy.

BackgroundTo investigate the expression and role of special AT-rich sequence-binding protein-2 (SATB2) in laryngeal squamous cell carcinoma (LSCC) tissue and cell line (HEp2), and to evaluate the clinical and prognostic significance of SATB2 protein in patients with LSCC.MethodsThe expression of SATB2 was examined in LSCC tissue and HEp2 cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of SATB2 on the proliferation and tumor progression ability of HEp2 cells. Tumor formation assay in nude mice was used to analyze the effect of SATB2 on the tumorigenicity of HEp2 cells.ResultsThe status of SATB2 protein in carcinoma tissues is much lower than that in paracarcinoma tissues. The overall survival of the patients with high SATB2 expression was significantly higher than the low SATB2 expression group. Lower or negative SATB2 expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of SATB2 in HEp2 cells inhibited cell proliferation and tumor progression ability, and down-regulation of SATB2 showed the opposite effects. Over-expression of SATB2 repressed the tumorigenicity of HEp2 cells by in vivo experiments. Moreover, multivariate analysis suggested that SATB2 expression might be an independent prognostic indicator for the survival of LSCC patients after curative surgery.ConclusionsSATB2 might involve in the development and progression of LSCC as a tumor suppressor, and thereby may be a valuable prognostic marker for LSCC patients.

MicroRNAs are critical regulators of the development and progression of laryngeal squamous cell carcinoma (LSCC). However, the role of microRNA-154 (miR-154) in the development and progression of LSCC has not been clarified. We found that down-regulated miR-154 expression in LSCC tissues was associated with poorer prognosis in LSCC patients. MiR-154 over-expression inhibited the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest, which were reversed by miR-154 inhibition. MiR-154 targeted GALNT7 expression by reducing GALNT7-regulated luciferase activity in LSCC cells while up-regulating GALNT7 mRNA transcription in LSCC tissues and cells. GALNT7 silencing significantly attenuated the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest. Finally, intravenous treatment with lentivirus for miR-154, but not scrambled control miRNA, significantly restrained the growth of implanted LSCC Hep-2 tumors and decreased the tumor mass by reducing GALNT7 expression in mice. Therefore, miR-154 may serve as a novel prognostic marker and therapeutic target for LSCC.

The present study is targeted at investigating the effects of long intergenic non-protein coding RNA 847 (LINC00847) on the malignant biological behaviors of laryngeal squamous cell carcinoma (LSCC) cells, and the mechanisms. Quantitative real-time PCR and Western blotting were conducted for detecting the expressions of LINC00847, microRNA-181a-5p (miR-181a-5p) and zinc finger E-box binding homeobox 2 (ZEB2) in LSCC cell lines and tissue samples. BrdU, cell counting kit-8, scratch wound healing, Transwell and flow cytometry assays were utilized for detecting cell proliferation, migration, invasion, and cell cycle progression. Dual-luciferase reporter gene, RNA binding protein immunoprecipitation (RIP), and RNA pull-down assays were utilized to investigate the interaction among LINC00847, miR-181a-5p, and ZEB2. The subcellular location of LINC00847 was determined by RNA fluorescence in situ hybridization (RNA-FISH) assay. Tumor growth was evaluated using a xenograft model of nude mice. It was revealed that LINC00847 expression was increased in LSCC tissues, and its high expression was associated with lymph node metastasis and poor differentiation. LINC00847 was mainly located in the cytoplasm of LSCC cells, and LINC00847 overexpression promoted LSCC cell proliferation, migration, invasion, and accelerated the cell cycle progression while knocking down LINC00847 had the opposite effects in vitro and inhibited the tumor growth in vivo. LINC00847 directly targeted miR-181a-5p and negatively modulated miR-181a-5p expression. ZEB2 was a target gene of miR-181a-5p, and was positively and indirectly modulated by LINC00847. Our data suggest that LINC00847 promotes LSCC progression by regulating the miR-181a-5p/ZEB2 axis.

Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor and its expression is associated with the development of malignant tumor types. However, to the best of our knowledge, there is no information on the role of ECT2 in the development and progression of laryngeal squamous cell carcinoma (LSCC). The present study aimed at investigating the expression pattern and potential role of ECT2 in the development and progression of LSCC. The expression of ECT2 in 81 pairs of LSCC and adjacent non-tumor tissues was characterized by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. ECT2 expression was upregulated in LSCC tissues and associated significantly with poor differentiation, higher stages, lymph node metastasis and poor survival in the sample population. The relative expression levels of ECT2 mRNA transcripts were correlated with the intensity of anti-ECT2 staining in 25 ECT2+LSCC specimens selected randomly. These results indicated that ECT2 expression was crucial for the progression of LSCC and may serve as a biomarker for the diagnosis and prognosis of LSCC.

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most frequent malignant neoplasms of head and neck. Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with LSCC are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for LSCC from the aspect of circRNA-miRNA-mRNA interaction. Methods: We investigated the expression of circRNAs in 3 paired LSCC tissues and adjacent non-tumor tissues by microarray analysis. Differentially expressed circRNAs were identified between LSCC tissues and non-cancerous matched tissues, including 527 up-regulated circRNAs 414 down-regulated circRNAs. We focused on hsa_circ_0059354, which is located on chromosome 20 and derived from RASSF2, and thus we named it circRASSF2. Results: circRASSF2 was found to be significantly upregulated in LSCC tissues and LSCC cell lines compared with paired adjacent non-tumorous tissues and normal cells. Moreover, knockdown of circRASSF2 significantly inhibited cell proliferation and migration in vitro, which was blocked by miR-302b-3p inhibitor. Bioinformatics analysis predicted that there is a circRASSF2/miR-302b-3p/IGF-1R axis in LSCC progression. Dual-luciferase reporter system validated the direct interaction of circRASSF2, miR-302b-3p, and IGF-1R. Western blot verified that inhibition of circRASSF2 decreased IGF-1R expression. Furthermore, silencing circRASSF2 suppressed LSCC growth in vivo. Importantly, we demonstrated that circRASSF2 was upregulated in serum exosomes from LSCC patients, and correlated with cancer metastasis. Altogether, silencing circRASSF2 suppresses progression of LSCC by interacting with miR-302b-3p and decreasing inhibiting IGF-1R expression. Conclusion: In conclusion, these data suggest that circRASSF2 is a central component linking circRNAs to progression of LSCC via a miR-302b-3p / IGF-1R axis. Funding: The research was supported by the grants from the National science Foundation of china (81402234, 81241085, 81372902), and the postdoctoral scientific research developmental fund (LBH-Q16157). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The study protocol was approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University.

LAMP1 expression is associated with malignant behaviours and predicts unfavourable prognosis in laryngeal squamous cell carcinoma

Heat shock protein47 (HSP47) is a 47kDa collagen binding protein that has a close relationship with the development and progression of tumours. However, little is known concerning the expression profile of HSP47 in laryngeal squamous cell carcinoma (LSCC) patients and there is still insufficient data concerning the underlying mechanisms. The aim of the present study was to explore the expression of HSP47 in LSCC and provide an overview of its association with tumourigenicity and clinical prognosis. The expression of HSP47 in LSCC and adjacent non-cancerous laryngeal tissues was assessed via western blotting and immunohistochemical studies. The prognostic significance of HSP47 expression was analysed using a Kaplan-Meier survival curve. To investigate the influence of HSP47 on the viability, invasion and apoptosis of a LSCC cell line, we performed an invitro analysis with plasmid vectors and small interfering RNA (siRNA). Our results showed that HSP47 protein expression in the LSCC tissues was markedly decreased compared to that noted in the adjacent non-cancerous tissues, and low expression of HSP47 was correlated with poor prognosis in LSCC patients. Upregulation of HSP47 via plasmid vectors inhibited the proliferation, reduced the invasive ability, increased the sensitivity to cisplatin chemotherapy, promoted apoptosis, and induced the G1phase arrest of LSCC cells invitro. The expression of apoptosis-regulating proteins was also altered when HSP47 was upregulated, involving increased expression of cleaved caspase-7/-8/-9, PARP, and Bax and decreased expression of Bcl-2. Our present data suggest that HSP47 is an important prognostic factor and an attractive therapeutic target in LSCC due to its influence on the biological behaviour of LSCC cells.

Background: Circular RNAs (circRNAs) are a class of non-coding RNAs (ncRNAs) broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with laryngeal squamous cell carcinoma (LSCC) are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for LSCC from the aspect of circRNA-microRNA (miRNA)-mRNA interaction.Methods: We investigated the expression of circRNAs in three paired LSCC tissues and adjacent non-tumor tissues by microarray analysis. Differentially expressed circRNAs were identified between LSCC tissues and non-cancerous matched tissues, including 527 up-regulated circRNAs and 414 down-regulated circRNAs. We focused on hsa_circ_0059354, which is located on chromosome 20 and derived from RASSF2, and thus we named it circRASSF2.Results: circRASSF2 was found to be significantly up-regulated in LSCC tissues and LSCC cell lines compared with paired adjacent non-tumorous tissues and normal cells. Moreover, knockdown of circRASSF2 significantly inhibited cell proliferation and migration in vitro, which was blocked by miR-302b-3p inhibitor. Bioinformatics analysis predicted that there is a circRASSF2/miR-302b-3p/ insulin-like growth factor 1 receptor (IGF-1R) axis in LSCC progression. Dual-luciferase reporter system validated the direct interaction of circRASSF2, miR-302b-3p, and IGF-1R. Western blot verified that inhibition of circRASSF2 decreased IGF-1R expression. Furthermore, silencing circRASSF2 suppressed LSCC growth in vivo Importantly, we demonstrated that circRASSF2 was up-regulated in serum exosomes from LSCC patients. Altogether, silencing circRASSF2 suppresses progression of LSCC by interacting with miR-302b-3p and decreasing inhibiting IGF-1R expression.Conclusion: In conclusion, these data suggest that circRASSF2 is a central component linking circRNAs to progression of LSCC via an miR-302b-3p/IGF-1R axis.

New evidence suggests that abnormal expression of circular RNA (circRNA) is associated with the development of human cancers. This study aims to reveal circMYOF roles in the malignant phenotype of laryngeal squamous cell carcinoma (LSCC). The expression of circMYOF, microRNA (miR)-145-5p, and orthodenticle homeobox 1 (OTX1) was detected by quantitative real-time PCR. Cell proliferation, migration, invasion, and apoptosis were determined using colony formation assay and EdU assay, wound healing assay, transwell assay, and flow cytometry, respectively. Protein expression was examined by western blot analysis. Cell glycolysis was assessed by detecting glucose consumption and lactate production. Mice xenograft models were constructed to evaluate the regulation of circMYOF on LSCC tumorigenesis. The regulatory relationships among circMYOF, miR-145-5p, and OTX1 were identified using dual-luciferase reporter assay and RIP assay. Serum exosomes were isolated to confirm the existence of circMYOF in LSCC patients. CircMYOF was upregulated in LSCC tissues and cells, and its knockdown suppressed LSCC cell growth, metastasis, and glycolysis, as well as inhibited LSCC tumor growth. MiR-145-5p had decreased expression in LSCC, and it could be sponged by circMYOF. The inhibition effect of circMYOF lentivirus short hairpin RNA (sh-circMYOF) on LSCC progression was restored by the inhibitor of miR-145-5p (in-miR-145-5p). Also, OTX1 was targeted by miR-145-5p and was positively regulated by circMYOF. MiR-145-5p could repress LSCC progression, and OTX1 overexpression also eliminated this effect. In addition, we found that circMYOF was significantly overexpressed in the serum exosomes of LSCC patients. Our data revealed that circMYOF contributed to LSCC progression by promoting cell growth, metastasis, and glycolysis through miR-145-5p/OTX1 axis.