Proliferation Of Laryngeal Squamous Cell Carcinoma Cells Research Articles

Hypermethylation-Mediated lncRNA MAGI2-AS3 Downregulation Facilitates Malignant Progression of Laryngeal Squamous Cell Carcinoma via Interacting With SPT6

Long noncoding RNAs (lncRNAs) have an effect on the occurrence and progression of a considerable number of diseases, especially cancer. Existing research has suggested that MAGI2 antisense RNA 3 (MAGI2-AS3) takes on a critical significance in the development of hepatocellular carcinoma and lung cancer. However, the functions of MAGI2-AS3 in laryngeal squamous cell carcinoma (LSCC) remain unclear. In this study, MAGI2-AS3 expression level in LSCC tissue and cell lines was detected, and the effect of MAGI2-AS3 overexpressed on LSCC phenotypes and the possible influence mechanisms were examined. MAGI2-AS3 was downregulated in the tissues of LSCC patients versus non-tumor tissues, and it was correlated with advanced TNM (tumor, node, metastasis) stage and lymph node metastases, as indicated by the results of this study. MAGI2-AS3 inhibited the proliferation, migration, and invasion of LSCC cells in vitro and in vivo. Furthermore, the hypermethylation level of the MAGI2-AS3 promoter region was indicated by bisulfite genomic sequencing and methylation-specific polymerase chain reaction, such that MAGI2-AS3 expression was downregulated. Besides, MAGI2-AS3 promoter hypermethylation was regulated by DNA methyltransferase 1 (DNMT1), and MAGI2-AS3 expression was reversed by 5-Aza-2′-deoxycytidine (5-Aza). Moreover, the result of the RNA pull-down experiment suggested that 38 proteins were enriched in the MAGI2-AS3 group versus the control group in TU177 cells. To be specific, SPT6 (ie, a conserved protein) was enriched by fold change >10. SPT6 knockdown reduced the antitumor effect of MAGI2-AS3 in TU177 and AMC-HN-8 cells. Meanwhile, SPT6 overexpression inhibited the proliferation, metastasis, and invasion of TU177 and AMC-HN-8 cells. As revealed by the above findings, DNMT1-regulated MAGI2-AS3 promoter hypermethylation led to downregulated MAGI2-AS3 expression, such that the presence and progression of LSCC were inhibited in an SPT6 binding-dependent manner.

IGF2BP3 Regulates TMA7-mediated Autophagy and Cisplatin Resistance in Laryngeal Cancer via m6A RNA Methylation.

Translation machinery associated 7 homolog (TMA7) is closely related to proliferation-related diseases. However, the function and regulatory mechanism of TMA7 in laryngeal squamous cell carcinoma (LSCC) remain unclear. The present study aimed to investigate the effect of TMA7 on the occurrence and development of LSCC and to study the mechanism of TMA7. TMA7 is upregulated in LSCC tissues and associated with poor prognosis. After TMA7 downregulation, the autophagy level was increased, and the proliferation, migration, and invasion of LSCC cells were inhibited. The m6A methylated reader IGF2BP3 enhanced the stability of TMA7 and reduced the level of autophagy. TMA7 interacted directly with UBA2. Furthermore, the activation of the IGF2BP3-regulated TMA7-UBA2-PI3K pathway is the primary mechanism by which TMA7 inhibits autophagy and promotes the progression of LSCC. The current study revealed that IGF2BP3-mediated TMA7 m6A modification promotes LSCC progression and cisplatin-resistance through UBA2-PI3K pathway, providing new insights into the autophagy-related mechanism, potential biomarkers, and therapeutic targets for LSCC.

Roles and mechanisms of m6A modification regulating RP11-426A6.5 in laryngeal squamous cell carcinoma

Objective: To investigate the roles of N6-methyladenosine (m6A) modification in regulating RP11-426A6.5 in the development of laryngeal squamous cell carcinoma (LSCC). Methods: The methylation and expression levels of lncRNAs were identified and important lncRNAs were screened utilizing long non-coding RNA (lncRNA) m6A methylation microarray. Cancer and para cancer tissue samples were taken from 48 LSCC patients hospitalized to the Department of Otolaryngology-Head and Neck Surgery of the Second Affiliated Hospital of Harbin Medical University between January and September 2017. Expression profiling microarray was performed in 3 of 48 LSCC samples, and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR) and quantitative real-time fluorescent PCR (qRT-PCR) were performed in the remaining 45 LSCC samples to verify the m6A modification and expression levels of RP11-426A6.5. Correlations between RP11-426A6.5 and clinical factors were anlysed. Laryngeal cancer cell line with low expression of RP11-426A6.5 was created in vitro using RNA interference (RNAi) technology. The 5-Ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment, wound healing experiment, and transwell invasion experiment were used respectively to measure the proliferation, migration, and invasion of LSCC cells. The effect of RP11-426A6.5 down-regulation on the growth of transplanted tumors in vivo was verified by nude mice tumorigenesis assay. The Cancer Genome Atlas (TCGA) database and sequence-based RNA adenosine methylation site predictor (SRAMP) website were used to predict the enzymes and corresponding methylation sites. MazF digestion was chosen to validate the binding sites. RNAi technology was used to observe the changes in cell function after interfering with the expression of the corresponding genes of the modified enzymes. MeRIP-qPCR was used to detect the level of RP11-426A6.5 m6A cell line treated with actinomycin D was used to observe the stability of RP11-426A6.5. Results: RP11-426A6.5 methylation and expression levels were significantly higher in LSCC tissues than those in paracancerous tissues (methylation levels: 23.828±4.975 vs 20.280±3.607; expression levels: 1.197±0.314 vs 1.015±0.170, all P values<0.05). RP11-426A6.5 expression levels were closely correlated with T stage (T1-2: 1.081±0.298 vs T3-4: 1.306±0.292, χ2=5.35, P<0.05). The postoperative survival of patients with high RP11-426A6.5 expressions was significantly lower than that of patients with low RP11-426A6.5 expression (P=0.046). Assays in vitro and in vivo showed that the downregulation of RP11-426A6.5 significantly decreased the proliferation, migration, and invasion abilities of LSCC cells and the growth of transplanted tumors. The binding of methyltransferase-like 3 (METTL3), an m6A-modified enzyme, to the corresponding methylation site of RP11-426A6.5 enhanced its stability and mediated its regulation of malignant behaviors of LSCC cells. Conclusions: RP11-426A6.5 can regulate the malignant behaviors of LSCC cells, which is mediated by the m6A modification process involving in the methyltransferase METTL3.

Overexpression of Laminin 5γ2 Chain Correlates with Tumor Cell Proliferation, Invasion, and Poor Prognosis in Laryngeal Squamous Cell Carcinoma.

Objective Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor. Laminin 5γ2 chain (LAMC2) was reported to be associated with tumorigenesis. This study explored the role of LAMC2 on LSCC progression by regulating the integrinβ1/FAK/Src/AKT pathway. Methods The level of LAMC2 in 46 LSCC patients was detected by qRT-PCR and western blot. Then the relationship between LAMC2 expression and LSCC malignancy as well as prognosis was analyzed, and the effect of LAMC2 expression on LSCC patient survival was also analyzed using the Kaplan–Meier survival curves. Afterwards, the LSCC cells were transfected with LAMC2 overexpression and knockdown vectors, the effect of LAMC2 on LSCC cell viability, proliferation ability, cell cycle, cell migration, and invasion were detected by CCK-8, colony formation, flow cytometry, wound healing, and Transwell assays. The expression of EMT-related biomarkers and integrin β1/FAK/Src/AKT signaling-related proteins was detected by western blot. Moreover, the effect of LAMC2 on LSCC tumor growth was evaluated by in vivo xenograft experiments and western blot. Results LAMC2 was expressed at high level in LSCC tissues and associated with poor prognosis. LAMC2 overexpression increased TU177 cell viability, proliferation ability, promoted cell cycle, cell migration, and invasion capacity. The expression of N-cadherin, vimentin, and integrinβ1/FAK/Src/AKT related proteins was increased, while the expression of E-cadherin protein was decreased. When the LAMC2 knockdown in AMC-HN-8 cells had opposite effects. Furthermore, shLAMC2 decreased tumor volume and the expression of LAMC2, Ki-67 and integrinβ1, but increased the expression of E-cadherin in LSCC tumor-bearing mice. Conclusion The findings suggested that LAMC2 was overexpressed in LSCC and correlated with poor prognosis. LAMC2 knockdown inhibited LSCC progression by regulating the integrinβ1/FAK/Src/AKT signaling pathway. Therefore, LAMC2 could be a target for LSCC therapy.

KRT17 Accelerates Cell Proliferative and Invasive Potential of Laryngeal Squamous Cell Carcinoma (LSCC) through Regulating AKT/mTOR and Wnt/β-Catenin Pathways.

Background Laryngeal squamous cell carcinoma (LSCC) is a prevalent malignant tumor of the head and neck with a dismal prognosis. Keratin17 (KRT17) has been proven to serve as an oncogene in various cancers, but it has never been explored in LSCC. We proposed to assess the impact and possible mechanisms of KRT17 in the development of LSCC. Methods Quantitative reverse transcription-PCR (qRT-PCR) was utilized to examine the mRNA levels. The Kaplan–Meier method was used to calculate the relationship between KRT17 expression and survival curves in LSCC patients. Cell counting kit-8 (CCK-8), colony formation, and flow cytometry assays were utilized to estimate LSCC cell proliferation. The migration and invasion abilities of LSCC cells were ascertained by wound-healing and transwell assays. Immunohistochemical and western blot assays were utilized to appraise protein levels. The xenograft tumor model was used to determine the effect of KRT17 on tumor growth. Results In the present study, KRT17 was extremely high in LSCC tissues and cells and correlated with a poor prognosis. Inhibition of KRT17 weakens cell proliferative, migratory, and invasive abilities in LSCC and contributes to cell cycle arrest. Besides, we approved that knockdown of KRT17 extraordinarily restrained the xenograft tumor growth in vivo. We preliminarily investigated the role of KRT17 on the AKT/mTOR and Wnt/β-catenin signaling axes and found that these signaling pathways were largely blocked by KRT17 deletion. Conclusion Collectively, we uncovered that exhaustion of KRT17 suppresses LSCC progression through coordinating AKT/mTOR and Wnt/β-catenin signaling axes, illustrating KRT17 as a promising biomarker for making strides in LSCC treatment.

CircSERPINA3 promoted cell proliferation, migration, and invasion of laryngeal squamous cell carcinoma by targeting miR‐885‐5p

CircSERPINA3 has been shown to be upregulated in laryngeal squamous cell carcinoma (LSCC); however, whether it regulates the development of LSCC and the specific molecular mechanism remains unclear, which is to be explored in this study. Expressions of circSERPINA3, miR-885-5p, and Malic enzyme 1 (ME1) in LSCC tissues or cell lines were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulation of circSERPINA3 on the biological behavior of LSCC cells was confirmed by loss and gain experiments (cell counting kit-8, transwell, and colony formation assay). The correlation between circSERPINA3/ME1 and miR-885-5p was predicted and confirmed by bioinformatics analysis, dual-luciferase reporter assay, and qRT-PCR. The effect of circSERPINA3/miR-885-5p axis on the biological behavior of LSCC cells and expressions of epithelial-mesenchymal transition-related proteins was confirmed by rescue experiments. CircSERPINA3 and ME1 was upregulated in LSCC tissues, whereas miR-885-5p was downregulated. MiR-885-5p was the target gene of circSERPINA3, whereas ME1 was the target gene of miR-885-5p. Silent circSERPINA3 suppressed viability, invasion, migration, colony formation, and expression of ME1, claudin-4, snail, and vimentin but elevated expression of miR-885-5p and E-cadherin, whereas overexpressed circSERPINA3 was the opposite. However, miR-885-5p inhibitor or mimic reversed the effects of silent circSERPINA3 or overexpressed circSERPINA3. Collectively, circSERPINA3 promotes proliferation, migration, and invasion of LSCC cells by targeting miR-885-5p.

SNHG20 promotes the development of laryngeal squamous cell carcinoma via miR-342-3p/MTDH axis

Long noncoding RNAs (lncRNAs) are important players in laryngeal squamous cell carcinoma (LSCC). However, the function of the long noncoding RNA small nucleolar RNA host gene 20 (SNHG20) in LSCC is hardly known. We therefore analyzed the role of this lncRNA in LSCC. Our data showed that SNHG20 was significantly overexpressed in LSCC cell lines and human LSCC tissue. SNHG20 significantly promoted cell proliferation, migration and invasion of LSCC cells. The actions of SNHG20 are likely mediated by miR-342-3p expression, which results in increased expression of MTDH. Finally, the results of in vivo models confirmed that SNHG20 promotes LSCC progression through modulating miR-342-3p and MTDH expression. Taken together, our study demonstrates that SNHG20/miR-342-3p/MTDH axis participates in LSCC progression.

High CBX8 Expression Leads to Poor Prognosis in Laryngeal Squamous Cell Carcinoma by Inducing EMT by Activating the Wnt/β-Catenin Signaling Pathway.

BackgroundIn this study, we detected the expression of chromobox protein homolog 8 (CBX8) in laryngeal squamous cell carcinoma (LSCC) and its influence on the occurrence and progression of LSCC.MethodsPancancer analysis of CBX8 was analyzed by TCGA database and its expression in LSCC.The expression of CBX8 in 30 pairs of LSCC and adjacent tissues was analyzed by quantitative real-time PCR(qRT-PCR)and immunohistochemical assays, and its association with the prognosis and clinicopathological features of LSCC was further evaluated. A CBX8 knockdown model was constructed in AMC-HN-8 and Hep2 cell lines. The effects of CBX8 on LSCC cell proliferation, migration, invasion and apoptosis were detected by CCK8,EdU,wound healing, Transwell and flow cytometry assays. Levels of apoptosis-related protein, WNT/β-catenin signaling pathway and epithelial to mesenchymal transition (EMT) proteins, including Bax, Bcl2, β-catenin, DKK1, GSK3β, N-cadherin, E-cadherin and Snail1, in LSCC cells were detected by Western blotting.ResultsCBX8 was overexpressed in LSCC. High expression of CBX8 in LSCC patients led to shorter overall survival and correlated with tumor stage and lymphatic metastasis. After CBX8 knockdown, the proliferation of AMC-HN-8 and Hep2 cells slowed, and the number of EdU-positive cells decreased. Wound healing slowed down, and the number of Transwell invading cells decreased. The percentage of apoptotic cells increased. The expression levels of Bcl2, β-catenin, N-cadherin and Snail11 proteins were significantly reduced in the CBX8 knockdown cells, while Bax, DKK1, GSK3β and E-cadherin significantly increased with their corresponding controls.ConclusionCBX8 is highly expressed in LSCC and induces the EMT process by activating the Wnt/β-catenin signaling pathway to promote LSCC cell proliferation and migration and inhibit apoptosis, resulting in poor prognosis.

Long Noncoding RNA Hotair Promotes the Progression and Immune Escape in Laryngeal Squamous Cell Carcinoma through MicroRNA-30a/GRP78/PD-L1 Axis.

Homeobox (HOX) transcript antisense RNA (Hotair) is elevated in many cancers significantly. However, the oncogenic role of Hotair in human laryngeal squamous cell carcinoma (LSCC) is still unknown. Thus, we explored the expression profile of Hotair and its function in LSCC. We observed high expression levels of Hotair in six LSCC cell lines compared to the human nasopharyngeal epithelial cell line. Knockdown of Hotair inhibited proliferation and enhanced apoptosis of Tu212 and Hep-2 cell lines in vitro. Moreover, the overexpression of hsa-miR-30a-5p inhibited the expression of GRP78 and PD-L1, but Hotair overexpression in LSCC cells rescues both proteins. Furthermore, the impacts of hsa-miR-30a-5p upregulation on the apoptosis and proliferation of LSCC cells were rescued by overexpression of Hotair. Finally, we combined si-Hotair and a VEGF inhibitor to treat LSCC cells in vitro or in vivo and surprisingly observed a significant inhibition of LSCC growth. In summary, these results indicate that Hotair displays an oncogenic role in both malignancy and immune escape in LSCC related to hsa-miR-30a-5p/GRP78/PD-L1 signaling. Therefore, Hotair may be a potential target for treating LSCC.

Circular RNA MTCL1 promotes advanced laryngeal squamous cell carcinoma progression by inhibiting C1QBP ubiquitin degradation and mediating beta-catenin activation

BackgroundCircular RNAs (circRNAs) are involved in regulatory processes of ubiquitination and deubiquitination in various tumors at post-transcriptional epigenetic modification level. However, the underlying mechanism and its biological functions of circRNAs in the advanced laryngeal squamous cell carcinoma (LSCC) remain obscure.MethodsRNA sequencing and quantitative real-time PCR (qRT-PCR) assays were applied to screen for circRNAs differentially expressed in LSCC tissues and cell lines. The candidate RNA-binding proteins and target signalling pathway were detected by RNA pull-down and mass spectrometry, in situ hybridization (ISH), immunohistochemistry (IHC), qRT-PCR assays, and bioinformatics analysis. The functional roles of these molecules were investigated using in vitro and in vivo experiments including EdU, transwell, wound healing, western blot assays, and the xenograft mice models. The molecular mechanisms were identified using RNA pull-down assays, RNA immunoprecipitation (RIP), Co-IP, ISH, Ubiquitination assay, bioinformatics analysis, and the rescue experiments.ResultsHere, we unveil that microtubule cross-linking factor 1 circRNA (circMTCL1, circ0000825) exerts its critical oncogenic functions by promoting complement C1q-binding protein (C1QBP)-dependent ubiquitin degradation and subsequently activating Wnt/β-catenin signalling in laryngeal carcinoma initiation and development. Specifically, circMTCL1 was remarkably up-regulated in the paired tissues of patients with LSCC (n = 67), which predicted a worse clinical outcome. Functionally, circMTCL1 exerted oncogenic biological charactersistics by promoting cell proliferative capability and invasive and migrative abilities. Ectopic circMTCL1 augumented cell proliferation, migration, and invasion of LSCC cells, and this effect could be reversed by C1QBP knocking down in vitro and in vivo. Mechanistically, circMTCL1 directly recruited C1QBP protein by harboring the specific recognized sequence (+ 159 − + 210), thereby accelerating the translation of C1QBP expression by inhibiting its ubiquitin–proteasome-mediated degradation. Importantly, the direct interaction of C1QBP with β-catenin protein was enhanced via suppressing the β-catenin phosphorylation and accelerating its accumulation in cytoplasm and nucleus.ConclusionOur findings manifested a novel circMTCL1-C1QBP-β-catenin signaling axis involving in LSCC tumorigenesis and progression, which shed new light on circRNAs-ubiquitous acidic glycoprotein mediated ubiquitin degradation and provided strategies and targets in the therapeutic intervention of LSCC.

Long intergenic non-protein coding RNA 847 promotes laryngeal squamous cell carcinoma progression through the microRNA-181a-5p/zinc finger E-box binding homeobox 2 axis

ABSTRACT 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.

MCTS1 promotes laryngeal squamous cell carcinoma cell growth via enhancing LARP7 stability.

MCTS1 Re-Initiation and Release Factor (MCTS1) has been characterised as an oncoprotein in some cancers. In this study, we explored the expression of MCTS1 in laryngeal squamous cell carcinoma (LSCC) and its regulatory effects on the proliferation and cell-cycle progression of tumour cells, as well as the underlying mechanisms. The data from the Cancer Genome Atlas was used to analyse MCTS1 expression and its correlation with survival outcomes in LSCC patients. Subsequent in vitro cellular and molecular studies were performed based on representative LSCC cell lines. Results showed that the upregulation of MCTS1 in LSCC is linked to poor progression-free survival (PFS) and disease-specific survival (DSS). In TU177 and AMC-HN-8 cells, MCTS1 exerted positive regulations on cell viability, colony formation, cell cycle progression, and the expression of CDK1, CDK2, cyclin A2, and cyclin B1. Co-IP assay confirmed mutual interaction between MCTS1 and LARP7, mainly in the cytoplasm. Cycloheximide (CHX) chase and co-IP assay of ubiquitination showed that MCTS1 could increase LARP7 protein half-life and reduce its poly-ubiquitination. LARP7 overexpression enhanced the viability and colony formation of LSCC cells and also elevated the expression of CDK1, CDK2, cyclin A2, and cyclin B1. In addition, its overexpression partly reversed the negative influence of MCTS1 knockdown. In summary, this study confirmed that the expression of MCTS1 might be an indicator of unfavourable prognosis for patients with LSCC. Mechanically, it promotes LSCC cell viability and proliferation via interacting with LARP7 and reducing its proteasomal-mediated degradation.

Circular RNA ZNF609 promotes laryngeal squamous cell carcinoma progression by upregulating epidermal growth factor receptor via sponging microRNA-134-5p

ABSTRACT Emerging evidence has revealed that aberrantly expressed circular RNAs (circRNAs) play vital roles in tumorigenesis and progression of diverse human malignancies. CircZNF609 was found to be involved in hepatocellular carcinoma, but the role and underlying mechanism of circZNF609 in laryngeal squamous cell carcinoma (LSCC) remain unclear. This study aimed to explore the molecular mechanism of circZNF609 in LSCC. qRT-qPCR was performed to detect the expression of circZNF609 and microRNA-134-5p (miR-134-5p) in LSCC. Colony formation assay, CCK-8 assay, BrdU incorporation assay, clone formation assay, transwell invasion assay and Western blot analysis were performed to evaluate LSCC cell proliferation, as well as the expression of proliferating cell nuclear antigen (PCNA) and MMP-2. Luciferase reporter assay, target gene prediction and screening were used to validate downstream target genes of circZNF609 and miR-134-5p. EGFR expression was detected by Western blot analysis and RT-qPCR. Nude mice were used to detect tumor changes. CircZNF609 was upregulated in LSCC and associated with poor survival of LSCC patients. Knockdown of circZNF609 inhibited LSCC proliferation, invasion and the expression of PCNA and matrix matalloproteinases-2 (MMP-2). CircZNF609 can regulate miR-134-5p to upregulate epidermal growth factor receptor (EGFR). In addition, knockdown of EGFR or overexpression of miR-134-5p could reverse the tumor-promoting effects of circZNF609 in LSCC. In LSCC tissues, circZNF609 was negatively correlated with miR-134-5p and positively correlated with EGFR. CircZNF609 promotes the progression of LSCC via the miR-134-5p/EGFR axis, which might be the therapeutic target of LSCC.

LncRNA NCK1-AS1 Promotes Malignant Cellular Phenotypes of Laryngeal Squamous Cell Carcinoma via miR-137/NCK1 Axis.

Increasing evidence demonstrates that many long noncoding RNAs (lncRNAs) are implicated with the development of laryngeal squamous cell carcinoma (LSCC). As shown by bioinformatics analysis, lncRNA non-catalytic region of tyrosine kinase adaptor protein 1-antisense 1 (NCK1-AS1) is upregulated in tissues of head and neck squamous cell carcinoma. The study aimed to explore the role and mechanism of NCK1-AS1 in LSCC. NCK1-AS1 expression in LSCC cells was evaluated by reverse transcription qPCR. The viability, proliferation, invasion, migration, and apoptosis of LSCC cells with indicated transfection were evaluated by CCK-8 assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, wound healing assays, and TUNEL assays, respectively. Subcellular fractionation assays were performed to evaluate the cellular distribution of NCK1-AS1 and NCK1. NCK1 protein level in LSCC cells with indicated transfection was quantified by western blotting. The binding relation between miR-137 and NCK1-AS1 (or NCK1) were determined using RNA immunoprecipitation assays and luciferase reporter assays. NCK1-AS1 was highly expressed in LSCC cell lines. NCK1-AS1 depletion suppressed LSCC cell viability, proliferation, invasion, and migration while enhancing cell apoptosis. NCK1, an adjacent gene of NCK1-AS1, is also highly expressed in LSCC cells and was positively regulated by NCK1-AS1. Moreover, NCK1-AS1 interact with miR-137 to upregulate NCK1 expression. NCK1 was the downstream target of miR-137 and was negatively correlated to miR-137. In addition, overexpressed NCK1 reversed the suppressive impact of NCK1-AS1 depletion on malignant behaviors of LSCC cells. NCK1-AS1 contributes to LSCC cellular behaviors by upregulating NCK1 via interaction with miR-137.

Long non-coding RNA AGAP2-AS1 promotes cell proliferation and invasion through regulating miR-193a-3p/LOXL4 axis in laryngeal squamous cell carcinoma

ABSTRACT Laryngeal squamous cell carcinoma (LSCC) is an aggressive malignancy with highly mortality rate. Long non-coding RNA (lncRNA) AGAP2-AS1 is an identified oncogene in several types of cancers. However, the role of AGAP2-AS1 in LSCC remains unclear. The expression levels of AGAP2-AS1 in LSCC tissues and cell lines were measured using qRT-PCR. AGAP2-AS1 was knocked down in LSCC cells through transfection with siRNA-AGAP2-AS1. Cell proliferation and invasion were detected using MTT and transwell assays. Dual-luciferase reporter gene assay was performed to confirm the interaction with AGAP2-AS1 and downstream genes. Our results showed that AGAP2-AS1 expression was remarkably increased in human LSCC tissues and cell lines. Knockdown of AGAP2-AS1 significantly inhibited the proliferation and invasion of LSCC cells. In addition, AGAP2-AS1 sponged miR-193a-3p and regulated its expression in LSCC cells. Inhibition of miR-193a-3p reversed the effects of AGAP2-AS1 knockdown on LSCC cells. Furthermore, Lysyl oxidase-like 4 (LOXL4) was a target gene of miR-193a-3p and the role of miR-193a-3p was mediated by LOXL4. In conclusion, these findings suggest that knockdown of AGAP2-AS1 inhibited the proliferation and invasion of LSCC cells through regulating the miR-193a-3p/LOXL4 axis.

Circ_0044520 regulates the progression of laryngeal squamous cell carcinoma via the miR-338-3p/ROR2 axis.

Laryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumor in head and neck. Circular RNA_0044520 (circ_0044520) expression is increased in LSCC. However, the molecular mechanism of circ_0044520 remains unknown. The expression of circ_0044520, microRNA-338-3p (miR-338-3p) and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). Cell viability, cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and Flow cytometry assay, respectively. Western blot examined the protein levels of PCNA, Cyclin D1, Bax, Bcl-2 and ROR2 cells. The relationship between miR-338-3p and circ_0044520 or ROR2 was verified by Dual-luciferase reporter assays. The xenotransplantation model was established to study the role of circ_0044520 in vivo. The expression of circ_0044520 and ROR2 was increased in LSCC tissues and cells, while the expression of miR-338-3p was decreased. Circ_0044520 can sponge miR-338-3p, and ROR2 is the target of miR-338-3p. In vitro complement experiments showed that knockdown of circ_0044520 significantly inhibited malignant behavior of LSCC, while co-transfection of miR-338-3p inhibitor partially up-regulated this change. In addition, miR-338-3p can inhibit the proliferation of LSCC cells, while co-transfection of overexpression of ROR2 can partially reverse this change. Mechanically, circ_0044520 regulates ROR2 expression in LSCC cells by sponging miR-338-3p. In addition, in vivo studies have shown that down-regulation of circ_0044520 inhibits tumor growth. Circ_0044520 silencing plays anti-proliferation and pro-apoptosis roles in LSCC cells by regulating the miR-338-3p/ROR2 axis, suggesting that the circ_0044520/miR-338-3p/ROR2 axis may be a potential regulatory mechanism for the treatment of LSCC.

LncRNA CASC15 upregulates cyclin D1 by downregulating miR-365 in laryngeal squamous cell carcinoma to promote cell proliferation

BackgroundThis study investigated the role of lncRNA CASC15 in laryngeal squamous cell carcinoma (LSCC).MethodsThis study included 58 LSCC patients. Both tumor (LSCC) and adjacent (within 3 cm around tumors) non-tumor tissues from 3 different sites of each patient were collected. CCK-8 assay was used to determine cell proliferation. The expression levels of proteins and mRNAs were determined by Western blotting analysis and qRT-PCRs, respectively.ResultsCASC15 was upregulated in LSCC and high expression levels of CASC15 predicted poor survival. In LSCC tissues, CASC15 was negatively correlated with miR-365 but positively correlated with cyclin D1. In LSCC cells, overexpression of CASC15 resulted in downregulation of miR-365 and upregulation of cyclin D1. Overexpression of miR-365 did not affect the expression of CASC15 but downregulated cyclin D1. Overexpression of Cyclin D1 did not affect the expression of miR-365 and CASC15. Overexpression of CASC15 and cyclin D1 led to promoted, while overexpression of miR-365 led to inhibited LSCC cell proliferation. In addition, overexpression of miR-365 reduced the effects of overexpression of CASC15.ConclusionTherefore, CASC15 upregulates cyclin D1 by downregulating miR-365 in LSCC to promote cell proliferation.

The ETS1-LINC00278 negative feedback loop plays a role in COL4A1/COL4A2 regulation in laryngeal squamous cell carcinoma.

The present study aimed to investigate LINC00278 expression in laryngeal squamous cell carcinoma (LSCC) and its involvement in the process of proliferation, migration, and invasion, providing a rationale for mining potential diagnostic and therapeutic targets of LSCC. Univariate and multivariate Cox regression analyses were performed to identify optimal prognostic lncRNAs. MTS, colony formation, wound healing, and Transwell invasion assays were used to determine the effects of LINC00278 overexpression on the proliferation, migration, and invasion of cancer cells. The expressions of signaling pathway-related proteins and epithelial-mesenchymal transition (EMT) marker proteins were detected using western blot. The chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were performed to demonstrate the binding of ETS proto-oncogene 1, transcription factor (ETS1), and LINC00278 promoter region. The molecular targets of LINC00278 were identified by RNA sequencing analysis and co-expression analysis. Kaplan-Meier analysis and CIBERSORT algorithm were used to analyze survival and immune cell infiltration based on LINC00278, COL4A1, and COL4A2. Multivariate Cox regression was used to establish a six-gene prognostic model. LINC00278 expression was low in LSCC tissues, and it was significantly associated with the TNM (tumors/nodes/metastases) stage (p<0.001), lymphatic metastasis (p<0.01), and pathological differentiation (p<0.01). LINC00278 overexpression significantly reduced LSCC cell proliferation, migration, and invasion in TU686, TU177, and AMC-HN-8 cell lines. E-cadherin protein expression was increased, while N-cadherin, Vimentin, Zeb1, and Snail protein expression was decreased in the LINC00278 group, compared to the pcDNA3.1 group. Additionally, in AMC-HN-8 and FaDu cell lines, the LINC00278-treated group had significantly lower p-AKT and p-mTOR protein levels than the control group. ETS1 is a direct transcriptional regulator of the LINC00278 gene based on luciferase reporter assays and ChIP experiments. Western blot analysis demonstrated that high LINC00278 expression inhibited both ETS1 expression and phosphorylation. COL4A1/COL4A2 were identified as potential downstream targets of LINC00278. Meanwhile, the LINC00278/COL4A1/COL4A2-dominated low-risk group showed higher antigen-presenting activity and a higher immune score than the high-risk group. The findings indicated that ETS1 upregulated LINC00278 expression on the Y chromosome, which in turn inhibited LSCC growth in vivo and in vitro by inhibiting the AKT/mTOR signaling pathway via downregulation of COL4A1/COL4A2.

Hsa_circ_0023305 Enhances Laryngeal Squamous Cell Carcinoma Progression and Modulates TRPM7 via miR-218-5p Sponging.

Recently, circular RNAs have been shown to function as critical regulators of many human cancers. However, the circRNA mechanism in laryngeal squamous cell carcinoma (LSCC) remains elusive. Recent investigations using bioinformatics analysis revealed high expression of hsa_circ_0023305 in LSCC tissues compared to normal tissues. Furthermore, we discovered that hsa_circ_0023305 expression level was positively correlated to tumor/node/metastasis (TNM) stage as well as lymph node metastasis in LSCC. Moreover, higher hsa_circ_0023305 levels were correlated to poorer LSCC patient outcomes. Knockdown of hsa_circ_0023305 significantly inhibited LSCC cell proliferation, invasion, and migration abilities. Our team validated that hsa_circ_0023305 functioned as a miR-218-5p sponge from a mechanistic perspective, targeting the melastatin-related transient receptor potential 7 (TRPM7) in LSCC cells. TRPM7 regulates a nonselective cation channel and promotes cancer proliferation and metastasis. Our data demonstrated that miR-218-5p was downregulated in LSCC and that miR-218-5p upregulation repressed LSCC proliferation and invasion both in vivo and in vitro. Additionally, we found that hsa_circ_0023305-mediated upregulation of TRPM7 inhibited miR-218-5p and contributed to LSCC migration, proliferation, and invasion. In summary, these data propose a new mechanism by which the hsa_circ_0023305/miR-218-5p/TRPM7 network enhances LSCC progression.

ALKBH5-mediated m6A modification of lncRNA KCNQ1OT1 triggers the development of LSCC via upregulation of HOXA9.

It has been shown that N6‐methyladenosine (m6A) modification is involved in the development of complex human diseases, especially in the development of cancer. Our research investigated the role and mechanism of the m6A modification of lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) in Laryngeal squamous cell carcinoma (LSCC) progression. Microarray analysis was used to quantitatively detect the m6A apparent transcriptional modification level of lncRNA in LSCC tissue. Methylated RNA immunoprecipitation‐qPCR (MeRIP‐qPCR), in situ hybridization (ISH) and quantitative real‐time PCR (qRT‐PCR) were used to examine the m6A modification and expression of KCNQ1OT1. In addition, in vivo and in vitro experiments have tested the effects of KCNQ1OT1 knockdown on the proliferation, invasion and metastasis of LSCC. Mechanically, we found the N6‐methyladenosine (m6A) demethylase ALKBH5 mediates KCNQ1OT1 expression via an m6A‐YTHDF2‐dependent manner and KCNQ1OT1 could directly bind to HOXA9 to further regulate the proliferation, invasion and metastasis of LSCC cells. In general, our research indicates that ALKBH5‐mediated m6A modification of KCNQ1OT1 triggers the development of LSCC via upregulation of HOXA9.