Cell Carcinoma Of Larynx Research Articles

MYBPC1 is a key regulator for laryngeal carcinoma formation.

Laryngeal carcinoma represents one of the most common types of tumor of the respiratory tract. The aim of the present study was to evaluate the functions of myosin-binding protein C1 (MYBPC1) in the progression of laryngeal carcinoma and to unravel the potential underlying molecular mechanism(s). Significantly differentially expressed mRNAs and miRNAs were analyzed, and potential genes were verified using clinically recruited patients with laryngeal carcinoma. The human laryngeal carcinoma cell lines TU686, TU212 and AMC-HN-8, as well as the control nasopharyngeal epithelial cell line NP69, were selected for the functional analysis of MYBPC1. The interaction between MYBPC1 and miR-451a was also explored in depth. The functions of MYBPC1 in the laryngeal carcinoma cell lines were examined using colony formation assay, cell proliferation and invasion assays, and via measuring the extent of apoptosis. The intracellular function of MYBPC1 was subsequently confirmed by constructing an in vivo xenograft model through the subcutaneous injection of laryngeal carcinoma cells into 4-week-old male nude mice. Compared with normal tissue, MYBPC1 was found to be the most significantly downregulated gene, whereas activating transcription factor-2 (ATF-2) was the most significantly upregulated one. At the same time, miR-451a was found to be the most significantly downregulated miRNA in laryngeal squamous cell carcinoma tissues. According to the WHO classification system, we found that the level of MYBPC1 was significantly decreased in grade IV tissues compared with grade II and grade III tissues, a finding that was consistent with the observed activity of miR-451a. MiR-451a was found to cause a marked enhancement of the activity of MYBPC1 in TU212 cells, which in turn was attenuated by ATF overexpression, suggesting that miR-451a could indirectly modulate the function of MYBPC1 through the ATF2-dependent signaling axis. MYBPC1 suppressed the invasion of cells induced by ATF2 in laryngeal carcinoma cells. Moreover, subcutaneous injection of MYBPC1 to construct an in vivo xenograft mouse model enabled rescue of the mice from laryngeal carcinoma formation. Taken together, the results of the present study have shown that MYBPC1 fulfills a pivotal role in laryngeal carcinoma formation, and these findings may provide both a new avenue for research planning and a potential therapeutic target for laryngeal carcinoma.

Nerolidol assists Cisplatin to induce early apoptosis in human laryngeal carcinoma Hep 2 cells through ROS and mitochondrial-mediated pathway: An in vitro and in silico view.

The objective of this study was to examine Nerolidol (NER) and Cisplatin (CIS) performed against human laryngeal carcinoma (Hep 2) cells. We evaluated the effect of NER, CIS, and NER + CIS on cell viability, cell migration, oxidative stress, mitochondrial membrane depolarization, nuclear condensation, apoptotic induction, and DNA damage in Hep 2 cells. We used the MTT assay to assess the cytotoxicity effect of NER and CIS on Hep 2 cells in terms of morphological alterations. Present results demonstrated that IC50 values of NER and CIS have potential cytotoxicity against Hep 2 cells. NER effectively inhibited cell viability, increased reactive oxygen species generation, apoptotic induction, and DNA damage in Hep 2 cells. In addition, the docking study evaluated the structural binding interaction of NER with PI3K/Akt and PCNA protein. Furthermore, NER with PI3K/Akt, PCNA has a higher crucial score and affinity. Present results infer that NER could be used to target signaling molecules in anticancer studies. PRACTICAL APPLICATIONS: Nerolidol is a dietary phytochemical with high biological activity that can find in a variety of plants. Many researchers focused on Nerolidol to treat various diseases including cancer. However, there is no studies exist on laryngeal cancer. This study uses Nerolidol and Cisplatin to generate oxidative stress and stimulate apoptosis and DNA damage in human laryngeal cancer cells. Based on present findings, Nerolidol could be a choice of anticancer medication, either alone or in combination against oral squamous cell carcinomas in both in vitro and in vivo experimental systems.

Novel prognostic matrisome-related gene signature of head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is a common malignancy of the mucosal epithelium of the oral cavity, pharynx, and larynx. Laryngeal squamous cell carcinoma (LSCC) and oral squamous cell carcinoma are common HNSCC subtypes. Patients with metastatic HNSCC have a poor prognosis. Therefore, identifying molecular markers for the development and progression of HNSCC is essential for improving early diagnosis and predicting patient outcomes. Methods: Gene expression RNA-Seq data and patient clinical traits were obtained from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma (TCGA-HNSC) and Gene Expression Omnibus databases. Differentially expressed gene (DEG) screening was performed using the TCGA-HNSC dataset. Intersection analysis between the DEGs and a list of core matrisome genes obtained from the Matrisome Project was used to identify differentially expressed matrisome genes. A prognostic model was established using univariate and multivariate Cox regression analyses, least absolute shrinkage, and selection operator (LASSO) regression analysis. Immune landscape analysis was performed based on the single-sample gene set enrichment analysis algorithm, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, prognostic value, receiver operating characteristic curve analysis, and gene mutation analyses. Immunohistochemical results regarding prognostic protein levels were obtained from the Human Protein Atlas. Single-gene RNA-sequencing data were obtained from GSE150321 and GSE172577 datasets. CCK-8 and Transwell assays were used to confirm cell proliferation and migration. Results: A total of 1,779 DEGs, including 939 upregulated and 840 downregulated genes, between tumor and normal samples were identified using the TCGA-HNSC microarray data. Intersection analysis revealed 52 differentially expressed matrisome-related genes. After performing univariate and multivariate Cox regression and LASSO analyses, a novel prognostic model based on six matrisome genes (FN1, LAMB4, LAMB3, DMP1, CHAD, and MMRN1) for HNSCC was established. This risk model can successfully predict HNSCC survival. The high-risk group had worse prognoses and higher enrichment of pathways related to cancer development than the low-risk group. Silencing LAMB4 in HNSCC cell lines promoted cell proliferation and migration. Conclusion: This study provides a novel prognostic model for HNSCC. Thus, FN1, LAMB4, LAMB3, DMP1, CHAD, and MMRN1 may be the promising biomarkers for clinical practice.

Combination RSL3 Treatment Sensitizes Ferroptosis- and EGFR-Inhibition-Resistant HNSCCs to Cetuximab.

Head and neck squamous cell carcinomas (HNSCCs) are a type of cancer originating in the mucosal epithelium of the mouth, pharynx, and larynx, the sixth most common cancer in the world. However, there is no effective treatment for HNSCCs. More than 90% of HNSCCs overexpress epidermal growth factor receptors (EGFRs). Although small molecule inhibitors and monoclonal antibodies have been developed to target EGFRs, few EGFR-targeted therapeutics are approved for clinical use. Ferroptosis is a new kind of programmed death induced by the iron catalyzed excessive peroxidation of polyunsaturated fatty acids. A growing body of evidence suggests that ferroptosis plays a pivotal role in inhibiting the tumor process. However, whether and how ferroptosis-inducers (FINs) play roles in hindering HNSCCs are unclear. In this study, we analyzed the sensitivity of different HNSCCs to ferroptosis-inducers. We found that only tongue squamous cell carcinoma cells and laryngeal squamous cell carcinoma cells, but not nasopharyngeal carcinoma cells, actively respond to ferroptosis-inducers. The different sensitivities of HNSCC cells to ferroptosis induction may be attributed to the expression of KRAS and ferritin heavy chain (FTH1) since a high level of FTH1 is associated with the poor prognostic survival of HNSCCs, but knocked down FTH1 can promote HNSCC cell death. Excitingly, the ferroptosis-inducer RSL3 plays a synthetic role with EGFR monoclonal antibody Cetuximab to inhibit the survival of nasopharyngeal carcinoma cells (CNE-2), which are insensitive to both ferroptosis induction and EGFR inhibition due to a high level of FTH1 and a low level of EGFR, respectively. Our findings prove that FTH1 plays a vital role in ferroptosis resistance in HNSCCs and also provide clues to target HNSCCs resistant to ferroptosis induction and/or EGFR inhibition.

Transcriptome analysis of well-differentiated laryngeal squamous cell carcinoma cells in below-background environment.

BackgroundPreliminary research has shown an inhibited growth rate of well-differentiated laryngeal squamous cell carcinoma cells (FD-LSC-1) in below-background radiation (BBR), but how the cells respond to this environmental stress and the potential mechanisms are yet unknown. The current study aimed to reveal the molecular differences in cells grown under BBR conditions and normal radiation at the transcriptional level.MethodsThe expression profiles between FD-LSC-1 cells grown in a deep underground laboratory and above ground laboratory collected on day 4 were investigated by whole-transcriptome analysis, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). Functional analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were then implemented for differentially expressed (DE) mRNAs and target genes of lncRNAs and circRNAs. Co-expression levels and the Bayesian network of DE genes were subsequently constructed, and the reliability of expression patterns were validated by quantitative real-time polymerase chain reaction (PCR).ResultsThe study identified a total of 671 mRNAs, 286 lncRNAs, 489 circRNAs, and 6 miRNAs as significantly expressed in response to the environmental stress. The GO annotations regarding the biological processes category were mainly biological regulation, metabolic process, response to stimulus, cell cycle, and modification process. The KEGG enrichment analysis indicated that TGF-β and Hippo signaling played a crucial role in the transcriptional regulation of FD-LSC-1 cell growth under background radiation. Further network construction suggested that the enriched KEGG pathways affected this process by regulating cell proliferation-related genes including SMAD, SMAD7, CDH1, EGR1, and BMP2.ConclusionsBelow-background radiation can lead to transcriptional changes in FD-LSC-1 cells cultured in the deep underground. The inhibitory growth effect is associated with multiple biological processes as well as canonical pathways of proliferation.

Anticancer Potential of Hesperidin against HEp-2 Laryngeal Carcinoma Cell Line in Comparison to Doxorubicin

BACKGROUND: Doxorubicin (DOX) is a drug that is frequently used to treat a variety of cancers. Unfortunately, in many situations, it is ineffective, and raising the dosage is restricted due to systemic toxicity. An important strategy to minimize the toxic effects of the above cited drug is to use co-adjuvant. A citrus flavonoid hesperidin (Hesp) has emerged as promising anticancer natural product and proved to be potent antioxidant agent. It suppresses cancer cell replicating by triggering apoptosis and cell cycle arrest. AIM: The study’s goal was to investigate anticarcinogenic effects of Hesp in comparison with DOX against HEp-2 laryngeal carcinoma cell line. MATERIALS AND METHODS: Five groups of HEp-2 cell line were included, two groups were subjected to Hesp and the other two groups were subjected to DOX, which was used as a reference drug, in addition to a control untreated group. Expression of Bcl-2 and p53 genes was evaluated. Furthermore, the cell cycle arrest and apoptotic induction were assessed. RESULTS: Hesp exerted anti-proliferative effects against HEp-2 cells which increase in time dependent manner. Gene profile analysis revealed highly statistically significant decrease of anti-apoptotic Bcl-2 expression and highly statistically significant increase of tumor suppressor gene p53 expression (p ˂ 0.01 and p ˂ 0.0001, respectively) for both tested drugs. CONCLUSIONS: Hesp proved potential anticancer effects with reducing cancer cell viability in HEp-2 cell line through cell cycle arrest and apoptotic mechanism. It could be used as a prodrug or coadjuvant in treatment of oral cancer.

MicroRNA-375 inhibits laryngeal squamous cell carcinoma progression via targeting CST1

ObjectiveThis study aims to explore the effect and mechanism of miR-375 in Laryngeal Squamous Cell Carcinoma (LSCC) cell progression. MethodsLSCC cells (LSC-1 and TU177) were transfected with miR-375-mimic, miR-375-inhibitor or miR-375-mimic+oe-CST1. The expression of miR-375, CST1, MMP-2, and MMP-9 was measured. The effect of miR-375-mimic, miR-375-inhibitor or miR-375-mimic+oe-CST1 on cell biological functions, including cell proliferation, migration, invasion, and apoptosis, was also assessed. The potential relationship between CST1 and miR-375 was predicted by Jefferson software and validated by dual luciferase reporter gene assay. ResultsDownregulated miR-375 expression was found in LSCC cells. Overexpression of miR-375 inhibited the viability and migration and promoted apoptosis of LSCC cells. Jefferson database and dual luciferase reporter gene assay confirmed that miR-375 directly targeted CST1. Overexpression of CST1 could reverse the anti-cancer effect of miR-375 overexpression in LSCC cells. ConclusionCollected evidence showed that miR-375/CST1 axis was implicated in LSCC progression. Level of evidenceLevel 3

MiRNA-21-5p Accelerates EMT and Inhibits Apoptosis of Laryngeal Carcinoma via Inhibiting KLF6 Expression.

The incidence of laryngeal carcinoma accounts for 1 to 5% of systemic malignancies and ranks second among head and neck malignancies. Screening more effective targets are meaningful for the treatment of laryngeal carcinoma. The purpose was to research the action of miR-21-5p in the occurrence of laryngeal carcinoma. Genecards combined with g:profiler was used for cluster analysis to predict gene-related miRNAs. Q-PCR assay was performed for measuring the level of miR-21-5p and Kruppel-like factor 6 (KLF6). miR-21-5p-mimic, miR-21-5p-inhibitor and sh-KLF6 were transfected using LipofectamineTM 2000. Both CCK-8 and EdU experiments were undertaken to detect cell proliferation ability. Western blot was used to detect apoptosis and epithelial-mesenchymal transition (EMT) related proteins. Wound healing assay and transwell assay were undertaken for migration and invasion, respectively. Three online software (ENCORI, miRWalk, and miRDB) were applied to screen the downstream of miR-21-5p. At the same time, a dual-luciferase reporter experiment was processed to verify the binding. Finally, a rescue experiment was applied to reveal the mediating role of miR-21-5p and KLF6. MiR-21-5p expressed highly in laryngeal carcinoma tissues and cell lines. Knockdown of miR-21-5p reduced the EMT, while enhancing apoptosis of laryngeal carcinoma cell lines. MiR-21-5p targeted KLF6 with negative relationships. The rescue assay results confirmed that sh-KLF6 rescued the action of miR-21-5p knockdown in developing laryngeal carcinoma cells. MiR-21-5p promotes the occurrence and development of laryngeal cancer by targeting KLF6. This finding may provide new insights into miRNA as a biomarker for diagnosing and treating laryngeal carcinoma in the future.

Circular RNA circ-ABCB10 Promotes Proliferation and Inhibits Apoptosis of Laryngeal Carcinoma by Inhibiting KLF6.

Objective To explore the effect of circular RNA circ-ABCB10 on the proliferation and apoptosis of laryngeal carcinoma via inhibiting KLF6. Methods RT-qPCR assay was adopted to detect the expression of circ-ABCB10 and KFL6 in laryngeal carcinoma tissues and cell lines. Cell counting kit-8 (CCK-8) and clone formation assay were employed to detect laryngeal cancer cell viability and proliferation when circ-ABCB10 was silenced or upregulated. In this study, the apoptosis rate was detected by flow cytometry and the protein expression was detected by Western blotting. Wound healing and cross-hole invasion were used to study the migration and invasion of laryngeal cancer cells when circ-ABCB10 was silenced or upregulated. Results The results of RT-qPCR detection indicated that the expression of circ-ABCB10 in all three laryngeal carcinoma cells was downregulated by 3.2 times compared with that of HaCat cells. There is low expression of circ-ABCB10 in most laryngeal carcinoma tissues, the diagnostic cutoff value of circ-ABCB10 is 0.0008, the area under the curve is 0.718, the sensitivity is 0.981, and the specificity is 0.556. The expression level of KLF6 in laryngeal carcinoma is on the rise, which is significantly higher compared to healthy tissues (P < 0.05); 48 hours after transfection, RT-qPCR analysis confirmed the transfection efficiency, and upregulation of circ-ABCB10 could significantly promote cell proliferation. Compared with the control group, silencing circ-MTCL1 could inhibit cell proliferation, overexpression of circ-ABCB10 promoted cell migration, and downregulation of circ-ABCB10 significantly inhibited cell movement (P < 0.001). Upregulation of circ-ABCB10 significantly enhanced the invasiveness and motility of laryngeal cancer cells, while downregulation of circ-ABCB10 was the opposite. Compared with the KLF6 NC group, KLF6 level increased significantly in the KLF6 group, while cell viability, colony formation, scratch healing rate, invasive cell number, and Bcl-2 expression level decreased significantly in the KLF6 group, while apoptosis rate and Bax expression level increased significantly (P < 0.05). KLF6 level in the si-circ-ABCB10+anti-KLF6 group was significantly lower than that in the si-circ-ABCB10+anti-KLF6-NC group (P < 0.05). Meanwhile, the cell activity, colony formation number, cell scratch healing rate, number of invaded cells, and Bcl-2 all indicated an upward trend, while the cell apoptosis rate and Bax expression indicated a downward trend (P < 0.05). Conclusion The expression of circ-ABCB10 in laryngeal carcinoma was significantly higher compared to that in paracancerous tissues. Silencing circ-ABCB10 could significantly inhibit the growth and proliferation of laryngeal adenocarcinoma cells, while overexpression of circ-ABCB10 could significantly promote the growth of laryngeal adenocarcinoma cells, probably by inhibiting KLF6 to enhance the proliferation of laryngeal carcinoma and inhibit apoptosis.

Notch1 signaling modulates hypoxia-induced multidrug resistance in human laryngeal cancer cells.

Laryngeal carcinoma is one of the common malignant tumors of the head and neck. Multidrug resistance (MDR) remains a critical problem in the chemotherapy of patients with laryngeal cancer. This study aims to clarify the role and mechanisms of Notch1 signaling in MDR induced by hypoxia in laryngeal cancer cells. Laryngeal carcinoma cells were cultured under normoxia or hypoxia. Notch1 expression was inhibited by small interfering RNA (siRNA). The mRNA expression of Notch1, Hes1, Hey1, MDR1 and survivin was analyzed by real-time PCR. The protein expression of Notch1, the Notch1 intracellular domain (N1ICD), MDR1/P-gp and survivin was analyzed by Western blotting. Current research has shown that hypoxia can upregulate Notch1 expression and Notch1 signaling activity. Furthermore, suppression of Notch1 expression effectively downregulated Notch1 signaling activity and the expression of the MDR and survivin genes in laryngeal cancer cells under hypoxic conditions (P < 0.05). The Cell Counting Kit-8 (CCK-8) assay results confirmed that the sensitivity of hypoxic laryngeal cancer cells to a variety of drugs could be upregulated by suppressing Notch1 expression (P < 0.05). Additionally, flow cytometry (FCM) showed that suppression of Notch1 expression significantly increased drug-induced apoptosis and intracellular rhodamine 123 (Rh123) accumulation in hypoxic laryngeal carcinoma cells (P < 0.05). Notch1 signalling could be regarded as a pivotal regulator of hypoxia-induced MDR in laryngeal cancer cells through the regulation of survivin-mediated apoptosis resistance and MDR1/P-gp-mediated drug transport.

Effect of Glut-1 and HIF-1α double knockout by CRISPR/CAS9 on radiosensitivity in laryngeal carcinoma via the PI3K/Akt/mTOR pathway.

Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of 18 F-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1α and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1α and/or Glut-1 in the presence of irradiation. HIF-1α and/or Glut-1knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1α and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the PI3K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1α and Glut-1. HIF-1α and/or Glut-1knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1α and/or Glut-1knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.

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.

LncRNA CCAT1 facilitates the proliferation, invasion and migration of human laryngeal squamous cell carcinoma cells via the miR-218-5p/BMI1.

Long non-coding RNAs (LncRNAs) are vital in the treatment of laryngeal squamous cell carcinoma (LSCC). This study estimated the mechanism of lncRNA CCAT1 (CCAT1) in LSCC cells. The expression of CCAT1 in the human laryngeal mucosal epithelial cells (HLCs) and LSCC cells (Hep-2 and TU177) was detected. CCK-8 and Transwell assays were used to evaluate the cell proliferative, migrative, and invasive abilities, respectively. The subcellular localization of CCAT1 was verified by RNA-FISH and cytoplasmic isolation assays. The targeted relationship among CCAT1, miR-218-5p, and BMI1 was verified by dual-luciferase assay. Expressions of miR-218-5p and BMI1 were detected by RT-qPCR. Our results depicted that CCAT1 was highly-expressed in Hep-2 and TU177 cells. Silencing CCAT1 inhibited the proliferation, migration, and invasion of Hep-2 and TU177 cells. Mechanically, CCAT1 regulated the BMI1 expression by competitively binding to miR-218-5p as a competing endogenous RNA (ceRNA), and thus facilitated the growth of Hep-2 and TU177 cells. Downregulation of miR-218-5p or upregulation of BMI1 inhibited the inhibitory effect of silencing CCAT1 on Hep-2 and TU177 cell proliferation, invasion, and migration. In conclusion, our study elicited that lncRNA CCAT1 facilitated the proliferation, migration, and invasion of Hep-2 and TU177 cells by sponging miR-218-5p and regulating the downstream BMI1.

Foxhead box D1 promotes the partial epithelial-to-mesenchymal transition of laryngeal squamous cell carcinoma cells via transcriptionally activating the expression of zinc finger protein 532

ABSTRACT The presence of cervical lymph node metastases has been considered as the most important adverse prognostic factor for patients with laryngeal squamous cell carcinoma (LSCC). However, the underlying mechanisms remain to be fully revealed. In this study, we explored the expression profile of Foxhead box D1 (FOXD1), its association with epithelial-to-mesenchymal transition (EMT), and its downstream targets in LSCC. Bioinformatic analysis was performed based on the LSCC subset of The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma (TCGA-HSNC) and Chromatin immunoprecipitation (ChIP)-seq data from Cistrome Data Browser. LSCC cell lines AMC-HN-8 and TU212 were used for in vitro studies. Results showed that FOXD1 upregulation was associated with poor prognosis of LSCC. FOXD1 knockdown reduced N-cadherin and Vimentin expression but increased E-cadherin expression in AMC-HN-8 cells. Its overexpression showed opposite effects in TU212 cells. FOXD1 could bind to the promoter of ZNF532 and activate its transcription. ZNF532 overexpression enhanced the invasion of both AMC-HN-8 and TU212 cells. In comparison, its knockdown significantly impaired their invasion. ZNF532 knockdown nearly abrogated the alterations of EMT markers caused by FOXD1 overexpression. Its overexpression largely rescued the phenotypes caused by FOXD1 knockdown. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that ZNF532 correlated genes are largely enriched in extracellular matrix regulations. LSCC patients with high ZNF532 expression (top 50%) had a significantly worse progression-free survival. In summary, this study confirmed that FOXD1 promotes partial-EMT of LSCC cells via transcriptionally activating the expression of ZNF532.

Optimization of endothelial growth factor receptor monoclonal antibody-gold nanorods photothermal therapy for laryngeal squamous cell carcinoma

ABSTRACT It reported that heat generated by near-infrared laser irradiation of gold nanorods (AuNRs) effectively inhibited tumor cells, and the conjugate of epidermal growth factor receptor monoclonal antibody (EGFRmAb) and gold nanorods could selectively binded to the surface of cancer cell membrane expressing EGFR. However, there are few research reports on EGFRmAb-AuNRs in laryngeal squamous cell carcinoma. Therefore, our study aimed to investigate the photothermal effect of EGFRmAb modified AuNRs in inducing tumor cell death in an animal model of laryngeal squamous cell carcinoma. We showed that the conjugates of EGFRmAb and AuNRs selectively entered laryngeal squamous cell carcinoma cells. We analyzed the parameters of laser irradiation by controlling the near-infrared to optimize the condition and procedure of targeted treatment in nude mice treated with EGFRmAb and AuNRs. In addition, we examined the safety of the combined therapy. Test results showed that EGFRmAb-AuNRs inhibited the growth of Hep-2 and CNE-2 cells but not normal epithelial cells, and the semi-inhibitor concentration of EGFRmAb in Hep-2 and CNE-2 cells was 4 pmol/ml and 2 pmol/ml, respectively. AuNRs injected into the tumor and irradiated by near-infrared laser effectively inhibited tumor growth in nude mice without toxic effect in mice. We further confirmed that the apoptosis and necrosis rates of tumor cells in mice were highest under 3 W/cm2 near-infrared laser irradiation and AuNRs minimum concentration of 280 μg/kg. In conclusion, we developed a new method of targeting EGFRmAb combined with AuNRs to achieve photothermal effect in the treatment of laryngeal squamous cell carcinoma.

SLC3A2 inhibits ferroptosis in laryngeal carcinoma via mTOR pathway

ObjectiveThis study aimed to explore the mRNA and protein expression of SLC3A2 in laryngeal carcinoma cells and tissues, and functional regulatory mechanism of SLC3A2 in cell ferroptosis of laryngeal carcinoma.MethodsWe chose the key gene-SLC3A2 of DEGs from TCGA by bioinformatics analysis, and then we constructed stable knockdown of SLC3A2 in laryngeal carcinoma cells. MTT assay and clonogenic assay were used to determine cell viability and cell growth, respectively. The mRNA and protein expression were determined by RT-qPCR and western blotting, respectively. Xenograft tumor model was used to determine the role of SLC3A2 in tumor growth.ResultsThe results of limma analysis recovered that 92 genes were involved in both upregulated DEGs and high risk of poor prognosis, whereas 36 genes were involved in both downregulated DEGs and low risk of poor prognosis. Pathway enrichment analysis indicated that mTOR signaling pathway and ferroptosis exerted a role in regulating these intersection genes. Moreover, SLC3A2 is a key gene in ferroptosis in laryngeal carcinoma. SLC3A2 is highly expressed in laryngeal carcinoma tissues and cells. Patients with high SLC3A2 expression exerted poor survival. SLC3A2 deficiency inhibited cell proliferation and foci formation. Furthermore, knockdown of SLC3A2 expression induced the efficacy of ferroptosis and suppressed ferroptosis related proteins expression. Mechanically, SLC3A2 deficiency facilitated ferroptosis through upregulating the expression of mTOR and P70S6K, whereas inhibited p-mTOR and p-P70S6K expression in laryngeal carcinoma cells. SLC3A2 deficiency inhibited tumorigenesis in nude mice.ConclusionOur study suggests that SLC3A2 negatively regulates ferroptosis through mTOR pathway in laryngeal carcinoma.

The Antitumoral Effect of Paris Saponin II on Head and Neck Squamous Cell Carcinomas Mediated via the Nitric Oxide Metabolic Pathway.

Paris saponin has shown great therapeutic value in cancer therapy. We used isolated Paris saponin II (PSII), an active component of Paris saponin, and demonstrated its antitumor effect on human head and neck squamous cell carcinoma cell lines. Additionally, we investigated its mechanisms of action in vivo by establishing a xenograft mouse model. The results showed that PSII had presented strong anticancer effects on both hypopharyngeal malignant tumor cell lines (FaDu) and laryngeal carcinoma cell lines (Tu212 and Tu686). In addition, we successfully isolated and cultured the head and neck squamous stem cells and the primary fibroblasts to perform metabonomics studies. The results showed that RPII remarkably decreased energy metabolism, and type III nitric oxide synthase 3 (NOS3) may be a target to block tumor growth. Furthermore, we found that PSII inhibited HNSCC proliferation and metastasis by inhibiting the nitric oxide metabolic pathway. Overall, these results demonstrated that PSII is a potent anticancer agent, and the metabonomics analysis is a valuable tool to investigate and establish the antitumor effects of traditional Chinese medicines.

Study on Forsythin promoting apoptosis of laryngeal carcinoma cells by regulating miRNA-1469

Objective The paper aimed to explore the mechanism of Forsythin regulating miRNA expression in laryngeal carcinoma cells, and to clarify the molecular biological mechanism of Forsythin regulating miRNA to promote the apoptosis of laryngeal carcinoma cells, providing theoretical and experimental basis for clinical application of Forsythin as an anti-laryngeal cancer treatment drug. Methods: A miR-1469 low-expression laryngeal carcinoma cell line was established. Western blot and flow cytometry were applied to detect the effect of Forsythin on cell apoptosis. Western blot was employed to detect the effects of Forsythin on P53 protein, P53 low expression, and P53 overexpression in laryngeal carcinoma cells, as well as the effects on overexpression of miRNA-1469, and on double low expression of P53 and Mcl1. Real-time PCR method was used to detect the effect of miR-1469 on p53 low expression in laryngeal carcinoma cells. Results: Flow cytometry detection of cell apoptosis showed that, after the cells with low miR-1469 expression were treated with Forsythin, the apoptosis rate was significantly reduced. Western blot detection showed that, compared with the Control group, the expression level of miR-1469 was significantly reduced after Forsythin administration in Hep2 cells with low expression of P53. Compared with the idle Control group, the apoptosis level of laryngeal carcinoma cells in Hep2 cells with low expression of P53 was significantly reduced. In Hep2 cells transfected with P53 overexpression plasmids, apoptosis level of laryngeal carcinoma cells increased. Compared with the idle Control group, the apoptosis level of laryngeal carcinoma cells in the single-transformed P53 shRNA group decreased, while the apoptosis level of the double-transformed miR-1469 mimic+P53 shRNA group increased again. After drug treatment, the apoptosis level of the single-transformed P53 shRNA group decreased, while the apoptosis level of the double-transformed Mcl1 shRNA+P53 shRNA group increased again. Conclusion: Forsythin can promote the apoptosis of laryngeal carcinoma cells by up-regulating the expression of miR-1469 and then down-regulating the expression of Mcl1. The drug can up-regulate the expression of miR-1469 by elevating the expression of P53. miR-1469 can promote the apoptosis of laryngeal carcinoma cells by inhibiting the expression of its downstream target gene Mcl1.

Epigenetic network of EZH2/SFRP1/Wnt in the epithelial-mesenchymal transition of laryngeal carcinoma cells.

Enhancer of Zeste Homologue 2 (EZH2) as a histone methyltransferase epigenetically regulates laryngeal carcinoma (LGC) progression. The present study sought to explore the role and mechanism of EZH2 in the epithelial-mesenchymal transition (EMT) of LGC cells. Expressions of EZH2, secreted frizzled-related protein 1 (SFRP1), and trimethylation of lysine 27 on histone H3 (H3K27me3) in LGC tissues or cells were detected via reverse transcription quantitative polymerase chain reaction (qRT-PCR) and western blotting. Upon transfection of si-EZH2, si-SFRP1, oe-SFRP1, or H3K27me3 upregulation, cell viability was assessed via cell counting kit-8, protein levels of E-cadherin, N-cadherin, β-catenin, c-Myc, and Cyclin D1 were determined via western blotting, and Vimentin expression was determined via immunofluorescence. The enrichment level of H3K27me3 in the SFRP1 promoter was measured via chromatin immunoprecipitation-PCR. EZH2 was highly expressed in LGC tissues and cells. Silencing EZH2 repelled the EMT of LGC cells. Mechanically, EZH2 upregulated H3K27me3 in the SFRP1 promotor to inhibit SFRP1 expression, and SFRP1 overexpression inactivated the Wnt pathway. H3K27me3 upregulation or SFRP1 downregulation reversed the inhibition of silencing EZH2 in the EMT of LGC cells. Overall, EZH2 upregulated H3K27me3 in the SFRP1 promoter to inhibit SFRP1 expression and activate the Wnt pathway, thereby facilitating the EMT of LGC cells.