Circ_0000052/miR-382-3p axis induces PD-L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma.

A better understanding of the mechanisms underlying PD-L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD-L1 in forty-five HNSCC archival samples was determined by qRT-PCR. The biological function associated with malignant behaviour was assessed by PD-L1 depletion, miR-382-3p re-expression and regulation of circ_0000052. The interactions of PD-L1-miRNA and miRNA-circRNA were determined by qRT-PCR, Western blot analysis, dual-luciferase reporter assays and RNA immunoprecipitation assays. PD-L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD-L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN-γ/JAK2/STAT1 signalling pathway can induce PD-L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD-L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR-382-3p and alleviates its repression of PD-L1. This leads to overexpression of PD-L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR-382-3p/PD-L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti-PD-L1 therapy may improve personalized disease management.

The Diagnosis and Management of Advanced Endolymphatic Sac Tumor.

Endolymphatic sac tumor (ELST) is a group of low-grade malignant tumors originating from the endolymphatic sac of the inner ear. It is rare in the clinic and has the biological characteristics of slow growth and local aggression. Due to the lack of specificity in the clinical manifestations of patients with ELST, many cases have entered the advanced stage at the time of diagnosis. However, there are still great challenges in the treatment of advanced ELSTs. Here, the authors describe a case of advanced ELST, which relapsed after 2 operations. This time, the authors chose the transotic approach for tumor resection, which achieved the goal of complete resection of the tumor, and the patient recovered smoothly after surgery. There were no surgical complications and no tumor recurrence after the follow-up. Through literature review and our own experience, the authors suggest that complete surgical resection is the first choice for both primary and recurrent advanced ELSTs. The choice of a reasonable surgical approach is the key to ensuring complete resection of the tumor, while preoperative angiography and embolization, fine treatment of important structures during surgery, and postoperative long-term follow-up are equally important for patients with advanced ELST to obtain a good prognosis.

Erratum: Overexpression of lncRNA H19/miR-675 promotes tumorigenesis in head and neck squamous cell carcinoma: Erratum.

[This corrects the article DOI: 10.7150/ijms.16571.].

Total Resection of Cerebellopontine Angle Meningioma via Presigmoid Transmastoid Approach: An Otologist's Perspective.

PURPOSE: Cerebellopontine angle meningiomas (CPAMs) are benign tumors that arise from the dura mater of the petrosal surface of the temporal bone, lateral to the trigeminal nerve. This study aimed to describe 1 case of CPAMs violating the mastoid and highlight the unique superiority of the presigmoid transmastoid approach for this type of CPAMs from an otologist's perspective. METHODS: One case of specific CPAMs treated by total resection via presigmoid transmastoid approach in otomicrosurgery was described. RESULTS: A patient was referred for the left intracranial space-occupying lesion found in physical examination. Surgical resection via presigmoid transmastoid approach was performed and there was no sign of recurrence of tumor 2 years after the operation. CONCLUSIONS: Presigmoid transmastoid approach in otomicrosurgery is suitable for CPAMs invading the mastoid. It is suggested that neurosurgeons and ear surgeons should comprehensively analyze the type and extent of the tumor and flexibly adopt surgical methods to ensure it is the best for patients.

A Case of Atypical Skull Base Osteomyelitis Secondary to Otitis Media Due to Delayed Diagnosis.

ABSTRACT: Skull base osteomyelitis that is secondary to otitis media is extremely rare in the modern antibiotic era. The authors present an 84-year-old male with atypical skull base osteomyelitis that developed from otitis media during the COVID-19 pandemic due to delayed diagnosis and partial treatment which is blamed for development of skull base osteomyelitis. The atypical presentations of skull base osteomyelitis pose a diagnostic challenge. This case highlights that even otitis media is a potentially fatal infection in older patients with diabetes. Early diagnosis and aggressive management of skull base osteomyelitis are of upmost importance and will ensure a more favorable prognosis.

A Rare Instance of Primary Oncocytic Schneiderian Papilloma of Middle Ear and Eustachian Tube With a Combined Trans Oto and Nasal Approach Resection.

Oncocytic Schneiderian papillomas are rare tumours which usually arise in the sinonasal region. This paper presents, to the authors' knowledge, the first reported case of oncocytic Schneiderian papilloma arising primarily from the middle ear and eustachian tube. The resection of the tumor was performed with an endoscopic approach of combined trans oto and nasal. Oncocytic Schneiderian papilloma in the middle ear and eustachian tube is extremely rare as a primary lesion and challenging to manage. Very few documents have provided guide of resection using the endoscopic approach when this tumor extends to involve the eustachian tube. Our study illustrates that the endoscopic approach of combined trans oto and nasal is a good choice for tumor resection of middle ear and eustachian tube.

Overexpression of lncRNA H19/miR-675 promotes tumorigenesis in head and neck squamous cell carcinoma.

There is accumulating evidence indicating that long non-coding RNA H19 and its mature product miR-675 play essential roles for tumor growth and progression. However, their prognostic value in human head and neck squamous cell carcinoma (HNSCC), particular in laryngeal carcinoma, remains to be elucidated. In this study, we observed that both H19 and miR-675 were significantly overexpressed in a cohort of 65 primary tumor samples and two HNSCC cell lines. Importantly, when paired with patient follow-up data, higher expression of either H19 or miR-675 was significantly correlated with higher risk of patient relapse, and associated with worse overall survival and poor disease-free survival. Knockdown miR-675 caused significant reduction of cell viability, migratory and invasive capabilities. Taken together, these results suggest that the strong correlation of H19 overexpression together with higher miR-675 and lymph node metastases could be useful predictive markers, indicating a potentially therapeutic strategy for HNSCC patients.

Constitutive secretion of Interleukin-4 dictates CD133+ side population cells to resist drug treatment and cell death.

PURPOSE: In the present study, we made an attempt to elucidate the role of oversecretion of interleukin-4 (IL-4) in cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC). METHODS: HNSCC samples were analyzed for the presence of CSCs by flow cytometry. In addition, we have performed drug and apoptosis resistance assays to determine the role of IL-4 in CSCs. RESULTS: HNSCC samples contained 3.3% of CD133+ cancer stem like side population (SP) cells in HNSCC which displayed infinite cell proliferation and they had high self-renewal capacity. These CD133+ cells displayed enhanced expression of IL-4, which promoted multidrug and apoptosis resistance. After neutralizing IL-4, the CD133+ SP cells became more sensitive to drug treatment and apoptosis. CONCLUSIONS: Our data suggest that the autocrine secretion of IL-4 is a potential target for the development of novel anticancer drugs to prevent the CSCs-mediated therapy failure and tumorigenesis.

Salmonella typhimurium mediated delivery of Apoptin in human laryngeal cancer.

An effective cancer therapeutic should target tumours specifically with limited systemic toxicity. Here, we transformed an attenuated Salmonella typhimurium (S. typhimurium) with an Apoptin expressing plasmid into a human laryngeal carcinoma cell line. The expression of the inserted gene was measured using fluorescence and immunoblotting assays. The attenuated S. typhimurium-mediated Apoptin significantly decreased cytotoxicity and strongly increased cell apoptosis through the activation of caspase-3. The process was mediated by Bax, cytochrome c and caspase-9. A syngeneic nude murine tumour model was used to determine the anti-tumour effects of the recombinant bacteria in vivo. Systemic injection of the recombinant bacteria with and without re-dosing caused significant tumour growth delay and reduced tumour microvessel density, thereby extending host survival. Our findings indicated that the use of recombinant Salmonella typhimurium as an Apoptin expression vector has potential cancer therapeutic benefits.

Expression of PD-L1 and CD4+ tumor-infiltrating lymphocytes predict survival in head and neck squamous cell carcinoma.

The clinical efficacy of immune checkpoint blockade has been recently demonstrated in a variety of cancer types. The aim of the present study was to characterize the expression profile of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1 (PD-L1) in head and neck squamous carcinoma (HNSCC). A total of 63 patients with HNSCC were enrolled in the present study. CD3+ and CD4+ TILs and the expression of PD-L1 were detected by immunohistochemistry. PD-L1 mRNA levels were evaluated by reverse transcription-quantitative PCR analysis. The association of TILs and PD-L1 with patient clinicopathological characteristics was also assessed. CD3+ and CD4+ TILs were detected in 100% of the samples. CD3+ was the predominant subset of TILs. PD-L1 was expressed in 53 of 61 (86%) patients when a score of ≥1 on tumor cells was considered positive and in 28 patients (45.2%) when a score of >5 on tumor cells was considered positive. PD-L1 mRNA levels were determined to be significantly correlated with PD-L1 protein expression. Survival analysis demonstrated that high CD4+ TILs were associated with improved overall survival (OS) and disease-free survival (DFS), and furthermore, the association of high PD-L1 expression with unfavorable OS and DFS was statistically significant. Multivariate analysis identified CD4+ TILs and PD-L1 as prognostic markers for HNSCC. The results of the present study suggested that increased CD4+ TILs in HNSCC may be associated with improved outcomes, while high expression of PD-L1 may indicate unfavorable OS and DFS; thus, these factors may serve as predictors of the response to immune checkpoint therapy.

Long non-coding RNA ANRIL promotes proliferation, clonogenicity, invasion and migration of laryngeal squamous cell carcinoma by regulating miR-181a/Snai2 axis.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is the common cancer with poor prognosis. Long non-coding RNA (lncRNA) ANRIL has been proven to play an important role in many cancers. However up to now, the role of ANRIL in LSCC is still poorly understood. The present study aimed to investigate the role and underlying mechanisms of ANRIL and miR-181a in LSCC. METHODS: Expression of ANRIL, miR-181a and Snai2 in both LSCC tissues and cells was determined by qRT-PCR. CCK-8 assay, colony formation assay, flow cytometry analysis and transwell assay were conducted to detect cell proliferation, clonogenicity, apoptosis, invasion and migration, respectively. The binding between ANRIL and miR-181a, as well miR-181a and Snai2 was confirmed by dual luciferase reporter assay. Western blotting was used to determine the protein levels of Snail, Slug, E-cadherin, N-cadherin and Vimentin. RESULTS: ANRIL was up-regulated while miR-181a was down-regulated in LSCC tissues. ANRIL was negatively correlated with miR-181a and was positively correlated with Snai1 and Snai2. Dual luciferase reporter assay showed ANRIL could directly sponge miR-181a to counteract its suppression on Snai2, serving as a positive regulator of Snai2. Either knockdown of ANRIL or overexpression of miR-181a significantly inhibited the proliferation, clonogenicity, invasion, migration and epithelial mesenchymal transformation (EMT), as well as promoted the apoptosis of LSCC cells, and knockdown of miR-181a reversed the effects. CONCLUSION: Inhibition of ANRIL could suppress cell proliferation, clonogenicity, invasion and migration, as well as enhance cell apoptosis of LSCC cells through regulation of miR-181a/Snai2 axis, indicating that ANRIL might be a promising therapeutic target during the treatment of LSCC.

Clinical evaluation of balloon dilation eustachian tuboplasty surgery in adult otitis media with effusion.

CONCLUSION: BDET might be effective for the patients with OME, and proved to be an efficacious and mini-invasive treatment for OME. OBJECTIVES: To evaluate the therapeutic benefits of balloon dilation eustachian tuboplasty (BDET) in the treatment of adult patients with otitis media with effusion (OME) caused by eustachian tube dysfunction (ETD). METHODS: After informed consent, eight adult patients with OME were included in this study. After investigated patients' case history and oto-function, all patients underwent BDET treatment. Then four criteria including tympanic membrane, pure tone audiometry (PTA), tympanometry, and subjective symptoms were adopted to evaluate the therapeutic benefits of BDET. RESULTS: None of the involved patients complained of problems or complications during the post-operative period, or with absence of pain and bleeding after the operation. Prominent post-operative improvement was observed in tympanic membrane and otoscopic appearance. In addition, cure rates after 3 months and 6 months post-operatively were gradually increased.

Significance of ATP-binding cassette transporter proteins in multidrug resistance of head and neck squamous cell carcinoma.

According to the cancer stem cell theory, a small subpopulation of cancer cells, known as cancer stem cells (CSCs), exist that are self-renewing and are involved in tumor invasion, metastasis and recurrence. A number of studies have reported that certain cancer cells are able to efflux the Hoechst 33342 dye. These cells are termed side population (SP) cells and share characteristic features of CSCs. The results of the present study revealed that 2.7% of primary head and neck squamous cell carcinoma (HNSCC) cells were SP cells. This was reduced to 0.7% following treatment with verapamil. The immunofluorescence and reverse transcription polymerase chain reaction analysis revealed that SP cells have an enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC subfamily G, member 2 (ABCG2), which has been identified to be actively involved in drug exclusion. Similarly, the mRNA level of the oncogene B lymphoma Mo-MLV insertion region-1 and the stem cell surface proteins nestin and octamer-binding transcription factor-4 were highly expressed in the SP cells compared with the non-SP cells. In addition, it was demonstrated that HNSCC SP cells exhibited increased proliferation and were highly resistant to multiple drugs. These findings suggest that the presence of CSCs, such as SP cells, may be responsible for chemotherapy failure and tumor relapse in patients with HNSCC. Therefore, the identification of a novel therapeutic drug that could effectively target CSCs may help to eradicate refractory tumors.

Prognostic value of TROP2 in human nasopharyngeal carcinoma.

BACKGROUND: There is increasing evidence demonstrating the role of human trophoblast cell surface antigen 2 (TROP2) in cancer development and progression. However, their prognostic value in Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) remains to be elucidated. METHOD: The prognostic significances of TROP2 and Ki-67 were determined by immunohistochemistry in 58 NPC samples. TROP2 mRNA expression level and biological functions were evaluated. The presence of EBV was assessed using in situ hybridization. Analyses were conducted on the association between each of these variables as well as clinical outcome. RESULTS: TROP2 was exhibited over expression in 64% of NPC samples and significantly associated with highly proliferative tumor cells (P = 0.05) and lymph node metastases (P = 0.03). Overexpression of TROP2 significantly correlated with worse overall survival (P = 0.026) and poor disease-free survival (P = 0.021). By univariate analysis, high expression of TROP2 significantly correlated with patients with distant metastases, Ki-67 and EBV infection. Multivariate analysis further revealed that TROP2 along with Ki-67 and distant metastasis are independent prognostic predictors for NPC patients. CONCLUSION: Our findings have demonstrated that overexpression of TROP2 appears to be an independent predictor for poor clinical outcome in NPC. The strong correlation of overexpression of TROP2 with Ki-67 and distant metastases indicates a potentially therapeutic strategies targeting TROP2 for NPC patients.

Gene expression profiling via bioinformatics analysis reveals biomarkers in laryngeal squamous cell carcinoma.

The present study aimed to identify key genes and relevant microRNAs (miRNAs) involved in laryngeal squamous cell carcinoma (LSCC). The gene expression profiles of LSCC tissue samples were analyzed with various bioinformatics tools. A gene expression data set (GSE51985), including ten laryngeal squamous cell carcinoma (LSCC) tissue samples and ten adjacent non-neoplastic tissue samples, was downloaded from the Gene Expression Omnibus. Differential analysis was performed using software package limma of R. Functional enrichment analysis was applied to the differentially expressed genes (DEGs) using the Database for Annotation, Visualization and Integrated Discovery. Protein-protein interaction (PPI) networks were constructed for the protein products using information from the Search Tool for the Retrieval of Interacting Genes/Proteins. Module analysis was performed using ClusterONE (a software plugin from Cytoscape). MicroRNAs (miRNAs) regulating the DEGs were predicted using WebGestalt. A total of 461 DEGs were identified in LSCC, 297 of which were upregulated and 164 of which were downregulated. Cell cycle, proteasome and DNA replication were significantly over-represented in the upregulated genes, while the ribosome was significantly over-represented in the downregulated genes. Two PPI networks were constructed for the up- and downregulated genes. One module from the upregulated gene network was associated with protein kinase. Numerous miRNAs associated with LSCC were predicted, including miRNA (miR)-25, miR-32, miR-92 and miR-29. In conclusion, numerous key genes and pathways involved in LSCC were revealed, which may aid the advancement of current knowledge regarding the pathogenesis of LSCC. In addition, relevant miRNAs were also identified, which may represent potential biomarkers for use in the diagnosis or treatment of the disease.

Abnormal Wnt signaling and overexpression of ABCG2 contributes to drug efflux properties of side population cells in nasopharyngeal carcinoma.

The presence of cancer stem cells (CSCs) has major implications in the choice of cancer treatment strategy and is responsible for tumor relapse. CSCs have been isolated and characterized in several types of cancer; however, studies concerning the CSCs from nasopharyngeal carcinoma (NPC) are limited. Thus, the present study was designed to isolate and characterize the cancer stem-like side population (SP) cells from NPC samples. The fluorescence-activated cell sorting (FACS)-based Hoechst 33342 dye exclusion technique identified that 3.9% of cells from NPC samples were cancer stem-like SP cells. Upon treatment with verapamil (ABC transporter inhibitor), the percentage of SP cells was significantly reduced to 0.7%, which confirms that the ABC transporter protein exhibits a significant role in drug exclusion. Fluorescence microscopy analysis revealed that the FACS purified SP cells showed increased expression of ABCG2 (ATP transporter protein), Oct-4 and CD44 (stem cell surface protein). Furthermore, these SP cells exhibited increased mRNA expression of ABCG2 and anti-apoptotic factor Bmi-1, which contribute to multi-drug resistance and increased cell survival rate. Notably, the Wnt/ß-catenin signaling pathways are altered in SP cells. In addition, using reverse transcription­quantitative polymerase chain reaction analysis it was observed that the cells exhibited increased expression of DKK1 and AXIN2. In conclusion, data from the present study clearly demonstrated that the presence of cancer stem-like SP cells from NPC may be responsible for chemotherapeutic drug resistance, tumor recurrence and invasion.

Small interfering RNA survivin and GRIM-19 co-expression salmonella plasmid inhibited the growth of laryngeal cancer cells in vitro and in vivo.

OBJECTIVE: To investigate the inhibitory effect of plasmid-based survivin-specific short hairpin RNA and GRIM-19 on the growth of Hep-2 laryngeal cancer cells. METHODS: The plasmid expressing survivin-specific short hairpin RNA (shRNA) and GRIM-19 (p-siRNA survivin/GRIM-19) was prepared and transfected into Hep-2 cells with Lipofectamine 2000. The mRNA and protein expression of surviving and GRIM-19 were measured with RT-PCR and western blot assay, respectively. MTT assay was employed to detect the proliferation of Hep-2 cells, and flow cytometry and AO/EB assay were done to determine the apoptosis of Hep-2 cells. RESULTS: In the p-siRNA survivin/GRIM-19, the mRNA and protein expression of survivin was markedly reduced by 54.4% and 42.2%, and the reduction in protein expression of surviving was more obvious than that in the p-siRNA survivin group (37%) (P<0.05). The protein expression of GRIM-19 was markedly enhanced when compared with the control group (P<0.01). MTT assay revealed the proliferation of Hep-2 cells undergoing transfection with p-siRNA survivin/GRIM-19 was markedly inhibited, and the inhibition rate was as high as 79%, which was higher than that in the psi-survivin group (45%) and p-GRIM-19 group (35%). AO/EB assay and flow cytometry indicated that the apoptotic cells in the p-siRNA survivin/GRIM-19 group were dramatically increased as compared to the psi-survivin group and p-GRIM-19 group. CONCLUSION: The p-siRNA survivin/GRIM-19 has marked decrease in survivin expression and dramatic increase in GRIM-19 expression. Moreover, silencing of survivin and over-expression of GRIM-19 can significantly inhibit the growth and induce the apoptosis of Hep-2 in vitro and in vivo.

[Construction of the nucleic vaccine pVVP3L-18HN and its antitumor effect on human laryngeal carcinoma].

OBJECTIVE: Nucleic vaccine of pVVP3IL-18HN expressing apoptin gene, Newcastle disease virus HN gene and IL-18 gene were constructed to observe the combinative antitumor effect of the above three genes. METHODS: Eukaryotic expression plasmid pVVP3IL-18HN was constructed by inserting apoptin gene and fragment comprising fused IL-18HN gene and IRES promoter into the downstream of CMV promoter of vector pVAX1. The expression of inserted gene was identified by RT-PCR, indirect immunofluorescence and Western-blot. The recombinant plasmid was introduced into Hep-2 cells by liposome, then suppression rate of Hep-2 of different time and different quantity was calculated according to MTT results. RESULT: The recombinant plasmid of pVVP3IL-18HN suppressed Hep-2 successfully and its suppression rate was up to 61.9% with 20 microg/ml, incubation of 72 hours. CONCLUSION: The nucleic vaccine constructed pVVP3IL-18HN had antitumor effect on Hep-2. It may can be used to the therapy and research of laryngeal carcinoma.

[Antitumor research on mouse melanoma with combined application of Newcastle disease virus and its HN gene].

BACKGROUND & OBJECTIVE: Although Newcastle disease virus (NDV) shows antitumor effect on many tumors, its mechanism is unclear. Hemagglutinin-neuraminidase (HN) gene was found to play an important role in NDV antitumor effect and HN protein located on tumor cell surface. This research was to evaluate the possibility of HN protein as a foreign antigen of tumor cell and the antitumor effect of the combined application of HN gene and NDV. METHODS: C57BL/6 mice were subcutaneously inoculated with 2 x 10(5) B16 tumor cells in the right hindlimb. Combination group: on 2nd day post-inoculation, the recombinant plasmid containing HN gene was injected intramuscularly in the left hindlimb; on 7th day post-inoculation, 2 x 10(9) pfu NDV was administrated intratumorally. The alone HN gene group, NDV group, and PBS control group were treated as above. The antitumor effect was observed through tumor suppression rate, the antitumor mechanisms were researched with specific cytotoxic T lymphocyte (CTL) assay, and the expression determination of HN protein, ICAM-I, and CD48 on the B16 tumor cells. RESULTS: The antitumor efficacy of the combined application of NDV and its HN gene increased compared with NDV,and its HN gene alone, the tumor suppression rates were 82.8%, 41.0%, and 56.6%; the specific CTL activity were 18.4%, 10.1%, and 4.4%, respectively. Furthermore, the expression of HN gene had been detected, and the expression of ICAM-I and CD48 were up-regulated on the tumor cells after NDV injection. CONCLUSION: HN protein located on the surface of tumor cells and mediated the specific repulsion to tumor cells; the antitumor efficacy increased after the combined application of NDV and its HN gene.