N6-Methyladenosine reader HNRNPC-mediated downregulation of circITCH prevents miR-224-3p sequestering and contributes to tumorigenesis in nasopharyngeal carcinoma.

BACKGROUND: N6-Methyladenosine (m6A) RNA methylation modulators are implicated in nasopharyngeal carcinoma (NPC). Circular RNAs (circRNAs) stimulate/inhibit the development of NPC by sponging microRNAs (miRNAs). Herein, m6A modifications affecting the circRNA/miRNA axis in NPC were explored. METHODS: Twenty prognostic m6A RNA methylation regulators were identified from 504 head/neck squamous cell carcinoma and 44 normal samples from The Cancer Genome Atlas (TCGA). Differentially expressed miRNAs were screened from the TCGA and Gene Expression Omnibus (GEO) databases. RNA-binding protein (RBP)-circRNA and circRNA-miRNA interactive pairs were verified using RBPmap and RNAhybrid, respectively. The RBP/circRNA/miRNA network was constructed using Cytoscape. Furthermore, CircITCH (hsa_circ_00059948), HNRNPC, and miR-224-3p expressions were detected by western blotting and quantitative polymerase chain reaction. The role of circITCH in NPC was examined using apoptosis, scratch wound healing, transwell invasion, and cell counting kit-8 assays. Finally, CircITCH-miR-224-3p and circITCH-HNRNPC interactions were assessed by dual-luciferase reporter and RNA-immunoprecipitation (RIP) assays, respectively. RESULTS: Bioinformatics analysis revealed that high pathological grade, late-stage tumors, and low survival were associated with increased HNRNPC expression. MiR-224-3p was upregulated in NPC and sequestered by circITCH. Construction of the RBP/circRNA/miRNA network highlighted the HNRNPC/circITCH/miR-224-3p axis. In vitro experiments demonstrated decreased circITCH expression and increased HNRNPC and miR-224-3p expressions in NPC. In NPC cells overexpressing circITCH, HNRNPC and miR-224-3p expressions were significantly decreased. Dual-luciferase assays demonstrated a targeting relationship between circITCH and miR-224-3p, and RIP assays demonstrated interaction of HNRNPC targets with circITCH. CONCLUSION: CircITCH overexpression inhibited NPC progression by sequestering miR-224-3p, and HNRNPC reduced circITCH expression through direct interaction.

Tumor suppressor functions of miRNA-375 in nasopharyngeal carcinoma through inhibition of ubiquitin-specific protease 1 expression.

BACKGROUND: Nasopharyngeal carcinoma (NPC) development involves many genetic alterations. This study profiled differentially expressed microRNAs (DE-miRNAs) and selected miR-375 for further study. METHODS: DE-miRNAs were screened using online databases and subjected to various analyzes. miR-375 mimics with negative control (NC) cDNA, and a ubiquitin-specific protease 1 (USP1) as well as a NC group were transfected into NPC cells for analysis by quantitative PCR, western blotting, wound healing, Transwell, flow cytometry, cell counting kit-8 (CCK-8), and luciferase gene reporter assays. RESULTS: Among these DE-miRNAs, miR-375 was downregulated and miR-21 was upregulated in NPC cells. Bioinformatical analysis identified USP1 as a potential target gene of miR-375. Increased USP1 expression was associated with poor survival of head and neck cancer patients. The luciferase assay confirmed miR-375 binding to the USP1 3'-untranslated region (UTR), while the transfection experiment confirmed miR-375 expression reduced USP1 expression. USP1 overexpression reversed the anti-tumor activity of miR-375 in NPC cells as determined by tumor cell migration, invasion, apoptosis, and viability assays. In addition, USP1 overexpression activated phosphoinositide 3-kinase (PI3K) signaling, whereas a selective PI3K inhibitor (S2739) could reverse the effects of USP1 on NPC cells in vitro. CONCLUSIONS: miR-375 and miR-21 are both related to NPC and miR-375 can target USP1. Further experiments revealed that up-regulated miR-375 expression led to USP1 down-regulation, and miR-375 overexpression suppressed PI3K/Akt signaling and inhibited NPC cell migration and invasion, but promoted NPC cell apoptosis.

Frequent overexpression of PDK1 in primary nasopharyngeal carcinoma is associated with poor prognosis.

OBJECTIVE: Pyruvate dehydrogenase kinase 1 (PDK1) is reported to be up-regulated and play multiple functions in several cancers. Herein, we investigated its roles in primary nasopharyngeal carcinoma. METHODS: Expression of PDK1 protein was examined by immunohistochemical staining in 102 samples of primary nasopharyngeal carcinoma (pNPC) and 31 samples of chronic nasopharyngitis. The relationships of PDK1 expression levels with clinical features and prognosis in NPC patients were analyzed. RESULTS: PDK1 protein expression was significantly greater in pNPC tissues than in the chronic nasopharyngitis tissues (P<0.01). In addition, overexpression of PDK1 was associated with advanced clinical stage (P<0.01), advanced N classification (P=0.024), and distant metastasis (P=0.048). Kaplan-Meier survival analysis demonstrated that patients with higher PDK1 expression had significantly shorter overall survival (P=0.006), disease-free survival (P=0.015), loco-regional relapse-free survival (P=0.008), and distant metastasis-free survival (P=0.017). Furthermore, multi-variate analysis showed that PDK1 expression was an independent prognostic factor in pNPC patients. CONCLUSION: PDK1 is frequently upregulated in pNPC and may serve as a prognostic marker.

Down-regulation of succinate dehydrogenase subunit B and up-regulation of pyruvate dehydrogenase kinase 1 predicts poor prognosis in recurrent nasopharyngeal carcinoma.

Succinate dehydrogenase subunit B (SDHB) and pyruvate dehydrogenase kinase 1 (PDK1) play key roles in the regulation of growth and survival of various cancers. This study aimed to investigate expression of SDHB and PDK1 in recurrent nasopharyngeal carcinoma (rNPC) tissues and analyzed the association of SDHB and PDK1 expression with the clinical significance and potential prognostic implication of rNPC. Immunohistochemistry was performed to determine the expression of SDHB and PDK1 in tissues in primary NPC (pNPC) and rNPC patients. Our results revealed that expression of SDHB in rNPC was significantly lower than that in pNPC, while the expression of PDK1 was higher compared to pNPC. The expression levels of SDHB and PDK1 were associated with T stage, N stage, clinical stage, and metastasis of rNPC. Survival analysis showed that patients with low SDHB expression had a significantly shorter overall survival time than those with high SDHB expression. Patients with high PDK1 expression had a shorter survival time than patients with low PDK1 expression. Multivariate analysis showed that the expression of SDHB and PDK1 was an independent predictor for the survival of patients with rNPC. Our results demonstrated that down-regulation of SDHB and up-regulation of PDK1 may be novel biomarkers for predicting advanced tumor progression and unfavorable prognosis in rNPC patients.

Expression of reversion-inducing cysteine-rich protein with kazal motifs and matrix metalloproteinase 9 in middle ear squamous cell carcinoma.

OBJECTIVE: Reversion-inducing cysteine-rich protein with kazal motifs (RECK) may negatively regulate matrix metalloproteinase 9 (MMP9) activity and suppress tumor invasion and metastasis. The aim of this study was to investigate whether RECK and MMP were involved in regulating middle ear carcinoma invasion and metastasis. METHODS: RECK and MMP9 were measured in 30 middle ear squamous cell carcinoma tissues and 20 adjacent normal external ear canal skin tissues using immunohistochemical analysis. RESULTS: The positive rate of RECK expression in the middle ear squamous cell carcinoma was much lower than that in the normal external ear canal skin. In contrast, the positive rate of MMP9 expression was higher in middle ear squamous cell carcinoma tissue than that in normal external ear canal skin tissue. The expressions of RECK and MMP9 were correlated with the histological grade and tumor stage, but not with patient age or gender. CONCLUSIONS: RECK and MMP9 are involved in middle ear squamous cell carcinoma and may serve as markers to evaluate progression and metastasis.

[The CT study of relation between the height of middle concha and paranasal sinusitis].

OBJECTIVE: To evaluate the relation between the incidence of sinusitis and the position of the inferior border of the middle concha related to the semilunar hiatus. METHOD: Clinical data of 94 cases (185 sides of paranasal sinus) diagnosed by CT detection, operative findings and clinical features were analyzed. The middle concha was divided into 3 types according the position of its inferior border related to the semilunar hiatus: superior hiatus type (the inferior border of the middle concha superior to the semilunar hiatus), hiatus type (the inferior edge of the middle concha at the level of the semilunar hiatus) and inferior hiatus type (the inferior edge of the middle concha inferior to the semilunar hiatus). Statistic analysis were taken for comparing the incidence of sinusitis among the position of the middle concha and other anatomical variations such as deviation of nasal septum, pneumatization of middle concha, paradoxical curve of the middle concha, variations of the uncinate process, ethmoidal bulla enlargements, Haller cells and agger cell pneumatization. RESULT: There was no significant difference of the anatomic variations by comparing the superior hiatus type together with the hiatus type versus the inferior hiatus type (P > 0.05). But the incidence of sinusitis in each type was remarkably different, the superior hiatus type and hiatus type had more sinusitis than the inferior hiatus type. Furthermore, the second and third type of sinusitis in the superior hiatus type and hiatus type weighted over the inferior hiatus type (P < 0.01), while the first type didn't (P > 0.05). CONCLUSION: There is no association between the position of the middle concha and the anatomic variations of the nasal cavity and paranasal sinus. The poorly developed middle concha may acts as a risk factor for sinusitis and nasal polyps.