Ru Wang

Microarray Gene Expression Analysis of Tumorigenesis and Regional Lymph Node Metastasis in Laryngeal Squamous Cell Carcinoma

Background Laryngeal squamous cell carcinoma (LSCC) is the most common type in head and neck squamous cell carcinoma (HNSCC), and the development and progression of LSCC are multistep processes accompanied by changes of molecular biology. Objective The purpose of this study was to investigate the molecular basis of tumorigenesis and regional lymph node metastasis in LSCC, and provide a set of genes that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies. Methods A total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays, and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner. The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC. Results Analysed by Illumina mRNA microarrays, there were 361 genes significantly related to tumorigenesis while 246 genes significantly related to regional lymph node metastasis in LSCC. We found that the six genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4) were most frequently differently expressed functional genes related to tumorigenesis while eIF3a and RPN2 were most frequently differently expressed functional genes related to regional lymph node metastasis in LSCC. The expressions of these genes were also validated by qRT-PCR. Conclusions The research revealed a gene expression signature of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma. Of the total, the deregulation of several genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4, EIF3a and RPN2) were potentially associated with disease development and progression. The result will contribute to the understanding of the molecular basis of LSCC and help to improve diagnosis and treatment.

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Knockdown of CUG-binding protein 1 induces apoptosis of human laryngeal cancer cells.

To investigate the role of CUG‐binding protein 1 (CUGBP1) in human laryngeal cancer, we employed lentivirus‐mediated short hairpin RNA (shRNA) to knockdown CUGBP1 expression in Hep‐2 cells. Depletion of CUGBP1 remarkably inhibited the proliferation of Hep‐2 cells. CUGBP1 knockdown induced cell cycle arrest in S phase, especially in the sub‐G1 phase, representing apoptotic cells. Knockdown of CUGBP1 in Hep‐2 cells markedly increased the expression of LIP and cleavage of PARP, which could contribute to apoptosis. Thus CUGBP1 has a critical role in modulating cell growth and apoptosis, and serves as a potential therapeutic target in laryngeal cancer.

Dysregulation of zinc finger protein, X-linked (ZFX) impairs cell proliferation and induces apoptosis in human oral squamous cell carcinorma

Zinc finger protein, X-linked (ZFX) is a transcriptional factor involved in many physiological processes such as embryonic stem cell survival and self-renewal. Though ZFX dysfunctions have been identified in variant human diseases and especially in cancers, its pathological roles have not been fully addressed. Here, we explored the relationship between ZFX expression and squamous cell carcinoma (SCC) of the tongue. We found that ZFX expression was significantly higher in tongue SCC tumors as compared to tumor-adjacent normal tissues. Furthermore, ZFX knockdown impeded cell proliferation, impaired colony formation ability, and lead to cell cycle arrest while induced cell apoptosis in human tongue squamous cell carcinoma cell line Tca-8113. Our results provide evidence suggesting that ZFX overexpression is associated with the development of tongue SCC and ZFX knockdown is a potential treatment for tumor suppression.

Tumor necrosis factor superfamily member 13 is a novel biomarker for diagnosis and prognosis and promotes cancer cell proliferation in laryngeal squamous cell carcinoma.

Tumor necrosis factor superfamily member 13 (TNFSF13) modulates cell proliferation and apoptosis and participates in the pathogenesis of solid tumors, but its role in laryngeal cancer development is not clearly defined. In order to investigate whether TNFSF13 can be used as a biomarker for diagnosis and prognosis in laryngeal squamous cell carcinoma (LSCC) and the role of TNFSF13 in laryngeal cancer carcinogenesis, we conducted immunohistochemistry and ELISA assays to evaluate the expression level of TNFSF13 in laryngeal cancer patients and the contrast. We also conducted experiments on the functional study of TNFSF13 in vitro. We found that the expression levels of TNFSF13, ki-67, and NF-κB p65 in LSCC tumor tissues were higher than those in vocal polyp and para-carcinoma tissues. The Spearman rank correlation analysis showed that the expression of TNFSF13 had a positive correlation with the expression of ki-67 and NF-κB p65. Cox regression analysis and Kaplan–Meier plots confirmed the expression level of TNFSF13 was a prognostic factor for LSCC. Moreover, the serum TNFSF13 level was significantly higher in LSCC patients than in the controls, and the serum expression level of TNFSF13 can distinguish LSCC from healthy people, precancerosis, or laryngeal benign tumor. In addition, functional study of TNFSF13 in vitro revealed that knockdown of TNFSF13 inhibited cell proliferation by inducing G1 phase cell cycle arrest in Hep-2 cells. In conclusion, TNFSF13 may be a potential novel molecular target for diagnosis and prognosis in human LSCC, and therapies that target TNFSF13 may have clinical significance for the treatment of LSCC.

Effect of microRNA-203 on tumor growth in human hypopharyngeal squamous cell carcinoma

MicroRNAs (MiRNAs) have been recognized to regulate cancer initiation and progression in carcinogenesis as either oncogenes or tumor suppressor genes, but their role in hypopharyngeal cancer development is not clearly defined. To determine whether miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma, we conducted experiments on the functional study of miRNA-203 and identification of miRNA-203 regulated target genes in hypopharyngeal cancer cells. We found that cell proliferation and cell colony-forming increased more in the miRNA-203 up-regulated cancer cells than in the negative control cancer cells. Up-regulation of miRNA-203 accelerated cell cycle progression in hypopharyngeal cancer cells. TP63 and B3GNT5 mRNAs were identified and validated as targets of miRNA-203. However, transwell assay and wound scratch assay showed that miRNA-203 did not involve in invasion and metastasis in hypopharyngeal cancer cells. According to the results, we conclude that miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma. These results provide the convincing evidence for the first time that up-regulation of miRNA-203 contributes to the malignancy of hypopharyngeal squamous cell carcinoma, possibly through down-regulating TP63 and B3GNT5.

The value of narrow band imaging combined with stroboscopy for the detection of applanate indiscernible early-stage vocal cord cancer

Abstract Background: Narrow band imaging (NBI) and stroboscopy are non-invasive techniques to detect the malignant lesions of the vocal cord. This study was to assess the diagnostic value of combined endoscopic analysis in the applanate indiscernible early-stage vocal cord cancer. Methods: A total of 110 patients with 160 suspicious vocal cord malignant lesions were included in this retrospective study. Stroboscopy was immediately performed after NBI and white light endoscopy (WLE) were performed in all patients. Excisional biopsy was performed to examine histopathology examination. Results: We found that the diagnostic specificity and PPV were higher in the NBI and WLE combined with stroboscopy group than in the NBI and WLE group without stroboscopy (88.9% vs 72.5%, 88.4% vs 60.9%). However, the diagnostic sensitivity was not significantly different in those two groups (69.3% vs 67.7%). Conclusion: NBI and WLE combined with stroboscopy is a promising method to detect early-stage vocal cord cancer with the advantage of clinical feasibility and diagnostic specificity.

The up-regulation expression of APRIL is a marker of glottic malignant disease

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) family. Recent studies have implied that APRIL is closely related to solid tumors and hematological tumors, indicating that APRIL could be a potential marker to diagnose glottic malignant disease. The purpose of this study was to investigate the difference of the APRIL mRNA and protein expression in glottic malignant disease, corresponding adjacent non-neoplastic tissues and glottic benign lesion, and detect the influence of different clinical parameter in glottic carcinoma. The APRIL mRNA expression in the glottic carcinoma, corresponding adjacent non-neoplastic tissues and glottic polypus tissue samples from patients was detected by qRT-PCR. Moreover, we studied the APRIL protein expression in pathological sections of other patients with glottic carcinoma or glottic polypus using immunohistochemistry. All the patients with different clinical parameter underwent surgery. Using qRT-PCR, we revealed an up-regulation of APRIL mRNA expression in glottic carcinoma as compared to glottic polypus and corresponding adjacent non-neoplastic tissues, but no significant difference with T stages, histopathological differentiation grade or lymph node metastasis in glottic carcinoma. The result of the immunohistochemistry was the same, with no influence of different clinical parameter in glottic carcinoma. These results strongly suggest that APRIL could be a potential diagnosed marker to distinguish glottic malignant disease from glottic benign lesion, and it may play an important role in the development of glottic malignant disease.

Transcobalamin I: a novel prognostic biomarker of neoadjuvant chemotherapy in locally advanced hypopharyngeal squamous cell cancers

Background Hypopharyngeal squamous cell carcinoma (HPSCC) is an aggressive head and neck squamous cell carcinoma with poor prognosis. Neoadjuvant chemotherapy (NACT) followed by concurrent chemoradiotherapy could provide better efficacy in HPSCC treatment. Identification of predictive biomarkers is critically needed to improve selection of patients who derive the most benefit from NACT. The aim of this study was to investigate whether transcobalamin I (TCN1) could be a novel predictive biomarker for NACT in HPSCC. Methods We collected biopsy specimens from 102 patients with primary locally advanced HPSCC. Messenger RNA (mRNA) and protein expression levels of TCN1 were analyzed using quantitative polymerase chain reaction and immunohistochemistry, respectively. The relationship between TCN1 expression, chemotherapy sensitivity, and clinical outcome was assessed using univariate Kaplan–Meier survival analyses and multivariate analysis with covariate adjustments. Furthermore, we knocked down TCN1 by small interfering RNA (siRNA) in HPSCC cell FaDu, tested the effects of TCN1 knockdown on cisplatin toxicity by MTT assay, and detected cisplatin-induced apoptosis by Western blotting. Results TCN1 expression was significantly lower in NACT-sensitive patients than nonsensitive patients at protein level (p=0.013) and mRNA level (p<0.001), indicating that low TCN1 expression predicts better NACT treatment response. Furthermore, TCN1 was an independent prognostic biomarker for both overall survival (p=0.047) and disease-free survival (p=0.05) in advanced HPSCC patients. In addition, in vitro experiments showed that genetic silencing of TCN1 using siRNA sensitized FaDu cells to cisplatin treatment with increased cell apoptosis. Conclusion Low expression of TCN1 might be a novel prognostic biomarker for predicting NACT sensitivity and clinical outcome in local advanced HPSCC patients.

Identification of microRNAs associated with medullary thyroid carcinoma by bioinformatics analyses

The present study aimed to investigate the microRNA (miRNA) profile in human medullary thyroid carcinoma (MTC) tissue. The GSE40807 data profile was downloaded from the Gene Expression Omnibus database. Following preprocessing, differentially expressed microRNAs (DEMs) between MTC and healthy tissues were identified. Based on the obtained DEMs, transcription factor (TF)‑miRNA and miRNA‑target gene regulatory association pairs were predicted. Finally, functional enrichment analysis was performed on target genes of DEMs. Fifteen upregulated and 17 downregulated DEMs were identified. In the constructed TF‑miRNA regulatory network, hsa‑miR‑9‑5p was regulated by 9 TFs and hsa‑miR‑1 was regulated by 8 TFs. TFs of nuclear factor of κ light polypeptide gene enhancer in B‑cells 1 (NF‑κB1) and v‑myc avian myelocytomatosis viral oncogene homolog (MYC) regulated 4 and 3 DEMs, respectively. In the miRNA‑target gene regulatory network, hsa‑miR‑1, hsa‑miR‑9‑5p, hsa‑miR‑96‑5p and hsa‑miR‑590‑5p were most upregulated. The target genes of these 4 miRNAs were primarily enriched in the mitogen activated protein kinase (MAPK) signaling pathway. Therefore, MAPK signaling pathway may serve important roles in MTC progression. In conclusion, the DEMs hsa‑miR‑1 and hsa‑miR‑9‑5p, and TFs of NF‑κB1 and MYC may be used as biomarkers for the diagnosis and treatment of MTC.