MicroRNA-145 inhibits growth of laryngeal squamous cell carcinoma by targeting the PI3K/Akt signaling pathway.

Purpose: In this study, we used a nude mouse model of human laryngeal squamous cell carcinoma (LSCC) to investigate inhibition of tumor growth by microRNA-145 (miR-145) and the mechanisms underlying this inhibition. Methods: Tumors were established in nude mice by transplantation of the LSCC AMC-HN-8 cell line. Forty-eight nude mice were randomly divided into groups of eight mice each and treated with high (1.0 optical density [OD]) or low (0.5 OD) doses of miR-145, or relevant control treatments. Tumor growth was observed in each group and used to calculate the inhibition rate. Routine pathological and electron microscopic examinations were used to determine tumor apoptosis and proliferation. Changes in levels of miR-145 and PI3K and Akt protein levels were also analyzed. Results: MiR-145 inhibited LSCC growth in a dose-dependent manner, as tumor growth was significantly inhibited in mice injected intratumorally with high-dose miR-145 compared with both the untreated and low-dose miR-145 groups (p<0.05). Pathological examination showed increased tumor necrotic and apoptotic changes in treated mice, which was confirmed by electron microscopy. PI3K and Akt protein expression were significantly lower in tumors treated with high-dose miR-145 group compared with those in the untreated and low-dose miR-145 groups (p<0.05). Conclusions: MiR-145 was associated with inhibited tumor growth in a nude mouse model of LSCC. The underlying mechanism may be inhibition of the PI3K/Akt signaling pathway, which regulates tumor growth, invasion, and metastasis and also plays an important role in tumor angiogenesis and proliferation of tumor stem cells. MiR-145 may act as a tumor suppressor gene and is a promising candidate for cancer treatment.

LZTS2 promoter hypermethylation: a potential biomarker for the diagnosis and prognosis of laryngeal squamous cell carcinoma.

BACKGROUND: LZTS2 (leucine zipper tumor suppressor 2), a candidate tumor suppressor gene, suppresses cell growth and plays a vital role in the carcinogenesis and development of tumors. No studies to date have described methylation of the LZTS2 promoter in human cancers, including LSCC (laryngeal squamous cell carcinoma). Therefore, the aim of this study was to explore the relationship between LZTS2 promoter methylation and risk of LSCC. METHODS: In our study, LZTS2 promoter methylation levels in LSCC tumor and adjacent normal tissues from 96 patients were measured using quantitative methylation-specific polymerase chain reaction (qMSP) assays. RESULTS: The qMSP analyses revealed that LZTS2 promoter methylation levels in the LSCC tumor samples were significantly higher than those in paired adjacent healthy tissue samples. Furthermore, LZTS2 methylation levels were elevated in smokers, advanced T classified, and clinically staged patients, as well as in patients with lymph node metastases. In addition, Kaplan-Meier survival curves results showed that overall survival of LSCC patients with hypomethylated LZTS2 promoters was significantly higher than that in patients with hyper-methylated LZTS2 promoters (log-rank test P = 0.028). Meanwhile, the area under the receiver operating characteristic curve was 0.920. The diagnostic threshold value for LZTS2 methylation was 11.63% (94.7% sensitivity and 80.4% specificity). CONCLUSIONS: LZTS2 promoter hypermethylation is associated with risk, progression, and prognosis of LSCC in a cohort of 96 human subjects; LZTS2 promoter hypermethylation is a candidate diagnostic and prognostic biomarker for LSCC.

Long non-coding RNA AC026166.2-001 inhibits cell proliferation and migration in laryngeal squamous cell carcinoma by regulating the miR-24-3p/p27 axis.

Long non-coding RNA (lncRNA) AC026166.2-001 was found to be down-regulated in laryngeal squamous cell carcinoma (LSCC) tissues and metastatic neck lymph nodes. Decreased AC026166.2-001 was associated with poorer prognosis and may act as a novel biomarker for LSCC patients. In this study, AC026166.2-001 was overexpressed by a lentivirus vector and down-regulated by a small interfering RNA (siRNA). The results of real-time cell analysis (RTCA) and a plate colony formation assay showed that AC026166.2-001 inhibited LSCC cell proliferation and the clone-forming capacity. Cell cycle distribution and related protein changes were measured by flow cytometry. AC026166.2-001 arrested the cell cycle at the G1 phase and induced apoptosis. In addition, AC026166.2-001 decreased cell migration as measured by wound healing assays and transwell migration assays. Moreover, luciferase reporter assay and Western blotting results suggested that AC026166.2-001 acts as a sponge of miR-24-3p and regulates the expression of p27 and cyclin D1. The in vivo results showed that AC026166.2-001 significantly suppressed the growth of LSCC xenografts and promoted apoptosis. We validated the molecular mechanisms underlying AC026166.2-001 in LSCC. This is the first report of AC026166.2-001 acting as a tumor suppressor in LSCC by regulating the miR-24-3p/p27 axis.

The Clinical Signification of Claudin-11 Promoter Hypermethylation for Laryngeal Squamous Cell Carcinoma.

BACKGROUND Claudin-11 (CLDN11) is frequently silenced by its promoter hypermethylation. Previous studies have shown that CLDN11 promoter hypermethylation is a potential biomarker for diagnosing various cancers. The aim of this study was to investigate CLDN11 promoter methylation and its potential relevance to clinicopathologic features and prognosis of patients with laryngeal squamous cell carcinoma (LSCC). MATERIAL AND METHODS Using the quantitative methylation-specific polymerase chain reaction (qMSP), CLDN11 promoter methylation was measured in 91 tumor tissues and their paired adjacent normal tissues, and the relationship between CLDN11 methylation and clinicopathologic features was evaluated. A receiver operating characteristic (ROC) curve was created to assess diagnostic values, and the Kaplan-Meier survival analysis was used to evaluate the association between CLDN11 methylation and prognosis of patients with LSCC. RESULTS Our results showed significantly elevated promoter methylation of CLDN11 in tumor tissues compared to their adjacent tissues (p=1.227E-16). CLDN11 promoter methylation also increased in patients with lymph node metastasis (p=0.009), advanced clinical stage (p=9.26E-06) and higher T classification (p=0.003). The area under the ROC curve (AUC) of CLDN11 was 0.884 (95% CI=0.835-0.932, p<0.01). The Kaplan-Meier analysis indicated that high CLDN11 promoter methylation levels were associated with poor overall survival of LSCC patients (log-rank test, p=0.007). CONCLUSIONS We demonstrated that CLDN11 promoter hypermethylation is a frequent event in LSCC, and contributes to metastasis and progression of LSCC. Thus, CLDN11 could be a potential biomarker for diagnosis and prognosis of LSCC patients.

SHISA3 Promoter Methylation Is a Potential Diagnostic and Prognostic Biomarker for Laryngeal Squamous Cell Carcinoma.

The purpose of this study was to evaluate the contribution of SHISA3 promoter methylation to laryngeal squamous cell carcinoma (LSCC). SHISA3 promoter methylation status and expression were determined using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (qRT-PCR) in 93 paired LSCC and adjacent normal tissues, respectively. Furthermore, the regulatory function of the SHISA3 promoter fragment was analyzed using a luciferase reporter assay. The results reveal that there is a significant increase in SHISA3 methylation in LSCC tissues compared with corresponding nontumor tissues (P = 4.58E - 12). The qRT-PCR results show a significant association between SHISA3 methylation and expression in LSCC (P = 1.67E - 03). In addition, the area under the receiver operating characteristic curve was 0.91. Consequently, a log-rank test and multivariate Cox analysis suggest that SHISA3 promoter hypermethylation is a predictor of poor overall survival for LSCC (log-rank P = 0.024; HR = 2.71; 95% CI = 1.024-7.177; P = 0.047). The results indicate that SHISA3 promoter hypermethylation might increase the risk of LSCC through regulation of gene expression and is a potential diagnostic and prognostic biomarker for LSCC.