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Objective: To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC). Methods: The expressions of CDK6 mRNA in fresh LSCC specimens, the adjacent normal mucosa of LSCC, and cell lines of LSCC were detected with quantitative real time polymerase chain reaction, pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells, and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells. Flow cytometry was employed for cell cycle analysis. SPSS17.0 software was used to analyze the data. Results: CDK6 was highly expressed in LSCC (t =14.01, P =0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression, with a significant difference ( HR =3.236, P <0.001). Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group ( P <0.01), while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group ( P >0.05). A (490) values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group, 0.51±0.13 in siRNA TU-212 group; 0.98±0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group. Colonies were 55±4 in siRNA Hep-2 group, 51±3 in siRNA TU-212 group, 108±6 in control Hep-2 group and 105±7 in control TU-212 group, namely, cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group. Cells cycle was blocked in G0/G1 phase (G0/G1: 65.20%±10.12% in siRNA Hep-2 group; 63.42%±8.97% in siRNA TU-212 group; 45.31%±7.55% in control Hep-2 group; and 42.37%±7.28% in control TU-212 group), and cells decreased obviously in S phase (S: 25.39%±5.51% in siRNA Hep-2 group; 27.21%±5.43% in siRNA TU-212 group; 42.87%±6.85% in control Hep-2 group; and 44.76%±7.02% in control TU-212 group). Compared with miR-497 group, cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all P <0.05). Conclusions: CDK6 expression in LSCC is upregulated, functioning as an oncogene. High expression of CDK6 is a predictor for poor prognosis. miR-497, functioning as a tumor suppressor gene, inhibits the growth of LSCC by targeting CDK6.

MicroRNAs are critical regulators of the development and progression of laryngeal squamous cell carcinoma (LSCC). However, the role of microRNA-154 (miR-154) in the development and progression of LSCC has not been clarified. We found that down-regulated miR-154 expression in LSCC tissues was associated with poorer prognosis in LSCC patients. MiR-154 over-expression inhibited the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest, which were reversed by miR-154 inhibition. MiR-154 targeted GALNT7 expression by reducing GALNT7-regulated luciferase activity in LSCC cells while up-regulating GALNT7 mRNA transcription in LSCC tissues and cells. GALNT7 silencing significantly attenuated the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest. Finally, intravenous treatment with lentivirus for miR-154, but not scrambled control miRNA, significantly restrained the growth of implanted LSCC Hep-2 tumors and decreased the tumor mass by reducing GALNT7 expression in mice. Therefore, miR-154 may serve as a novel prognostic marker and therapeutic target for LSCC.

Objective: To study the roles of miR-497 and PlexinA4 in the progression of laryngeal squamous cell carcinoma. Methods: The expressions of miR-497 and PlexinA4 in fresh tumor specimens and adjacent normal mucosa tissues as well as in cell lines of laryngeal squamous cell carcinoma (LSCC) were detected with qRT-PCR and immunohistochemistry. The association of miR-497 and PlexinA4 expressions with clinicopathologic factors and their prognostic values in LSCC were evaluated PlexinA4 siRNA and pcDNA3.1 (+ )/PlexinA4 plasmid were transfected into the LSCC and measured by Transwell to evaluate their effect on the invasion of LSCC. Results: miR-497 was low expression in LSCC, which related to pathological differentiation, while PlexinA4 mRNA was high expression in LSCC. Kaplan-Meier method showed that the prognosis of patients with high miR-497 expression was better than that of patients with low miR-497 expression (χ(2)=10.342, P =0.001); . Cox regression analysis showed that miR-497 was an independent prognostic factor for LSCC. The double luciferase reporter gene showed that the variation of the fluorescence activity of wild type PlexinA4 was significantly different from that of the control group ( P <0.01). In Hep-2 and TU212 cell line, the number of cells with PlexinA4 siRNA passing through the compartments was 70.00±10.85 and 85.00±6.45, significantly higher than control ( F values were 30.251 and 23.936, both P <0.05), the number of cells with pcDNA3.1 (+ ) /PlexinA4 was 170.56±11.95 and 142.00±10.43, also significantly less than control (F values were 35.104 and 29.643, both P <0.05). Conclusion: The expression of miR-497 in LSCC is decreased, indicating poor prognosis, which is as an independent risk factor for prognosis of LSCC. miR-497 may modulate LSCC invasion through PlexinA4.