The Study Of MiR-210 And Target Gene E2F3 In Laryngeal Squamous Cell Carcinoma

Objective:To study the expression of miR-210 and E2F3 in LSCC cell lines, and prove that E2F3 wasdirect target gene of miR-210.And investigate the relationship of expression of miR-210 andE2F3 with clinicopathological factors in laryngeal squamous cell carcinoma. It also have thepotential of providing theoretical basis for the diagnosis, prognosis prediction, and treatmentof laryngeal squamous cell carcinoma.Methods:1. measure expression level of miR-210 and E2F3 in two human laryngeal carcinoma celllines hep-2 and TU177 by qRT-PCR. Moreover, luciferase reporter assays showed that E2F3was a direct target of miR-210;2. using qRT-PCR to analyze the expression of miR-210 and E2F3 mRNA in 20 paired offresh carcinoma tissues and fresh normal epithelium beside carcinoma tissues;3. immunohistochemical staining was used to detect the expressions of E2F3 protein in 136laryngeal squamous cell carcinoma tissues and 79 adjacent normal tissues. The correlations ofexpression of E2F3 protein, clinical stage, age, gender, tumor type, T stage, lymph nodemetastasis, smoking and differentiation degree with clinicopathological factors of laryngealsquamous cell carcinoma are analyzed byχ2test or Fisher’s exact test;4. overall survival rate is computed by Kaplan-Meier estimate. The log-rank test was used tocompare survival curves by obtaining aχ2value;5. a multivariate Cox proportional hazard regression model (Cox model) was used to test theprognostic effect of all factors on study. All data were analyzed using SPSS software version16.0.Results:1. The expression of miR-210 is higher in hep-2 cell line with lower expression of E2F3,comparing with TU177 cell line with higher expression of E2F3, Luciferase reporter assaysshowed that E2F3 was a direct target of miR-210;2. The expression of miR-210 negatively correlated with E2F3 in laryngeal squamous cellcarcinoma;3. The positive rates of E2F3 protein in the adjacent normal tissues and laryngeal squamouscell carcinoma tissues were 27.85% (22/79) and 90.44%(123/136) respectively. The expression of E2F3 protein in laryngeal squamous cell carcinoma was higher than that innormal mucosa(P＜0.05). The nuclear expression of E2F3 protein in laryngeal squamous cellcarcinoma tissues was correlated with the clinical stage, age, tumor type, and differentiationdegree (P＜0.05). And it was not correlated with the T stage, lymph node metastasis, smokingand gender(P＞0.05). In addition, very high level nuclear expression of E2F3 protein wasfound in almost all subglottic laryngeal squamous cell carcinomas analysed;4. In univariate analysis, clinical stage, the nuclear expression of E2F3 protein, lymph nodemetastasis, tumor type and T stage were associated with survival in laryngeal squamous cellcarcinoma patients. However, there were no association between age, gender, differentiationdegree, smoking, the cytoplasmic expression of E2F3 protein and the overall survival oflaryngeal squamous cell carcinoma patients;5. In multivariate analysis, T stage, lymph node metastasis, and the nuclear expression ofE2F3 protein are independent prognostic factors of laryngeal squamous cell carcinomapatients (P＜0.05).Conclusion:1. The expression of miR-210 is high level in laryngeal squamous cell carcinoma cell lines,and E2F3 is showed as a direct target of miR-210.2. The expression of miR-210 and E2F3 mRNA were highly up-regulated in fresh laryngealsquamous cell carcinoma tissues, the expression of miR-210 was negatively corelated withE2F3 in LSCC;3. The expression of E2F3 protein had a relationship with the clinical stage, age, tumor type,and differentiation degree. It may have the important significance in the laryngealtumorigenesis and progression;4. Clinical stage and the nuclear expression of E2F3 protein reflect prognosis of patients withlaryngeal squamous cell carcinoma. E2F3 protein may be a sensitive biomarker for theprognosis predict of laryngeal squamous cell carcinoma.