| Chapter1Clinical significance of Tiaml expression in laryngeal carcinomaObjective:To evaluate the expression of Tiam1in tissue specimens of laryngeal carcinoma, and further correlate Tiaml expression with clinicopathological parameters and prognosis of laryngeal carcinoma.Methods:Tiaml expression in98primary laryngeal carcinoma tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patients’survival. Additionally,12paired laryngeal carcinoma tissues were evaluated for Tiaml expression by Western blotting.Results:Western blotting indicated that Tiaml expression levels in laryngeal carcinoma tissues were significantly higher than that in the corresponding paraneoplastic tissues (P<0.001). Immmunohistochemistry staining revealed that Tiam1was detected in all primary tumor samples, moreover, Tiaml overexpression was significantly correlated with lymph node metastasis (P<0.001), clinical stage (P=0.027), histological grade (P=0.020), and recurrence (P=0.003). Survival analysis demonstrated that high Tiaml expression was significantly correlated with shorter disease-free survival (P=0.001) and overall survival (P<0.001). When combined the Tiaml expression and lymph node status, Kaplan-Meier survival showed that patients with Tiaml overexpression/lymph node metastasis (+) had both shorter disease-free and overall survival than others (both P<0.001). Multivariate Cox proportional hazards model analysis confirmed that lymph node metastasis (P=0.001) and Tiaml overexpression (P=0.020) were statistically significant, independent predictor of prognosis for patients with laryngeal carcinoma.Conclusion:This study demonstrated that Tiaml protein expression was obviously increased in laryngeal carcinoma tissue samples, and Tiaml protein overexpression was associated with lymph node metastasis, recurrence, and poorer prognosis in laryngeal carcinoma patients. These results suggested that Tiaml might contribute to laryngeal carcinoma progression, and represent as a novel prognostic indicator as well as a potential therapeutic target for laryngeal carcinoma.Chapter2Investigation of the regulation of Tiaml on the proliferation, migration and invasion of the laryngeal carcinoma in vitroObjective:Our previous study has demonstrated that Tiaml is overexpressed in laryngeal carcinoma, and Tiaml protein overexpression was correlated with clinical stage, lymph node metastasis, tumor recurrence, and poorer prognosis in laryngeal carcinoma. Hence, we will upregulate the Tiaml expression in cell lines of laryngeal carcinoma, and observe the effect of Tiaml upregulation on the proliferation, migration and invasion of laryngeal carcinoma in vitro.Methods:Tiaml/C1199plasmid was used to transfect into the laryngeal carcinoma cell line Hep-2, and the Hep-2cell line of stable high Tiaml expression was obtained by G418screening. Western blotting, Immunofluorescence of cells and Immunohistochemistry of cells were used to validate the gene upregulation efficiency of Tiaml in laryngeal carcinoma cell line Hep-2. MTT cell viability assay, plate clone formation assay, fluorescence-activated cell sorting analysis, invasion and migration assay were carried out to observe the effects of Tiam1upregulation on the proliferation, cell cycle, apoptosis, migration and invasion of laryngeal carcinoma cell line Hep-2in vitro.Results:Tiam1/C1199plasmid was successfully transfected into the Hep-2cell line, and the Hep-2cell line of stable high Tiam1expression (Hep-2/Tiam1) was successfully obtained by G418screening. Compared with the group of Hep-2and cell line with null vector transfection (Hep-2/Mock), there was no significant statistical difference in proliferation among3groups (P>0.05), and there was also no significant statistical difference in percentage of G1phase, S phase and G2phase (P>0.05), but the apoptotic rate of Hep-2/Tiaml cells was significantly decreased (2.50±1.33vs.5.61±1.08,5.83±1.02)(P=0.021). Moreover, Tiaml upregulation led to significantly increased migration [Hep-2/Tiam1group, migration distance (147.00±26.06) vs Hep-2, Hep-2/Mock control groups, migration distance (83.33±23.18) and (96.33±16.01), P=0.028] and invasion (68.33±6.66vs43.00±8.89,45.00±7.55; P=0.013) of laryngeal carcinoma Hep-2cells.Conclusion:Upregulation of Tiaml has no effect on the proliferation of Hep-2cells in vitro, but can increase the migration and invasion of Hep-2cells in vitro, suggestting that Tiam1contributes to the laryngeal carcinoma progression.Chapter3Investigation of the regulation of Tiaml on the proliferation of the laryngeal carcinoma in vivoObjective:To further confirm the above results obtained in vitro, we observe the effect of Tiam1upregulation on the proliferation of laryngeal carcinoma in vivo.Methods:Tiaml/C1199plasmid was used to transfect into the laryngeal carcinoma cell line Hep-2, and the Hep-2cell line of stable high Tiaml expression was obtained by G418screening. Western blotting was used to validate the gene upregulation efficiency of Tiaml in laryngeal carcinoma cell line Hep-2. Stable clones were used to establish laryngeal carcinoma metastatic xenograft mouse model. Hematoxylin-eosin staining was used to identify xenografted tumors. Immunohistochemistry and Western blotting was used to investigate the protein expression of Tiam1in xenografted tumors.Results:Tiam1/C1199plasmid was successfully transfected into the Hep-2cell line, and the Hep-2cell line of stable high Tiaml expression (Hep-2/Tiam1) was successfully obtained by G418screening, which was further successfully used to establish laryngeal carcinoma metastatic xenograft mouse model. Hematoxylin-eosin staining confirmed that it is Hep-2xenograft tumor. There was no significant statistical difference among3groups (Hep-2, Hep-2/Mock and Hep-2/Tiaml) on the tumor volume and tumor weight of xenografted tumors (both P>0.05). Immunohistochemistry and Western blotting demonstrated that Tiaml expression was significantly increased in xenografted tumors of Hep-2/Tiam1compared with Hep-2and Hep-2/Mock (P<0.05).Conclusion:Upregulation of Tiam1has no effect on the proliferation of Hep-2cells in vivo. |
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