OAZ1基因稳定表达SCC15细胞株的构建及其对舌鳞状细胞癌上皮间质化的影响

Establishment of SCC15 cell line stably expressing human OAZ1 gene and its influence on the epithelial mesenchymal transition of tongue squamous cell carcinoma

目的探索鸟氨酸脱羧酶抗酶(OAZ1)对舌鳞癌细胞株SCC15上皮间质化的影响及相关机制。方法构建框移位点突变的OAZ1过表达慢病毒表达载体p LVX-Neo-OAZ1-IRES-Zs Green,包装病毒并感染SCC15细胞,采用有限稀释法挑取荧光强度较高的单克隆细胞株;利用RT-PCR和Western blot检测OAZ1以及上皮间质化相关基因(E-cadherin、Vimentin、N-cadherin、Zeb1、Snail、Twist1、TGF-β1、Smad1)的m RNA水平和蛋白水平。结果成功构建了OAZ1基因稳定表达的SCC15细胞株,且OAZ1的过表达可以抑制Vimentin、N-cadherin、TGF-β1的m RNA和蛋白水平表达;此外,OAZ1可以促进Smad1的蛋白水平降低。结论 OAZ1可以抑制舌鳞癌细胞的上皮间质化,其机制可能涉及TGF-β1信号通路和Smad1信号通路的抑制。

Objective To investigate the role of OAZ1 in epithelial mesenchymal transition（EMT） in tongue squamous cell carcinoma cell lines SCC15 and explore its related mechanism. Methods The lentivirus vector p LVXNeo-OAZ1-IRES-Zs Green in which the target gene OAZ1 was modified at the frameshift site was constructed, packaged lentivirus and infected SCC15 cells, and the monoclonal cell lines with high fluorescence intensity were obtained by limited diluted method. Real-time PCR and Western blot was used to detect the expression of OAZ1 and the genes（E-cadherin, Vimentin, N-cadherin, Zeb1, Snail, Twist1, TGF-β1, Smad1） related with EMT. Results The SCC15 cell line with stable expression of OAZ1 gene was successfully constructed. The overexpression of OAZ1 inhibited the protein level and m RNA level of the Vimentin, N-cadherin, TGF-β1. Furthermore, the OAZ1 would promote the degradation of Smad1. Conclusion OAZ1 can inhibit the EMT of tongue squamous cell carcinoma cell lines through the possible mechanism related with the inhibition of TGF-β1and Smad1 pathway.