N端乙酰转移酶家族敲低细胞系构建及功能研究

Generation and Function Evaluation of HepG2 Stable Cell Lines with Knocking-down Different N-alphaacetyltransferase Family Members

N端乙酰转移酶在肝癌细胞中的研究很少,为更好地研究其功能,本研究构建了全部6种已知的N端乙酰转移酶单基因敲低细胞系.首先设计PLKO载体适应的RNAi序列,将正确的RNAi质粒采用病毒包装并转染肝癌细胞,采用嘌呤霉素筛选,获得稳定细胞株后RT-PCR检测敲低效率,用细胞增殖曲线法检测细胞增殖.RT-PCR显示N端乙酰转移酶敲低细胞系构建成功,细胞增殖实验证实Naa10、Naa20、Naa30、Naa40敲低能够抑制细胞生长,该敲低细胞系的建立为进一步研究N端乙酰化的功能提供了稳定可靠的细胞模型.

Protein amino-terminal acetylation mediated by N-alpha-aeetyltransferase is critical for cel- lular processes; however, the dysfunction of the N-alpha-acetyltransferase family in hepatocellular carcino- ma is poorly understood. Here we generated six stable cell lines for knocking down each N-alpha-acetyl- transferase （Naal0 to Naa60） separately, and evaluated the effects of knockdown and their loss-of-function on cell proliferation in HepG2 cells. Our RT-PCR results showed that we have successfully established all six knockdown stable cell lines for single N-alpha-acetyltransferase using HepG2 cells. The available Naa20 antibody also showed the protein level of Naa20 was dramatically reduced. Furthermore, the cell proliferation assays indicated that suppressing each expression of Naa10-Naa40 downregulated the cell prolif- eration rates. Taken together, our works provide helpful cell models for further study the function of N-al- pha-acetyltransferase in the future.