MFRN1过表达对293T细胞生长及线粒体功能的影响

Overexpression of MFRN1 in 293T Cell and Its Effects on the Cell Proliferation and Mitochondrial Function

Mitoferrin-1(MFRN1)是溶质携带蛋白家族的一员,是线粒体内膜上最主要的铁运输蛋白。为了观察MFRN1过表达对293T细胞生长及线粒体功能的影响,采用包装慢病毒和感染293T细胞的方法构建MFRN1稳定过表达的细胞模型(293T-MFRN1),并对其线粒体功能进行评价。构建的稳定细胞株293T-MFRN1中MFRN1转录水平及蛋白表达水平显著增加(P<0.01),MFRN1过表达细胞线粒体内总铁的含量明显增加(P<0.05);电镜显示线粒体结构损伤,线粒体嵴减少、空泡化;线粒体能量代谢受抑制,细胞ATP含量显著下降,腺苷酸池总量(ATP+ADP+AMP)比对照组减少约40%;细胞生长受到抑制,在细胞培养48 h后高表达组细胞数量显著低于对照组;细胞内羟自由基(·OH)含量增加;线粒体DNA发生明显的氧化损伤。以上表明,MFRN1表达量的稳定对细胞功能极为重要,表达量异常增加将导致细胞线粒体功能障碍、细胞增殖抑制。

Mitoferrin-1（MFRN1） is a member of the solute carrier family as an essential iron importer, which is located in the mitochondrial inner membrane. In order to investigate the effect of MFRN1 overexpression in 293 T cell line on cell proliferation and mitochondrial function, a MFRN1 stably overexpressed cell line was generated by lentiviral-mediated MFRN1 transfected into 293 T cell line, then the mitochondrial function was measured. It was found that the level of MFRN1 expression was significantly increased in the MFRN1 stably overexpressed 293 T cell line（P0.01）. Furthermore, it was observed that the total iron loading was markedly elevated in the overexpressed cell line（P0.05）. In addition, it was also found that the mitochondrial structure was damaged with mitochondrial cristae reduced and vacuolated, as well as energy metabolism in mitochondrion was inhibited. It was indicated that the cellular ATP content markedly decreased and total adenine nucleotide pool reduced by 40% compared to controls. Even-tually, it was showed that the proliferation of overexpressed cell line were inhibited with significant reduction of cell counts, whereas the content of cellular hydroxyl free radical（·OH） increased and oxidative damage of mitochondrial DNA occurred. It was concluded that MFRN1 played important roles in the cellular function, and the aberrant expression of MFRN1 could result in the mitochondrial dysfunction and inhibition of cell proliferation.