The analysis of checkpoint mechanism of cell cycle in liver cancer cells

利用AAV载体进行基因靶向，建立了内源性TP53等位基因外显子2被破坏的HepG2细胞（p53+/-）克隆。然而，TP53同源敲除HepG2细胞的克隆尚未获得。这些结果得出了一个假设，即p53可能对HepG2细胞的增殖至关重要，但使用siRNA敲低p53的表达并不会降低细胞增殖。另一方面，我们发现p53+/-细胞持续表达p53的亚型deltaN40p53。由于deltaN40p53在肝癌中的功能知之甚少，我们通过多种方法研究了deltaN40p53在HepG2细胞中的功能。我们的研究发现，deltaN40p53增强p21的表达，诱导G1/S阻滞增加，即在HepG2细胞中，当p53表达降低时，deltaN40p53弥补了p53的功能，抑制肿瘤细胞增殖，诱导细胞衰老。

Using gene targeting with AAV vectors, we established hetero knock-out HepG2 cells clones (p53+/-) of which Exon2 in endogeneous TP53 alleles were destroyed. However, TP53 homo knock-out HepG2 cells clones have not been obtained. These results derived a hypothesis that p53 might be essential for cell proliferation in HepG2 cells, but knock down of expression of p53 using siRNA did not decrease cell proliferation.On the other hand, we found that p53+/- cells constantly expressed deltaN40p53, an isoform of p53. Since the function of deltaN40p53 has been known little in liver cancer, we investigated the function of deltaN40p53 in HepG2 cells by using several methods. Our studies revealed that deltaN40p53 enhanced the expression of p21 and induced an increase of G1/S arrest, that is, deltaN40p53 made up for the function of p53 and suppressed tumor cell proliferation and induced cellular senescence in HepG2 cells when the expression of p53 was reduced.