Cell Cycle-dependent Expression Of CtIP In HCC Cell Lines And Primary Study Of Its Ubiquitization

Objectives To solve the conflict of CtIP expression between levels of tissue andcell lines，we detected the expression changes of CtIP in different phases of cell cycleusing the technology of cell synchronization. To search for the mechanism of CtIPubiquitization，We also detect the influence of SIAH1on CtIP and cell proliferation，apoptosis and cell cycle changes of HCC cell lines.Methods1、 We induced cell synchronization using Thymidine, which is also called TdR.Real-time quantitive PCR(qRT-PCR) was used to detect the changes of CtIPexpression before and after cell synchronization.2、 The technology of RNA interfering was used to transiently knock down theexpression of SIAH1in HCC cell line HepG2. We utilized qRT-PCR andimmunoblotting to evaluate the SIAH1transfecion efficiency and to detect thechanges of CtIP expression.3、 Flow cytometry was used to evaluate the efficiency of cell synchronization andchoose the optimal time point of TdR incubation, and predominantly to observethe changes of apoptosis and cell cycle in HCC cell lines after RNA interferingfor SIAH1. We utilized WST-1which is also called CCK8to detect proliferationof HCC cell lines after SIAH1knocking down.ResultsDuring G1phase of cell cycle, the expression of CtIP in HCC cell line HepG2islower than that in the normal liver cell line L02. However, it turns to the opposite when cell cycle progresses to S and G2phase. We subsequently evaluated theexpression dynamics of CtIP and SIAH1when cells were passing through the G1/Sboundary. We have found that both CtIP and SIAH1increse dominantly after G/Sboundary with a summit at2hours after TdR releasing, and then descend rapidly tonormal level similar to G1phase. At the level of mRNA, SIAH1-deficient HepG2performs higher expression of CtIP comparing to the wild-type cell line. However, wehave not detected any changes at protein level. We also investigated the influence ofsiRNA-SIAH1on biological behavior of HepG2. Conparing to wild type cell line,SIAH1-deficient HepG2presents decreased level of cell proliferation.Conclusion The dynamic expressions of CtIP and SIAH1correlate with cell cycleprogression, therefore they may participate in cell cycle regulation and phase-specifichomologous recombination pathway during DSB. SIAH1remarkably affects theproliferation of cell line HepG2, and also has influence on cell apoptosis to somedegree. With regard to its function for CtIP ubiquitination, these findings raise thepossibility that SIAH1may has more functions like transcription and proliferationregulation other than the only mechanism of protein degradation.