Effects Of Rock2on Signal Transduction During S Phase And Hepatocellular Carcinoma Cells

Objective:1. To investigate the effects of Rock2on proliferation and apoptosisof hepatocellular carcinoma Huh-7and HepG2cell lines.2. To analyze the regulatoryeffects and mechanism of Rock2on cell cycle checkpoint Cdc25A.3. To clarify theupper regulatory kinase of Rock2. Finally,to identify the signaling pathway of Rock2in S phase checkpoint and the effects on hepatocellular carcinoma cells.Methods:1. Hepatocellular carcinoma Huh-7and HepG2cell lines weretransfected with shRock2. Reverse transcriptase polymerase chain reaction andWestern blot assays were used to detect the Rock2mRNA and protein levels,respectively. Huh-7and HepG2cell proliferation was measured by MTT assay,change of cell cycle was detected by PI staining method through flow cytometry andapoptosis was demonstrated by Annexin V apoptosis kit.2. The expression of Rock2and Cdc25A protein in51pairs of hepatocellular carcinoma tissue were detected byWestern blot. shRock2plasmids were constructed and selected. shRock2plasmid wasstably transfected into hepatocellular carcinoma Huh-7and HepG2cells. Theexpression of Cdc25A protein were determined by Western blot. Based on the Rock2interfering sequences, we designed primers and changed the4bases indicated viasite-specific mutagenesis. The Rock2mutant plasmid was verified by sequencing andwas transfected into stable Rock2knockdown cells. Cdc25A protein expression wasdetected by Western blot and cell proliferation was measured by MTT assay.Theexpression of Chk1/Chk2protein was also detected in Rock2stable knockdown cells.The interaction between Rock2and Cdc25A was measured by coimmunoprecipitationand the colocalization between Rock2and Cdc25A was detected by confocal laserscanning microscopy. To explore the mechanism of Rock2regulating Cdc25Athrough ubiquitin proteasome system.3. To design the phosphorylated peptides ofRock2phosphorylation sites induced by DNA damage and prepare the correspondingspecific antibodies. Wild-type protein and the corresponding mutant proteins wereanalyzed using the specific phosphorylation antibodies. Rock2phosphorylation siteswere detected by Western blot when ionizing radiation induced DNA damage. When ATR/ATM expression was reduced and DNA damage was induced, the change ofRock2phosphorylation sites was detected by Western blot. Rock2mutant plasmidwas transfected into shRock2cells and exposed to IR. The growth of hepatomacarcinoma cells were measured by MTT assay.Results:1. The Rock2mRNA and protein levels both noticeably decreased inshRock2transfected Huh-7and HepG2cells. Cell proliferation was blocked in theshRock2cells compared to the control groups (P<0.05), and no significant differencewas observed between the untreated and siControl cells (P>0.05). Knockdown ofRock2promoted cell-cycle arrest at the G0/G1phase. The earlier apoptosis rate ofshRock2Huh-7and HepG2cells increased. This value was significantly higher thanthose of the Control groups (P<0.05).2. Rock2and Cdc25A were aberrantlyupregulated in HCC tumors and that they demonstrated a significantly positivecorrelation. The expression of Cdc25A protein was significantly downregulated inRock2stable knockdown cells. The expression of Chk1and Chk2were not changedfollowing knockdown of Rock2. These co-immunoprecipitation results verified thatRock2bound to Cdc25A. The confocal laser scanning microscopy showed that Rock2and Cdc25A colocalized in HCC cells. Rock2regulates Cdc25A through ubiquitinproteasome system.3. Rock2phosphorylation site S1121was detected when IRinduced to DNA damage. The upper kinase of Rock2was ATM. Rock2mutant couldimpact the growth of HCC.Conclusion: Rock2could regulate the biological function of HCC. Furthermore,Rock2plays important roles on signal transduction during S phase and this mayprovide a new target gene for treatment and gene expression regulation of HCC.