Effects Of The Cell Cycle And The MRNA Expression Of G1/S Checkpoint Regulatory Gene In Liver Cells Of Rats Induced By Sub-chronically VCM

ObjectiveDetect the cell cycle at different phase distribution in hepatocytes of rats induced by sub-chronically VCM with applied flow cytometry, and detect the changes of G1/S checkpoint regulatory genes (including negative regulation factors TP53, CDKN1A, CDKN2A and positive regulation factors CCNA2, CCND1, CCNE1, CDK2, CDK4) mRNA expression in rat liver cells that were effected by VCM, from the field of cell cycle regulation, to explore the possible carcinogenic mechanism of VCM.MethodsSixty-four Sprague Dawley (SD) rats were randomly divided into negative control group and three experimental groups exposed to5,25, and125mg/kg VCM. The rats were treated with clean air and different doses VCM by intraperitoneal injection with three times a week for twelve weeks, and rats were killed at the end of6weeks and12weeks respectively. Flow cytometry (FCM) was applied to detect cell cycle and apoptosis rates in hepatocytes, HE stain was used to observe the morphological changes in the liver tissue of rats, Real-time quantitative PCR method was applied to detect the changes of G1/S checkpoint regulatory genes mRNA expression which including negative regulation genes (TP53, CDKN1A and CDKN2A) and positive regulation genes (CCNA2, CCND1, CCNE1, CDK2and CDK4) in liver cells, Real-time quantitative PCR method was applied to detect the changes of miR-122a expression.Results The distribution of cell cycle show that, exposed for6weeks, the percentage of GO/G1phase in5,25mg/kg VCM experimental groups were (69.73±10.94) and (74.32±9.29) which were higher than the control group(56.13±10.65)(P<0.05). The percentage of S phase in25mg/kg VCM experimental group (1.80±2.11) was statistically significant difference from the control group (12.82±5.85)(P<0.05).Exposed to VCM for12weeks, the percentage of GO/G1phase did not showed significant difference compared to control group (P>0.05) while the percentage of S phase in125mg/kg VCM exposed group (14.91±4.43) was statistically significant difference from the control group (6.68±2.64)(P<0.05).The apoptosis rates of liver cells in rats did not change significantly (P>0.05) exposed to6weeks and12weeks.HE stain of liver tissue showed, the control group and5mg/kg VCM group were both no hepatocyte degeneration or cell necrosis, but the25mg/kg VCM group’s results were hepatocyte degeneration, including fatty degeneration, vacuolar degeneration, the125mg/kg VCM group observed hepatocyte degeneration were aggravated, including fatty degeneration, vacuolar degeneration, ballooning degeneration and so on.Exposed to VCM of liver cells for6weeks, the level of mRNA expression of CCND1, CDK4and CDK2showed that VCM exposure groups are lower than the control group. Compared with the control group, the125mg/kg VCM exposure group is decreased significantly (P<0.05). No significant changes of the mRNA expression of TP53, CDKN1A, CDKN2A, CCNA2and CCNE1.Exposed to VCM of liver cells for12weeks, the CDKN1A mRNA expression increased with the increasing doses. Compared with the control group, the125mg/kg VCM exposure group is higher significantly (P<0.05). TP53, CDKN2A, CCNA2, CCND1, CCNE1, CDK2and CDK4gene mRNA expression did not change significantly.Exposed to VCM of lymphocytes for12weeks, the mRNA expression of G1/S checkpoint regulatory genes TP53, CDKN1A, CDKN2A, CCNA2, CCND1, CCNE1, CDK2and CDK4did not change significantly, and the difference were not statistically significant.Exposed to VCM of liver cells for12weeks, the level of miR-122a expression decreased with the increasing doses. Compared with the control group, the VCM exposure groups did not change significantly, and the difference were not statistically significant (P>0.05).ConclusionsThe liver is one of target organs in rats exposed to VCM. We observed pathological changes such as portal area larger, fibrosis and cell nucleus swelling.VCM may induce GO/G1phase and S phase arrest of hepatocytes in rats.VCM affect the function of G1/S phase checkpoint.VCM did not affect the apoptosis rates of liver cells in rats.G1arrest induced by VCM through down-regulate CCND1, CDK2and CDK4gene mRNA expression.G1arrest induced by VCM might not be related with the negative regulation of CDKN1A.VCM did not affect the level of miR-122a expression in liver cells of rats.