The Expression Of POLD1 In Hepatocellular Carcinoma And The Study Of Nitidin Chrolide Intervention

AIMTo investigate the expression of DNA polymerase delta catalytic subunit gene (POLD1) and its encoding protein P125 in hepatocellular carcinoma (HCC) and its significance. And to investigate the mode of regulation of p53 on POLD1 and observe the intervention effect of nitidine chloride d on POLD1 pathway in hepatoma cells.METHODSReverse transcription polymerase chain reaction (RT-PCR) technique was used to detect the expression level of POLD1 insurgically resected HCC tissues in 20 cases, and P125 was detected by western blot. Short hairpin RNA(shRNA) sequences targeting human wild type p53 gene were designed and constructed recombinant plasmid pGPU6/GFP/Neo-p53 shRNA. SMMC-7721 cells were transfected with the recombinant plasmid and negative control vector respectively. Stable transfected clone was selected using G418. The proliferation was detected with MTT assay and clone form assay. The expression of POLD1 mRNA was detected by RT-PCR. P125 protein expression was detected by western bloting. In addition, proteomics was studied using SELDI technology. After treatment with NC, the proliferation of SMMC-7721 was detected with MTT assay and clone form assay. RT-PCR and western blot were used to detect genes and their proteins expression of regulating POLD1 pathway factors respectively.RESULTSThe expression level of POLD1 mRNA was clearly observed in hepatomatic tissues compared with matched tumor-adjacent noncancerous tissues (0.70±0.34 compared with 0.37±0.23, P<0.05). The level of P125 expression in hepatomatic tissues was significantly higher than in tumor-adjacent tissues (0.63±0.19 compared with 0.39±0.21, P<0.05), which similared with POLD1 expression. In addition, P125 expression was correlated with tumor size and clinical stages, but not correlated with HBV infection, age and sex.POLD1 gene and protein expression, which were significantly lower in high p53 expression cell line than the empty vector cell line, were significantly higher in low p53 expression cell line than transfected unrelated sequence shRNA cell line. MTT assay results showed that growth rate of p53 shRNA group was significantly faster than the control group, and pEGFP-p53 group was slower than the control group.The shape and the number of colonies in pGPU6/GFP/neo-p53 shRNA group were larger than the control group. In addition, local convergence was found between colonies. In contrast, the number of colonies in transfected pEGFP-p53 cells was less than empty vector cell line, and colony formation decreased significantly. 14 protein peaks were significant changes after treatment with NC in SMMC-7721 cells, which related to the signal transduction proteins, apoptosis, cell cycle, metabolism, immunity, transcription, DNA replication and repair pathways.Compared with the negative control, POLD1 mRNA expression did not change in NC group. However,0.3mg/L NC can significantly reduce the expression of P125 in SMMC-7721 cells, and 0.15mg/L NC was no significant difference. Compared with negative control group, NC significantly increased p53 mRNA expression (P<0.01).0.3 to 2.4mg/L NC could increase the expression of P53 protein in different degree, the levels of 0.3 and 0.6mg/L NC were P<0.05 too, and 1.2mg/L and 2.4mg/L NC were P<0.01, which consistent with p53 mRNA expression. It is worth noting,0.15mg/L NC did not increase P53 protein expression, but showing a downward trend, which is not coincidence with the p53 mRNA expression.0.15mg/L NC had no inhibitory effect on the P125, but 0.3mg/L NC inhibited P125 expression following P53 was upragulated. These results illustrated that there could be other regulatory factors affecting the translation of p53 mRNA, which deserved our continued in-depth study. p53 mRNA expresse was significantly increased in dominant inactivation of p53 cells after treatment with 1.0mg/L NCDefferent concentration of NC had defferent effect on P21 expression in SMMC-7721 cells.2.4mg/L (P<0.01) and 1.2 mg/L (P<0.05) NC significantly increased P21 expression. Interestingly,0.5,0.3 and 0.6mg/L NC had no significant effect on P21 protein in liver cells, but 0.3 and 0.6mg/L NC could significantly increase the P53 protein expression. Further studies shown that 1.2, and 2.4mg/L NC significantly increased the expression of p21 mRNA (P<0.01), but 0.6mg/L NC had no significant effect on the expression of p21 mRNA and 0.3, and 0.15mg/L NC reduced p21 mRNA expression. The expression of p21 mRNA was significantly decreased in SMMC-7721 cells when p53 was silenced, which indicated that the cell line had normal p53-p21 pathway. After treated with 1.0mg/L NC for 48h, p21mRNA expression was increased significantly in p53-interfered cells, which was higher than that of p53 overexpression cell lines. Different concentrations of NC could regulate C-myc, E2F, CyclinD and RB-1 in various degree, which 0.15 and 0.3mg/L NC group could upregulate CyclinD and C-myc expression significantly (P<0.05), 0.15mg/L and 0.6mg/L NC upregulated E2F expression significantly (P<0.05, P<0.01),0.6mg/L NC upregulated CyclinD expression significantly (P<0.01) and 0.15～0.6mg/L NC upregulated RB-1 expression significantly (P<0.01). However,0.3mg/L NC had no significant effect on C-myc and E2F. Compared with the control group,0.15,0.3 mg/L NC significantly reduced CyclinE mRNA expression (P<0.05), but 0.6mg/L NC increased CyclinE mRNA expression.0.15,0.3 and 0.6mg/L NC, compared with the control group, had significant downward effect on expression of CDK4 mRNA (P<0.05). Within range of 0.15, 0.3 and 0.6 mg/L dose, NC had a concentration- and time-dependent inhibition effect on cell proliferation. Cell morphology was observed under the light microscope. Cells gradually become irregular after exposing to NC. Because of the poor adhesion, most cells showed shrunken and round, even death, and the higher concentration of NC, the more dead cells. Colony-forming assay results showed that NC (0.15,0.30 and 0.60mg/L) treated cells for 10 d, colony size and formation rate decreased too. NC could significantly improve the G2/M phase cell percentage, so that S, G0/G1 phase cells reduced proportionally. NC treatment group were significantly higher apoptosis rate (P<0.01), the apoptosis rate increased with increasing concentration, but also showing a concentration dependent manner.CONCLUSION:The positive rate of POLD1 mRNA and P125 in HCC is significantly correlated with the pathological staging. POLD1 may be used as a diagnostic maker and a potential therapeutic target. p53 can inhibit the expression of POLD1. The cell proliferation and colony formation of liver cancer changes with the expression of p53, which the reason may be due to fluctuations in the presence of POLD1 to some extent inhibited the expression of the DNA replication blocked on. NC in a certain range of concentration in a dose- and time- dependent inhibition of liver cell proliferation. NC block the cell cycle G2/M phase and induce apoptosis. NC and non-reliance by relying on co-induction of p21 increases p53 pathway, thus blocking cell cycle; by upregulating the expression of p53 inhibition of P125, the lack of DNA replication DNA replication DNA replication enzyme 8 key enzyme catalytic subunit and is inhibited; p53, RB-1, C-myc, E2F, CyclinD, CyclinE, CDK2 and CDK4 together to complete NC-induced apoptosis.