【作者】 欧贤红；

【导师】 刘华钢； 徐恒；

【作者基本信息】 广西医科大学 ， 药理学， 2011， 博士

【摘要】 目的探讨DNA聚合酶δ催化亚基基因(DNA polymerase delta catalytic subunit gene 1, POLD1)及其编码蛋白P125在原发性肝癌中的表达及意义,同时探讨p53对POLD1的调控模式,并观察氯化两面针碱对肝癌细胞中调控POLD1通路的干预作用。方法选用原发性肝癌患者手术切除标本20例,分别采用反转录聚合酶链反应(RT-PCR)法和western blot法检测POLD1基因表达,同时分析POLD1基因产物- P125蛋白与临床特征的相关性。设计合成针对p53基因的短发夹RNA(short hairpin RNA, shRNA),并构建pGPU6/GFP/Neo-p53 shRNA重组质粒。分别将PGPU6/GFP/Neo-p53 shRNA、无关序列对照质粒pGPU6/GFP/Neo-NC shRNA、真核表达载体pEGFP-p53和空载体pEGFP-C1转染至SMMC-7721细胞,G418抗性筛选,获得阳性克隆。MTT法测定生长曲线,克隆形成实验观察细胞的增殖,RT-PCR法及western blot法分别检测POLD1的mRNA及蛋白表达。运用SELDI技术对NC作用后SMMC-7721的蛋白组学进行研究。NC处理SMMC-7721细胞,采用MTT法和克隆形成实验观察细胞的增殖,RT-PCR法和/或荧光定量PCR及western blot法分别检测POLD1调控通路中各调控基因的表达和蛋白表达情况,并采用RNA干扰技术对其进行验证。结果与癌旁组织相比,肝癌组织中POLD1基因表达明显升高(0.70±0.34compared with 0.37±0.23, P<0.05);其编码蛋白P125表达与POLD1基因表达一致,在肝癌组织中的表达显著高于癌旁组织(0.63±0.19 compared with0.39±0.21,P<0.05)。P125蛋白的阳性表达率与肿瘤大小和临床分期有关(P<0.05),而与HBV是否感染、性别及年龄无明显关系(P>0.05)。p53低表达细胞系中的POLD1基因及蛋白P125表达比转染无关序列shRNA细胞系明显升高,而p53高表达细胞系中的POLD1基因及蛋白P125表达比转染空载体的细胞系降低。采用MTT法检测p53低表达细胞系和p53高表达细胞系的生长规律,p53低表达细胞系的生长速度明显比对照组快,而p53高表达细胞系的生长速度慢于对照组。稳定转染pGPU6/GFP/neo-p53 shRNA的SMMC-7721细胞的集落较大,局部集落间呈汇合形式,且集落数目多于对照组；相反,稳定转染pEGFP-p53的SMMC-7721细胞的集落数少于转染空载体的细胞,集落形成能力明显下降。NC处理SMMC-7721后筛选出有统计学意义的差异表达蛋白质峰14个,这些差异蛋白主要跟信号转导、细胞凋亡、细胞周期、代谢、免疫、转录、DNA复制及修复等通路有关。与阴性对照相比,NC作用SMMC-7721细胞后POLD1 mRNA的表达无明显变化。但0.3mg/L NC可明显降低SMMC-7721细胞P125表达水平,而0.15mg/L NC对SMMC-7721细胞P125的表达没有显著性差异。与阴性对照组比,NC明显提高p53 mRNA表达(P< 0.01).0.3～2.4mg/L NC均能不同程度的上调P53蛋白表达水平,其中0.3～0.6mg/L NC上调p53表达水平显著性差异相似(P<0.05),1.2mg/L和2.4mg/L NC与0mg/L NC相比显著性差异水平为P<0.01,结果与mRNA表达吻合。值得注意的是,0.15mg/L NC对P53蛋白表达水平不但不上调,而且还呈现下调趋势,这与该浓度NC上调p53 mRNA表达不吻合。结合NC对P125的表达结果,0.15mg/L NC对P125并无抑制作用,0.3mg/L NC上调P53,继而抑制P125表达。说明有可能存在其他调控因子影响p53 mRNA的翻译,这一现象值得我们继续深入研究。1.0mg/L NC处理p53显性失活的SMMC-7721细胞,p53 mRNA表达明显高于未处理的模型组7721-p53i,但并未回复到原有水平,表明1.0mg/L NC可诱导p53上调。NC作用SMMC-7721细胞48h后,不同程度的影响P21蛋白表达,其中2.4mg/L (P<0.01)和1.2mg/L (P<0.05) NC明显上调P21蛋白表达。有意思的是,0.5,0.3,0.6mg/L NC对肝癌细胞中的P21蛋白并无明显作用,但0.3,0.6mg/L NC却能明显上调细胞中的P53蛋白。因此我们进一步检测了p21 mRNA的表达情况。Real-time PCR结果显示,1.2和2.4mg/L NC对p21 mRNA表达明显增强(P<0.01),但0.6mg/L NC对p21 mRNA表达无明显影响,将NC浓度降至0.3mg/L或0.15mg/L, p21 mRNA表达反而降低。在p53干扰的SMMC-7721细胞中,p21mRNA表达明显降低,表明细胞存在正常的p53-p21途径。p53干扰的SMMC-7721细胞经1.0mg/L NC处理48h后,p21 mRNA表达显著性升高,甚至高于p53显性细胞组。不同浓度NC可不同程度地上调C-myc、E2F、CyclinD和RB-1,其中0.15、0.3mg/L NC对C-myc和CyclinD的上调及0.15mg/L NC对E2F的上调均有显著性差异(P<0.05),0.6mg/L NC对E2F、CyclinD的上调和0.15～0.6mg/LNC对RB-1的上调均有极显著性差异(P<0.01),但0.3mg/L NC对C-myc、E2F的作用与对照组比无显著性差异,但呈上调趋势。与对照组相比,0.15、0.3mg/L NC显著性下调CyclinE mRNA表达(P<0.05),但0.6mg/L NC对CyclinE mRNA的表达不降反升。0.15～0.6mg/L NC对CDK4 mRNA的表达与对照组相比均有显著性下调作用(P<0.05)。0.15～0.6 mg/L剂量范围内对细胞的抑制作用呈现浓度和时间依赖性。光学显微镜观察细胞形态,发现在NC作用过程中,细胞形态逐渐变得不规则,大部分细胞贴壁不良,出现皱缩、变圆、脱落死亡,且随着浓度增加,死亡细胞增多。集落形成实验结果显示,NC(0.15,0.30,0.60mg/L)处理细胞10d后,细胞克隆大小及集落形成率随药物浓度增大而减小。NC可明显提高G2/M期的细胞比例,使S、G0/G1期的细胞比例减少。NC处理组的细胞凋亡率显著高于对照组(P<0.01),凋亡率随浓度增大而增高,也呈现出浓度依赖性。结论原发性肝癌与POLD1基因表达有关,检测其蛋白产物P125有利于判断肝癌的恶性程度。POLD1的表达受p53的抑制。肝癌细胞的增殖及集落形成能力随着p53表达波动的原因可能与p53的存在一定程度上抑制了POLD1的表达,使DNA复制受阻有关。NC在一定浓度范围内可剂量、时间依赖性抑制肝癌细胞增殖。NC阻滞了细胞周期G2/M期,诱导细胞凋亡。NC通过依赖和非依赖p53途径共同诱导p21上调,从而阻滞细胞周期；通过上调p53抑制P125表达,使DNA复制因缺乏DNA复制主要酶DNA复制酶δ的催化亚基而受到抑制；p53、RB-1、C-myc、E2F、CyclinD、CyclinE、CDK2、CDK4共同完成NC诱导的细胞凋亡。更多还原

【Abstract】 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.更多还原