【作者】 谢淑丽；

【导师】 全成实；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 信号蛋白分子RhoC在肿瘤细胞中的异常表达近年来引起了人们的广泛重视,但现有的研究多集中在RhoC调控肌动蛋白细胞骨架影响肿瘤转移方面,还有许多关键性的问题如RhoC在细胞增殖、凋亡、分化和耐药性等方面的作用知之不多。本课题以人肝癌细胞Bel7402为对象,研究RhoC基因对肝癌细胞生物学行为的影响及其相关的分子机制,为靶向RhoC的肝癌基因治疗奠定理论和实验基础。免疫细胞化学法检测RhoC基因在肝癌细胞和正常肝细胞中的表达;RNAi基因沉默肝癌细胞RhoC,RT-PCR和Western blot检测基因沉默效果;MTT法、平板克隆和软琼脂克隆形成实验以及AgNOs染色检测细胞增殖;划痕实验和细胞铺展实验检测细胞迁移侵袭能力;流式细胞术检测细胞周期、细胞凋亡、细胞膜通透性和线粒体膜电位变化;RT-PCR检测细胞周期蛋白、凋亡相关蛋白、MMPs表达;明胶酶谱分析检测MMPs活性;测量IC50值评价肝癌细胞化疗药物敏感性;As2O3诱导实验检测肝癌细胞分化水平;基因转染实验观测RhoC高表达所致的正常肝细胞恶性转化倾向。实验结果显示:RhoC在肝癌细胞中表达高于正常肝细胞,基因沉默RhoC可以显著抑制肝癌细胞增殖和转移,促进肝癌细胞大量凋亡,增加肝癌细胞对化疗药物环磷酰胺和长春新碱的敏感性,促进肿瘤细胞分化和诱导分化;RhoC转化正常肝细胞可使之出现恶性变倾向。进一步机理研究揭示:RhoC基因沉默对肝癌细胞增殖的作用可能与其调控Cyclin D1、CDK4表达上调及P16和P21表达下调有关;对肝癌细胞凋亡的作用可能与其调控Bcl-2/Bax基因、增加细胞膜通透性和降低线粒体膜电位有关;对迁移和侵袭的作用与其调控MMP-2和MMP-9基因表达有关。本研究表明,RhoC在肝癌细胞增殖、分化、凋亡、迁移及对化疗药物敏感性等方面起一定的作用,这种作用可能与其调控相关的分子有关。更多还原

【Abstract】 Rho protein is an important member of Ras superfamily, which plays a diverse role in cell biology, including cell growth, differentiation and metastasis. RhoC is an important member of Rho GTPases. Recent researches showed the overexpression of RhoC in a great number of malignant tumors, and its activation could lead to enhanced tumor metastasis through specific signal transduction pathway. The high level expression of RhoC is closely correlated to tumor metastasis. Many studies suggested RhoC as a promising index in assessing tumor malignancy, with a possibility of acting as a therapeutic target for cancer gene therapy. However, research proofs on the role of RhoC in tumors are not strong enough to support RhoC as a therapeutic target, because there are still a lot of evidences should be provided and a lot of important mechanisms should be elucidated. This research systemically studied the effects of RhoC gene silencing in hepatocellular carcinoma (HCC) develpoment and cell differentiation. Also the role that RhoC plays in biological features and drug sensitivity in HCC cells were investigated. The importance of this research was to investigate the possibility of RhoC as a therapeutic target in HCC gene therapy.Immunohistochemistry was applied to detect RhoC expression in HCC cells. Eukaryotic expression plasmid pU6mRFP was employed as RNA interference vector; specific DNA sequence was synthesized to construct pU6mRFP siRNA-RhoC and pU6mRFP siRNA-scramble as negative control. Human HCC cell line Bel7402 was transfected to detect RhoC inhibiting efficiency. Lipotransfect was conducted to transfect Bel7402 cells, and its efficiency was assessed through laser confocal microscope. Semi-quantitative RT-PCR was used to detect mRNA level, Western blot was applied to detect RhoC protein expression. G418 was used to select stable transfection clone. MTT, cell nuclear AgNORs stain, plate clone formation and soft agar clone formation test were conducted to determine cell proliferation and growth feature. FACS was applied to determine cell cycle, cell apoptosis, cell membrane and mitochondrial membrane potential. As2O3 induction test, alkaline phosphate stain and biochemical analysis were employed to determine cell differenciation level. Cell migration capacity was determined through injury healing and cell spreading test. Zymography was used to detect MMPs activity. Semi-quantitative RT-PCR was performed to determine the expression level of cell cycle proteins, apoptosis associating proteins, MMPs and VEGF. IC50 values were measured to determine the sensitivities of anticancer drugs to HCC cells. Normal hepatocellular cell line HL7702 was transfected with RhoC to test RhoC transformation capacity. The results showed as followings:1. RNA interfering vector targetting RhoC was successfully constructed, with inhibiting effeciency in transfected Bel7402 cells as high as 85%.2. The effects of RhoC gene silencing in the proliferation of HCC and its mechanism. (1)The cultured cell proliferation capacity was compared between RNAi group (Bel7402 cell transfected with pU6mRFP siRNA-RhoC), negative control group (Bel7402 cells transfected with pU6mRFP siRNA-scramble) and parental Bel7402 cells. From the fourth day on, the proliferation speed in RNAi group was notably lower than in parental Bel7402 and negative control groups (0.41±0.10 vs. 0.73±0.11 and 0.71±0.07,p<0.05). These phenomena lasted to the end of the 7 day experiment. There was not remarkable difference between the two control groups. (2)Cell cycle analysis showed that G0-G1 cell percentage in RNAi group was significantly higher than in parental Bel7402 group (73.14±5.93% vs.57.05±5.97% and 52.99±4.80%,p<0.05). G2-S cell percentage was notably lower in RNAi group than in parental Bel7402 cells (24.86±3.66% vs.38.81±3.77% and 47.00±4.80%,P<0.01).There was not significant difference between two control groups.(3)Plate clone formation test showed that there was rare clones in RNAi group under naked eye observation, and more clones were formed in control groups. Statistical analysis showed that both clone number and clone formation efficiency in RNAi group were notably lower than in control groups (20.33±5.42% vs. 70.58±10.10% and 69.83±14.77%,p<0.01), suggesting RhoC gene silencing could notably inhibit the potential of cell growth. (4)AgNORs stain test showed that average cell stain particle in RNAi group was significantly lower than in parental Bel7402 and negative control(1.23±0.35 vs. 3.47±0.93 and 3.17±0.78,p<0.01), demonstrating the reduced cell proliferation capacity. (5)Soft agar clone formation test showed that clone formation number and percentage in RNAi group were significantly higher than in the control groups(0.133±0.115% vs. 2.867±0.987% and 2.533±1.007%,p<0.01), while the differences between two control groups were not remarkable (p>0.05), showing that RhoC silencing changed the non-anchor growth capacity of tumor cells. (6)Compared with Bel7402 and negative control groups, the following growth associated gene expressions were significantly decreased: CyclinD1(0.45±0.21 vs. 1.25±0.24 and 1.12±0.15,p<0.05)and CDK4(0.55±0.08 vs. 1.18±0.32 and 1.10±0.29,p<0.05). The following genes were notably increased: P16 ( 1.07±0.23 vs. 0.36±0.12 and 0.35±0.13,p<0.01)and P21(0.42±0.12 vs. 0.17±0.06 and 0.19±0.08,p<0.05). No remarkable changes were found in the following genes: CyclinA, CyclinG1, P19 and P27.3.The effects of RhoC gene silencing on Bel7402 cell apoptosis and its mechanism.(1)The results of cell apoptosis detected through FACS showed that the apoptosis ratio in RNAi group was significantly higher than in parental Bel7402 and negative control groups (21.00±2.23% vs. 6.47±1.64% and 4.63±0.47%,p<0.01). (2) Typical DNA fragment“ladder”for cell apoptosis could be found in RNAi group in gel electrophoresis analysis, suggesting there was great amount of cell apoptosis in RNAi group. There was not such DNA fragment“ladder”be found in parental Bel7402 and negative control groups, suggesting that there was not great amount cell apoptosis in control groups. (3)Wright stain test showed that there was lot of apoptosis cells in RNAi group, showing RhoC silencing could induce tumor cell death. (4)The membrane permeability in RNAi group was remarkably higher than in parental 7402 and negative control ones(30.70±5.63% vs.13.73±1.30% and 14.97±1.74%,p<0.01). (5)In RNAi group, the mitochondrial membrane potential was notably lower than in control ones ( 35.30±2.26% vs.65.60±5.54% and 65.37±5.61% , p<0.01 ) . (6)Compared with Bel7402 and negative control groups, Bcl-2 gene expression in RNAi group was significantly lower(0.28±0.15 vs. 0.96±0.21 and 1.03±0.24,p<0.05),and Bax gene expression was notably higher(1.09±0.21 vs. 0.26±0.10 and 0.25±0.07,p<0.01).4.Effects of RhoC gene silencing on Bel7402 cell differenciation and induced-differenciation. (1) Alkaline phosphates (ALP) stain showed that the stain color in RNAi group was more than in control ones. ALP activity in RNAi group was significantly higher than in parental Bel7402 and negative control groups (65.33±11.93u/L vs. 35.67±6.03u/L and 37.33±6.11u/L,p<0.05) .γ-GT activity was notably higher than in control groups ( 12.67±3.06u/L vs. 23.33±5.03u/L and 24.33±5.13u/L,p<0.05).(2)Under As2O3 induction, ALP activity in RNAi group was significantly higher than in parental 7402 and negative controls(123.67±24.58u/L vs. 56.67±11.93u/L and 54.67±10.26u/L,p<0.05).γ-GT activity was notably lower than in control ones(6.33±1.53u/L vs. 11.67±2.52u/L and 10.67±2.08u/L,p<0.05).5.Influence of RhoC gene silencing on Bel7402 invasion and metastasis. (1)Twenty four hours after scratch, the relative cell migration in RNAi group was significantly lower than in parental Bel7402 and negative control ones(32%±6% vs. 64%±13% and 67%±11%,p<0.01). (2)After 2 hours of cell spreading, the spreading ratio in RNAi group was notably lower than in parental 7402 and negative control ones(32.67±12.01% vs. 65.67±11.93% and 63.33±13.87%,p<0.05). (3)Zymography analysis showed that gelatinase activities could be found in parental Bel7402 and negative control ones, but not in RNAi group, suggesting the less production of MMPs with the silencing of RhoC gene. (4)MMP2 expression in RNAi group was significantly lower than in parental 7402 and negative control ones(0.26±0.10 vs. 1.01±0.23 and 1.03±0.27,p<0.01). MMP9 expression could not be detected in RNAi group, but with considerable level expression in control ones.6.Influence of RhoC on Bel7402 cell drug sensitivity. The value of IC50 for cyclophosphamide in RNAi group was significantly lower than in parental 7402 and negative control ones ( 838.54±69.22μg/ml vs. 1178.72±116.73μg/ml and 1168.36±168.68μg/ml ,p<0.01).Significant decrement of IC50 for vincristine could be found in RNAi group than in control ones(9.57±2.42μg/ml vs. 47.51±12.62μg/ml and 56.96±7.56μg/ml,p<0.01).7.The transformation effects of RhoC on normal liver cell HL7702. (1) The RhoC expression level in PcDNA3-RhoCgroup(HL7702 cells transfected with PcDNA3-RhoC) was significantly higher than in HL7702 and PcDNA3(HL7702 cells transfected with PcDNA3) groups(1.11±0.23 vs. 0.35±0.07 and 0.34±0.09,p<0.01), and Western blot detection proofed this difference. (2)From the third day on of cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than in HL7702 and PcDNA3 groups(0.83±0.10 vs. 0.54±0.11 and 0.58±0.55,p<0.05). (3)The clone formation efficiency in PcDNA3-RhoC group was significantly higher than in HL7702 and PcDNA3 group(64.27±8.57% vs. 42.33±6.70% and 40.93±5.78%,p<0.01).(4) AgNORs stain test showed that the average stain particles in PcDNA3-RhoC group was notably higher than in HL7702 and PcDNA3 groups(2.77±0.40 vs. 1.07±0.23 and 1.13±0.25, P<0.01). (5)Soft agar clone formation in PcDNA3-RhoC group could be detected, but could not in the HL7702 and PcDNA3 groups. (6)The ALP stain was more intensive in PcDNA3-RhoC group than in HL7702 and PcDNA3 groups.In summary, the overexpression of RhoC in hepatocellular carcinoma cells can control tumor cell proliferation and metastasis through regulating the gene expressions involving cell growth and migration. RhoC gene silencing can inhibit proliferation and metastasis capacity of tumor cells, as well as enhancing the sensitivity of chemical therapy drugs such as cyclophosphamide and vincristine. This research lays a foundation to selecting RhoC as target molecule in tumor gene therapy both theoretically and experimentally.更多还原