N-ras与epiregulin联合干扰对肝癌细胞增殖的抑制效果研究
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摘要
肝癌发生和发展具有复杂的分子机制,有许多癌基因和信号传导通路参与其中,多靶点联合治疗可能为肝癌的治疗提供一种新的途径。RNA干扰可以实现多个靶点同时的特异性抑制,而且没有传统化学疗法带来的毒副作用,是一种有潜力的治疗手段。基于本研究室在N-ras干扰治疗肝癌研究的基础上,我们寻找到一个参与N-ras干扰后的补偿途径调控基因epiregulin。该基因与N-ras基因联合发挥协同效应,对epiregulin与N-ras进行双干扰,可以达到更好的治疗效果。
一、瞬时及稳定干扰N-ras基因对肝癌细胞HepG2基因表达谱的影响
1.RT-PCR法和Western blot法检测瞬时及稳定干扰后N-Ras RNA及蛋白水平的变化。结果显示瞬时和稳定干扰后,HepG2细胞中N-Ras的RNA以及蛋白水平相对于未处理以及mock处理组而言都有明显的降低。灰度扫描结果显示抑制率均约为60%。
2.芯片实验检测瞬时及稳定干扰N-ras后变化的基因。与mock-siRNA处理组相比,N-ras-siRNA处理24小时后上调的基因为185个(ratio＞1.5),下调的基因为206个(ratio＜0.67);N-Ras-siRNA干扰48小时后上调的基因为291个,下调的基因为248个;与HepG2/mock细胞相比,N-Ras稳定干扰HepG2/N-ras(-)细胞上调的基因为482个,下调的基因的基因为524个。相对于瞬时干扰,稳定干扰后基因表达变化的幅度大。
3.用DAVID生物信息学资源数据库进行基因功能富集分析。稳定干扰N-ras后的功能富集分析中出现了cell motility,actin filament-based process,acincytoskeleton organization and biogenesis等关键词。瞬时干扰N-ras 48小时后的功能富集分析中出现了regulation of epithelial cell proliferation,regulation of cellproliferation,cell adhesion等关键词。瞬时干扰N-ras 24小时后的功能富集分析中出现了cell cycle,regulation of progression through cell cycle,regulation of cell cycle等关键词。说明N-ras干扰后细胞周期、细胞增殖、细胞运动和细胞骨架等方面相关的基因发生了变化。
4.用Pathway miner进行变化基因的通路分析。稳定干扰N-ras以及顺时干扰N-ras 48小时后,发生变化的通路相同,为focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton以及regulation of actin cytoskeleton,而瞬时干扰后24小时后变化的通路略有不同,依次为focal adhesion,cell cycle,MAPK signalingpathway以及wnt signaling pathway。
5.Gene Cluster 3.0以及Java TreeView对六张芯片结果进行了聚类分析。与稳定干扰N-ras后的基因表达谱相比,瞬时干扰后的两组芯片的基因表达谱更为接近。变化趋势较为明显的ClusterⅠ-Ⅳ主要参与的细胞生物学过程依次为angiogensis和growth,Catalytic activity,Inflammatory response和cell adhesion以及Secretory pathway。
6.用RT-PCR法验证三组芯片结果。稳定干扰N-ras变化的基因中我们验证的有HDGFRP3,GPNMB,VRK1,MKI67,TM4SF3,EREG以及ACTG2。N-ras干扰48小时组中我们挑选的基因为ACTC和FN1,N-ras瞬时干扰24小时组中我们挑选的基因为EREG,芯片结果和RT-PCR结果较为符合。
7.观察N-ras干扰对HepG2细胞形态的影响。N-ras-siRNA干扰HepG2细胞后,细胞形态变得狭长,而mock-siRNA处理组的细胞形态没有明显的变化。
二、筛选与N-ras联合干扰具有协同效果的基因
1.RT-PCR法验证ACTG2-siRNAs的干扰效果后,用SRB法检测N-ras-siRNA与ACTG2-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的ACTG2-siRNA1或N-ras-siRNA干扰后细胞的存活率分别为43.5%和66.5%。将浓度减半(各50nM),联合干扰后细胞的存活率为67.5%,比抑制单基因略高,提高浓度(各100nM)后,联合干扰后细胞的存活率降为28%。ACTG2-siRNA2的情况与ACTG2-siRNA1大体相同。
2.RT-PCR法验证EREG-siRNA的干扰效果后,用SRB法检测N-ras-siRNA与EREG-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的EREG-siRNA1或N-ras-siRNA干扰后的细胞存活率分别为49%和41%。将浓度减半(各50nM),联合干扰后细胞的存活率为36%,比抑制单基因有明显降低,提高浓度(各100nM)后,联合干扰后细胞的存活率降为15%。EREG-siRNA2的情况与EREG-siRNA1大体相同。进一步用SRB法检测epiregulin受体EGFR的小分子抑制剂AG1478对N-ras干扰的细胞增殖抑制增效作用。结果表明与对照组和mock-siRNA干扰组相比,N-ras干扰组对AG1478更加敏感。
3.RT-PCR法验证R-ras-siRNAs的干扰效果后,用SRB法检测N-ras-siRNA与R-ras-siRNAs联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的R-ras-siRNA1或N-ras-siRNA干扰后细胞的存活率分别为43.5%和48%。将浓度减半(各50nM),联合干扰后细胞的存活率为39.5%,比抑制单基因有明显降低,提高浓度(各100nM)后,细胞的存活率降为16%。
4.RT-PCR法验证TM4SF1-siRNA的干扰效果后,用SRB法检测N-ras-siRNA与TM4SF1-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,干扰N-ras或TM4SF1后细胞的存活率分别为43.5%和65%。将浓度减半(各50nM),细胞的存活率为66.5%,提高浓度(各100nM)后,细胞的存活率降为28.5%。
三、N-ras与epiregulin联合干扰效果以及机理探讨
1.克隆形成实验检测N-ras与epiregulin联合干扰效果。结果表明N-ras-siRNA+EREG-siRNA1(各50nM)组与N-Ras-siRNA(100nM)以及EREG-siRNA1(100nM)组相比克隆形成数少量降低。N-Ras-siRNA+EREG-siRNA1(各100nM)与N-Ras-siRNA(100nM)以及EREG-siRNA1(100nM)组相比克隆形成数显著降低。此结果与N-ras与epiregulin联合干扰的SRB实验结果相符。
2.流式细胞术检测N-ras以及epiregulin干扰对细胞周期分布的影响。结果表明,干扰N-ras与epiregulin均可增加G0/G1期细胞,降低S期细胞。与对照细胞相比,干扰N-ras或epiregulin使G0/G1期细胞分别增加了14.7%和11.9%。联合干扰N-ras和epiregulin使G0/G1期细胞增加到20.4%。结果证明单干扰N-ras或epiregulin均可造成一定的G0/G1期阻滞,而联合干扰会增强这种效应。
3.Western blot检测N-ras以及epiregulin联合干扰对MAPK以及PI3K-Akt通路蛋白的影响。实验表明,干扰N-ras或epiregulin均可使ERK1/2和Akt的磷酸化水平降低,而总ERK1/2及Akt蛋白水平保持不变。干扰N-ras或epiregulin也可使c-myc蛋白表达降低。联合干扰N-ras和epiregulin会增强上述效应。
4.Western blot检测N-ras以及epiregulin联合干扰对G1期蛋白的影响。实验结果表明,干扰N-ras或epiregulin均可降低Rb的磷酸化水平以及cyclin D1蛋白的表达水平,升高p21的表达水平。联合干扰N-ras和epiregulin会增强上述效应。这个结果支持了第2部分的实验结果,并进一步证明联合干扰N-ras和epiregulin可以通过对G0/G1期的阻滞来发挥作用。
综上所述,基因芯片结果表明,N-ras干扰可对HepG2细胞的增殖、细胞周期、细胞粘附以及细胞骨架相关基因和通路产生影响。联合干扰N-ras和epiregulin对HepG2细胞增殖有协同抑制作用。Epiregulin可作为肝癌联合治疗的新靶点,联合干扰N-ras与epiregulin可作为治疗肝细胞癌的新方法。
Hepatocellular carcinomas(HCC) is a major challenge because of the complicated molecular mechanisms of hepatocarcinogenesis and the involvement of multiple oncogenes and crucial signaling pathways,multiple-targeted therapy may be a new option for HCC treatment.Combined RNAi can be a therapy specific enough to allow the use of multiple RNAi targets at the same time,without the toxic effects often observed in chemotherapeutics.Based on our former study,N-ras gene was chosen to be a target for RNA interference treatment of HCC.We hypothesize that the up-regulated genes after knockdown of N-Ras may be involved in the compensatory mechanisms of cell growth.We examined whether dual knockdown of N-ras and the compensatory gene displayed a greater inhibitory effect of HepG2 cell growth.
Part 1.The alteration of gene expression profile in HepG2 cells after transient and stable transfection of N-ras-siRNA
1.The RNA and protein levels of N-ras after both transient and stable transfection of N-ras-siRNA were detected by RT-PCR and Western blot.The expressions of N-Ras in both mRNA and protein levels were significantly decreased by transient and stable transfection of N-Ras-siRNA,N-Ras proteins were significantly decreased up to 60% in both experiments.
2.Microarry analysis of differentially expressed genes after transient and stable transfection of N-Ras-siRNA.In microarray analysis,the increase and decrease of 1.5-fold signal intensity are regarded as a significant change in mRNA expression.In transient 24h transfected HepG2 cells,185 genes were up-regulated and 206 genes were down-regulated.After 48h transient transfection,291 genes were up-regulated and 248 genes were down-regulated.In stable transfected HepG2/N-ras(-) cells,482 genes were up-regulated and 524 genes were down- regulated.
3.Gene function enrichment analysis of differentially expressed genes using DAVID Bioinformatics Resources database.There was a statistically significant overlap between differentially expressed genes after stable transfection of N-ras-siRNA and the Gene Ontology categories "cell motility","actin filament-based process" and "acin cytoskeleton organization and biogenesis".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-ras-siRNA(48h) are "regulation of epithelial cell proliferation","regulation of cell proliferation" and "cell adhesion".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-Ras-siRNA(24h) are "cell cycle","regulation of progression through cell cycle" and "regulation of cell cycle".
4.Pathway analysis of differentially expressed genes using Pathway Miner.The altered pathways identified in stable and transient transfected(48h) HepG2 cells were focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton and regulation of actin cytoskeleton,the altered pathways identified in transient transfected (24h) HepG2 cells were focal adhesion,cell cycle,MAPK signaling pathway and wnt signaling pathway.
5.Hierarchical cluster analysis of genes exhibiting significantly modulated expression after both transient and stable transfection of N-ras-siRNA.The gene function enrichment analysis showed that the Gene Ontology go with ClusterⅠ-Ⅳare "Organ morphogenesis","Catalytic activity","Inflammatory response" and "Secretory pathway".
6.RT-PCR validation of microarray results.The genes chosn for RT-PCR validation are HDGFRP3,GPNMB,VRK1,MKI67,TM4SF3,EREG and ACTG2 after N-ras stable knockdown,ACTC and FNI after N-ras transient knockdown(48h),EREG after N-ras transient knockdown(24h).The relative mRNA levels of these genes are consistent between RT-PCR and microarray experiments.
7.The morphology change of HepG2 cells after N-ras-siRNA treatment is observed. Part 2.The effect of dual knockdown of N-ras and other genes on HepG2 cell growth.
We picked ACTG2,EREG,R-ras and TM4SF1 for this part of the research.Among them,combined silence of N-ras and epiregulin achieved greater effect on suppressing HepG2 cell growth.SRB assay showed that the survival rates of HepG2 cells treated with Mock-siRNA,N-ras-siRNA,EREG-siRNA1 and N-ras-siRNA combined with EREG-siRNA1 were 78%,49%,41%and 15%,respectively.
Part 3.The molecular mechasim of the growth suppression after dual knockdown of N-ras and epiregulin.
1.Colony formation assay was used to examine the synergistic effect of combined inhibition.Compared with untreated cells,the rates of colony formation in HepG2 cells after treated with mock-siRNA(100nM),N-Ras-siRNA(100nM),EREG-siRNA1 (100nM),N-Ras-siRNA and EREG-siRNA1(50nM each),N-Ras-siRNA and EREG-siRNA1(100nM each) are 81%,55%,58%,46%and 14%respectively.The results were consistent with the SRB assay in Part 2.
2.Flow cytometric analysis revealed that the treatment of HepG2 cells with N-ras-siRNA resulted in a 14.7%increase of cells at G0/G1 phase compared with control cells.Under same conditions,cells at G0/G1 phase were enhanced up to 11.9% by the treatment of EREG-siRNA1 compared to untreated cells.Moreover,dual knockdown of N-ras and epiregulin exhibited a 20.4%increase of cells at G0/G 1 phase, suggesting that dual knockdown of N-ras and epiregulin resulted in G0/G1 arrest and that the inhibition of cell growth was associated with the disturbation of cell cycle progression.
3.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of ERK1/2 and Akt with unchanged expressions of ERK1/2 and Akt proteins.The dual knockdown of N-ras and epiregulin had enhanced reductions of ERK1/2 and Akt phosphorylations.The similar effect was observed on the expression of c-myc downstream of ERK1/2.
4.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of Rb and the expression of cyclin D1.The dual knockdown of N-ras and EREG had enhanced reductions of p-Rb and cyclin D1. N-ras-siRNA and EREG-siRNA1 significantly increased the expression of p21.The dual knockdown of N-ras and epiregulin had the enhanced effect.
In summary,we developed an approach that uses gene expression profile analysis to identify targets for combinatorial treatment.We demonstrate that inhibition of N-Ras and epiregulin achieves synergistic effect in suppressing hepatoma HepG2 cells growth. Our findings imply that molecular multi-targeted therapy of oncogenes can be an effective treatment for HCC.
一、瞬时及稳定干扰N-ras基因对肝癌细胞HepG2基因表达谱的影响
1.RT-PCR法和Western blot法检测瞬时及稳定干扰后N-Ras RNA及蛋白水平的变化。结果显示瞬时和稳定干扰后,HepG2细胞中N-Ras的RNA以及蛋白水平相对于未处理以及mock处理组而言都有明显的降低。灰度扫描结果显示抑制率均约为60%。
2.芯片实验检测瞬时及稳定干扰N-ras后变化的基因。与mock-siRNA处理组相比,N-ras-siRNA处理24小时后上调的基因为185个(ratio＞1.5),下调的基因为206个(ratio＜0.67);N-Ras-siRNA干扰48小时后上调的基因为291个,下调的基因为248个;与HepG2/mock细胞相比,N-Ras稳定干扰HepG2/N-ras(-)细胞上调的基因为482个,下调的基因的基因为524个。相对于瞬时干扰,稳定干扰后基因表达变化的幅度大。
3.用DAVID生物信息学资源数据库进行基因功能富集分析。稳定干扰N-ras后的功能富集分析中出现了cell motility,actin filament-based process,acincytoskeleton organization and biogenesis等关键词。瞬时干扰N-ras 48小时后的功能富集分析中出现了regulation of epithelial cell proliferation,regulation of cellproliferation,cell adhesion等关键词。瞬时干扰N-ras 24小时后的功能富集分析中出现了cell cycle,regulation of progression through cell cycle,regulation of cell cycle等关键词。说明N-ras干扰后细胞周期、细胞增殖、细胞运动和细胞骨架等方面相关的基因发生了变化。
4.用Pathway miner进行变化基因的通路分析。稳定干扰N-ras以及顺时干扰N-ras 48小时后,发生变化的通路相同,为focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton以及regulation of actin cytoskeleton,而瞬时干扰后24小时后变化的通路略有不同,依次为focal adhesion,cell cycle,MAPK signalingpathway以及wnt signaling pathway。
5.Gene Cluster 3.0以及Java TreeView对六张芯片结果进行了聚类分析。与稳定干扰N-ras后的基因表达谱相比,瞬时干扰后的两组芯片的基因表达谱更为接近。变化趋势较为明显的ClusterⅠ-Ⅳ主要参与的细胞生物学过程依次为angiogensis和growth,Catalytic activity,Inflammatory response和cell adhesion以及Secretory pathway。
6.用RT-PCR法验证三组芯片结果。稳定干扰N-ras变化的基因中我们验证的有HDGFRP3,GPNMB,VRK1,MKI67,TM4SF3,EREG以及ACTG2。N-ras干扰48小时组中我们挑选的基因为ACTC和FN1,N-ras瞬时干扰24小时组中我们挑选的基因为EREG,芯片结果和RT-PCR结果较为符合。
7.观察N-ras干扰对HepG2细胞形态的影响。N-ras-siRNA干扰HepG2细胞后,细胞形态变得狭长,而mock-siRNA处理组的细胞形态没有明显的变化。
二、筛选与N-ras联合干扰具有协同效果的基因
1.RT-PCR法验证ACTG2-siRNAs的干扰效果后,用SRB法检测N-ras-siRNA与ACTG2-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的ACTG2-siRNA1或N-ras-siRNA干扰后细胞的存活率分别为43.5%和66.5%。将浓度减半(各50nM),联合干扰后细胞的存活率为67.5%,比抑制单基因略高,提高浓度(各100nM)后,联合干扰后细胞的存活率降为28%。ACTG2-siRNA2的情况与ACTG2-siRNA1大体相同。
2.RT-PCR法验证EREG-siRNA的干扰效果后,用SRB法检测N-ras-siRNA与EREG-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的EREG-siRNA1或N-ras-siRNA干扰后的细胞存活率分别为49%和41%。将浓度减半(各50nM),联合干扰后细胞的存活率为36%,比抑制单基因有明显降低,提高浓度(各100nM)后,联合干扰后细胞的存活率降为15%。EREG-siRNA2的情况与EREG-siRNA1大体相同。进一步用SRB法检测epiregulin受体EGFR的小分子抑制剂AG1478对N-ras干扰的细胞增殖抑制增效作用。结果表明与对照组和mock-siRNA干扰组相比,N-ras干扰组对AG1478更加敏感。
3.RT-PCR法验证R-ras-siRNAs的干扰效果后,用SRB法检测N-ras-siRNA与R-ras-siRNAs联合干扰对HepG2细胞增殖的影响。结果表明,浓度为100nM的R-ras-siRNA1或N-ras-siRNA干扰后细胞的存活率分别为43.5%和48%。将浓度减半(各50nM),联合干扰后细胞的存活率为39.5%,比抑制单基因有明显降低,提高浓度(各100nM)后,细胞的存活率降为16%。
4.RT-PCR法验证TM4SF1-siRNA的干扰效果后,用SRB法检测N-ras-siRNA与TM4SF1-siRNA联合干扰对HepG2细胞增殖的影响。结果表明,干扰N-ras或TM4SF1后细胞的存活率分别为43.5%和65%。将浓度减半(各50nM),细胞的存活率为66.5%,提高浓度(各100nM)后,细胞的存活率降为28.5%。
三、N-ras与epiregulin联合干扰效果以及机理探讨
1.克隆形成实验检测N-ras与epiregulin联合干扰效果。结果表明N-ras-siRNA+EREG-siRNA1(各50nM)组与N-Ras-siRNA(100nM)以及EREG-siRNA1(100nM)组相比克隆形成数少量降低。N-Ras-siRNA+EREG-siRNA1(各100nM)与N-Ras-siRNA(100nM)以及EREG-siRNA1(100nM)组相比克隆形成数显著降低。此结果与N-ras与epiregulin联合干扰的SRB实验结果相符。
2.流式细胞术检测N-ras以及epiregulin干扰对细胞周期分布的影响。结果表明,干扰N-ras与epiregulin均可增加G0/G1期细胞,降低S期细胞。与对照细胞相比,干扰N-ras或epiregulin使G0/G1期细胞分别增加了14.7%和11.9%。联合干扰N-ras和epiregulin使G0/G1期细胞增加到20.4%。结果证明单干扰N-ras或epiregulin均可造成一定的G0/G1期阻滞,而联合干扰会增强这种效应。
3.Western blot检测N-ras以及epiregulin联合干扰对MAPK以及PI3K-Akt通路蛋白的影响。实验表明,干扰N-ras或epiregulin均可使ERK1/2和Akt的磷酸化水平降低,而总ERK1/2及Akt蛋白水平保持不变。干扰N-ras或epiregulin也可使c-myc蛋白表达降低。联合干扰N-ras和epiregulin会增强上述效应。
4.Western blot检测N-ras以及epiregulin联合干扰对G1期蛋白的影响。实验结果表明,干扰N-ras或epiregulin均可降低Rb的磷酸化水平以及cyclin D1蛋白的表达水平,升高p21的表达水平。联合干扰N-ras和epiregulin会增强上述效应。这个结果支持了第2部分的实验结果,并进一步证明联合干扰N-ras和epiregulin可以通过对G0/G1期的阻滞来发挥作用。
综上所述,基因芯片结果表明,N-ras干扰可对HepG2细胞的增殖、细胞周期、细胞粘附以及细胞骨架相关基因和通路产生影响。联合干扰N-ras和epiregulin对HepG2细胞增殖有协同抑制作用。Epiregulin可作为肝癌联合治疗的新靶点,联合干扰N-ras与epiregulin可作为治疗肝细胞癌的新方法。
Hepatocellular carcinomas(HCC) is a major challenge because of the complicated molecular mechanisms of hepatocarcinogenesis and the involvement of multiple oncogenes and crucial signaling pathways,multiple-targeted therapy may be a new option for HCC treatment.Combined RNAi can be a therapy specific enough to allow the use of multiple RNAi targets at the same time,without the toxic effects often observed in chemotherapeutics.Based on our former study,N-ras gene was chosen to be a target for RNA interference treatment of HCC.We hypothesize that the up-regulated genes after knockdown of N-Ras may be involved in the compensatory mechanisms of cell growth.We examined whether dual knockdown of N-ras and the compensatory gene displayed a greater inhibitory effect of HepG2 cell growth.
Part 1.The alteration of gene expression profile in HepG2 cells after transient and stable transfection of N-ras-siRNA
1.The RNA and protein levels of N-ras after both transient and stable transfection of N-ras-siRNA were detected by RT-PCR and Western blot.The expressions of N-Ras in both mRNA and protein levels were significantly decreased by transient and stable transfection of N-Ras-siRNA,N-Ras proteins were significantly decreased up to 60% in both experiments.
2.Microarry analysis of differentially expressed genes after transient and stable transfection of N-Ras-siRNA.In microarray analysis,the increase and decrease of 1.5-fold signal intensity are regarded as a significant change in mRNA expression.In transient 24h transfected HepG2 cells,185 genes were up-regulated and 206 genes were down-regulated.After 48h transient transfection,291 genes were up-regulated and 248 genes were down-regulated.In stable transfected HepG2/N-ras(-) cells,482 genes were up-regulated and 524 genes were down- regulated.
3.Gene function enrichment analysis of differentially expressed genes using DAVID Bioinformatics Resources database.There was a statistically significant overlap between differentially expressed genes after stable transfection of N-ras-siRNA and the Gene Ontology categories "cell motility","actin filament-based process" and "acin cytoskeleton organization and biogenesis".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-ras-siRNA(48h) are "regulation of epithelial cell proliferation","regulation of cell proliferation" and "cell adhesion".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-Ras-siRNA(24h) are "cell cycle","regulation of progression through cell cycle" and "regulation of cell cycle".
4.Pathway analysis of differentially expressed genes using Pathway Miner.The altered pathways identified in stable and transient transfected(48h) HepG2 cells were focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton and regulation of actin cytoskeleton,the altered pathways identified in transient transfected (24h) HepG2 cells were focal adhesion,cell cycle,MAPK signaling pathway and wnt signaling pathway.
5.Hierarchical cluster analysis of genes exhibiting significantly modulated expression after both transient and stable transfection of N-ras-siRNA.The gene function enrichment analysis showed that the Gene Ontology go with ClusterⅠ-Ⅳare "Organ morphogenesis","Catalytic activity","Inflammatory response" and "Secretory pathway".
6.RT-PCR validation of microarray results.The genes chosn for RT-PCR validation are HDGFRP3,GPNMB,VRK1,MKI67,TM4SF3,EREG and ACTG2 after N-ras stable knockdown,ACTC and FNI after N-ras transient knockdown(48h),EREG after N-ras transient knockdown(24h).The relative mRNA levels of these genes are consistent between RT-PCR and microarray experiments.
7.The morphology change of HepG2 cells after N-ras-siRNA treatment is observed. Part 2.The effect of dual knockdown of N-ras and other genes on HepG2 cell growth.
We picked ACTG2,EREG,R-ras and TM4SF1 for this part of the research.Among them,combined silence of N-ras and epiregulin achieved greater effect on suppressing HepG2 cell growth.SRB assay showed that the survival rates of HepG2 cells treated with Mock-siRNA,N-ras-siRNA,EREG-siRNA1 and N-ras-siRNA combined with EREG-siRNA1 were 78%,49%,41%and 15%,respectively.
Part 3.The molecular mechasim of the growth suppression after dual knockdown of N-ras and epiregulin.
1.Colony formation assay was used to examine the synergistic effect of combined inhibition.Compared with untreated cells,the rates of colony formation in HepG2 cells after treated with mock-siRNA(100nM),N-Ras-siRNA(100nM),EREG-siRNA1 (100nM),N-Ras-siRNA and EREG-siRNA1(50nM each),N-Ras-siRNA and EREG-siRNA1(100nM each) are 81%,55%,58%,46%and 14%respectively.The results were consistent with the SRB assay in Part 2.
2.Flow cytometric analysis revealed that the treatment of HepG2 cells with N-ras-siRNA resulted in a 14.7%increase of cells at G0/G1 phase compared with control cells.Under same conditions,cells at G0/G1 phase were enhanced up to 11.9% by the treatment of EREG-siRNA1 compared to untreated cells.Moreover,dual knockdown of N-ras and epiregulin exhibited a 20.4%increase of cells at G0/G 1 phase, suggesting that dual knockdown of N-ras and epiregulin resulted in G0/G1 arrest and that the inhibition of cell growth was associated with the disturbation of cell cycle progression.
3.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of ERK1/2 and Akt with unchanged expressions of ERK1/2 and Akt proteins.The dual knockdown of N-ras and epiregulin had enhanced reductions of ERK1/2 and Akt phosphorylations.The similar effect was observed on the expression of c-myc downstream of ERK1/2.
4.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of Rb and the expression of cyclin D1.The dual knockdown of N-ras and EREG had enhanced reductions of p-Rb and cyclin D1. N-ras-siRNA and EREG-siRNA1 significantly increased the expression of p21.The dual knockdown of N-ras and epiregulin had the enhanced effect.
In summary,we developed an approach that uses gene expression profile analysis to identify targets for combinatorial treatment.We demonstrate that inhibition of N-Ras and epiregulin achieves synergistic effect in suppressing hepatoma HepG2 cells growth. Our findings imply that molecular multi-targeted therapy of oncogenes can be an effective treatment for HCC.
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