【作者】 阮健；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 研究背景和目的原发性肝细胞癌(Hepatoccllular carcinoma,HCC)，是一种严重影响人类健康的恶性肿瘤。我国是世界上肝癌高发区之一，发病率为20．37／10万，位于常见肿瘤的第3位。据2002年全球最新统计，肝癌发病率在常见癌症中排行第6，而病死率则排第3位，每年发病人数为62．6万例，新增5．7％，共有59．8万例死亡，其中82％的病例在发展中国家，中国占55％。虽然目前临床上可用于治疗肝癌的手段比较多，包括手术切除、肝移植、血管介入、消融技术、放化疗等，且在肝癌的药物治疗上取得重大突破，但手术切除仍被公认为肝癌获得根治的最好手段。然而，即使是根治性切除，5年内仍有60～70％的病人出现转移复发，而局部治疗的转移复发率更高。如何预防肿瘤转移复发已成为提高肝癌生存率的关键。因此,寻找预测肿瘤转移倾向、判断肿瘤预后韵标志物，探索预防和治疗的有效途径，从而降低死亡率，提高肝癌患者的5年生存率，这是肝癌研究的重点内容，也是肿瘤防治研究中的一个难点。GPC3(Glypican-3，磷脂酰肌醇蛋白聚糖3，又称MXR7、OCI-5、GTXR2-2)蛋白是硫酸乙酰肝素糖蛋白(Heparan sulfate proteoglycan,HSPG)家族中的一员。GPC3的突变导致细胞增殖和细胞凋亡失去平衡，可能是肿瘤发生的重要原因。GPC3还可结合肝素结合型蛋白如细胞粘附分子、基质成分、生长因子、酶和酶抑制物，参与调节细胞增殖、分化、粘附和迁移等过程，还可能参与抑制或调节大部分中胚层组织和器官生长的过程。目前研究发现GPC3参与Wnt、Hedgehog (Hh)、FGF、IGF、BMP、SMAD、TGF-β等多个与肿瘤发生发展密切相关的信号通路的调节。研究表明GPC3在肝细胞癌、大肠癌、恶性黑色素瘤、Wilm’s瘤、成神经细胞瘤和肝胚细胞瘤高表达,而在肺腺癌、卵巢癌、乳腺癌、间皮瘤中表达显著下调,提示GPC3在肿瘤的发生发展中起着重要的作用。RNA干扰(RNA interference,RNAi)是将与目的基因mRNA序列互补的小的双链RNA导入靶细胞,促使mRNA降解,诱使细胞表现出特定基因缺失的表型,从而高效特异性阻断体内特定基因表达。其特征为：①RNAi是指通过反义RNA与正链RNA形成双链RNA,特异性地抑制靶基因的现象,它通过人为地引入与内源靶基因具有相同序列的双链RNA(有义RNA和反义RNA),从而诱导内源靶基因的mRNA降解,达到阻止基因表达的目的。②RNAi是指体外人工合成的或体内的双链RNA (dsRNA)在细胞内特异性的将与之同源的mRNA降解成21nt-23nt的小片段,使相应的基因沉默。③RNAi是将与靶基因的mRNA同源互补的双链RNA(dsRNA)导入细胞,能特异性地降解该mRNA,从而产生相应的功能表型缺失,属于转录后水平的基因沉默(post-transcriptional gene silencing, PTGS)。④RNAi存在瀑布放大效应,每个细胞仅需几分子siRNA就可产生RNAi效应,并可达到缺失突变体表型的程度。相比普通siRNA,具有短发夹结构的双链RNA,稳定性比siRNA更好,而且能延长目的基因沉默的时间。本研究拟应用RNA干扰技术将靶向GPC3基因的siRNA瞬时转染肝癌离体细胞株huh-7,观察GPC3基因表达沉默后对肝癌细胞株huh-7生物学特性的影响,初步探讨GPC3基因在肝癌细胞增殖、凋亡、迁移中的作用。研究的目的在于给肝癌GPC3基因治疗的可行性提供初步依据。设计靶向GPC3基因的siRNA,瞬时转染肝癌离体细胞huh-7,观察GPC3基因的表达情况。1. siRNA的设计应用siRNA设计软件初筛出9对siRNA,再通过BLAST分析剔除与其他编码基因有同源性的siRNA,最后经RNA结构分析软件分析将对应的基因序列位于二级结构的siRNA去掉,选出两条对靶向GPC3的siRNA(包括GPC3-siRNA-1161、GPC3-siRNA-516)。2.瞬时转染肝癌huh-7细胞采用脂质体转染技术将GPC3-siRNA转染huh-7细胞,转染48小时后进行相应检测。3.各项指标的检测用real-time PCR技术检测干扰后huh-7细胞的GPC3表达量,然后用Western-blot检测干扰后GPC3的蛋白表达水平,Annexin V-FITC/PI双染经流式细胞仪检测细胞凋亡率,荧光显微镜观察细胞形态,MTT法检测细胞增殖情况,划痕实验检测细胞迁移情况。4.统计学方法应用SPSS 13.0软件进行统计学处理,计量资料以均数±标准差(x(_)±s)表示。MTT细胞增殖检测、细胞凋亡检测及细胞迁移实验均采用析因设计方差分析比较各组间差异；P<0.05为有统计学意义。1. GPC3 mRNA表达的测定real-time PCR结果显示,导入设计的siRNA(包括GPC3-siRNA-1161、GPC3-siRNA-516)的huh-7细胞GPC3表达水平为未转染的huh-7细胞的0.56±0.07和0.10±0.06,P值：0.000,F值：257.640。2.GPC3蛋白的表达的测定Western blot结果显示,GPC3-siRNA-1161组、GPC3-siRNA-516组、NC-siRNA-173组和NS组的GPC3蛋白相对表达量分别为0.51±0.06、0.15±0.05、1.08±0.04和1.03±0.01,P值：0.000,F值：314.814。3.细胞凋亡率的测定结果显示转染72小时后GPC3-siRNA-1161组的细胞凋亡率为23.13±0.39%,GPC3-siRNA-516组细胞凋亡率为24.90±0.28%,均显著高于未转染的huh-7组的细胞凋亡率(12.24±1.23%)(P值：0.000,F值：139.553)。然而值得注意的是,转染后96小时后两干扰组与未转染组间虽然仍有统计学差异,但差异已变小(GPC3-siRNA-1161组：28.11±0.12%,GPC3-siRNA-516组：29.37+0.34%,未转染组：25.87±1.85%；P值：0.029,F值：5.085)。4.细胞增殖的测定结果显示转染后48小时和72小时GPC3-siRNA-1161组和GPC3-siRNA-516组的活细胞数显著低于未转染组,以72小时最为明显(GPC3-siRNA-1161组：0.962±0.025,GPC3-siRNA-516组：0.871±0.034,未转染组：1.953±0.043；P值：0.000,F值：819.992)。转染96小时后,两干扰组与未转染组间的差异仍具有统计学意义,但已明显变小(GPC3-siRNA-1161组：1.863±0.058,GPC3-siRNA-516组：1.816±0.052,未转染组：2.842±0.013；P值：0.002,F值：294.267)。并且两干扰组96小时的活细胞数较72小时增长超过2倍。5.细胞划痕实验检测GPC3基因沉默后对肝癌细胞体外运动迁移能力的影响。实验结果显示：不同时间点迁移细胞数有显著性差异(F=1594.373,P=0.000)；GPC3-siRNA-1161组和GPC3-siRNA-516组细胞迁移数明显低于NS组和NC-siRNA-173组,差异具有显著性(F=125.549,P=0.000)；不同细胞在不同时间迁移细胞数有显著性差异(F=24.233,P=0.000)。1.通过siRNA设计软件初筛-BLAST分析-RNA结构分析软件分析这3步进行siRNA设计,是一种快速、简便、有效的方法。2.应用RNAi技术,将靶向GPC3基因的siRNA转染肝癌细胞(huh-7),能成功介导GPC3基因沉默,达到靶向封闭GPC3基因的目的。结果显示,GPC3基因的沉默使huh-7细胞增殖及迁移能力下降、凋亡率增高。提示该基因在肝癌细胞生物学行为调控中可能起着关键作用,因而,以GPC3基因为靶基因的基因治疗对肝癌可能有效。3.瞬时转染靶向GPC3基因的siRNA可以高效地敲低huh-7细胞的GPC3基因表达,但发挥RNAi的时间较短,从结果分析,转染72小时后,siRNA的干扰效率开始降低,并逐渐失效。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which seriously threat the body health of human being. China is among the high incidence area of HCC, with an incidence of 20.37 per 100 thousand which is the third highest incidence in common tumors. According to the statistical data collected in 2002, HCC’s incidence ranked the sixth in common cancers while its mortality ranked the third; the annual incidence cases of HCC was 626 thousand which showed a 5.7% increase, while the number of death was 598 thousand, among which 82% of the cases appeared in developing country, especially China enjoyed 55%. Though many methods including surgical resection, liver transplantation, vascular intervention, ablation technology, radiotherapy, chemotherapy, can be performed in HCC in clinic practice,more than one half of HCC patients had the micrometastasis failing to dectect before radical surgery,which may be the direct reason of metastasis or recurrence of HCC after surgery. Metastasis and recurrence have become the crux of further improvement on survival rate of HCC patients. Therefore, to seek and predicate HCC’s metastasis and recurrence tendency, judge prognosis of HCC patients is the key and focus of HCC’s research, as well as its emphasis and difficulty. Glypican-3(Glypican-3, also called MXR7, OCI-5, and GTXR2-2) is a member of HSPG (heparan sulfate proteoglycan) family. In the way of combining with heparin binding proteins such as cell adhesion molecules, matrix components, growth factors, enzyme and enzyme inhibitors, GPC3 participates in the control of cell proliferation, differentiation, adhesion, migration and so on, even possibly involved in the inhibition or control of most tissues and organs’ growth in mesoderm. The mutation of GPC3 leads to imbalance of cell proliferation and apoptosis, which may be the key reason of tumor development. So far research shows that GPC3 participates in the control of signal transduction pathway which closely related to tumor development such as Wnt,Hedgehog (Hh),FGF,IGF,BMP,SMAD,TGF-βand so on. Researches also show that GPC3 is highly expressed in HCC, colorectal carcinoma,Wilm’s tumor, neuroblastoma and hepatoblastoma, while loses its expression in lung adenocarcinoma, Ovarian cancer, breast cancer, mesothelioma, which suggests that GPC3 is a worth-expecting specific marker of multiple tumors.Double-stranded RNA (dsRNA) can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs, a process known as RNA interference. Small interference RNA(siRNA) or shot hairpin RNA(shRNA) both are the medium of RNAi.There are several characters of RNAi,including specificity of sequence which can discriminate mutation gene sequence with normal gene sequence,high efficiency and amplification effect.shRNA is more stable than siRNA and also can extent the time of target gene silencing.This research intent to test GPC3’s influence on biological behavior of huh-7 cells by transient transfecting the GPC3-targeted siRNA into huh-7 cells and observing the changes of huh-7 cells on cell proliferation,cell apoptosis and cell migration.The object of the study is to provide the evidence to the feasibility of hepatocellular carcinoma gene therapy. Design GPC3-targeted siRNA,transient transfect huh-7 cells,and detect the intracellular expression of GPC3 gene.1. Design of siRNA Apply siRNA design software for screening out of nine pairs of siRNA,and then remove the siRNA which are homologous with the other encoding genes,the final select two pairs of GPC3-targeted siRNA.2. Transient transfection Transfect GPC3-siRNA into huh-7 cells by lipofection technique,and detect after 48h.3. Detecting Expression of GPC3 mRNA was detected by real-time PCR,expression of GPC3 protein was identified by Western blot,cell apoptosis rate was evaluated by flow cytometry, cell proliferation was detected by MTT assay,and cell migration was detected by wound healing assay.4. Statistical methods The difference of GPC3 protein and mRNA expression rate was analyzed by Chi-square test and Wilcoxon rank sum test respectively.The difference among groups on cell proliferation,cell apoptosis and cell migration was analyzed by Factorial design analysis of variance.1. Expression of GPC3 mRNA The result showed that the expression of GPC3 mRNA of siRNA interfered huh-7 cells of two groups was further lower than control huh-7 cells group.2. Expression of GPC3 protein The result displayed that protein level of siRNA transfected huh-7 cells of two groups was lower than control huh-7 cells group.3. Apoptosis The result of 48hrs and 72hrs after transfection demonstrated that apoptosis rate of two experimental groups was higher than control group.However,there are smaller difference between two experimental groups and control group at the time point of 96hrs. 4. Cell proliferation The result of 48hrs and 72hrs after transfection also show that OD490nm detected by Spectrophotometer of two experimental groups is lower than of control group.Small difference can be seen in 96hrs among groups.5. Cell migration The result of wound healing assay displayed that the number of migratiing cells of experimental groups were significantly lower than the control group.1. Three-step method of design siRNA is very feasible and effective.2. Application of RNAi technology,GPC3-targeted siRNA can induce GPC3 gene silencing to achieve the purpose of closure of GPC3 gene.The results showed that the biological behavior of huh-7 cells interfered by GPC3-targeted siRNA changed significantly,including decline of cell proliferation and cell migration and increase of cell apoptosis.lt suggested that GPC3 gene may play a key role on regulation of biological behavior of hepatoma cells.3. Transient transfection of GPC3-targeted siRNA can efficiently knock out the GPC3 gene expression of huh-7 cells,but only last for a short time.更多还原