摘要
肝细胞癌(hepatocellular carcinoma, HCC)是世界上最常见的恶性肿瘤之一,以亚洲和非洲高发。近些年西方发达国家的HCC发病率也呈上升趋势,尤其是美国和加拿大。我国是世界上HCC高发地区之一,每年发病人数约为30万人,位于常见肿瘤的第3位。此外,我国肝癌的年死亡率为20.40/10万,其中城市为19.98/10万,农村为23.59/10万,分别居恶性肿瘤死亡率的第二位和第一位。手术切除仍然是治愈早期肝癌的最佳选择,然而,即使是根治性切除之后,60%-70%的患者在术后5年内会发生转移和复发。尽管一些临床因素(如肿瘤分化程度、肿瘤大小、是否脉管侵润等)与患者预后密切相关,但是HCC的发生发展的分子机制目前仍不明确,因此,探索肝癌的发病基础及转移复发的相关因素,寻找肝癌早期诊断、预测转移复发的生物指标和靶向治疗的靶点,是目前肿瘤学研究的迫切任务之一。
硫酸乙酰肝素蛋白多糖(HSPGs)是一类携带一条或多条硫酸乙酰肝素链的蛋白。这些肝素链是由长线状的葡糖醛酸聚合物/N-乙酰氨基葡糖重复双糖单位组成。链的长度高度均可变,大部分双糖单位都受N-去乙酰化、去硫酸化、差向异构化和O-去硫酸化的调节。去乙酰化诱导了硫酸乙酰肝素链产生高强度的负电荷,从而允许硫酸乙酰肝素糖蛋白以较为杂乱的方式与带有正电荷区域的蛋白产生相互作用,其中包括了一些所谓的“肝素结合生长因子”,如成纤维细胞生长因子(FGFs), Wnt, Hedgehogs (Hhs)和骨形态发生蛋白(BMPs)。磷脂酰肌醇蛋白聚糖(GPC)是硫酸乙酰肝素糖蛋白家族中的一员,它的成员通过磷脂酰肌醇锚(GPI)连接于细胞表面。哺乳动物的基因组包括了GPC家族的六个成员：(GPC1至GPC6),还有其他两个成员在果蝇中发现(dally和dlp)[10,111
GPC3基因定位于人染色体Xq26.1区,编码的GPC3蛋白的相对分子质量约为70Kda,由580个氨基酸组成。GPC3的核心蛋白由两个部分组成,分别40kDa的GPC3氨基端(N-)片段及30kDa的GPC3羧基端(C-)片段。1996年,Pilia等首先报道了GPC3基因突变和功能缺失的患者出现了过度生长综合征(SGBS),这是一种X连锁异常的疾病,主要表现为产前、产后胎儿的过度生长,并伴有广泛的内脏和骨骼的异常,同时还会增加胚胎源性肿瘤发生的可能性[14]。随后的研究又表明,GPC3的突变会导致细胞凋亡和细胞增殖的比例失调,而这可能是肿瘤发生发展的重要原因之一。GPC3还可与肝素结合型蛋白相结合,如基质成分、细胞粘附分子、酶和酶抑制物、生长因子等,参与调节细胞分化、增殖、粘附和迁移等生理过程等,除此之外,其还可能参与调节一部分中胚层组织和器官发育的过程。随后有大量的研究报道表明GPC3在肝细胞癌、恶性黑色素瘤、Wilm's瘤、大肠癌、肝胚细胞瘤和成神经细胞瘤高表达,而在间皮瘤、乳腺癌、肺腺癌以及卵巢癌中表达显著下调,提示GPC3在肿瘤的发生发展中起着重要的作用。
本课题组前期已完成以下的研究工作：应用免疫组化及组织芯片技术,通过检测36例原发性肝细胞癌组织和33例原发性肝癌癌旁组织以及一些其他常见肿瘤组织和其癌旁组织中GPC3蛋白的表达情况。结果表明：(1)36例原发性肝细胞癌组织及33例原发性肝细胞癌癌旁组织中GPC3蛋白的表达率分别为66.17%(24/36),3.0%(1/33),两者之间差异有显著性；(2)GPC3蛋白在原发性肝细胞癌、肺鳞癌、癌旁慢性浅表性胃炎组织中表达,而乳腺癌、胃癌、胰腺癌、卵巢癌、宫颈癌、胆管细胞癌组织中无表达；(3)原发性肝细胞癌组织中GPC3蛋白表达与患者性别、年龄、病理分级无显著相关性。随后我们收集和分析了61例原发性肝细胞癌患(HCC)者术后标本切片,比较GPC3高表达患者与GPC3低表达患者之间的生存时间差异。结果表明,GPC3的表达与接受手术患者的转移/复发高度相关,GPC3高表达的术后患者发生转移或者复发的风险是低表达患者的3.214倍。生存分析显示GPC3高表达的肝癌患者预后显著差于低表达者。此外,多变量分析提示GPC3在HCC中是一个独立的预后预测参数[20]。同时,我们还采用了酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay)法分别检查HCC30例,其他肝脏源性肿瘤8例,其他肝脏疾病(如肝炎、肝硬化、肝良性肿瘤)13例,以及25例正常人血清标本中GPC3及甲胎蛋白的表达。结果显示,血清GPC3蛋白水平诊断原发性肝细胞癌的敏感性和特异性分别为50%和92%,联合甲胎蛋白检测可以使诊断敏感性从50%提升至73.3%。GPC3蛋白水平的增高提示原发性肝细胞癌患者肿瘤分化低、临床TNM分期晚、肝硬化程度明显。以上临床数据提示GPC3在肝癌发生发展中起着重要作用,故我们采用micro RNA对肝癌发生的分子机理进行研究。在研究中,我们发现miR-219-5p在83例HCC肿瘤组织中及3株肝癌细胞株中显著下调,其与HCC患者的预后、病理分级、肿瘤大小密切相关；在细胞学上,miR-219-5p能抑制细胞增殖。进一步研究表明,miR-219-5p负性调节GPC3表达,在肝癌中发挥抑癌作用。综上所述,GPC3蛋白是具有前景的特异性HCC肿瘤标志物,可用于HCC的诊断及判断预后,并可能与肿瘤转移相关。
在前期研究的工作基础上,拟应用RNA干扰和基因转染技术进一步探讨GPC3在肝癌的发生发展及侵袭过程中的作用。针对来源于同一遗传背景,但具有不同转移潜能的肝癌细胞株MHCC-97H和MHCC-97L,应用载体转染方法使GPC3在细胞中表达下调或上调,来观察GPC3在体外肝癌细胞增殖、侵袭、转移中的作用；同时,拟将转染后细胞注射至裸鼠皮下,观察肿瘤生长速度,从而明确GPC3在体内肝癌形成过程中的作用。本文旨在探讨GPC3作为肝癌的肿瘤标志物及治疗靶点的可能性,为肝癌患者的早期诊断、预测预后及治疗手段提供理论基础。
方法
1.检测肝癌细胞株MHCC-97H和MHCC-97L的GPC3表达水平
通过western blot和荧光定量PCR检测两株细胞的GPC3蛋白/基因表达量,以一株正常肝细胞作为对照,从而验证高低转移能力的细胞株的GPC3表达情况,为下一步实验提供依据。
2.上调MHCC-97L的GPC3表达量以观察其对肝癌细胞生物学行为的影响
上一步实验已证明MHCC-97L的GPC3表达量较低,故采用基因重组技术及限制性内切酶技术构建表达载体p-EGFP-C3-GPC3,经鉴定后通过脂质体lipo2000转染至MHCC-97L中,通过流式细胞仪筛选带荧光的细胞,经G418培养后建立稳定转染GPC3的细胞株,最后通过western blot和荧光定量PCR检测GPC3在细胞表达情况。以空载体p-EGFP-C3及未转染细胞作为对照,通过MTT、细胞克隆、transwell等实验,观察三组细胞的生物学行为差异。最后,通过裸鼠成瘤实验观察三组细胞体内的肿瘤增殖能力。
3.下调MHCC-97H的GPC3表达量以观察其对肝癌细胞生物学行为的影响
首先设计了4对靶向GPC3的shRNA,合成后插入pGenesil-1shRNA表达载体,通过脂质体分别转染MHCC-97H细胞,经G418筛选后建立稳定转染细胞株,使用western blot及荧光定量PCR检测各组GPC3表达情况,筛选表达量最低一组用作后续实验。以空载体及未转染细胞作为对照,通过MTT、细胞克隆、transwell等实验,观察三组细胞的生物学行为差异。最后,通过裸鼠成瘤实验观察三组细胞体内的肿瘤增殖能力。
4.GPC3影响肝癌细胞生物学行为的机制探讨
我们选取Akt通路作为研究对象,通过WB来检测通路上的相关分子(包括Akt和磷酸化Akt)在各组细胞中的表达。
5.统计学处理
应用SPSS19.0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、肿瘤体积观察均采用析因设计方差分析比较各组间差异；对比转染前后和干扰前后肝癌细胞株的GPC3蛋白和RNA水平、平板克隆形成实验、流式细胞术细胞周期实验、Transwell侵袭实验、Akt和p38蛋白和RNA表达水平采用单因素方差分析；多重比较采用LSD法,P<0.05为有统计学意义。
结果
1.GPC3在肝癌细胞株MHCC-97H和MHCC-97L中的表达
结果表明,以正常肝细胞作为对照,肝癌细胞MHCC-97H的GPC3的蛋白及核酸表达水平较高,差异具有显著性(P=0.000)；而与MHCC97H相比较,MHCC-97L的GPC3表达水平较低,差异具有显著性(P=0.000)。
2.稳定表达GPC3对肝癌细胞株生物学行为的影响
持续使用G418进行筛选后,转染p-EGFP-C3-GPC3和p-EGFP-C3细胞形成明显克隆,然后进行扩增,最终成功构建稳定表达GPC3的MHCC-97L细胞和空白载体细胞,分别命名为MHCC-97L/GPC3和MHCC-97L/con。通过western blot和qPCR进行检验,结果表明,MHCC-97L/GPC3的GPC3表达水平较MHCC-97L/con和未转染的MHCC-97L显著升高(P=0.000),而MHCC-97L/cpn和未转染MHCC-97L细胞间的GPC3表达水平无差异(P>0.05)。
通过MTT法和平板克隆实验检测各组细胞体外增殖情况。MHCC-97L/GPC3、 MHCC-97L/con和未转染的MHCC-97L在不同时间点的增殖速度有显著差异(P<0.05)。MHCC-97L/GPC3随着时间的推移,增殖速度明显加快,与对照组比较有显著统计学差异(P=0.000),而MHCC-97L/con和未转染的MHCC-97L的增殖速度则无显著差异(P>0.05)。此外,平板克隆形成实验结果表明,与MHCC-97L/con和未转染的MHCC-97L相比,MHCC-97L/GPC3的细胞活力显著提高(P=0.000)。
细胞体外迁移和侵袭小室检测稳定表达特异性GPC3基因后细胞迁移侵袭能力。迁移实验结果表明,MHCC-97L/GPC3细胞体外运动能力明显高于MHCC-97L/con和未转染的MHCC-97L细胞(P=0.000)；此外,侵袭实验结果也表明,与MHCC-97L/con和未转染的MHCC-97L细胞相比,MHCC-97L/GPC3细胞的侵袭能力亦是明显增高(P=0.000)。
裸鼠成瘤实验检测稳定表达GPC3基因后细胞体内成瘤能力。将裸鼠随机分成3组,分别皮下注射MHCC-97L/GPC3, MHCC-97L和MHCC-97L/con细胞,然后观察21天,结果发现MHCC-97L/GPC3注射组裸鼠皮下肿瘤体积明显大于对照组(P=0.000)。21天后处死裸鼠,将肿瘤称重,MHCC-97L/GPC3, MHCC-97L和MHCC-97L/con三组的肿瘤平均重量分别为2.987±0.168g,1.801±0.015g和1.758±0.040g, MHCC-97L/GPC3组的瘤重显著大于MHCC-97L和MHCC-97L/con组,有显著统计学差异(P=0.000),而MHCC-97L和MHCC-97L/con组之间的瘤重则无显著差异(P>0.05)。
3.GPC3基因表达沉默对肝癌细胞株生物学行为的影响
MHCC97-H细胞分别转染pGenesil-1-GPC3-shRNA1,2,3,4和pGenesil-1-negative,随后持续使用G418进行筛选,最终成功构建稳定表达GPC3的MHCC-97L细胞和空白载体细胞,分别命名为MHCC97-H/GPC3-shRNA1,2,3,4和MHCC97-H/con-shRNA。通过western blot和qPCR进行检验,结果表明,MHCC97-H/GPC3-shRNA1的GPC3表达水平较MHCC97-H/con-shRNA和未转染的MHCC-97H显著下降(P=0.000),而MHCC97-H/con-shRNA和未转染MHCC-97H细胞间的GPC3表达水平无差异(P>0.05)。因此选择MHCC97-H/GPC3-shRNA1作进一步的研究对象。
通过MTT法和平板克隆实验检测各组细胞体外增殖情况。MHCC97-H/GPC3-shRNA1、MHCC97-H/con-shRNA和未转染的MHCC-97H在不同时间点的增殖速度有显著差异(P=0.000)。MHCC97-H/GPC3-shRNA1的增殖速度较慢,与对照组比较有显著统计学差异(P=0.000),而MHCC97-H/con-shRNA和未转染的MHCC-97H的增殖速度则无显著差异(P>0.05)。此外,平板克隆形成实验结果表明,与MHCC97-H/con-shRNA和未转染的MHCC-97H相比,MHCC97-H/GPC3-shRNA1的细胞活力显著减弱(P=0.000)。
细胞体外迁移和侵袭小室检测GPC3基因沉默后肝癌细胞迁移侵袭能力。迁移实验结果表明,MHCC97-H/GPC3-shRNA1细胞体外运动能力明显低于MHCC97-H/con-shRNA和未转染的MHCC-97H细胞(运动实验迁移细胞数目分别为4.647±1.319、14.774±1.112和15.770±2.025,P=0.000)；此外,侵袭实验结果也表明,与MHCC97-H/con-shRNA和未转染的MHCC-97H细胞相比,MHCC-97L/GPC3细胞的侵袭能力亦是明显降低(透膜细胞数分别为4.421±1.536,15.902±1.450,和14.374±2.015,P=0.000)。
裸鼠成瘤实验检测GPC3基因敲除后细胞体内成瘤能力。将裸鼠随机分成3组,分别皮下注射MHCC97-H/GPC3-shRNA1, MHCC-97H和MHCC97-H/con-shRNA细胞,然后观察21天,结果发现MHCC97-H/GPC3-shRNA1注射组裸鼠皮下肿瘤体积明显小于对照组(P=0.000)。21天后处死裸鼠,将肿瘤称重,MHCC97-H/GPC3-shRNA1, MHCC-97H和MHCC97-H/con-shRNA三组的肿瘤平均重量分别为1.541±0.096g,2.424±0.095g和2.402±0.080g, MHCC97-H/GPC3-shRNA1组的瘤重显著小于MHCC-97H和MHCC97-H/con-shRNA组,有显著统计学差异(p=0.000),而MHCC-97H和MHCC97-H/con-shRNA组之间的瘤重则无显著差异(P>0.05)。
4.GPC3过表达(或沉默)能促进(或抑制)Akt通路的活化
结果表明,当GPC3表达沉默时,MHCC97-H/GPC3-shRNA1细胞的磷酸化Akt较对照组显著下降(P=0.000)；而当GPC3表达上调时,MHCC-97L/GPC3细胞的磷酸化Akt水平则较对照组显著升高(P=0.000)。
结论
1.利用质粒介导的基因转染技术成功构建了GPC3高表达的肝癌细胞株,初步探讨了GPC3的表达对肝癌细胞在体内和体外的生物学行为的影响,发现GPC3能促进肝癌细胞增殖、迁移及侵润能力。
2.同时利用质粒介导的基因转染技术成功构建了GPC3表达沉默的肝癌细胞株,进一步验证了GPC3对肝癌细胞生物学行为有着重要的正性调节作用,是肝癌的促癌因子。
3.GPC3过表达(或沉默)能促进(或抑制)磷酸化Akt的表达,故认为,GPC3促进肝癌细胞侵袭转移作用的部分机制可能是通过促进Akt通路的活化来实现的。
创新之处
1.成功建立了GPC3过表达细胞株MHCC-97L/GPC3和GPC3表达沉默细胞株MHCC97-H/GPC3-shRNAl,为进一步深入研究GPC3在肝癌发生发展的过程中的作用提供了有效的途径。
2.初步探索了GPC3促进肝癌侵袭转移的作用机制,提示GPC3的促肿瘤生长作用和Akt通路存在密切的联系,GPC3促进肝癌侵袭转移作用的部分机制可能与活化Akt通路有关。
Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide, with a high prevalence in Asia and Africa.An increasing trend in the incidence of HCC in the Unite State and Canada. China is among the high incidence area of HCC, with The number of HCC patients is increasing by300,000a year which is the third highest incidence in common tumors. In addition, HCC's mortality ranked the second in cities and first in rural areas. Surgical resection remains the standard choice of treatment for patients in the early stage of HCC. However, even with radical resection,60-70%of patients develop metastasis and recurrence within5years of surgery (3). Although several clinicopathological features, including a poorly differentiated phenotype, large-sized tumor, and portal venous invasion have been found to contribute to the poor prognosis in HCC patients before operation, the underlying molecular mechanisms of the development of HCC remain unclear. Thus, it is urgent to study the pathogenesis of HCC.
Heparan sulfate proteoglycans(HSPGs) are proteins that carry one or more heparin sulfate(HS) chains. These chains consist of long linear polymers of glucuronic acid/N-aceltylglucosamine repeating disaccharide unit. The length of the chains is highly variable, and a significant proportion of the disaccharides are modified by N-deacetylation/N-sulfation, epimerization and O-sulfation. The sulfation confers a highly negative charge to HS chains that allows HSPGs to interact in a rather promiscuous way with proteins that display positively charged domains, including the so called "heparin-binding growth factor" such as fibroblast growth factors (FGFs), Wnts, Hedgehogs(Hhs) and bone morphogenetic proteins(BMPs). Glypicans are a family of HSPGs whose members are bound to the cell surface by a glycosylphosphatidylinositol(GPI) anchor. The mammalian genome contains six members of glypican family (GPC1to GPC3),and two members of this family have been found in Drosophila(dally and dlp).
In1996Pilia et al reported that the Glypican-3gene (GPC3) displays loss of function mutations in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre-and post-natal overgrowth, a broad spectrum of visceral and skeletal abnormalities, and an increased risk for the development of embryonic tumors. Subsequent research indicated that mutation of GPC3resulted in disequilibrium of cell proliferation and apoptosis, and caused tumorigenesis. GPC3also could bind to adhesion factor, component part of matrix, growth factor, enzyme, engage in the regulation of cell proliferation, differentiation, adhesion and migration. GPC3is highly expressed in HCC, colon cancer, melanoma, Wilms' tumor, neuroblastoma, hepatoblastoma cells. On the other hand, GPC3is silenced in breast cancer, mesothelioma, epithelial ovarian cancer and lung adenocarcinoma, which suggested that GPC3played an important role in tumor development.
In our previous study, GPC3protein expression was detected in the human common tumor and human normal tissue by using tissue chip. The result indicated that GPC3is high expressed in the tissues of HCC, lung squamous carcinoma, chronic superficial gastritis. On the other hand, GPC3is silenced in gastric carcinoma, breast cancer, epithelial ovarian cancer, pancreatic carcinoma, cervical carcinoma, and cholangiocarcinoma. In addition, expression of GPC3is higher in HCC than that in adjacent tissue. GPC3protein expression was not related with age, gender, pathological grade of HCC patients. Expression of GPC3and its relationship with clinicopathological factors were determined by immunohistochemical analysis in61primary HCC patients. The potential prognostic value of GPC3was investigated by comparing the survival time between HCC patients with high and low GPC3expression. The results demonstrated that GPC3expression was closely related with metastasis/recurrence in an HCC patient who can receive the operation. The risk of metastasis/recurrence after surgery in an HCC patient with high GPC3expression was increased to3.214as compared to that of an HCC patient with low GPC3expression. Survival analysis showed that HCC patients with high GPC3expression had a significantly shorter overall survival time than HCC patients with low GPC3expression. Further, multivariate analysis showed that GPC3expression was a significant, independent prognostic parameter for HCC patients. At the same time, we also found that GPC3level of serum is high in HCC patients than that in liver benign tumor patients. The sensitivity and specificity for HCC diagnosis were:GPC350%and92%, GPC3+AFP73.3%and95%. Furthermore, we found that miR-219-5p was significantly downregulated in83HCC tissues and three HCC cell lines, compared to their non-tumor counterparts. MiR-219-5p expression correlated with tumor size, histological differentiation, and overall survival time in HCC patients. We also found that miR-219-5p could inhibit cell proliferation in vitro and arrest cell cycle at the Gl to S transition. Further research demonstrated that miR-219-5p reduced both the mRNA and protein levels of GPC3. These findings indicated that miR-219-5p exerts tumor-suppressive effects in hepatic carcinogenesis through negative regulation of GPC3expression. Above all, GPC3is a promising HCC tumor marker, which involved in tumor metastasis.
On the basis of previous work, RNA interference (RNAi) with a GPC3small hairpin RNA (GPC3shRNA) and pEGFP-Cl/GPC3Plasmid were used to identify the effects of GPC3on the regulation of malignant behaviors of HCC. MHCC97-H and MHCC97-L, had similar genetic background and were thus selected as a cell model for in vitro and in vivo experiments. This article shows a research and analysis that whether GPC3can be used as a new target for early HCC diagnostic marker and targeted therapy.
Methods
1. Detection of GPC3expression in MHCC-97H and MHCC-97L
Western blot and qPCR were used to examined the GPC3expression of MHCC-97H and MHCC-97L.
2. Effect of GPC3overexpression on the biological behaviors of MHCC-97L
MHCC97-L cells were grown to60-70%confluency and transfected with different vectors (p-EGFP-C3-GPC3, p-EGFP-C3) using Lipofectamine2000(Invitrogen). Stable transfectants, named MHCC97-L/GPC3and MHCC97-L/neo, were selected in a medium containing400μg/ml G418for14days and later maintained in the medium with100μg/ml G418. qRT-PCR and Western blotting were applied to analyze GPC3mRNA and protein levels, respectively. The biological behaviors of tranfectant were investigated by plate colony formation assay, MTT assay, transwell assay in vitro and subcutaneous tumor model in nude mice assay in vivo.
3. Effect of GPC3knockdown on the biological behaviors of MHCC-97L
According to the manufacturer's protocol, MHCC97-H cells were grown to60-70%confluency and transfected with different vectors (pGenesil-1-GPC3-shRNA1,2,3,4, pGenesil-1-negative) using Lipofectamine2000(Invitrogen). Stable transfectants, named MHCC97-H/GPC3-shRNA1,2,3,4and MHCC97-H/negative-shRNA, were selected in a medium containing400μg/ml G418for14days and later maintained in the medium with200μg/ml G418. qRT-PCR and Western blotting were applied to analyze GPC3mRNA and protein levels, respectively. One group that transfected GPC3-shRNA would be chosen for further comparison of biological behaviors.
4. The mechanism that GPC3affect the biological behaviors of HCC
We analyzed the activation of Akt pathway by studying the levels of pAkt phosphorylation in cells growing both in presence or absence of serum. We detected Akt expression using western blot and qPCR.
5. Statistical analysis
SPSS19.0software (Abbott Laboratories, North Chicago, IL) was used for statistical analysis. Results of qRT-PCR, western blot analysis, plate clone formation assay and the Transwell assay were assessed using one-way ANOVA. MTT assay, subcutaneous tumo model in nude mice were analyzed by Factorial analysis of variance square analyze Differences were considered statistically significant when P<0.05.
Result
1. GPC3expression of HCC cell lines MHCC-97H and MHCC-97L
Compared with normal liver cell, GPC3was more strongly expressed in the highly metastatic MHCC97-H cells, and more weakly expressed in the non-metastatic, MHCC97-L cell line.
2. The effect of overexpression of GPC3on the biological behaviors of HCC
The subclone HCC cancer cell line MHCC-97L, stable expressing GPC3+EGFP and neo+EGFP respectively, were successfully selected, named as MHCC-97L/GPC3, MHCC-97L/con. Compared with the MHCC-97L/neo and MHCC-97L, the GPC3expression level of MHCC-97L/GPC3was significantly increased (P=0.000).
We examined cell proliferation in vitro using MTT and plate clone formation assay. The results indicated that the growth of MHCC-97L/GPC3cells in vitro was markedly increased after the transfection of GPC3+EGFP (P=0.000). This indicates a positive relation between the expression of GPC3and the rate of hepatocellular cancer cell growth. In addition, MHCC-97L/GPC3cells, compared with MHCC-97L and MHCC-97L/con cells, had a significant enhanced in their ability to form colonies, and their ability to form colonies correlated with GPC3expression (P=0.000).
We examined cell migration and invasion activity in vitro using transwell migration and invasion assay. MHCC-97L/GPC3displayed remarkable increase in migration ability as compared with either MHCC-97L/con or MHCC-97L (P<0.05, for both). The migration cells of MHCC-97L/GPC3was15.480±1.561, while those of the MHCC-97L and MHCC-97L/con groups were5.103±1.994and4.174±0.898, respectively. Reexpression of GPC3cause significantly enhanced migration of MHCC-97L cells. Invasion assays were carried out using Matrigel-coated Transwell culture chambers. After24h invading cells were counted using image analysis. MHCC-97L/GPC3showed much higher invasion activities than either MHCC-97L or MHCC-97L/con (P=0.000, respectively). The invasion cells of the MHCC-97L/GPC3,MHCC-97L, MHCC-97L/con groups were12.521±1.620,4.707±1.131and4.569±1.502, respectively. Reexpression of GPC3led to a significant increase in the invasion of MHCC-97L cells.
The effect of GPC3on in vivo tumor growth was assessed by the subcutaneous injection of MHCC-97L/GPC3, MHCC-97L or MHCC-97L/con cells for21days. The results indicated that remarkable increase of tumor size was observed in the MHCC-97L/GPC3group as compared to that of the control group (P=0.000). By day21after cell injection, the average tumor weight (n=4) of the MHCC-97L/GPC3, MHCC-97L and MHCC-97L/con groups was2.987±0.168g,1.801±0.015g and1.758±0.040g, respectively (P=0.000), indicating that reexpression of GPC3in hepatocellular cancer cells enhanced their tumorigenic potential.
3. The effect of silence of GPC3on the biological behaviors of HCC
We successfully construct Stable transfectants, named MHCC97-H/GPC3-shRNA1,2,3,4and MHCC97-H/con-shRNA. The qPCR and western blotting were applied to analyze GPC3mRNA and protein levels, respectively. The results indicated that cells transfected with pGenesil-l-GPC3shRNAl showed a significantly reduced transcription of GPC3mRNA when compared with that of vector control and negative transfectants, respectively. The reduction in mRNA transcription was not detectable in cells transfected with pGenesil-l-GPC3-shRNA3and pGenesil-l-GPC3-shRNA4. In addition, Western blot analysis showed a remarkable reduction of GPC3protein levels in the MHCC97-H cell lines transfected with pGenesil-l-GPC3-shRNAl.Thus, MHCC97-H/GPC3-shRNA1and MHCC97-H/con-shRNA were chosen for further comparison of biological behaviors.
We examined cell proliferation in vitro using MTT and plate clone formation assay. The results indicated that the growth of MHCC97-H/GPC3-shRNA1cells in vitro was markedly decreased (P=0.000). This indicates a positive relation between the expression of GPC3and the rate of hepatocellular cancer cell growth. In addition, MHCC97-H/GPC3-shRNAl cells, compared with MHCC97-H/con-shRNA and MHCC97-H cells, had a significant reduction in their ability to form colonies, and their ability to form colonies correlated with GPC3expression (P=0.000).
We examined cell migration and invasion activity in vitro using transwell migration and invasion assay. MHCC97-H/GPC3-shRNA1displayed remarkable decrease in migration ability as compared with either MHCC97-H/con-shRNA or MHCC-97H (P=0.000, for both). The migration cells of MHCC97-H/GPC3-shRNAl were4.421±1.536, while those of the MHCC-97H and MHCC97-H/con-shRNA groups were14.374±2.015and15.902±1.450, respectively. Downregulation of GPC3cause significantly attenuated migration of MHCC-97H cells. Invasion assays were carried out using Matrigel-coated Transwell culture chambers. After24h invading cells were counted using image analysis. MHCC97-H/GPC3-shRNAl showed much lower invasion activities than either MHCC-97H or MHCC97-H/con-shRNA (P=0.000, respectively). The invasion rates of the MHCC-97H, MHCC97-H/con-shRNA and MHCC97-H/GPC3-shRNAl groups werel5.770±2.025,14.774±1.112, and4.647±1.319, respectively. Silence of GPC3led to a significant decrease in the invasion of MHCC-97H cells.
The effect of GPC3on in vivo tumor growth was assessed by the subcutaneous injection of MHCC97-H/GPC3-shRNA1, MHCC-97H or MHCC97-H/con-shRNA cells for21days. The results indicated that remarkable decrease of tumor size was observed in the MHCC97-H/GPC3-shRNAl group as compared to that of the control group (P=0.000). By day21after cell injection, the average tumor weight (n=4) of the MHCC97-H/GPC3-shRNA1, MHCC-97H or MHCC97-H/con-shRNA groups was1.541±0.096g,2.424±0.095g and2.402±0.080g, respectively (P=0.000), indicating that downregulation of GPC3in hepatocellular cancer cells attenuated their tumorigenic potential.
4. GPC3overexpression (or silence) promoted(or inhibited) the activation of Akt signal pathway
We found that whereas this pathway was constitutively active in control cells even in serum withdrawal conditions, GPC3silence induced decrease of phospho-Akt basal levels. In the other hand, compared to the control cells, GPC3reexpression promoted the incease of phospho-Akt.
Conclusions
1. We established cell line MHCC-97L/GPC3that stable transfected GPC3through using the plasmid transfection mediated by liposomal transfection reagent and found that GPC3could enhance HCC cell proliferation, invasion and migration.
2. We established GPC3downregulation cells MHCC-97H through using the plasmid transfection mediated by liposomal transfection reagent and found that knockdown of GPC3could decrease HCC cell proliferation, invasion and migration.
3. GPC3reexpression (or downregulation) induces and promotion (or inhibition) of the pro-survival Akt signaling pathway.
Novelty
1. In this study, we determined that GPC3expression plays an important role on the biological behaviors of HCC cells. Our results provide new clues regarding the mechanism involved in the modulation induced by GPC3of HCC cells growth and survival.
2. Now we demonstrate that GPC3is able to enhance the Akt over-activation detected in HCC cells. Thus, GPC3may promote HCC invasion and growth by modulating Akt signaling in part.
引文
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