Gαs在肝癌中的表达情况及其高表达对肝癌细胞HepG2的影响

目的:探讨Gαs过表达对人类肝癌细胞HepG2生长、增殖的影响,及Gαs在人类肝癌中的表达情况,寻求有效的治疗靶点.方法:利用QpCR技术检测人类肝癌组织中Gαs基因水平的表达情况;利用Western blot检测人肝癌细胞HepG2及正常肝细胞HL-7702中Gαs蛋白水平的表达;利用脂质体法将Gαs质粒转染HepG2细胞;用MTT检测HepG2细胞增殖情况;流式细胞术检测细胞周期变化.结果:QPCR 结果显示在基因肝癌组织中Gαs表达高于癌旁组织;Western blot结果显示肝癌细胞中Gαs在蛋白水平表达高于肝正常细胞;Gαs质粒转染HepG2细胞后,Gαs表达增高,细胞增殖加快.结论:Gαs基因表达水平在肝癌组织中高于周围癌旁组织;Gαs的高表达可能与肝癌的发生发展相关联,其高表达促进HepG2细胞的生长与增殖.     

MYH9在人肝癌组织中的表达及其对肝细胞系增殖和凋亡的影响

目的探讨非肌肉肌球蛋白重链(MYH9)在肝癌组织中的表达及通过沉默MYH9基因对肝癌细胞系SMMC-7721的增殖及凋亡的影响。方法收集50组人肝癌组织及癌旁组织,选用人肝癌细胞系SMMC-7721和Hep G2及人正常肝细胞系LO2,免疫组织化学方法及Western blot检测肝癌组织及癌旁组织中MYH9蛋白的表达,Western blot检测SMMC-7721、Hep G2及LO2中MYH9蛋白的表达;将MYH9 siRNA转染SMMC-7721,CKK8法及流式细胞术检测沉默MYH9对肝癌细胞增殖及细胞凋亡的影响。结果 MYH9蛋白在肝癌组织中的表达明显高于癌旁(P0.05);MYH9蛋白在SMMC-7721及Hep G2中的表达均明显高于LO2(P0.05);沉默MYH9基因可抑制细胞增殖(P0.001),促进细胞凋亡(P0.05)。结论 MYH9蛋白在肝癌组织的表达显著高于癌旁组织;MYH9低表达能有效抑制肝癌细胞的增殖,促进其凋亡。     

RNAi沉默WT1基因对人肝癌细胞HepG2的影响

目的:探讨利用RNA干扰沉默WT1基因对人肝癌细胞HepG2细胞生长,凋亡的影响,为肝癌的基因治疗提供理论依据。方法:利用脂质体将siRNA真核表达载体转入HepG2细胞中,并检测转染效率;利用免疫细胞化学染色方法检测HepG2细胞中WT1蛋白水平的表达,测定基因沉默效果;用MTT法测定HepG2细胞生长曲线;电镜下观察细胞凋亡形态,流式细胞仪检测凋亡率。结果:利用脂质体为载体将siRNA真核表达载体转染HepG2细胞,转染率达70%以上,转染后细胞中WT1蛋白表达水平下降;RNA干扰沉默WT1基因可抑制细胞增殖,促进细胞凋亡。结论:靶向WT1的序列特异性siRNA可显著抑制WT1基因的表达;下调HepG2细胞WT1基因表达可抑制细胞增殖,促进细胞凋亡。     

Cripto-1基因沉默对人肝癌MHCC-97H细胞侵袭、迁移能力的影响

 目的: 探讨Cripto-1蛋白在人肝癌组织和肝癌细胞系中的表达,研究沉默Cripto-1基因后对肝癌细胞MHCC-97H侵袭和迁移能力的影响及可能的机制。方法: 采用Western blot检测11对肝癌组织及相应的癌旁组织和不同肝癌细胞株中Cripto-1蛋白的表达;收集80例肝癌石蜡标本,免疫组织化学方法检测Cripto-1蛋白的表达情况;Cripto-1 siRNA转染肝癌细胞MHCC-97H细胞后,real-time PCR和Western blot分别检测转染效率;Transwell迁移和侵袭实验分别检测细胞的迁移和侵袭能力;Western blot分别检测基质金属蛋白酶(MMP)-2、MMP-9蛋白的表达。结果: Western blot检测结果显示肝癌组织中Cripto-1蛋白的表达明显高于对应的癌旁组织,Cripto-1蛋白在各肝癌细胞系中均有表达,且表达量高于对照细胞,其中高侵袭性肝癌细胞MHCC-97H表达量最高;免疫组织化学结果显示Cripto-1蛋白在88.7%的肝癌组织中表达;Cripto-1 siRNA转染MHCC-97H细胞后能显著降低Cripto-1 mRNA和蛋白的表达水平,并且能降低其侵袭和迁移能力;Western blot结果表明,Cripto-1基因沉默后能抑制MMP-2和MMP-9蛋白的表达。结论: Cripto-1在肝细胞癌中的高表达可能与肝癌的发生发展密切相关,下调Cripto-1的表达可以抑制肝癌细胞侵袭和迁移能力,其机制可能与下调MMP-2、MMP-9的表达有关。     

食管癌相关基因4在肝细胞肝癌中的表达和功能

目的了解人食管癌相关基因4（ ECRG4）在人肝细胞肝癌组织和肝癌细胞系中的表达情况,探讨ECRG4对肝癌细胞增殖、凋亡以及迁移能力的影响。方法提取24例配对肝细胞肝癌组织和癌旁肝组织的总RNA以及正常肝细胞系QSG7701和肝癌细胞系HepG2的总RNA和总蛋白,分别利用即时荧光定量PCR（ RT-qPCR）和Western blot法检测ECRG4的表达;构建ECRG4的真核表达质粒ECRG4-pcDNA3.1,转染肝癌细胞系HepG2,进行体外的细胞增殖、凋亡以及迁移实验。结果与癌旁肝组织相比,在肝细胞肝癌组织中ECRG4 mRNA表达下调或缺失（98.5％,23／24,P＜0.01）;和正常肝细胞系QSG7701相比,ECRG4 mRNA在肝癌细胞系HepG2中的表达明显下调（ P＜0.05）并且其蛋白表达水平也明显降低。转染ECRG4表达质粒后HepG2细胞的增殖和迁移能力明显低于对照组（P＜0.05）,细胞凋亡率明显高于对照组（P＜0.05）。结论 ECRG4在肝细胞肝癌中表达下调,在体外肝癌细胞系中过表达ECRG4可抑制细胞的增殖和迁移,促进细胞凋亡,ECRG4可能是肝细胞肝癌潜在的抑癌基因,有望为肝细胞肝癌分子靶向治疗提供新策略。     

长链非编码RNA LINP1对肝癌细胞增殖、侵袭转移的影响

目的探究长链非编码RNA LINP1在肝癌组织中的表达及其对肝癌细胞增殖及侵袭转移能力的影响。方法使用qRT-PCR方法检测60例肝癌组织以及癌旁组织、肝癌细胞株及正常肝细胞中LINP1的表达情况。si-LINP1转染肝癌细胞敲低LINP1表达;CCK-8实验检测si-LINP1对肝癌细胞增殖影响;Western blot检测Wnt/β-catenin信号通路蛋白β-catenin和cyclinD1表达。结果 LINP1在肝癌组织中的表达水平高于癌旁组织,肝癌细胞高于正常肝细胞系Lo2(P0.05);敲低LINP1抑制肝癌细胞增殖及侵袭转移,抑制Wnt/β-catenin信号通路蛋白β-catenin和cyclinD1表达(P0.05)。结论敲低LINP1抑制肝癌细胞的增殖、侵袭和转移与抑制Wnt/β-catenin信号通路相关。     

Uba-2在肝癌组织中的表达及其对肝癌细胞增殖和侵袭转移能力的影响

目的探讨Uba-2在肝癌组织中的差异表达以及过表达和低表达时其对肝癌细胞增殖和侵袭能力的影响。方法 qRTPCR法检测肝癌组织中Uba-2 mRNA的表达;将Uba-2的过表达载体和siRNA表达载体转染至肝癌细胞Hep G2后,MTS实验检测Uba-2对肝癌细胞增殖能力的影响;细胞迁移/侵袭实验检测其对肝癌细胞侵袭转移能力的影响;Western blot法检测肝癌细胞Hep G2中TGF-β1~3及VEGF、Snail蛋白的表达。结果 Uba-2在肝癌组织中呈高表达,与癌旁肝组织中的表达差异有显著性(P0.01),qRT-PCR法检测Uba-2在Hep G2细胞中过表达或低表达;细胞增殖实验数据显示Uba-2表达水平与肝癌细胞Hep G2的增殖能力相关,siRNA干扰沉默Hep G2细胞中Uba-2的表达会抑制其增殖生长,细胞迁移/侵袭实验数据显示Uba-2过表达能促进肝癌细胞Hep G2的侵袭、转移能力,且与阴性对照组细胞相比差异有统计学意义;Western blot结果显示Uba-2的异常表达影响TGF-β2及VEGF、Snail蛋白表达水平。结论 Uba-2基因在肝癌组织中的高表达可能具有促进肝癌细胞增殖及侵袭转移的作用。     

RNA干扰技术沉默CDC25a基因对人肝癌细胞HepG2增殖的影响

 目的:研究细胞分裂周期素25a（cell division cycle 25a,CDC25a）基因沉默后对于人肝癌细胞系HepG2增殖的影响。同时探讨该影响发生的可能作用机制。 方法: 使用RNA干扰技术沉默人肝癌HepG2细胞的CDC25a基因,采用实时荧光定量PCR技术检测肝癌细胞中的CDC25a 及其作用基因cyclin E及CDK2的 mRNA表达水平,Western blotting检测CDC25a的蛋白表达水平,并采用MTT法、Giemsa染色法及流式细胞技术检测细胞的增殖情况。 结果: CDC25a 的mRNA及蛋白表达水平在RNA沉默组细胞中的表达低于阴性对照组及正常对照组细胞（P＜0.05）。Cyclin E及CDK2 的mRNA表达水平在沉默组低于阴性对照及正常对照组（P＜0.05）。MTT法、Giemsa染色法结果显示沉默组细胞增殖能力低于阴性对照组及正常对照组细胞（P＜0.05）,流式细胞技术结果显示沉默组细胞阻滞在G1期。 结论: LV-CDC25a-RNAi重组体感染HepG2细胞可以有效抑制CDC25a基因的表达,使人肝癌HepG2细胞增殖受到抑制,提示CDC25a基因可能是肝癌治疗的关键靶点。     

AIFM2抑制铁死亡介导肝癌细胞索拉菲尼耐药

目的 探讨AIFM2对肝癌细胞索拉菲尼耐药的影响及对铁死亡的调控。方法 采用实时定量PCR检测30例肝细胞癌组织与相应癌旁组织和肝癌细胞系及正常肝细胞系中AIFM2 mRNA的表达水平;采用浓度梯度法培养索拉非尼耐药肝癌细胞,sh-AIFM2转染肝细胞癌细胞以抑制AIFM2表达,sh-Ctrl作为对照,实时定量PCR检测转染效率,Western blot法检测转染后AIFM2蛋白的表达;CCK-8法检测细胞活力,克隆形成实验检测细胞的增殖能力;DCFH-DA染色法检测ROS表达,脂质过氧化试剂盒检测MDA的表达,微量还原型谷胱甘肽（GSH）试剂盒检测MDA表达,流式细胞术检测铁离子的表达。结果 AIFM2在肝细胞癌组织中相对癌旁高表达,AIFM2在肝癌细胞中的表达高于正常肝细胞系;AIFM2基因敲低促进肝癌耐药细胞活性的下降,并促进肝癌耐药细胞发生铁死亡,其中ROS、MDA表达增加,GSH表达降低,而铁离子表达并没有发生明显改变。结论AIFM2抑制铁死亡介导肝癌细胞索拉非尼耐药,联合应用AIFM2抑制剂和索拉非尼可能是治疗肝细胞癌耐药的有效途径。     

Perilipin 5在肝细胞癌中的表达及对HepG2细胞脂质代谢和生物学行为的影响

目的:探讨脂滴包被蛋白5(perilipin 5,Plin5)在肝细胞癌中的表达变化,以及对肝癌细胞HepG2脂质含量、氧化应激和生物学行为的影响。方法:采用免疫组化染色和RT-qPCR分析人肝细胞癌组织中Plin5的表达。通过腺病毒感染,在HepG2细胞中过表达Plin5,检测其对肝癌细胞活力、迁移和侵袭能力的影响。利用BODIPY 493/503染色和脂质定量分析确定Plin5对HepG2细胞脂质含量的影响,并利用试剂盒检测过表达Plin5的HepG2细胞中脂质过氧化和超氧化物歧化酶(SOD)活性。结果:与正常肝组织比较,人肝细胞癌组织中Plin5的表达明显降低(P 0. 05)。过表达Plin5能够抑制HepG2细胞的活力,降低HepG2细胞的体外迁移和侵袭能力(P 0. 01)。进一步研究发现,过表达Plin5能够促进HepG2细胞中的脂质蓄积,并降低氧化应激水平。结论:肝细胞癌中Plin5呈低表达。Plin5能够促进肝癌细胞中脂质蓄积,降低氧化应激水平,从而抑制肝癌细胞的迁移和生长。     

Sulindac对人肝癌细胞HepG2增殖及β-catenin表达的影响

目的　观察Sulindac对人肝癌细胞HepG2增殖、凋亡及β-catenin蛋白表达的影响,探讨Sulindac抗肝癌的可能机制。 方法　不同浓度的Sulindac作用HepG2细胞后,采用MTT实验检测细胞增殖抑制作用;采用Hoechst33258染色法检测Sulindac对HepG2细胞凋亡的影响;利用RT-PCR及Western blotting检测HepG2细胞在Sulindac作用后Wint通路中β-catenin的表达变化。 结果　Sulindac对人肝癌细胞HepG2有增殖抑制作用,且呈剂量时间依赖关系;Hoechst33258结果显示,Sulindac作用24 h后HepG2细胞凋亡数目明显增多;随着Sulindac浓度的增加,β-catenin mRNA及蛋白表达量逐渐下降。 结论　Sulindac能够抑制人肝癌细胞HepG2增殖,通过阻断Wnt信号传导通路,降低β-catenin表达,诱导人肝癌细胞HepG2的凋亡。     

Involvement of Hepatopoietin Cn in the development of human hepatocellular carcinoma

Hepatopoietin Cn (HPPCn) is a novel nuclear protein with the ability to promote liver regeneration. In the present study, we investigated the expression profile of HPPCn and its functional activity in human hepatocellular carcinoma (HCC) cell line and tissue samples. HPPCn expression was detected in HCC cell lines and 54 paired HCC carcinomas by immunochemical staining and Western blotting. The functional activity of HPPCn in cell lines was evaluated by MTT and colony formation assays and with nude mouse model. The correlation of HPPCn expression with clinicopathological characteristics of 54 HCC patients was also analyzed. Our results showed that HPPCn protein was prominently located within the nuclei of hepatocytes and the expression level was evidently increased in HepG2 and Bel7402 cell lines compared with L02 normal hepatocytes. HPPCn silencing by small interfering RNA greatly suppressed HepG2 cell proliferation and colony formation capacity and the inhibitory effect was also observed in a Balb/c-null mouse model. The silencing HPPCn expression effectively enhanced the apoptosis of HepG2 cells. In addition, HPPCn expression was detected in 48 of 54 (89%) human HCC tissues in sharp contrast with the corresponding non-tumor liver tissues. HPPCn protein was mainly accumulated in the tumor nucleus. The elevated expression of HPPCn protein in tumors was significantly associated with poor tumor cellular differentiation and present of vascular invasion. Patients with higher HPPCn expression in tumors had significantly shorter overall survival (OS) of both all of patients and the patients at the early stage. On multivariate Cox analysis, elevated expression of HPPCn in tumors was found to be an independent prognostic factor for OS. Therefore, these data suggest that HPPCn expression might be involved in the development of HCC and could be served as a promising biomarker.     

Wnt/β-catenin信号因子在HepG2以及L02细胞中的表达

目的:比较Wnt/β-catenin信号分子在HepG2和L02细胞中的表达,探讨Wnt/β-catenin信号转导通路在肝癌发生中的作用机制,为肝癌的防治提供新的思路。方法:选取Wnt/β-catenin信号转导通路中上下游关键因子Wnt1、Wnt4、β-catenin、cyclin D1以及c-myc等,应用RT-PCR的方法观察他们在正常肝脏L02细胞和肝癌HepG2细胞中的转录水平。用免疫细胞化学染色方法和Western blot检测研究Wnt/β-catenin信号转导通路中最关键的成员β-catenin在上述2个细胞株中的定位和定量表达。结果:在正常的L02肝细胞中,用RT-PCR的方法未检测到Wnt1、Wnt4、cyclin D1以及c-myc的mRNA转录,只有β-catenin的基因被转录表达。同时用免疫细胞化学染色观察到β-catenin在L02细胞膜处存在表达。而在HepG2肿瘤细胞中,不仅检测到β-catenin的基因转录,同时也检测到Wnt1、cyclin D1以及c-myc的mRNA转录,只有Wnt4未转录。用免疫细胞化学的方法观察β-catenin在肿瘤细胞中的表达明显增强,表现为胞膜着色减弱而胞质甚至是胞核的阳性染色。采用Western blot检测也验证了β-catenin蛋白在肿瘤细胞中的表达水平明显高于正常细胞。结论:Wnt/β-catenin信号转导通路在人的肝癌细胞HepG2中存在异常活性,Wnt1可能是导致信号通路激活的始动因素之一。     

Proteomic identification of RhoA as a potential biomarker for proliferation and metastasis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and there is an urgent need to discover novel factors that can act as biomarkers for prognostic assessment and therapeutic targets of HCC. In this study, highly purified plasma membrane proteins from clinical tissue samples were obtained using a strategy combining sucrose density gradient centrifugation and subsequent phase partition. Using a two-dimensional gel electrophoresis and MALDI-Q-TOF MS/MS-based proteomics approach, we identified 13 plasma membrane-associated proteins that were differentially expressed in HCC and normal liver tissues. Of those, RhoA was one of the most significantly upregulated proteins in HCC, and its overexpression was confirmed using Western blotting. Immunohistochemistry suggested a link between RhoA expression and poor differentiation and clinicopathologic stage. Suppression of RhoA expression in HepG2 and Hep3B cells by RNA interference led to significant inhibition of cell growth, induction of apoptosis, and a decrease in migration. Our data suggest that RhoA may serve as a potential biomarker and an attractive therapeutic target for HCC.     

Downregulation of cyclin D1 is associated with decreased levels of p38 MAP kinases,Akt/PKB and Pak1 during chemopreventive effects of resveratrol in liver cancer cells

Resveratrol is a naturally occurring phytoalexin with antioxidant activity. The chemopreventive effects of resveratrol against various types of cancer are well known, though the underlying molecular mechanisms of its action are still not identified. Hepatocellular carcinoma (HCC) is a one of the most lethal malignancies and there is no effective treatment till date. It is known that cyclin D1 is overexpressed in liver cancers. Accordingly we have studied the chemopreventive effects of resveratrol on cyclin D1 expression and the signaling pathways that regulate cyclin D1 in HepG2 cells. Flow cytometry and PCNA western blot data showed that resveratrol inhibits proliferation of HepG2 cells. Also, resveratrol treatment downregulated cyclin D1 as well as p38 MAP kinase, Akt and Pak1 expression and activity in HepG2 cells, suggesting that growth inhibitory activity of resveratrol is associated with the downregulation of cell proliferation and survival pathways. Furthermore, resveratrol treated cells showed increase in ERK activity suggesting possible sensitization to apoptosis. Thus in the present study, we report a three-dimensional relationship between the growth inhibitory effects of resveratrol – decrease in the levels of cyclin D1 – and downregulation of cell proliferation and survival pathways in HepG2 cells leading to cellular degenerative changes. These observations suggest that resveratrol has good potential as effective chemopreventive agent against liver cancer and warrant further studies.     

SD118-2对人HepG2细胞凋亡影响及机制探讨

目的观察化合物SD118-2对人HepG2细胞增殖、凋亡及细胞周期的影响。方法取对数生长期人HepG2细胞,分为对照组和给药组。给药组分别给予7 mg/L SD118-2处理12、24和48 h,对照组给予相同浓度DMSO。用MTT法检测细胞增殖率,DAPI荧光染色法观察SD118-2处理后细胞形态,流式细胞仪AnnexinⅤ-FITC/PI双标法检测细胞凋亡率及周期阻滞,用流式细胞仪JC-1染色法检测线粒体膜电位(ΔΨm),Western blot检测凋亡相关蛋白BCL-2、BAX、PARP和C-PARP表达。结果 SD118-2呈时间依赖性抑制人HepG2细胞生长、降低线粒体膜电位、诱导细胞凋亡、阻滞细胞G2期;抗凋亡蛋白BCL-2表达呈时间依赖性减少,促凋亡蛋白BAX、C-PARP表达呈时间依赖性增加;SD118-2对正常肝脏细胞增殖几乎无影响。结论化合物SD118-2具有诱导人HepG2细胞凋亡的作用,并能使细胞周期进程发生变化。     

重组腺相关病毒介导黑色素瘤分化相关基因7对人肝癌细胞体内外抑制效应的研究

目的观察PEG启动子调控腺相关病毒介导的黑色素瘤分化相关基因7（MDA-7）在体内外抑制肝癌细胞的生物学活性，探讨重组腺相关病毒介导MDA-7基因用于肝癌基因治疗的应用前景。方法构建重组腺相关病毒rAAV-PEG-MDA-7表达系统，体外感染人肝癌细胞株HepG2细胞。Western印迹检测转染细胞内MDA-7蛋白；MTT法检测细胞增殖抑制率；流式细胞术分析细胞周期和细胞凋亡变化。构建肝癌细胞HepG2裸鼠皮下移植瘤模型，尾静脉注射rAAV-PEG-MDA-7，观察其对肝癌生长的抑制作用；ELISA方法检测血浆MDA-7蛋白浓度；TUNEL法分析MDA-7对肿瘤细胞的凋亡诱导情况；免疫组织化学分析MDA-7在肿瘤组织中的表达。结果重组腺相关病毒rAAV-PEG-MDA-7可特异性转染HepG2细胞，MDA-7蛋白在HepG2细胞中高效表达，并呈时间依赖性。重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡，G0／G1期细胞百分比明显增多，G2／M期的细胞显著减少（P〈0、05）。全身系统性给予rAAV-PEG-MDA-7后，血清中可持续检测到MDA-7蛋白，且注射后2周浓度达高峰（200ng／ml）；肿瘤生长受抑制，抑瘤率为62％（P〈0、05）；免疫组化结果显示MDA-7在肿瘤组织中表达；TUNEL结果显示rAAV-PEG-MDA-7可诱导肿瘤细胞凋亡。结论构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统具有良好的肿瘤靶向性，通过抑制肝癌细胞增殖和诱导其凋亡发挥抗肝癌作用。     

The role of N-acetylglucosaminyltransferases V in the malignancy of human hepatocellular carcinoma

To investigate the role of N-acetylglucosaminyltransferases V (GnT-V) in the malignancy of human hepatocellular carcinoma (HCC), the GnT-V stably suppressed cell line HepG2 GnT-V/1564 was constructed from HepG2. The proliferation, migration, invasion, metastasis of HepG2 GnT-V/1564 was investigated both in vitro and in vivo. The clinical pathological significance of GnT-V expression was also studied in 140 cases of HCC tissues. This study showed that down-regulation of GnT-V inhibited the proliferation, migration and invasion of the HepG2 cells. In addition, GnT-V expression was shown in 138 cases of 140 (98.6%) HCC samples, in 3 cases of 31 (9.7%) in liver cirrhosis cases and in 1 cases of 20 (5.0%) in normal liver tissues. Besides, a higher level of GnT-V was observed more frequently in the advanced tumors with higher T stage and histological grade. These data suggested that GnT-V expression was positively related with malignancy in HCC and GnT-V may be both a differentiation marker and a potential target for the treatment of HCC.