Fbxw7在肝癌中的表达及与肝癌细胞增殖相关性研究

目的:探讨Fbxw7在肝癌中的表达及其与肝癌细胞增殖能力的相关性.方法:收集40例肝细胞癌组织及对应的癌旁组织,运用RT-PCR、免疫组化技术检测Fbxw7在肝癌及对应癌旁组织中的表达情况;real time RT-PCR技术检测正常肝细胞株LO2、肝癌细胞株Hep3B和SMMC-7721中Fbxw7mRNA的表达水平;平板克隆形成实验与裸鼠皮下成瘤实验检测不同Fbxw7 mRNA表达水平的细胞株体内外增殖能力.结果:Fbxw7 mRNA及蛋白在肝癌组织中表达水平显著低于对应癌旁组织(P＜0.05);Fbxw7蛋白低表达与高Edmonson分级和高TNM分期具有显著的相关性(P＜0.05);正常肝细胞株LO2中Fbxw7 mRNA的表达水平显著高于肝癌细胞株Hep3B和SMMC-7721 (P＜0.05);Fbxw7表达较低的细胞株形成的克隆数较多,且裸鼠皮下成瘤体积较大,结果均具有统计学差异(P＜0.05).结论:Fbxw7低表达与肝癌的恶性临床病理特征相关,并与肝癌细胞增殖能力相关.     

GRAMD4在肝细胞癌中的表达及临床意义

目的 检测GRAMD4在肝细胞癌中的表达情况,分析其与肝癌临床病理特征之间的相关性.方法 收集40例肝细胞癌及对应癌旁组织,运用RT-PCR、免疫组化技术检测GRAMD4在肝癌及对应癌旁组织中的表达情况,统计学分析GRAMD4mRNA表达与肝癌临床病理特征之间的相关性;应用RT-PCR技术检测人正常肝细胞LO2和肝癌细胞Hep3B、HepG2、SMMC-7721中GRAMD4 mRNA的表达.结果 GRAND4 mRNA及蛋白在肝癌组织中的表达显著高于对应的癌旁组织(P＜0.05);GRAMD4 mRNA的高表达与肿瘤体积增大、高Edmonson分级和高TNM分期呈明显正相关(P＜0.05);肝癌细胞株Hep3B、HepG2和SMMC-7721中GRAMD4 mRNA的表达较正常肝细胞LO2显著增加(P＜0.05).结论 肝癌细胞系及肝癌组织中GRAMD4表达上调,且GRAMD4的表达上调与肝癌的恶性病理特征相关.     

miR-130b在肝细胞癌中的表达及临床病理意义

目的探讨miR-130b在人肝细胞癌(HCC)中的表达、与临床病理特征的关系及可能机制。方法用实时定量PCR(qRT-PCR)检测miR-130b在86例HCC及癌旁组织、不同肝癌细胞系的表达;免疫组织化学染色检测miR-130b不同表达水平的HCC组织中上皮钙黏素(E-cadherin)、波形蛋白(vimentin)及过氧化物酶体增殖物激活受体γ(PPARγ)的表达情况;qRT-PCR检测miR-130b在LO2人正常永生化肝细胞及Hep G2、Hep3B、SMMC-7721、Hu7肝癌细胞系的表达水平;应用人工合成的miR-130b抑制物转染SMMC-7721细胞,TranswellTM实验检测SMMC-7721细胞侵袭能力变化;应用人工合成的miR-130b抑制物及PPARγ小干扰RNA(siRNA)转染SMMC-7721人肝癌细胞,qRT-PCR检测癌细胞中miR-130b、PPARγ、E-cadherin、vimentin的mRNA水平,Western blot法检测癌细胞中PPARγ、E-cadherin、vimentin的蛋白水平。结果 miR-130b在HCC组织中表达水平显著高于对应癌旁组织;肝癌组织中miR-130b异常表达与门静脉侵犯、原发肿瘤分级、肿瘤TNM分期显著相关;miR-130b在不同肝癌细胞系中表达均高于LO2细胞;miR-130b高表达组PPARγ及E-cadherin蛋白水平显著低于miR-130低表达组,而vimentin水平显著高于miR-130b低表达组,相关性分析结果显示肝癌组织中miR-130b与PPARγ蛋白、E-cadherin蛋白水平呈显著负相关,与vimentin蛋白水平呈显著正相关;抑制miR-130b水平,可上调PPARγ蛋白的水平,E-cadherin蛋白的表达显著增加,而vimentin蛋白表达显著降低,SMMC-7721细胞的侵袭能力降低。PPARγsiRNA可部分逆转miR-130b抑制物对SMMC-7721细胞的作用。结论 miR-130b在HCC组织中表达上调并与HCC恶性临床病理特征有关,miR-130b可能通过抑制PPARγ表达及诱导上皮间质转化促进肝癌细胞侵袭。     

miR-204通过下调Bcl-2和Sirt1表达抑制肝癌细胞生长

目的探讨微小RNA 204(miR-204)在肝细胞癌中的表达、临床意义及可能分子机制。方法收集手术切除的60例肝细胞癌及对应癌旁肝组织,实时定量PCR(qRT-PCR)检测miR-204在肝癌及癌旁组织中的表达,免疫组织化学染色检测miR-204下游潜在靶点Bcl-2与组蛋白脱乙酰酶1(Sirt1)的表达;用人工合成的miR-204模拟物转染人SMMC-7721肝癌细胞,MTT法及流式细胞术检测SMMC-7721细胞的增殖、凋亡的情况,qRT-PCR、Western blot法分别检测Bcl-2与Sirt1的mRNA和蛋白表达。结果 miR-204在肝癌组织中表达水平显著低于对应癌旁组织;肝癌组织中miR-204低表达与肿瘤大小、肿瘤个数、肿瘤TNM分期显著相关;miR-204低表达组Bcl-2与Sirt1蛋白表达显著高于miR-204高表达组,相关性分析结果显示肝癌组织中miR-204与Bcl-2、Sirt1蛋白表达呈显著负相关;miR-204可显著抑制SMMC-7721细胞的增殖并促进其凋亡,并下调Bcl-2与Sirt1的mRNA与蛋白表达水平。结论 miR-204在肝癌组织中表达下调并与肝癌恶性临床病理特征有关,miR-204抑制肝癌细胞增殖、促进其凋亡的作用可能与下调Bcl-2和Sirt1表达有关。     

Per2对肝癌患者预后及肝癌细胞生长和凋亡的作用

目的探讨周期昼夜节律调节因子2(Per2)在肝细胞肝癌(HCC)组织中的表达和对患者生存的影响,分析Per2对肝癌SMMC-7721细胞增殖和凋亡的影响。方法应用免疫组化法检测HCC组织和癌旁肝组织中Per2的表达;对肝癌细胞系SMMC-7721转染Per2真核表达质粒后,Western blot检测Per2蛋白表达,MTS法和流式细胞计量术检测细胞增殖和凋亡。结果 117例HCC组织中,Per2阳性表达率70.94%,显著低于对应癌旁肝组织的87.18%;癌组织中Per2染色评分为2.14±1.76,显著低于癌旁肝组织的6.39±3.84(P0.01);Per2表达与HCC患者的肿瘤直径、门脉侵袭和TNM分期相关(P0.05);Per2低表达的患者总体生存期(OS)和无复发生存期(RFS)较Per2高表达的患者短(P0.05)。转染Per2真核表达质粒组细胞与对照组细胞相比细胞增殖显著受抑制(P0.05),同时细胞凋亡水平显著增加(P0.05)。结论 HCC组织中Per2表达显著下调,Per2对肝癌的进展发挥了抑制作用,可能是通过抑制细胞增殖和促进细胞凋亡实现。     

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。     

Noxa基因表达与肝细胞癌的关系

目的探讨Noxa基因与人肝癌的临床病理关系及对人肝癌Hep G2细胞的增殖抑制和促凋亡作用。方法采用免疫组化法检测100例肝癌组织及癌旁组织中Noxa蛋白的表达,结合其临床病理参数和随访资料进行统计学分析。同时利用脂质体将真核表达载体p IRES2-EGFP-Noxa瞬时转染至人肝癌Hep G2细胞,RT-PCR检测转染后Noxa基因m RNA的表达,Western blot法检测转染后Noxa蛋白表达,MTT比色法测定细胞增殖情况,流式细胞仪检测细胞凋亡。结果免疫组化检测结果显示肝癌组织中Noxa阳性率为50%,明显低于癌旁正常肝组织(78%),两组相比差异有显著性(P0.05)。Noxa蛋白表达与肝癌分化程度及TNM分期有关(P0.05)。Hep G2细胞中成功表达Noxa基因,转染后m RNA及蛋白表达随时间延长持续上升,差异有统计学意义(P0.05)。与其它各组相比,转染24、48、72 h后,其吸光度值逐渐升高(P0.05),流式细胞仪检测结果显示,其24 h、48 h、72 h凋亡率分别为(15.5±0.9)%、(24.6±0.8)%和(35.4±0.7)%,组间差异有统计学意义(P0.05)。结论 Noxa表达与肝癌分化程度及TNM分期密切相关,其高表达可抑制人肝癌细胞Hep G2的增殖并促进其凋亡。     

人参皂苷Rg3对人肝癌细胞增殖、迁移、黏附和凋亡的影响及其作用机制

目的研究人参皂苷Rg3对不同肝癌细胞增殖、迁移、黏附和调亡的影响及其作用机制。方法以人肝癌细胞系Hep G2、QGY细胞以及人成纤维细胞系HEL细胞为研究对象,运用MTT、细胞黏附、Transwell以及流式细胞仪观察Rg3对肝癌细胞的增殖、黏附、侵袭和转移以及细胞凋亡的影响;结合Western blot检测Rg3作用于Hep G2和QGY细胞后CD44、VEGF蛋白的表达。结果 Rg3能特异性抑制Hep G2和QGY肝癌细胞增殖、黏附、侵袭转移并显著诱导细胞凋亡(P0.05);Hep G2和QGY肝癌细胞暴露于Rg3后细胞中CD44和VEGF蛋白表达显著下调(P0.05)。结论Rg3能抑制人肝癌细胞增殖、黏附、侵袭转移并诱导细胞凋亡,其功能与CD44和VEGF蛋白表达密切相关。     

靶向敲低核蛋白1抑制HepG2肝癌细胞的增殖及迁移

目的观察应激相关核蛋白1(Nupr1)在不同肝癌细胞系中的表达,通过靶向敲低Hep G2肝癌细胞Nupr1,观察其对肝癌细胞增殖及迁移的影响。方法通过实时定量PCR检测BEL-7402、QSG-7703、SMMC-7721、Hep G2肝癌细胞中Nupr1的表达。采用RNA干扰技术敲低Hep G2细胞中Nupr1的表达,通过MTT法及集落形成实验比较细胞的增殖能力的变化,用流式细胞术进行细胞周期分析,利用TranswellTM实验观察Nupr1对细胞迁移能力的影响。结果 Hep G2人肝癌细胞中Nupr1的表达显著高于其他肝癌细胞系。靶向Nupr1的2个短发夹RNA(shRNA)均能有效敲低Hep G2肝癌细胞中Nupr1的表达,抑制效率分别为72.25%和84.25%。Nupr1表达降低能够抑制细胞增殖,导致细胞周期发生G1期阻滞。Nupr1敲低显著降低细胞的迁移能力及集落形成能力。Western blot结果显示Nupr-1表达降低能够上调细胞周期负性调控因子p21和p27的表达水平。结论靶向敲低Nupr1抑制Hep G2肝癌细胞的增殖及迁移。     

MicroRNA-1对肝癌细胞BAG4表达的影响

目的观察microRNA-1(miR-1)对肝癌细胞系Hep3B死亡结构域沉默子(BAG4)的表达及细胞凋亡的影响。方法 Western blot检测肝癌组织中BAG4蛋白表达情况;将过表达miR-1的质粒转染肝癌细胞Hep3B,分别用RT-qPCR及Western blot检测BAG4 mRNA和蛋白的表达,流式细胞仪检测细胞凋亡;生物信息学软件分析miR-1和BAG4 3’UTR作用关系。结果 BAG4蛋白在肝癌组织中的表达明显高于癌旁组织;Hep3B转染miR-1过表达质粒后,相较于阴性对照(NC)组,BAG4mRNA和蛋白水平的表达均有所降低;早期凋亡率明显增高。生物信息学分析提示BAG4是miR-1潜在靶基因。结论 miR-1可以诱导肝癌细胞Hep3B凋亡,其作用可能和抑制BAG4表达有关。     

microRNA与肝癌

肝癌是世界上最常见的恶性肿瘤之一,其发病率和病死率极高。microRNA(miRNA)是一类单链的非编码RNA,通常在转录后水平调控基因的表达。最近多项研究表明miRNA在肝癌中发挥着重要作用,已经成为肝癌诊断、治疗中的一个靶标。     

miRNAs与肝细胞癌

最近发现一类小分子物质--microRNAs在肝细胞癌中异常表达,其中部分已被证实与肝细胞癌的发生发展密切有关.有些microRNAs与肝细胞癌的分型有关,提示microRNAs在肝细胞癌的诊断和预后判断中有着潜在的作用;有些microRNAs被证实为肝细胞癌发生的独立危险因素,为临床早期筛查和风险评估提供了分子标记,也为个体化治疗提供了分子依据.     

Expression of both hepatocellular carcinoma and cholangiocarcinoma phenotypes in hepatocellular carcinoma and cholangiocarcinoma components in combined hepatocellular and cholangiocarcinoma

Expression of phenotype markers of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) in HCC and CC components of 20 combined hepatocellular and cholangiocarcinomas (CHCs) of the liver was investigated immunohistochemically. Both HCC and CC components of all CHCs expressed at least one of the CC phenotype markers [cytokeratin (CK)-7, CK-19, and carbohydrate (CA) 19-9]. HCC components in 90% of CHCs and CC components in 95% of CHCs expressed at least one of these CC phenotype markers in more than 40% of cancer cells. HCC components in all CHCs expressed at least one of the HCC phenotype markers [hepatocyte antigen (HA), α-fetoprotein (AFP), and canalicular carcinoembryonic antigen]. HCC components in 90% of CHCs and CC components in 75% of CHCs expressed HA, AFP, or both. HCC components in 75% of CHCs and CC components in 60% of CHCs expressed HA, AFP, or both in more than 10% of cancer cells. The present results show that both HCC and CC components of most of the CHCs expressed both HCC and CC phenotypes, supporting the hypothesis that CHC originates from a hepatic progenitor cell capable of differentiating into hepatocytes and cholangiocytes.     

Adult-type hepatocellular carcinoma in the center of a fibrolamellar hepatocellular carcinoma

A large hepatic tumor was detected in the noncirrhotic liver of a 27-year-old female patient. The tumor was radiologically characterized by a peripheral mass encircling a central ovoid tumor, and was resected by an extended right hemihepatectomy. Histologic examination revealed that the peripheral and major component of the tumor represented a fibrolamellar hepatocellular carcinoma, whereas the central, well-demarcated tumor was a less well-differentiated adult-type hepatocellular carcinoma completely encircled by the former. Cells of the peripheral tumor mass abundantly expressed cytokeratin-7, typically present in the fibrolamellar variant, whereas no cytokeratin-7 immunoreactivity was found in the central tumor. To our knowledge, this is the first reported case of a not admixed but clearly separated evolution of these 2 histologic patterns within the same tumor, and suggests that the 2 types of hepatocellular carcinoma may share a common pathogenic pathway.     

Pedunculated hepatocellular carcinoma

A case of this rare exception to the common gross types of hepatocellular carcinoma is reported. The main clinico-pathological findings of this variant are summarized in the light of a review of the relevant literature. Emphasis is placed on the usefulness of the latest diagnostic procedures including echography and CT scans for an increased accuracy of the preoperative clinical diagnosis.     

Fibrolamellar hepatocellular carcinoma

Fibrolamellar hepatocellular carcinoma (FLM) is a rare tumour that typically presents in young adults. It occurs in non-cirrhotic liver and is usually associated with normal serum alpha-fetoprotein. It is defined by a triad of morphological features: polygonal tumour cells with eosinophilic cytoplasm, prominent macronucleoli and lamellar fibrosis. A central scar can be present. The principal differential diagnosis is conventional hepatocellular carcinoma (HCC), especially the scirrhous variant. Acinar differentiation and focal mucin production are common and can be confused with adenocarcinoma. Focal neuroendocrine differentiation can occur and can be mistaken for neuroendocrine tumours. FLM also shows distinctive features at the molecular level; many of the commonly observed abnormalities in conventional HCC like p53 and β-catenin mutations are not observed in FLM. The extent of cytogenetic changes and CpG island methylation is low in FLM compared to conventional HCC. FLM is an aggressive neoplasm with 5-year survival of around 50%. Although the outcome in FLM has been considered more favourable compared to conventional HCC, this is likely to be related to absence of cirrhosis rather than unique morphological features of the tumour.     

Sarcoidosis-associated hepatocellular carcinoma

Sarcoidosis is a systemic granulomatous inflammation of unknown etiology, and seems to involve the liver parenchyma in most cases. However, sarcoidosis-associated hepatocellular carcinoma is rare. We report here a case in which a hepatocellular carcinoma occurred within the liver, which was probably involved as a result of systemic sarcoidosis. A 57-year-old Japanese man had been followed up for 2 years because of diabetic nephropathy and sarcoidosis. On admission for pneumonia, imaging studies revealed an unexpected hepatic tumor. Histology revealed a hepatocellular carcinoma accompanied by T-lymphocytic infiltration and marked granulomatous inflammation, which was surrounding some tumor nodules. The background liver parenchyma exhibited a moderate degree of fibrosis with granulomatous inflammation. The patient had no other apparent liver disease such as viral hepatitis, steatohepatitis, or primary biliary cirrhosis. Therefore, in the present case, sarcoidosis may be considered the probable background etiology for hepatocarcinogenesis.     

Pathology of hepatocellular carcinoma

Rather rapid and brief over-view of pathology of hepatocellular carcinoma has been made, where the connection between morphology and virology or biochemistry is stressed as the main focus of today's pathology. Thus, existence of some relation between hepatitis virus and hepatocellualr carcinoma seems to be definite for us. However, it is still very difficult to decide whether the relationship is a direct or indirect one. High incidence of hepatocellular carcinoma also in Budd-Chiari's cirrhosis and schistoma-induced cirrhosis seems to suggest existence of the high risk type of cirrhosis for hepatocellular carcinoma development, irrespective of the cause of cirrhosis itself, although HB antigen might be playing some role in these cases especially in the latter.     

Cytology of hepatocellular carcinoma

Twenty-five cases of cytologically and histologically confirmed hepatocellular carcinoma (HCC) were cytomorphologically analyzed using May-Grünwald-Giemsa (MGG)-stained aspiration smears and supplemented with special stains. The cell types were categorized as well differentiated (18 cases), vacuolated (four cases), giant cell (one case), and poorly differentiated (two cases). Glycogen staining was positive in 80% of the cases and hence served as a reliable parameter of diagnostic importance in HCC. Cytoplasmic hyaline bodies (14.6%) and bile pigment (17%), when present, were other important features supporting the diagnosis of HCC. Vacuolation of the cell cytoplasm (80%) was possibly related to glycogen accumulation. The cause of nuclear vacuolation (60%) and the significance of nuclear argyrophilia as markers of abnormal cell growth remain to be studied.