丙型肝炎病毒非结构蛋白5A对干扰素α-2b诱导的信号传导和转录激活因子1磷酸化及核转移的影响

目的探讨丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）对干扰素α-2b诱导的Janus激酶-信号传导和转录激活子（JAK—STAT）信号传导途径中STAT1磷酸化及核转移的影响。方法用表达HCVNS5A的质粒（pCNS5A）转染Huh7细胞，应用免疫细胞化学技术检测HCVNS5A的表达，用免疫荧光和Western blot方法检测HCVNS5A对干扰素α-2b诱导的STAT1磷酸化和核转移的影响。结果转染了pCNS5A的Huh7细胞质可见HCVNS5A蛋白的表达；以干扰素α-2b诱导30min后，STAT1磷酸化及核转移在转染了表达HCVNS5A的质粒组比转染空白载体pRC／CMV组及未转染组减少，而未转染组及转染空白载体pRC／CMV组间无明显差别。结论表达HCVNS5A的质粒pCNS5A成功转染至Huh7细胞；HCV NS5A减弱干扰素α-2b诱导的STAT1的磷酸化及核转移，提示NS5A影响干扰素α-2b的JAKSTAT信号传导途径可能是HCV干扰素抵抗的机制之一。     

高转移肝癌MHCC97细胞中Twist基因变化与Wnt信号传导通路的关系

目的研究高转移肝癌MHCC97细胞中Twist基因表达与Wnt信号通路的关系。方法取对数生长期MHCC97细胞分为五组,A、B组分别加入0.05 mol/L氯化锂分别培养12、24 h,C、D组予0.1 mol/L氯化锂分别培养12、24 h激活Wnt通路,E组不干预。采用RT-PCR和荧光定量PCR方法观察各组Twist mRNA表达情况。结果两种检测方法均显示Twist mRNA在A、B、C、D组表达均明显高于E组,P〈0.05;A、B组,C、D组,A、C组,B、D组间比较,P均〉0.05。结论MHCC97细胞中Wnt通路激活后Twist基因表达明显升高未激活,即Twist基因表达与Wnt信号传导通路激活关系密切,Wnt传导通路激活与氯化锂浓度及作用时间无明显相关性。     

JAK/STAT信号通路调控血管平滑肌细胞病理生理功能的分子机制

转录信号传导子及激活子(STATs)信号转导途径是当前心血管分子生物学领域的研究热点.血管平滑肌细胞的增殖和迁移是血管增殖性疾病发病的核心环节,对其与STAT信号通路关系的研究,将为揭示血管新生内膜形成、血管重塑等病理机制提供新的研究方向,本文就以此信号通路为靶点,探讨其在血管平滑肌细胞的分子病理生理学作用特点.     

JAK/STAT信号通路调控血管平滑肌细胞病理生理功能的分子机制

转录信号传导子及激活子(STATs)信号转导途径是当前心血管分子生物学领域的研究热点。血管平滑肌细胞的增殖和迁移是血管增殖性疾病发病的核心环节,对其与STAT信号通路关系的研究,将为揭示血管新生内膜形成、血管重塑等病理机制提供新的研究方向,本文就以此信号通路为靶点,探讨其在血管平滑肌细胞的分子病理生理学作用特点。     

不同转移潜能人肝癌细胞系转录因子活性差异分析

目的分析不同转移潜能人肝癌细胞系细胞核内转录因子活性的差异，筛选与肝癌转移相关的转录因子。方法应用转录因子活性芯片技术，在功能水平分析三种不同转移潜能人肝癌细胞系（Hep3B、MHCC97L和MHCC97H）细胞核内转录因子活性的差异，并用电泳迁移率变动分析和蛋白免疫印迹实验验证芯片结果。结果在345个候选的转录因子中，筛选出7个活性差异转录因子。随人肝癌细胞转移潜能的增高（Hep3B〈MHCC97L〈MHCC97H），活性上调的转录因子有5个，包括p53、缺氧诱导因子-1α（HIF-1α）、核因子κb、信号传导及转录活化因子3（Stat3）和Sp1；活性下调的转录因子有2个，包括Rb和Smad3。结论转录因子活性异常与肝癌转移密切相关，本实验筛选出的转录因子可能有助于揭示肝癌转移的分子机制，并寻找新的预测指标及干预治疗的靶点。     

lncRNA NBAT1通过MAPK通路影响肝癌细胞增殖和转移的机制

目的 探讨lncRNA神经母细胞瘤相关转录物(NBAT)1对肝癌细胞增殖和转移的影响及分子机制。方法 取43例肝癌组织及癌旁组织，用实时荧光定量聚合酶链反应(RT-qPCR)检测lncRNA NBAT1表达水平；将肝癌细胞MHCC97H分为pcDNA组(转染NBAT1过表达载体质粒的阴性对照)、pcDNA-NBAT1组(转染NBAT1过表达载体质粒)、SB203580组[10μmol/L丝裂原活化蛋白激酶(MAPK)通路抑制剂SB203580]、Con组(不进行任何处理);四甲基偶氮唑蓝(MTT)比色法检测细胞增殖活性；克隆形成实验检测细胞克隆形成数；划痕实验检测细胞划痕愈合率；Transwell检测侵袭细胞数；Western印迹检测蛋白表达。结果 肝癌组织中lncRNA NBAT1的表达水平显著低于癌旁组织(P<0.05)。过表达lncRNA NBAT1后，肝癌细胞MHCC97H活性降低，肝癌细胞克隆形成数减少，划痕愈合率降低，侵袭肝癌细胞数减少，p-细胞外信号调节激酶(ERK)、p-p38和磷酸化c-Jun NH2-末端激酶(p-JNK)蛋白表达水平降低，差异均有统计学意义(...     

ADAMTS9基因联合去甲氧基姜黄素对肝癌细胞侵袭迁移及PI3K/PTEN/AKT信号的影响

目的探讨过表达带有血小板凝血酶敏感蛋白样模体的解联蛋白金属蛋白酶(ADAMTS)9基因或去甲氧基姜黄素(DMC)单独或联合对肝癌细胞侵袭迁移及磷脂酰肌醇3-激酶/第10号染色体同源缺失性磷酸酶-张力蛋白/蛋白激酶B(PI3K/PTEN/AKT)信号的影响。方法 Western印迹检测肝癌(HepG2)、MHCC 97-L和MHCC97-H细胞中ADAMTS9的蛋白表达。将MHCC97-H细胞分为对照组、pcDNA3.1-ADAMTS9组、DMC组和pcDNA3.1-ADAMTS9+DMC组4个处理组,其中pcDNA3.1-ADAMTS9的转染参照LipofectamineTM 2000转染说明,收集处理48 h的细胞,CCK8法、Transwell小室分别检测细胞活力及侵袭和迁移能力。Western印迹检测ADAMTS9、增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-2、MMP-9、PI3K、PTEN和p-AKT的蛋白表达。结果肝癌HepG2、MHCC97-L和MHCC97-H细胞中ADAMTS9表达均有不同程度的降低,与正常肝细胞HL-7702比较,差异具有统计学意义(P0.05)。pcDNA3.1-ADAMTS9组DAMTS9的蛋白表达显著高于对照组(P0.05)。pcDNA3.1-ADAMTS9组和DMC组细胞活力、侵袭和迁移能力及PCNA、MMP-2、MMP-9、PI3K和p-AKT的蛋白表达均显著低于对照组,PTEN蛋白表达显著高于对照组(P0.05),而pcDNA3.1-ADAMTS9+DMC组细胞活力、侵袭和迁移能力及PCNA、MMP-2、MMP-9、PI3K和p-AKT的蛋白表达均显著低于pcDNA3.1-ADAMTS9组和DMC组,PTEN蛋白表达显著高于pcDNA3.1-ADAMTS9组和DMC组(P0.05)。结论过表达ADAMTS9及DMC均可抑制肝癌细胞活力、侵袭和迁移能力,两者联用对细胞抑制作用更明显,机制可能与下调PCNA、MMP-2和MMP-9及PI3K/PTEN/AKT信号通路有关。     

PGC-1α活性调节的信号通路

很多信号通路参与了过氧化物增殖子激活-受体因子γ辅激活因子(PGC-1α)的表达和活性调节,如Ca2+依赖通路、能量依赖调节、激素、细胞周期蛋白依赖激酶(CDKs)等代谢刺激及后翻译修饰。其中,钙将神经肌肉的活动通过钙神经素等调控通路传输到相关基因的转录变化,发挥着第二信使的作用;腺苷酸-活化蛋白激酶(AMPK)等能量物质化学诱导PGC-1α;甲状腺素能够通过直接或间接的通路控制线粒体生物发生;CDKs参与细胞循环或转录控制;脱乙酰酶(SIRT1)和AMPK等翻译后修饰增加了PGC-1α活性。PGC-1α在协调线粒体生物发生和代谢基因信号网路中发挥了中枢作用。     

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

AIM： To investigate the effects of Chrysanthemum indicum extract （CIE） on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma （HCC） MHCC97H cell line. METHODS： Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide （PI）, mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS： CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION： CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.     

c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma

c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. Conclusion: c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.     

Contributions of lung tissue extracts to invasion and migration of human hepatocellular carcinoma cells with various metastatic potentials

Purpose The MHCC97 cell line contains two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potentials, for which the pulmonary metastatic rate was 100% vs 40% between the two human hepatocellular carcinoma (HCC) clones. In an effort to elucidate the mechanism of organ-specific metastasis, we studied the effect of lung extracts from C57BL/6 mice on migration and invasion using the MHCC97 cell line.Methods Determination of migration and invasion induced by lung extracts to MHCC97 cell lines was examined by chemoinvasion assay. The organization of cytoskeleton was tested using filamentous actin (F-actin) polymerization assay and flow cytometry. The activity of matrix metalloproteinase (MMPs) was analyzed by zymogram. Fluorescence double staining was employed for MMPs and F-actin colocalization in MHCC97-H cells induced by lung extracts.Results The number of cells in response to extracts of lung, liver, kidney, and spleen in MHCC97-H cells was 64±10, 6±2, 22±4, and 3±1, respectively. Of the extracts, lung extracts showed significant differences to promote the migratory and invasive ability for MHCC97-H cells(p<0.001). The number of cells in response to lung extracts was threefold higher in MHCC97-H than that of cells in MHCC97-L (p<0.001). Confocal laser scan microscope of MHCC97-H cells and MHCC97-L cells stimulated with lung extracts revealed pseudopodia formation around the cell front at the indicated time point. With the time increasing, the pseudopodia formation became increasingly more obvious and distinct. Compared with unstimulated cells, analysis of FACS showed a transient 1.9-fold and 1.7-fold increase in F-actin within 30 s in MHCC97-H cells and MHCC97-L cells, respectively. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension with lung extracts revealed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. After the cells were incubated with lung extracts, not only expressions of active and latent form of MMP-9 were upregulated, but that of latent form of MMP-2 was increased in the MHCC97-H cells and MHCC97-L cells. The levels of latent and active form of MMP-9(18.8±1.2, 100.1±1.1), and latent form of MMP-2(22.4±1.3) were much higher in MHCC97-H cells than those of MHCC97-L cells, which were 7.8±0.3, 40.8±2.2, and 8.2±0.4, respectively. MMP-9 was mainly localized perinuclear pool when the MHCC97-H cells were incubated with serum-free medium. After the cells were stimulated with lung extracts, MMP-9 were expressed and colocalized with F-actin at the front of extending pseudopodia.Conclusions Our results indicate that the expression of matrix metalloproteinase and the pseudopodia formation in MHCC97-H cells may correlate to the metastatic potential; thus, the host environment may contribute to the preferential metastasis of HCC cells to the lung depending on the high level of MMP-9 and migratory ability.     

C-met-siRNA在肝癌细胞株MHCC97-H侵袭和转移中的作用

目的探讨c—met-siRNA对肝细胞癌生长和侵袭的影响。方法采用脂质体法将pSuppressorRetro／c—met—siRNA导入包装细胞Phoenix A中获得含c-met-siRNA的逆转录病毒颗粒，进一步感染靶细胞MHCC97-H，Western blot检测c—Met的表达，通过生长曲线、运动试验及侵袭试验比较转染前后细胞的生长、运动及侵袭能力。结果肝细胞癌MHCC97-H细胞中c—Met的表达显著降低；细胞的生长、运动及侵袭均受到明显抑制。结论c—met—siRNA能抑制肝细胞癌细胞MHCC97-H的生长、运动及侵袭，这对肝细胞癌的生长、转移具有重要的临床意义。     

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM To establish clone cells with different metastaticpotential for the study of metastesis-related mechanisms.METHODS Cloning procedure was performed on parentalhepetocellular carcinoma (HCC) cell line MHCC97,andbiological characteristics of the target clones selected byin vivo screening were studied.RESULTS Two clones with high (MHCC97-H) and low(MHCC97-L) metastatic potential were isolated from theparent cell line.Compared with MHCC97-L,MHCC97-H hadsmaller cell size (average cell diameter 43μm vs 50 μm)and faster in vitro and in vivo growth rate (tumor celldoubling time was 34.2 h vs 60.0 h).The main ranges ofchromosomes were 55-58 in MHCC97-H and 57-62 inMHCC97-L.Boyden chamber in vitro invasion assaydemonstrated that the number of penetrating cells throughthe artificial basement membrane was (37.5±11.0) cells/field for MHCC97-H vs (17.7±6.3)/field for MHCC97-L.The proportions of cells In G0-G1 phase,S phase,andG2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65,0.28/0.25 and 0.16/0.10,respectively,as measured byflow cytometry.The serum AFP levels in nude mice 5 wkafter orthotoplc implantation of tumor tissue were (246±66)μg.L~(-1) for MHCC97-H and (91±66)μg.L~(-1) for MHCC97-L.The pulmonary metastatic rate was 100% (10/10) vs40% (4/10).CONCLUSION Two clones of the same geneticbackground but with different biological behaviors wereestablished,which could be valuable models forInvestigation on HCC metastasis.     

Calcitriol inhibits the growth of MHCC97 heptocellular cell lines by down-modulating c-met and ERK expressions.

BACKGROUND: Previous studies have demonstrated that Calcitriol or its analogs has an anti-tumour activity. This study was designed to determine the effect and mechanism of Calcitriol on MHCC-97 heptocellular cell lines. METHODS: MHCC97 cell lines were treated with Calcitriol of 10(-6) approximately 10(-9) M concentration and with Calcitriol in lipiodol ultra-fluid (LUF) respectively. The conditions of cell proliferation were analyzed by MTT method. The cell apoptosis and cycle were analyzed by using a flow cytometer. Hepatocyte growth factor (HGF) concentration in the cell supernatant was measured by using ELISA method. C-met and vitamin D receptor (VDR) mRNA in cells were determined by using RT-PCR method. VDR and ERK(1/2) proteins were determined by using Western Blotting method. RESULTS: Calcitriol inhibited the proliferation of MHCC97 cell lines with an accumulation of cells in the G(0)/G(1) phase and reduction of cells in S phase. Calcitriol dissolved in LUF resulted in a better and longer inhibitive effect on the cell lines than Calcitriol alone. MHCC97 cell lines secreted HGF and expressed c-met mRNA and ERK(1/2) proteins abundantly. Calcitriol remarkably inhibited the expressions of c-met mRNA and ERK(1/2) proteins. CONCLUSION: Calcitriol inhibits the growth of MHCC-97 heptocellular cell lines by down modulating c-met and ERK expressions.