Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.     

肝癌转移相关的核心岩藻糖基化 蛋白质表达谱的研究

通过比较研究不同转移潜能肝癌细胞系中核心岩藻糖基化蛋白质表达谱的差别,筛查与转移相关的重要糖蛋白 . 用 SDS- 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 、双向电泳 (2-DE) 和凝集素印迹技术联合基质辅助激光解吸飞行时间串联质谱 (MALDI-TOF-MS/MS) 分析,建立 3 种不同转移潜能人肝癌细胞系 Hep3B 、 MHCC97L 和 MHCC97H 的核心岩藻糖基化蛋白质表达图谱 . 比较研究发现,不同转移潜能肝癌细胞呈现不同的 SDS-PAGE/LCA 凝集素印迹图谱, MHCC-97H 和 MHCC-97L 在 35~45 ku 和 45~60 ku 间出现了 Hep3B 未见的条带 . 在核心岩藻糖基化蛋白质表达图谱中, Hep3B、 MHCC97L 和 MHCC97H 分别平均检测到 (55±7) 个蛋白质点 (n=3), (60±6) 个蛋白质点 (n=3), (61±4) 个蛋白质点 (n=3);以各自双向电泳图谱为参考胶,Hep3B、 MHCC97L 和 MHCC97H 分别与其匹配的平均匹配点数为 (25±3) 个 (n=3), (30±4) 个 (n=3), (28±3) 个 (n=3). 该图谱中,与 Hep3B 相比, MHCC97L 有 13 个点未匹配,其中 9 个点为 Hep3B( － )/MHCC97L(+); MHCC97H 有 9 个点未匹配,其中 6 个点为 Hep3B( － )/MHCC97H(+), MALDI-TOF-MS/MS 可鉴定出 Annexin1、 Keratin 8 等 12 种差异蛋白质 . 这些结果证实了不同转移潜能的肝癌细胞有明显的核心岩藻基化糖蛋白差异性表达 . 提示肝癌转移可能与这些差异糖蛋白及其核心岩藻糖基化有关 .     

Derepression of c‐Fos caused by MicroRNA‐139 down‐regulation contributes to the metastasis of human hepatocellular carcinoma

This study investigates whether the anti‐metastasis effect of microRNA‐139 (miR‐139) on hepatocellular carcinoma (HCC) is mediated through regulating c‐fos expression. The expression levels of miR‐139 and c‐fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR‐139 mimics was transfected into MHCC97H cells to overexpress miR‐139, and miR‐139 inhibitor was transfected into MHCC97L cells to down‐express miR‐139. The effect of overexpression or down‐expression of miR‐139 on c‐fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR‐139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR‐139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c‐Fos was significantly up‐regulated in MHCC97H. The overexpression of miR‐139 significantly inhibited the expression of c‐fos in MHCC97H cells, and the down‐expression of miR‐139 significantly promoted the expression of c‐fos in MHCC97L cells. miR‐139 suppressed the luciferase activity of the pGL‐FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c‐fos or overexpression of miR‐139 in MHCC97H cells reduced cell migration, whereas overexpression of c‐fos or depletion of miR‐139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR‐139 expression is down‐regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c‐Fos caused by miR‐139 down‐regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.     

自分泌运动因子在肝细胞癌侵袭及转移中的作用

目的:探讨自分泌运动因子(AMF)在人肝细胞癌侵袭和转移中的作用。方法:人肝细胞系LO2和人肝细胞癌细胞株MHCC97-H作为实验材料,检测二者AMF的表达水平;设计并合成针对AMF基因序列的双链小干扰RNA转染高转移性人肝癌细胞株MHCC97-H,Western blot检测AMF基因的蛋白的表达水平;通过MTT实验检测转染后细胞的增殖力;通过体外Transwell小室对比沉默AMF基因前后的肝癌细胞的迁移力和侵袭力;最后用细胞悬液皮下接种小鼠,观察沉默AMF基因前后肝细胞的成瘤能力。结果:AMF在MHCC97-H的表达量较高;将双链小干扰RNA转入MHCC97-H后,AMF的表达显著降低(P0.05);沉默AMF基因序列后,MHCC97-H的增殖力、迁移力和侵袭力均有明显下降(P0.05);用细胞悬液皮下接种小鼠沉默AMF基因的MHCC97-H形成的肿瘤体积小于对照组(P0.05)。结论:AMF基因可调节肝癌细胞的迁移和侵袭。     

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC.

Methodology/Principal Findings

A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor.

Conclusions/Significance

Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Bub1基因对人肝癌MHCC97-H细胞增殖、周期和凋亡的影响

目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。     

Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.     

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.     

过表达微小RNA miR-125b抑制肝癌细胞上皮-间质转换及促进细胞凋亡

微小RNA-125b（miR-125b）在许多恶性肿瘤的增殖、分化和凋亡等过程中具有很重要的作用,但miR-125b是否涉及肝癌的上皮 间质转换过程（EMT）还有待进一步研究。本研究通过构建过表达miR-125b的肝癌稳转细胞株,初步检测miR-125b对于肝癌的EMT过程和相关的TGF-β信号通路的影响,以及对于肝癌细胞凋亡的影响。以慢病毒载体pHRS-1cla EGFP 构建过表达miR-125b的载体质粒(pHRS-1cla-miR125b-CMV-EGFP),并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定,鉴定正确后,在293T细胞中进行慢病毒包装,浓缩病毒后,对MHCC97-H进行慢病毒感染并采用流式分选GFP阳性的细胞。实时定量PCR检测表明肝癌细胞稳转株MHCC97-H-PHRS-miR-125b-EGFP的miR-125b表达量是空载体转染组的6倍。Western印迹检测发现,与空载体对照组相比,MHCC97-H-PHRS-miR-125b-EGFP细胞中间质细胞标志α-SMA表达显著下调,上皮细胞标志E-cadherin表达显著上调,同样的,用Western印迹检测也发现MHCC97-H-PHRS-miR-125b-EGFP细胞中TGF-β信号通路关键下游分子Smad2和Smad4的表达显著下调,细胞凋亡检测结果表明,与对照组相比,过表达miR-125b的稳转株凋亡率增加到19.66%,加入TGF-β1后,过表达miR-125b的稳转株凋亡率进一步增加到74.7%。同样的,在体内治疗实验中,我们采用商品化的体内核酸转染试剂,在皮下肿瘤组织中过表达miR-125b mimics,结果表明miR-125b的过表达与肿瘤组织的凋亡成正相关性（r=0.83463,P < 0.01）,且免疫组化结果也表明,miR-125b过表达后,E-cadherin表达显著上调,α-SMA及Smad2和Smad4的表达显著下调。上述结果表明,我们成功构建了过表达miR-125b的肝癌细胞稳转株,并成功建立了肿瘤组织中过表达miR-125b mimics的动物模型,在体内外均观察到过表达miR-125b后对肝癌细胞EMT过程的抑制作用和对细胞凋亡的促进作用。相关研究结果加深了我们对miR-125b在肝癌中抑制肝癌发展作用机制的理解,及其作为潜在的治疗肝癌的新靶点的重要性。     

丹皮酚对肝癌MHCC97-H细胞PTEN、AKT表达的影响

目的:探讨丹皮酚(Paeonol,Pae)在体外对人肝癌MHCC97-H细胞PTEN、AKT表达的影响。方法:体外培养人肝癌MHCC97-H细胞,MTT法检测丹皮酚对MHCC97-H细胞的增殖抑制作用,RT-PCR法检测PTEN、Akt1、Akt2mRNA表达,West- ern Blot法检测PTEN、p-AKT蛋白的表达。结果:丹皮酚呈时间剂量依赖性抑制人肝癌MHCC97-H细胞的增殖;肝癌MHCC97-H细胞低表达PTEN,高表达AKT,丹皮酚能显著上调MHCC97-H细胞PTEN表达,下调AKT表达。结论:丹皮酚可上调抑癌基因PTEN的表达,下调致癌基因AKT的表达,抑制MHCC97-H细胞的增殖。     

Wogonin inhibits cell cycle progression by activating the glycogen synthase kinase-3 beta in hepatocellular carcinoma

BackgroundWogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.PurposeThe aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.MethodsHerein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.ResultsOur results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.ConclusionIn summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.     

Identification of metastasis candidate proteins among HCC cell lines by comparative proteome and biological function analysis of S100A4 in metastasis in vitro

Widespread metastasis of hepatocellular carcinoma (HCC) was a complex cascade of events, which is still beyond full appreciation. Screening key proteins, which play a critical role in metastasis, using high-throughput proteomics approach help discover valuable biomarkers and elucidate the mechanism of metastasis. This study was to find out some metastasis candidate proteins among HCC cell lines with various metastatic potential by comparative proteomics, and then further validate the biological function of these proteins in metastasis in vitro. The protein profiles of metastatic HCC cell lines (MHCC97H and MHCC97L) displayed obvious differences compared with nonmetastatic ones (Hep3B). Twenty-six metastasis candidate proteins, which were identified by on-line LC-ESI-MS/MS, such as S100 calcium-binding protein A4 (S100A4), annexin 1, etc., might have much application in diagnostic procedures and prognosis evaluation. S100A4, as a leading different metastasis candidate protein, which overexpressed only in the metastatic cells, was selected for further investigation. A series of assays related to invasion and metastasis in vitro, including cell motility, invasion, and matrix metalloproteinases (MMPs) secretion, were performed in MHCC97H/antisense recombinant plasmid to S100A4 (pcDNA3.1(+) AS S100A4) and the mock controls. All the data in the present study suggested that S100A4 might contribute to HCC invasion and metastasis through two paths of matrix metalloproteinase (MMP9) secretion regulation and strengthened motility and invasion properties.     

miR-217 inhibits invasion of hepatocellular carcinoma cells through direct suppression of E2F3

The poor prognosis of hepatocellular carcinoma (HCC) is mainly due to the development of invasion and metastasis. Recent data strongly suggests the important role of miRNAs in cancer progression, including invasion and metastasis. Here, we found miR-217 expression was much lower in highly invasive MHCC-97H HCC cells and metastatic HCC tissues. Restored miR-217 expression with miR-217 mimics inhibited invasion of MHCC-97H cells. Inversely, miR-217 inhibition enhanced the invasive ability of Huh7 and MHCC-97L cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated E2F3 was a novel direct target of miR-217. Moreover, E2F3 protein level was positively associated with HCC metastasis and functional analysis confirmed the positive role of E2F3 in HCC cell invasion. Our findings suggest miR-217 function as a potential tumor suppressor in HCC progression and miR-217-E2F3 axis may be a novel candidate for developing rational therapeutic strategies.     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.