eEF1A1 Overexpression Enhances Tumor Progression and Indicates Poor Prognosis in Hepatocellular Carcinoma

Liver is a major contributor of protein production physiologically. The aberrant state of protein synthesis leads to tumor progression. Eukaryotic elongation factor 1 alpha 1 (eEF1A1) is a major member of the eukaryotic elongation factor family that regulates protein synthesis. Although eEF1A1 plays an essential role in controlling the cell fate, its clinical significance in tumor development and progression has not been reported. Here, we aimed to uncover the expression and prognostic significance of eEF1A1 in hepatocellular carcinoma (HCC). Our data indicated that eEF1A1 expression was elevated in HCC cell lines and clinical samples at both the mRNA and protein levels. Immunohistochemistry revealed that eEF1A1 expression was upregulated in HCC samples compared with corresponding non-tumorous tissues. In 50 HCC cases with portal vein embolus, higher eEF1A1 immunoreactivity was detected in tumor metastases compared with the primary lesions. Kaplan–Meier analysis indicated that increased eEF1A1 expression was closely associated with unfavorable post-surgical overall and disease-free survival in 453 HCC patients. Moreover, multivariate analysis indicated eEF1A1 as an independent predictor for overall and disease-free survival. Collectively, our study suggests eEF1A1 as a novel prognostic biomarker and potential therapeutic target for HCC patients.     

Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels

Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6.     

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

牦牛B细胞淋巴瘤2相关蛋白A1的原核表达及体外活性

为研究牦牛(Bos grunniens) B细胞淋巴瘤2相关蛋白A1(B cell lymphoma 2 related protein A1,BCL2A1)的原核表达及体外活性。采用原核表达载体构建、细胞划痕实验、CCK-8法、透射电镜和实时荧光定量PCR等方法。结果显示,成功构建pET-32a-BCL2A1重组质粒,表达获得约33kDa的BCL2A1。终浓度为0.02μg/mL、0.2μg/mL、2.0μg/mL的BCL2A1均能使HepG2细胞活性显著降低(P < 0.05),并在一定程度上抑制细胞的迁移。2.0μg/mL BCL2A1导致HepG2细胞核固缩,胞质中高密度物质聚集,溶酶体吞噬形成致密的凋亡小体等。此外,细胞凋亡相关基因CASP9的mRNA水平在2.0 μg/mL组中显著上调(P < 0.05),CASP8的mRNA水平在0.2 μg/mL和2.0 μg/mL组中显著上调(P < 0.05),而CASP3和Cyt c的mRNA水平在3个浓度处理组均显著上调(P < 0.05)。这提示BCL2A1可能通过凋亡途径影响HepG2细胞活性,为进一步探索牦牛BCL2A1基因功能积累资料。     

A comparison of the antitumor effects of interferon-alpha and beta on human hepatocellular carcinoma cell lines

The antiviral, antiproliferative and immunomodulatory effects of type I interferons (IFNs) are well documented, however, few studies have been published concerning differences in the antitumor effects of IFN-alpha and beta. In the present study, differences in antitumor effect, including the antiproliferative effect, cell cycle change, apoptosis, and the IFN-stimulated gene (ISG) were examined by flow cytometry between IFN-alpha and beta on three human hepatocellular carcinoma (HCC) cell lines (HepG2, Huh7 and JHH4). The antiproliferative effect of both IFNs on the HCC cell lines was time- and dose-dependent, and IFN-beta was significantly stronger than IFN-alpha. The cell cycle effect by both IFNs was an S-phase accumulation, with IFN-beta having a tendency to increase the S-phase ratio more strongly than IFN-alpha, especially in Huh7. Apoptosis marker expression, Fas antigen and intracellular active caspase-3, was increased after the addition of IFNs, especially of IFN-beta. The expression of human leukocyte antigen-class I molecules, ISG-encoded protein, was increased after the addition of IFNs, especially of IFN-beta. These data suggest that IFN-beta has a greater antitumor effect than IFN-alpha on HCC of a very early stage in patients with chronic hepatitis C.     

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.     

Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress

eEF1A-1 and eEF1A-2 are two isoforms of translation elongation factor eEF1A. In adult mammalian tissues, isoform eEF1A-1 is present in all tissues except neurons, cardiomyocytes, and myotubes, where its isoform, eEF1A-2, is the only form expressed. Both forms of eEF1A have been characterized to function in the protein elongation step of translation, and eEF1A-1 is shown to possess additional non-canonical roles in actin binding/bundling, microtubule bundling/severing, and cellular transformation processes. To study whether eEF1A-2 has similar non-canonical functions, we carried out a yeast two-hybrid screening using a full sequence of mouse eEF1A-2 as bait. A total of 78 hits, representing 23 proteins, were identified and validated to be true positives. We have focused on the protein with the highest frequency of hits, peroxiredoxin I (Prdx-I), for in-depth study of its functional implication for eEF1A-2. Here we show that Prdx-I coimmunoprecipitates with eEF1A-2 from extracts of both cultured cells and mouse tissues expressing this protein, but it does not do so with its isoform, eEF1A-1, even though the latter is abundantly present. We also report that an eEF1A-2 and Prdx-I double transfectant increases resistance to peroxide-induced cell death as high as 1 mM peroxide treatment, significantly higher than do single transfectants with either gene alone; this protection is correlated with reduced activation of caspases 3 and 8, and with increased expression of pro-survival factor Akt. Thus, our results suggest that eEF1A-2 interacts with Prdx-I to functionally provide cells with extraordinary resistance to oxidative stress-induced cell death.     

FAM225A promotes sorafenib resistance in hepatocarcinoma cells through modulating miR-130a-5p–CCNG1 interaction network

Chemotherapy resistance is still a key hurdle in current hepatocellular carcinoma (HCC) treatment. Therefore, clarifying the molecular mechanisms contributing to this acquired resistance is urgent for the effective treatment of liver cancer. In this research, we observed that lncRNA FAM225A expression is dramatically up-regulated not only in HCC tissues and cell lines but also in sorafenib-resistant HepG2/SOR cells. Moreover, FAM225A knockdown significantly weakened HepG2/SOR cells resistance to sorafenib treatment by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Similar results were obtained from the tumor xenograft model in mice. Further mechanistic researches revealed that the direct interaction between FAM225A and miR-130a-5p, while miR-130a-5p negatively modulated Cyclin G1 (CCNG1) expression by targeting 3′UTR of CCNG1. MiR-130a-5p inhibition or CCNG1 overexpression could partially offset FAM225A knockdown-induced increased viability of HepG2/SOR cells in response to sorafenib challenge. Collectively, our findings provide evidence that FAM225A/miR-130a-5p/CCNG1 interaction network regulates the resistance of HCC cells to sorafenib treatment and could supply a possible strategy for restoring sorafenib sensitivity in HCC therapy.     

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.     

Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

Anion exchanger (AE) 2, belonging to the chloride–bicarbonate transporter family, has been reported to involve cell survival for hepatocellular carcinoma (HCC) cells. Our previous findings showed that AE2 gene was highly expressed in a poorly differentiated HCC cell line, HA22T/VGH. Additionally, treatment with 4,4′-diisothiocyanatostilbene-2,20-disulfonic acid (DIDS), an AE-specific inhibitor, significantly inhibited cell proliferation and induced cell apoptosis for the HA22T/VGH. To further investigate the biological functions of AE2 in human HCC, suppression of AE2 expression by the antisense oligonucleotide-AE2 (AS-AE2) was performed, and the cell viability, cell cycle regulation, and cell apoptosis for HCC cell lines were monitored. The results showed that AS-AE2 treatment could efficiently suppress the mRNA expression of AE2 for various differentiated HCC cells, including HA22T/VGH, SK-Hep-1, PLC/PRF/5, Hep3B, and HepG2. Moreover, AS-AE2 treatment significantly reduced cell viability, arrested cell cycle at sub-G1 phase, and induced cell apoptosis for the poorly differentiated HA22T/VGH, but not for other moderately or well-differentiated HCC cell lines. The findings indicated that AE2 may play an important role in the progression of HCC cells, and provide a new strategy for the development of therapeutic treatment against human HCC.     

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.     

Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines

Recent evidence suggests that reactive oxygen species (ROS) play an important role in the pathogenesis of various illnesses, and the ROS and antioxidant enzymes are highly associated with cell differentiation and diseases. In this study, we tested the hypothesis that specific antioxidant enzymes are differentially expressed in hepatocellular carcinoma (HCC) cell lines with various degrees of differentiation. We compared the expression of several antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GRx), and glutathione peroxidase (GPx) in five HCC cell lines with well (Hep G2 and Hep 3B) or poor (HA22T/VGH, HA55T/VGH, and SK-Hep-1) differentiation. Our results showed that both well-differentiated HCC cell lines expressed extremely higher CAT and GRx enzyme activities than all three poorly differentiated ones. Moreover, the protein and mRNA levels of CAT were much higher in two well-differentiated HCC cell lines than in all three poorly differentiated ones. Both well-differentiated HCC cell lines also showed a higher protein or mRNA expression of Cu/ZnSOD and MnSOD than three poorly differentiated ones. Our results demonstrate that specific antioxidant enzymes (especially, CAT and GRx) are differentially expressed in HCC cell lines with well or poor differentiation. These findings suggest that CAT and GRx are two potential differentiation markers for HCC.     

Mcl-1在胆盐（GCDA）诱导的肝癌细胞耐药中的作用

目的：探讨抗凋亡蛋白Mcl-1在GCDA诱导的肝癌细胞耐药中的作用及其机制。方法：培养3种肝癌细胞系,用免疫荧光法和Western blot技术检测Mcl-1的表达;GCDA±CYC处理HepG2细胞,采用Western blot技术检测Mcl-1的半衰期变化;用抗癌药物Irinotecan与GCDA对HepG2细胞进行处理,采用MTT法和Western blot技术分别检测细胞增殖抑制率和Mcl-1的表达变化;用RNA干扰技术下调Mcl-1,检测化疗药物对HepG2细胞的敏感性。结果：Mcl-1在肝癌细胞中广泛表达;GCDA能延长Mcl-1的半衰期至6h以上,并明显减弱化疗药物对抗凋亡蛋白Mcl-1的抑制作用,降低癌细胞的药物敏感性;RNA干扰下调Mcl-1能增加癌细胞的药物敏感性。结论：胆盐（GCDA）能诱导HepG2细胞产生耐药性,其作用机制可能是通过延长Mcl-1半衰期增加其蛋白稳定性和抗凋亡作用来促使肝癌细胞抗药的。     

The expression of tumor suppressor gene Cyld is upregulated by histone deacetylace inhibitors in human hepatocellular carcinoma cell lines

CYLD is a deubiquitinating enzyme that exerts a tumor suppressive function. Its downregulation or inactivation has been associated with the development of several types of malignancies including hepatocellular carcinoma (HCC). HCC cells display significantly lower Cyld expression compared to primary human hepatocytes, and Cyld downregulation can contribute to apoptotic resistance of HCC cells. Little is known about the mechanism of Cyld downregulation in human HCC cells. In the present study we explored the possible regulation of Cyld expression by histone deacetylases (HDACs) in human HCC cell lines. We demonstrated that the HDAC inhibitors suberoylanilide hydroxamic acid, sodium butyrate, and trichostatin A induced the upregulation of both mRNA and protein levels of CYLD in two different HCC cell lines, HepG2 and Huh7. Our results demonstrate the involvement of HDACs in the downregulation of Cyld expression in HCC cells and support and may improve the use of HDAC inhibitors for the treatment for HCC.