Six long noncoding RNAs as potentially biomarkers involved in competitive endogenous RNA of hepatocellular carcinoma.

To investigate lncRNAs acting as competing endogenous RNAs (ceRNAs) involved in oncogenesis and progression of HCC. Different expressed lncRNAs, microRNAs, and mRNAs (DElncRNAs, DEmiRNAs, DEmRNAs), downloaded from The Cancer Genome Atlas (TCGA) database, were identified by edgeR package. CeRNA network was constructed based on miRcode, TargetScan, and miRTarBase. Target DEmRNAs were annotated by KEGG pathway and GO analysis. Negatively correlated lncRNA-miRNA pairs were analyzed by Pearson correlation coefficient, simultaneously, overall survival (OS) were evaluated. The expression of these lncRNAs were examined in HCC cell lines and tissues through qRT-PCR. 1070 DElncRNAs, 147 DEmiRNAs and 1993 DEmRNAs were acquired. CeRNA network was successfully established, including 27 lncRNAs, 5 miRNAs, and 30 mRNAs significantly correlated with OS. The DEmRNAs were significantly enriched in "Cell Cycle" and "pathways in cancer". Six lncRNAs and 2 miRNAs were negatively correlated. These lncRNAs were validated by qRT-PCR. These observations will provide a novel perspective to elucidate HCC pathogenesis.

Detection of Hepatocellular Carcinoma Biomarker Golgi Protein 73 Based on Magnetic Enzyme-Linked Immunoassay.

The magnetic enzyme-linked immunoassay (MEIA) based on magnetic nanoparticles as the solid phase was reported in this work. Magnetic nanoparticles (MNPs) have represented perfectly suitable materials for a variety of biomedical and biotechnological applications. Therefore, we used MEIA based on magnetic nanoparticles to provide a screening method for fast analysis of serum Golgi protein 73 (GP73) in hepatocellular carcinoma (HCC) and healthy subjects and comparison was made with the enzyme-linked immunosorbent assay (ELISA) method. Several relevant conditions, including the concentration of anti-GP73 monoclonal antibody and HRP-anti-human GP73 monoclonal antibody, amount of immunomagnetic beads, and the incubation time, were determined and optimized. Finally, the MEIA was successfully established and validated by 79 HCC and 64 healthy subjects. The results showed this method achieved a detection limit of 0.78 ng/mL, which was more sensitive than ELISA. Furthermore, the sensitivity and specificity of the MEIA were 78.43% and 91.47%, respectively, which were higher than ELISA. The MEIA based on MNPs proved to be simple, sensitive, specific and time-saving, therefore holds great potential for development of a commercial kit in the future.

Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth.

Frizzled-7 protein plays a significant role in the formation of several malignant tumors. Up regulation of the Frizzled-7 in cancer cell lines is associated with nuclear accumulation of wild-type ß-catenin from the Wnt/ß-catenin pathway which is frequently activated in tumors. To analyze activity of the Frizzled-7 promoter in tumor cells, we constructed two recombinant plasmid vectors in which the Frizzled-7 promoter was used to drive the expression of green fluorescent protein (GFP) and Shiga-like toxin I (Stx1) (pFZD7-GFP/Stx1) genes. The Frizzled-7 protein was found to be expressed in the cancer cell lines but not in the normal cell lines. The GFP expression was restricted to the cancer cell lines and xenografts in the BALB/C mice but not to normal cell lines. Moreover, cell proliferation and tumor growth decreased significantly after transfection with the pFZD7-Stx1. Results from this study will help determine a highly effective strategy for gene therapy of tumors.

Preparation and characterization of anti-GP73 monoclonal antibodies and development of double-antibody sandwich ELISA.

BACKGROUND: Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variables representing tumor growth and invasiveness. The aim of this study was to prepare a pair of monoclonal antibodys (mAbs) against GP73 and develop a newly designed double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis of HCC. MATERIALS AND METHODS: Produced by prokaryotic expression, the purified recombinant GP73 (rGP73), produced by prokaryotic expression, was used to immunize the Balb/c mice. Two hybridoma cell lines against GP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titers of anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealed that anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully established and validated by 119 HCC and 103 normal serum samples. RESULTS: showed that the detection limit of this method could reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those of in healthy controls (mean=70.92 ng/ml). CONCLUSIONS: Results of our study not only showed that sGP73 levels of HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratory homemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide a solid foundation for subsequent clinical applications.

Anticancer agent icaritin induces apoptosis through caspase-dependent pathways in human hepatocellular carcinoma cells.

Icaritin is an active ingredient derived from the plant Herba epimedium, which exhibits various pharmacological and biological activities. However, the function, and the underlying mechanisms of icaritin on the growth of SMMC­7721 human hepatoma cells have yet to be elucidated. The present study aimed to investigate the function and underlying mechanisms of icaritin in the growth of SMMC­7721 cells. The cells were treated with varying concentrations of icaritin for 12, 24 and 48 h, respectively, prior to cytotoxic analysis. Apoptosis of SMMC­7721 cells following treatment with icaritin was measured using flow cytometry. The gene expression of mitochondria­ and Fas­mediated caspase­dependent pathways was detected by reverse transcription­quantitative polymerase chain reaction and western blotting. Statistical analysis was performed by Student's t­test and one­way analysis or variance. The present study demonstrated that treatment with icaritin significantly inhibited growth, and induced apoptosis of SMMC­7721 cells, in a time­ and dose­dependent manner. In addition, icaritin triggered the mitochondrial/caspase apoptotic pathway, by decreasing the Bcl­2/Bax protein ratio and increasing activation of caspase­3. Icaritin also activated the Fas­mediated apoptosis pathway, as was evident by the increased expression levels of Fas and activation of caspase­8. These data suggest that icaritin may be a potent growth inhibitor and induce apoptosis of SMMC­7721 cells through the mitochondria­ and Fas­mediated caspase­dependent pathways. The present study may provide experimental evidence for preclinical and clinical evaluations of icaritin for HCC therapy.

Gene therapy with biosynthetic nanoscale peptide Cecropin A driven by the survivin promoter for hepatocarcinoma cells.

The biosynthetic nanoscale peptide Cecropin A is postulated to disrupt microbial phospholipid membranes by forming stable or transient pores. We demonstrated previously that green fluorescent protein (GFP), driven by the survivin promoter, was expressed highly in HepG2 but not in LO2 cells when they were transfected with the recombinant plasmid reporter vector (pSURV-GFP). To investigate the selective killing effect of this survivin promoter-driven peptide toxin gene system on hepatocellular carcinoma (HCC) cells in vitro, the recombinant plasmid pSURV-Cecropin A was constructed. HepG2 and LO2 cells were then transfected with the recombinant plasmid, which was driven by the survivin promoter, and the effects of Cecropin A were evaluated. Forty-eight hours after transfection with pSURV-Cecropin A, the growth of HepG2 cells was inhibited significantly. This finding was confirmed further by immunoblotting, which revealed consistently suppressed expression of proliferating cell nuclear antigen (PCNA) and cysteinyl aspartate specific proteinase-3 (caspase-3). Data demonstrated that the plasmid carrying the gene for the Cecropin A fusion protein was constructed successfully, and that its specific expression in HepG2 cells could provide the basis for targeted gene therapy in HCC. The identification of novel gene therapies for cancer is highly desirable to reduce drug toxicity and improve therapeutic outcomes..

Inhibitory effect of biosynthetic nanoscale peptide Melittin on hepatocellular carcinoma, driven by survivin promoter.

Hepatic resection and orthotopic liver transplantation are the only potentially curative treatments for hepatocellular carcinoma (HCC) but are indicated only in a minority of patients. Biosynthetic nanoscale peptide Melittin (Mel) is postulated to disrupt microbial phospholipid membranes by formation of stable or transient pores. Survivin, a member of the inhibitor of apoptosis family, is transcriptionally upregulated in most malignant tissues but not in normal tissues. It has been reported that the survivin promoter activity is tumor-specific and makes it a good candidate for construction of gene therapy vectors. In the present study, a non-viral vector (pSURV-Mel), encoding Mel gene, was developed to evaluate its anti-tumor effect in HCC cell lines and in vivo in a mouse model of human HCC xenograft tumor. Our results showed that the survivin promoter is specifically activated in tumor cells, and the pSURV-Mel plasmid expressed Mel selectively in tumor cells and also induced cytotoxicity. Moreover, intratumoral Injection of pSURV-Mel significantly suppressed the growth of xenograft tumors. Mechanistically, pSURV-Mel induced cell death by an apoptosis-dependent pathway. All taken together, this study elucidates a relatively safe, highly effective and cancer specific gene therapy strategy for HCC. The mechanisms of non-viral vector-induced cell death which were revealed by this work will shed light on the construction of more powerful vectors for cancer therapy.

Targeting and eradicating hepatic cancer cells with a cancer-specific vector carrying the Buforin II gene.

OBJECTIVE: The aim of this study was to investigate the suppressive effects of Buforin II on the growth of HepG2 cells. To accomplish this, we created a recombinant plasmid (pSUR-Buforin2) in which the survivin promoter was modified to drive the Buforin II gene. METHODS: The DNA fragment encoding the Buforin II gene was obtained by gene synthesis and cloned into the pSUR-Luc plasmid behind the survivin promoter. The vector was subsequently transfected into HepG2 and LO2 cells. Cell proliferation was measured by the MTT assay, cell cytotoxicity detected by the LDH assay, and cell apoptosis determined by flow cytometry, DNA ladder assays, and immunoblot analysis. RESULTS: The pSUR-Buforin2 vector effectively suppressed the proliferation of HepG2 cells. The MTT and LDH assay demonstrated that under control of the survivin promoter, Buforin II was not expressed in LO2 cells, but it was expressed in tumor cells where cell death was also observed. AnnexinV-PI staining, DNA ladder assays, and western blots showed massive apoptosis in HepG2 cells transfected with pSUR-Buforin2. CONCLUSION: pSUR-Buforin2 can significantly inhibit the growth of HepG2 cells, resulting in increased cancer cell apoptosis. Thus, this newly designed plasmid might provide a potent and selective anticancer therapy.

Suicide gene therapy for hepatocellular carcinoma cells by survivin promoter-driven expression of the herpes simplex virus thymidine kinase gene.

The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant 'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Toxoplasma gondii: enzyme-linked immunosorbent assay based on a recombinant multi-epitope peptide for distinguishing recent from past infection in human sera.

Enzyme-linked immunosorbent assays (ELISAs) based on a recombinant multi-epitope peptide (rMEP) were used in an attempt to differentiate pregnant women with Toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). The recombinant expression vector pET-32c-MEP encoding MEP constructed previously was expressed in Escherichia coli and the rMEP was purified as a bioactive fusion protein. The IgG-ELISA and IgM-ELISA based on the purified rMEP were developed, and used to detect IgG and IgM antibodies against Toxoplasma gondii in human sera. Immunoblot assays showed that the purified rMEP could be strongly recognized by IgM antibodies in the pooled sera from women with acute profiles, and by IgG antibodies in the pooled sera from women with chronic profiles. ELISA results also proved that the reactivities of IgG and IgM antibodies differed significantly in sera from women with acute and chronic profiles. Compared with two commercial ELISA tests for seradiagnosis of toxoplasmosis, the total concordance (including positive and negative sera) of this rMEP-based assay was 93.2% and 95.7% for the detection of IgG and IgM antibodies, respectively. Our study suggests that the rMEP protein could be used as the diagnostic antigen to differentiate recent from past infections in human toxoplasmosis.