Simultaneous ammonia and nitrate removal by novel integrated partial denitrification-anaerobic ammonium oxidation-bioelectrochemical system.

The current study explored the performance of an integrated partial denitrification-anaerobic ammonium oxidation (anammox)-bioelectrochemical system on simultaneous removal of ammonia nitrogen and nitrate nitrogen. Different operational conditions were selected to optimize critical parameters of the process for improving nitrogen removal. The results indicated that more than 90 % of total inorganic nitrogen removal efficiency was achieved under the optimal conditions: ammonia nitrogen/nitrate nitrogen ratio of 1:2, external resistance of 200 Ω and inoculation volume ratio of anammox bacteria/denitrifying at 2:1. Improved nitrogen removal under the optimal conditions were confirmed by microbial community changes (Candidatus Brocadia and Thiobacillus) and enhanced of nitrogen metabolism-related genes (hao, hzsA/C and hdh). Increases of Limnobacter indicated an enhanced electron transfer efficiency. Overall, high-efficiency and stable nitrogen removal efficiency without nitrite nitrogen accumulation could be achieved by the integrated system under the optimal conditions, providing novel insights for simultaneous treatment of domestic wastewater and groundwater.

Study on the performance of sewage sludge biochar modified by nZVI to remove Cu(II) and Cr(VI) in water.

In this study, to simultaneously dispose of sludge and wastewater containing heavy metals, sludge biochar loaded with nano zero-valent-iron (nZVI) was prepared at 700 °C (nBC700) to remove Cr(VI) and Cu(II). The results showed the removal capacity of biochar was greatly improved by loading nZVI, and the adsorption capacities of biochar for Cu(II) and Cr(VI) increased by 251.96% and 205.18%. Pseudo-second-order kinetic and Sips isotherm models were fitted to the removal processes. Intraparticle diffusion models showed the removal process was controlled by surface diffusion and intraparticle diffusion. Competitive experiments showed Cr(VI) can compete with Cu(II) for active sites, but Cr(VI) was more easily removed by nBC700 through cation bridge. The removal mechanism illustrated removing Cu(II) mainly depended on complex precipitation, followed by reduction reaction, while Cr(VI) was on the contrary. This work provided effective data for sludge disposal and heavy metal removal.

Study on visible light photocatalytic performance of BiVO4 modified by graphene analogue boron nitride.

In this study, a variety of boron nitride (BN) modified BiVO4 (BN-BiVO4) composites with visible-light response were prepared and used to degrade tetracyclines (TCs), including tetracycline (TC) and oxytetracycline (OTC). When treating the TCs solution under visible light irradiation, 4BN-BiVO4 displayed high photocatalytic performance (87.1% for TC and 86.2% for OTC), which were 3.6 and 2.3 times than that of BiVO4, respectively. Photoluminescence spectroscopy (PL) and transient photocurrent proved that the combination of BN and BiVO4 effectively promotes the efficient separation of photogenerated electrons and holes in the material, resulting in enhanced photocatalytic activity. Further, radical trapping experiments in combination with electron spin resonance (ESR) revealed that ·OH radicals and holes were the predominant reactive species. Ultimately, the possible photocatalytic mechanism for TCs degradation was proposed on the basis of the experiments and characterization analysis. This study offers a new promising approach for the design of photocatalysts with visible-light response for efficient TCs elimination.

Performance and mechanism of tea waste biochar in enhancing the removal of tetracycline by peroxodisulfate.

In this work, tea waste biochar was prepared and used to activate peroxodisulfate (PDS) for the removal of tetracycline (TC) efficiently. And SEM, XRD, Raman, and FTIR were used to characterize the biochar. The effects of reaction conditions including initial pH, biochar dosage, and PDS concentration on the removal of TC were explored, and the result showed that compared with the biochar prepared at 400 °C and 500 °C, the biochar pyrolyzed at 600 °C (TBC600) had the highest TC removal performance due to its higher sp2 hybrid carbon content, richer defective structure, and stronger electron deliverability. Under the optimal dosage of PDS (4 mM) and TBC600 (0.8 g L-1), the removal efficiency of TC (10 mg L-1) reached 81.65%. After four cycles of TBC600, the removal rate could still reach 75.51%, indicating that TBC600 has excellent stability. In addition, quenching experiments and electron paramagnetic resonance (EPR) verified that the active oxygen including SO4·-, ·OH, O2·-, and singlet oxygen (1O2) was involved, among which 1O2 and OH were the main active substance in the TC removal. Therefore, this work provided a green and efficient persulfate activator and a method for recycling tea waste.

Nonylphenol photodegradation by novel ternary MIL-100(Fe)/ZnFe2O4/PCN composite under visible light irradiation via double charge transfer process.

Nonylphenol (NP) residues, as a typical endocrine disrupting chemical (EDC), frequently exist in sewage, surface water, groundwater and even drinking water, which poses a serious threat to human health due to its bioaccumulation. In order to remove NP, a series of MIL-100(Fe)/ZnFe2O4/flake-like porous carbon nitride (MIL/ZC) was synthesized through in-situ synthesis at room temperature. High performance of ternary MIL/ZC is used to degrade NP under visible light irradiation. The results show that 30MIL/ZC2 (20 wt.% ZnFe2O4) ternary composite had the best photocatalytic activity (99.84%) when the dosage was 30 mg. Further mechanism analysis shows that the excellent photocatalytic activity of 30MIL/ZC2 could be ascribed to the double charge transfer process between flake-like porous carbon nitride (PCN) and other catalysts in the ternary heterojunction, and the separation of photogenerated electron-hole pairs was more effective. In addition, the 30MIL/ZC2 also showed high stability after five cycles of the photodegradation reaction. Furthermore, the active substance (•O2-) was considered to be the main active substance in the NP degradation process. Based on the research results, the possible photocatalytic reaction mechanism of 30MIL/ZC2 ternary composite was proposed and discussed in detail.

Prognostic nomogram for hepatocellular carcinoma patients after transarterial chemoembolization based on des-γ-carboxy prothrombin reactivity and modified Response Evaluation Criteria in Solid Tumors.

AIMS: The aim of this study was to construct a nomogram that will predict the overall survival (OS) of hepatocellular carcinoma (HCC) patients after transarterial chemoembolization (TACE). MATERIALS AND METHODS: Imaging data, clinical characteristics, and serum des-γ-carboxy prothrombin (DCP) levels of 93 HCC patients treated with TACE were collected. Lasso regression, random forest, and other methods were used to screen the OS-related variables and construct the Cox prognosis model. The model was visualized by nomogram, and the net benefit of the clinical decision was assessed by decision curve analysis (DCA). RESULTS: It was found that DCP level after TACE was an important predictor of OS in HCC patients. The OS of the patients with lower serum DCP levels after TACE was significantly better than the group with higher levels (P = 0.003). The Cox prognostic model was constructed using four predictors including DCP reactivity (P = 0.001), modified Response Evaluation Criteria in Solid Tumors (mRECIST, P = 0.005), Child-Pugh class (P = 0.018), and portal vein thrombosis (P = 0.039). The C-index of the nomogram for OS of patients after TACE was 0.813. The clinical decision-making net benefits based on the nomogram were better than the decision-making based on the TNM stage system. CONCLUSION: DCP reactivity and mRECIST are the key predictors of prognosis in HCC patients that received TACE as their initial treatment. The nomogram constructed with these two indicators as the core could predict the OS of HCC patients after TACE and help in clinical decision-making.

Annexin A2 promotion of hepatocellular carcinoma tumorigenesis via the immune microenvironment.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with a dismal prognosis, especially when diagnosed at advanced stages. Annexin A2 (ANXA2), is found to promote cancer progression and therapeutic resistance. However, the underlining mechanisms of ANXA2 in immune escape of HCC remain poorly understood up to now. Herein, we summarized the molecular function of ANXA2 in HCC and its relationship with prognosis. Furthermore, we tentatively elucidated the underlying mechanism of ANXA2 immune escape of HCC by upregulating the proportion of regulatory T cells and the expression of several inhibitory molecules, and by downregulating the proportion of natural killer cells and dendritic cells and the expression of several inhibitory molecules or effector molecules. We expect a lot of in-depth studies to further reveal the underlying mechanism of ANXA2 in immune escape of HCC in the future.

Screening and Identification of Four Prognostic Genes Related to Immune Infiltration and G-Protein Coupled Receptors Pathway in Lung Adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a common malignant tumor with the highest morbidity and mortality worldwide. The degree of tumor immune infiltration and clinical prognosis depend on immune-related genes, but their interaction with the tumor immune microenvironment, the specific mechanism driving immune infiltration and their prognostic value are still not very clear. Therefore, the aim of this work was focused on the elucidation of these unclear aspects. METHODS: TCGA LUAD samples were divided into three immune infiltration subtypes according to the single sample gene set enrichment analysis (ssGSEA), in which the associated gene modules and hub genes were screened by weighted correlation network analysis (WGCNA). Four key genes related to immune infiltration were found and screened by differential expression analysis, univariate prognostic analysis, and Lasso-COX regression, and their PPI network was constructed. Finally, a Nomogram model based on the four genes and tumor stages was constructed and confirmed in two GEO data sets. RESULTS: Among the three subtypes-high, medium, and low immune infiltration subtype-the survival rate of the patients in the high one was higher than the rate in the other two subtypes. The four key genes related to LUAD immune infiltration subtypes were CD69, KLRB1, PLCB2, and P2RY13. The PPI network revealed that the downstream genes of the G-protein coupled receptors (GPCRs) pathway were activated by these four genes through the S1PR1. The risk score signature based on these four genes could distinguish high and low-risk LUAD patients with different prognosis. The Nomogram constructed by risk score and clinical tumor stage showed a good ability to predict the survival rate of LUAD patients. The universality and robustness of the Nomogram was confirmed by two GEO datasets. CONCLUSIONS: The prognosis of LUAD patients could be predicted by the constructed risk score signature based on the four genes, making this score a potential independent biomarker. The screening, identification, and analysis of these four genes could contribute to the understanding of GPCRs and LUAD immune infiltration, thus guiding the formulation of more effective immunotherapeutic strategies.

Highly immunosuppressive HLADRhi regulatory T cells are associated with unfavorable outcomes in cervical squamous cell carcinoma.

Regulatory T cells (Tregs) are crucial for the maintenance of peripheral tolerance, but they also limit beneficial responses through cancer-induced immunoediting. The roles of Treg subsets in cervical squamous cell carcinoma (CSCC) are currently unknown. Here, we aimed to perform an extensive study with an increased resolution of the Treg compartment in the peripheral blood and tumor tissues of CSCC patients. We first identified that an HLADRhi Treg population in the peripheral blood was significantly increased in CSCC patients compared to precancer patients and healthy donors. We found that HLADRhi Tregs express high levels of a panel of inhibition and activation markers and the TCR-responsive transcription factors BATF and IRF4. However, this Treg subset showed reduced calcium influx after TCR crosslinking. In addition, HLADRhi Tregs are highly proliferative and vulnerable to apoptosis. Further studies demonstrated that the HLADRhi Tregs display high levels of suppressive activity. Quantitative multiplexed immunohistochemistry revealed that an increase in the number of tumor-infiltrating HLADRhi Tregs is associated with unfavorable classical risk parameters of advanced disease stage and stromal invasion. Context-based quantification revealed that a high frequency of stromal HLADRhi Tregs in patients is significantly associated with worse progression-free survival. In the current study, we characterized a population of highly activated and immunosuppressive HLADRhi Tregs in CSCC patients. An increased HLADRhi Treg frequency may be a potential biomarker to stratify CSCC patients and evaluate therapeutic efficacies in personalized immuno-oncology studies.

Midair Ultrasound Fragrance Rendering.

We propose a system that controls the spatial distribution of odors in an environment by generating electronically steerable ultrasound-driven narrow air flows. The proposed system is designed not only to remotely present a preset fragrance to a user, but also to provide applications that would be conventionally inconceivable, such as: 1) fetching the odor of a generic object placed at a location remote from the user and guiding it to his or her nostrils, or 2) nullifying the odor of an object near a user by carrying it away before it reaches his or her nostrils (Fig. 1). These are all accomplished with an ultrasound-driven air stream serving as an airborne carrier of fragrant substances. The flow originates from a point in midair located away from the ultrasound source and travels while accelerating and maintaining its narrow cross-sectional area. These properties differentiate the flow from conventional jet- or fan-driven flows and contribute to achieving a midair flow. In our system, we employed a phased array of ultrasound transducers so that the traveling direction of the flow could be electronically and instantaneously controlled. In this paper, we describe the physical principle of odor control, the system construction, and experiments conducted to evaluate remote fragrance presentation and fragrance tracking.

Oncogenic secretory clusterin in hepatocellular carcinoma: Expression at early staging and emerging molecular target.

Secretory clusterin (sCLU) is associated with hepatocellular carcinoma (HCC) progression by contributing to angiogenesis, chemoresistance, cell survival, and metastasis. However, the sCLU expression at early stage of HCC progression remains to be clarified. In this study, the alteration of sCLU oncogenicity was firstly evaluated in HCC- and their para-cancerous- tissues. The incidence of sCLU expression in HCC was significantly higher than that in their non-tumorous tissues at message RNA (mRNA) or protein level, gradually increasing with tumor-node-metastasis (TNM) staging. Abnormal sCLU expression was associated with the poor differentiation, TNM stage, and considered as an independent prognostic factor for HCC patients. Furthermore, silencing sCLU gene transcription inhibited the colony formation and proliferation of HCC cells, with decreasing phosphorylation level of AKT and GSK-3ß in HCCLM3 cells in vitro and significantly suppressed the HCC xenograft growth in vivo, suggesting that sCLU with oncogenicity should be not only an early indicator but also novel potential molecular-targeted therapy for HCC.

MicroRNA-30c inhibits metastasis of ovarian cancer by targeting metastasis-associated gene 1.

BACKGROUND: It is important to find reliable molecular markers or biological targets that associate with ovarian cancer (OC) metastasis for diagnosis and treatment. In this study, researchers investigated the regulated chain of microRNA-30c (miR-30c) and metastasis-associated gene 1 (MTA1) in OC tissues and cells. MATERIALS AND METHODS: Expression of miR-30c and MTA1 was detected with quantitative real-time polymerase chain reaction and immunohistochemistry in 33 OC and matched adjacent tissues. MiR-30c mimics were synthetized and transfected into SKOV3 cells to target MTA1. The wound healing and transwell assays were detected to observe migration and invasion of transfected OC cells. RESULTS: Compared with matching normal ovarian tissues, the MTA1 expression was upregulated and localized in the cytoplasm, and the expression of miR-30c was significantly reduced. The expression intensity of MTA1 was correlated with the Federation of Gynecology and Obstetrics stage, tumor grade, and metastasis of OC. Transfecting miR-30c mimics could significantly reduce the expression of MTA1 in SKOV3 cells and obviously inhibit the migration and invasion of SKOV3 cells. CONCLUSION: MiR-30c and MTA1 abnormally expressed in OC, which may be related to metastasis of OC. In MiR-30c as a tumor suppressor gene, its expression in OC could lead to reduced expression of MTA1, which may be one of the mechanisms of metastasis of OC cells.

Abnormality of Wnt3a expression as novel specific biomarker for diagnosis and differentiation of hepatocellular carcinoma.

The member 3a of Wingless-type MMTV integration site family (Wnt3a) as an oncogene is overexpressed in many kinds of tumors with a worse outcome. However, the mechanism and alteration of Wnt3a expression in hepatocellular carcinoma (HCC) have not been clarified. In this study, the levels of Wnt3a expression were investigated in 80 HCC tissues or sera of 186 patients with chronic liver diseases. The incidence of hepatic Wnt3a expression in HCC tissues was 96.25 % and significantly higher (χ (2) = 48.818, P < 0.001) than that in their surrounding tissues (46.25 %). The higher level (>800 ng/L) of circulating Wnt3a expression was found in 92.5 % HCC patients and significantly related (P < 0.05) to alpha-fetoprotein (AFP) level, liver cirrhosis, hepatitis B virus infection, poor differentiation, tumor node metastasis, and extra-hepatic metastasis. The level of Wnt3a expression in HCC patients was obviously higher (P < 0.001) than that in any group of cases with benign liver diseases. The diagnostic specificity or the area under the receiver operating characteristic curve was 94.34 % or 0.994 in Wnt3a and 69.81 % or 0.710 in AFP for HCC, respectively. The present data suggested that Wnt3a expression associated with tumor progression should be a novel specific biomarker for diagnosis and differentiation of HCC.

Diagnostic and prognostic significance of secretory clusterin expression in patients with hepatocellular carcinoma.

The upregulation of secretory clusterin (sCLU) is associated with tumor progression by contributing to angiogenesis, chemo-resistance, cell survival, and metastasis. However, its diagnostic or prognostic values for hepatocellular carcinoma (HCC) still remain to be clarified. The average serum sCLU level analyzed by an enzyme-linked immunosorbent assay was significantly higher (P < 0.001) in HCC patients than that in any of cases with cirrhosis, chronic hepatitis, or healthy control. The area under receiver operating characteristic curve and diagnostic sensitivity were 0.75 and 74.7 % in sCLU, and 0.74 and 58.7 % in α-fetoprotein (AFP), respectively. The combining detections of sCLU and AFP rose up to 90.7 % for HCC diagnosis. In liver, sCLU by immunohistochemistry was significantly higher (P < 0.001) in the HCC (77.3 %) group than that in their para-cancerous group (33.3 %). Abnormal serum or tissue sCLU expression was closely associated with tumor-node-metastasis (TNM) classification of malignant tumors and lymph node metastasis, as an independent prognosis factor (hazard ratio, 2.287; 95 % confidence interval, 1.044-5.007; P = 0.039), and higher sCLU expression significantly correlated (χ (2) = 4.252, P = 0.039) with poor survival of HCC patients analyzed by multivariate Cox regression or Kaplan-Meier method, suggesting that abnormal sCLU expression associated with tumor progression could be a potential diagnostic and prognostic biomarker for HCC.

Up-regulation of annexin A2 expression predicates advanced clinicopathological features and poor prognosis in hepatocellular carcinoma.

Hepatic annexin A2 (ANXA2) orchestrates multiple biologic processes and clinical symptoms and plays a key role in development, metastasis, and drug resistance of lethal hepatocellular carcinoma (HCC). However, the prognostic significance of ANXA2 for HCC has not been elucidated up to now. In this study, ANXA2 was frequently found to be up-regulated in HCC tissues compared with benign liver disease (BLD) tissues, which was consistent with the results in serum samples and tissue specimens of patients with HCC. Furthermore, ANXA2 expression was significantly correlated with differentiated degree, intrahepatic metastasis, portal vein thrombus, and tumor node metastasis (TNM) staging. More importantly, increased ANXA2 level was first confirmed to be closely associated with shortened overall survival of HCC (χ (2) = 12.872, P = 0.005) and identified as an independent prognostic factor (hazard ratio 1.338, 95 % confidence interval (CI) 1.013 ~ 1.766, P = 0.040), suggesting that ANXA2 up-regulation might represent an acquired metastasis phenotype of HCC, help to screen out high-risk population for HCC, or more effectively treat a subset of postsurgical HCC patients positive for ANXA2.

Abnormal Expression of Golgi Protein 73 in Clinical Values and Their Role in HBV-Related Hepatocellular Carcinoma Diagnosis and Prognosis.

BACKGROUND: The up-regulation of hepatic Golgi protein 73 (GP73) is associated with the progression of hepatocellular carcinoma (HCC). However, the exact mechanism and clinical values of its diagnosis and prognosis still need to be clarified. OBJECTIVES: To investigate the clinical values of abnormal liver or circulating GP73 expression and their effect on HCC diagnosis and prognosis. MATERIALS AND METHODS: The expression of GP73 was investigated in 88 cancerous and self-control non-cancerous tissues using tissue microarrays with immunohisto- chemistry and was confirmed by Western blotting. Circulating GP73 levels were detected in the sera of 281 patients with liver diseases using enzyme-linked immunosorbent assay. RESULTS: The levels of circulating GP73 expression in the HCC group were higher than those in any group of benign liver diseases or controls. No significant difference was found between GP73 expression and patients' sex or age, tumor size, or AFP level except for those with hepatitis B virus (HBV) infection or distal metastasis (P < 0.05). The area under the receiver operating characteristic curve, sensitivity, and specificity for HCC diagnosis were 0.881, 78.34%, and 77.59% for GP73 levels over 70 µg/L or 0.754, 71.97%, and 84.48% for alpha-fetoprotein levels over 50 µg/L, respectively. The total incidence of GP73 plus alpha-fetoprotein was up to 87.26% for HCC. A positive GP73 result with brown particles was mainly located in the cytosol, with a few in the nucleus and none in the cell membrane, with abnormal expression in HCC tissues (480.7 ± 148.7) that was significantly higher (t = 10.730, P < 0.001) than those in their non-cancerous tissues (208.0 ± 66.1). The high GP73 expression in HCC was related to lymph node metastasis (χ(2) = 6.940, P = 0.008), gross classification (χ(2) = 6.311, P = 0.012), HBV (χ(2) = 4.803, P = 0.028), tumor node metastasis staging (χ(2) = 4.887, P = 0.027), and five-year survival (χ(2) = 5.206, P = 0.023). CONCLUSIONS: Abnormality of hepatic or circulating GP73 expression should be regarded as an emerging biomarker for HCC diagnosis and prognosis.

Encouraging microRNA-based Therapeutic Strategies for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide with poor prognosis. Most of the HCC patients died quickly because of the rapid tumor progression with no effective therapy except for liver resection or transplantation. Recently, microRNAs (miRNAs) have emerged as key factors involved in a series of biological processes ranging from embryogenesis to programmed cell death with aberrant expression profiles, potentially attractive diagnosis, prognosis or therapeutic applications for related-diseases. Accumulating evidences have indicated the roles of miRNAs as tumor suppressor in hepatocyte malignant transformation including development, differentiation, proliferation and tumorigenesis. This article reviews that the recent progress underlies the development of novel miRNA-based HCC therapeutic strategies in the coming future.

[Inhibitory effects of intervention of the TNFa/NF-kappaB signaling pathway activation on hepatoma cell proliferation].

OBJECTIVE: To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFa)/nuclear factor-kappa B (NF-kappaB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism. METHODS: A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats. Human subjects with various liver diseases were enrolled in the study, and serum and peripheral blood nuclear cells were collected for analysis. HepG2 cells were cultured in vitro and treated with anti-TNFa (monoclonal antibody, mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-kappaB to inhibit its activation. The liver cell line L02 was used as a control. Changes in protein and gene expression levels of NF-kappaB and TNFa were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR, respectively. Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay, respectively. RESULTS: TNFa and NF-kappaB expression showed increasing trends during the malignant transformation of rat hepatocytes, and the differential expression patterns showed association with histopathological alterations in the hepatocytes. Following treatment with the TNFa mAb, the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45% +/- 4.07% vs. 5.63% +/- 0.93%, q =10.07, P less than 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23% +/- 1.29% vs. untreated control cells: 59.00% +/- 1.02%, q =10.98, P less than 0.01). The decreased expression of TNFa and NF-kappaB in cell culture supernatants was positively correlated with the dose of treatment (r =0.89, P less than 0.01), with the most robust decreases being achieved with the highest concentrations ( P less than 0.01). NF-kappaB expression was significantly higher in the HepG2 cells than in the L02 cells, and transfection of p65-siRNA reduced the mRNA (93%) and protein (62%) levels and increased the cell apoptosis index (to 85%). CONCLUSION: Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFa/NF-kappaB signaling pathway, which promotes cell apoptosis and blocks cell cycling.

[Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis].

OBJECTIVE: To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms. METHODS: GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA. RESULTS: In all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001). CONCLUSION: GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.

Abnormal expression of insulin-like growth factor-I receptor in hepatoma tissue and its inhibition to promote apoptosis of tumor cells.

Abnormal signaling of insulin-like growth factor I receptor (IGF-IR) is associated with hepatocellular carcinoma, but the underlying molecular mechanisms remain largely unknown. The objective of this study was to investigate IGF-IR's role as a signaling molecule, its pathological alteration in hepatoma tissues, and its effect on hepatoma cell proliferation when inhibited. As measured by immunohistochemical analysis, the incidence of hepatic IGF-IR expression in cancerous tissue was 80.0 % (24 of 30), which was significantly higher (P < 0.05) than 43.3 % (13 of 30) occurrence in the surrounding tissue and the nondetectable (0 of 30) frequency in the distal cancerous tissue. Hepatoma IGF-IR expression was correlated to the differentiation degree and not to the number or size of tumors, HBV infection, and AFP level. The in vitro IGF-IR expression in hepatoma cells was down-regulated significantly by picropodophyllin, a specific kinase inhibitor, in a time- and dose-dependent manner. Cell proliferation was inhibited through typical mechanisms of promoting apoptosis and cell cycle arrest (G2/M phase). Up-regulation of IGF-IR in hepatocarcinogenesis suggests that the down-regulation of IGF-IR expression could be a specific molecular target for hepatoma cell proliferation.