Mutations in the NDV fusion protein HR4 region decreased fusogenic activity due to failed protein expression.

Newcastle disease virus (NDV) is the pathogen of a zoonosis that is primarily transmitted by poultry and has severe infectivity and a high fatality rate. Many studies have focused on the role of the NDV fusion (F) protein in the cell-cell membrane fusion process. However, little attention has been given to the heptad repeat region, HR4, which is located in the NDV F2 subunit. Here, site-directed mutants were constructed to study the function of the NDV F protein HR4 region and identify the key amino acids in this region. Nine conserved amino acids were substituted with alanine or the corresponding amino acid of other aligned paramyxoviruses. The desired mutants were examined for changes in fusogenic activity through three kinds of membrane fusion assays and expression and proteolysis through IFA, FACS and WB. The results showed that when conserved amino acids (L81, Y84, L88, L91, L92, P94, L95 and I99) were replaced with alanine, the fusogenic activity of the F protein was abolished, possibly because of failed protein expression not only on the cell surface but also inside cells. These data indicated that the conserved amino acids above in NDV F HR4 are critical for normal protein synthesis and expression, possibly for the stability of the F protein monomer, formation of trimer and conformational changes.

Identification and validation of the N6-methyladenosine RNA methylation regulator ZC3H13 as a novel prognostic marker and potential target for hepatocellular carcinoma.

N6-methyladenosine (m6A) RNA methylation has been implicated in various malignancies. This study aimed to identify prognostic signature based on m6A methylation regulators for hepatocellular carcinoma (HCC) and provide candidate targets for HCC treatment. In this study, the expression levels, prognostic values, correlation with tumor grades and genetic variations of m6A-related genes in HCC were evaluated using bioinformatics analyses. Interestingly, the results show that methyltransferase zinc finger CCCH-type containing 13 (ZC3H13) was expressed at a significantly low level in HCC. Survival outcome analysis suggested that significant correlations existed between ZC3H13 downregulation and poor overall survival (OS) and poor recurrence-free survival (RFS) in HCC patients. Therefore, ZC3H13 was chosen for further experimental validation. The expression of ZC3H13 in HCC cell lines was investigated by western blotting. Knockdown of ZC3H13 significantly enhanced the migration and invasion of HCC cells, as demonstrated by wound healing and transwell assays. Moreover, upregulating ZC3H13 repressed the growth of xenograft tumors in vivo. Functional and pathway enrichment analyses indicated that ZC3H13 might be involved in transcriptional dysregulation or the JAK-STAT signaling pathway in cancer. Additionally, ZC3H13 expression was significantly correlated with lymphocytes and immunomodulators. Therefore, ZC3H13 is a promising candidate as a novel biomarker and therapeutic target for HCC.

Bladder drug mirabegron exacerbates atherosclerosis through activation of brown fat-mediated lipolysis.

Mirabegron (Myrbetriq) is a ß3-adrenoreceptor agonist approved for treating overactive bladder syndrome in human patients. This drug can activate brown adipose tissue (BAT) in adult humans and rodents through the ß3-adrenoreceptor-mediated sympathetic activation. However, the effect of the mirabegron, approved by the US Food and Drug Administration, on atherosclerosis-related cardiovascular disease is unknown. Here, we show that the clinical dose of mirabegron-induced BAT activation and browning of white adipose tissue (WAT) exacerbate atherosclerotic plaque development. In apolipoprotein E-/- (ApoE-/-) and low-density lipoprotein (LDL) receptor-/- (Ldlr-/-) mice, oral administration of clinically relevant doses of mirabegron markedly accelerates atherosclerotic plaque growth and instability by a mechanism of increasing plasma levels of both LDL-cholesterol and very LDL-cholesterol remnants. Stimulation of atherosclerotic plaque development by mirabegron is dependent on thermogenesis-triggered lipolysis. Genetic deletion of the critical thermogenesis-dependent protein, uncoupling protein 1, completely abrogates the mirabegron-induced atherosclerosis. Together, our findings suggest that mirabegron may trigger cardiovascular and cerebrovascular diseases in patients who suffer from atherosclerosis.

TRAIL promotes hepatocellular carcinoma apoptosis and inhibits proliferation and migration via interacting with IER3.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce substantial cytotoxicity in tumor cells but rarely exert cytotoxic activity on non-transformed cells. In the present study, we therefore evaluated interactions between TRAIL and IER3 via co-immunoprecipitation and immunofluorescence analyses, leading us to determine that these two proteins were able to drive the apoptotic death of hepatocellular carcinoma (HCC) cells and to disrupt their proliferative and migratory abilities both in vitro and in vivo. From a mechanistic perspective, we determined that TRAIL and IER3 were capable of inhibiting Wnt/ß-catenin signaling. Together, these results indicate that TRAIL can control the pathogenesis of HCC at least in part via interacting with IER3 to inhibit Wnt/ß-catenin signaling, thus indicating that this TRAIL/IER3/ß-catenin axis may be a viable therapeutic target in HCC patients.

LncRNA MALAT1 mediates doxorubicin resistance of hepatocellular carcinoma by regulating miR-3129-5p/Nova1 axis.

Drug resistance is one of the major challenges for cancer therapies. In recent years, research on disease-related molecular signaling pathways has become the key ways to understand and overcome obstacles. Dysregulation of MALAT1 could regulate doxorubicin resistance of hepatocellular carcinoma (HCC), but how MALAT1 involving in managing doxorubicin resistance remains unclear yet. We aimed to elucidate the specific molecular mechanism of MALAT1 with doxorubicin resistance in HCC cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was engaged to detect the expression levels of MALAT1, miR-3129-5p and Nova1 mRNA; MTT, western blot, flow cytometry and luciferase reporter assays were executed to identify the influence of MALAT1 on doxorubicin resistance of HCC cells. Xenograft tumor model was created to confirm the biological function of MALAT1 in doxorubicin resistance of HCC cells in vivo. MALAT1 and Nova1 were upregulated, while miR-3129-5p expression was decreased in doxorubicin-resistant HCC tissues and cells. Knockdown of MALAT1 regulated doxorubicin resistance of HCC cells through inhibiting cell proliferation, migration, invasion and promoting apoptosis, but antisense miR-3129-5p released the functional effect of MALAT1 knockdown. Nova1, as a target gene of miR-3129-5p, reversed the results of miR-3129-5p expression and enhanced doxorubicin resistance of HCC cells. Xenograft tumor model suggested that dysregulation of MALAT1 regulated tumor growth and Nova1 to mediate doxorubicin resistance of HCC cells by as a sponge for miR-3129-5p in vivo. Elevation of LncRNA MALAT1 mediated doxorubicin resistance and the progression of HCC via a MALAT1/miR-3129-5p/Nova1 axis. This study would be expected to enrich the understanding of doxorubicin resistance of HCC and provide new ideas for HCC treatment strategies.

LncRNA SNHG16 aggravates tumorigenesis and development of hepatocellular carcinoma by sponging miR-4500 and targeting STAT3.

Hepatocellular carcinoma (HCC) is the most common type of primary liver tumor and becomes a lethal malignancy on account of high mortality and increasing incidence. A growing body of studies has proved that long noncoding RNAs (lncRNAs) participate in the development of diverse cancers. Although it has been commonly accepted that SNHG16 is a procancer gene in numerous cancers, the regulatory mechanism of SNHG16 in HCC still needs more explorations. In this study, our results delineated that SNHG16 presented much higher expression levels in HCC tissues and cells, particularly in advanced stages of HCC. Enhanced SNHG16 expression was strongly related to poor prognosis. SNHG16 facilitated HCC progression by promoting cell proliferation, migration, invasion, and epithelial-mesenchymal transition process as well as inhibiting cell apoptosis. SNHG16 served as a sponge for miR-4500 in HCC and miR-4500 neutralized the influences of SNHG16 knockdown on HCC. SNHG16 was confirmed to compete with signal transducer and activator of transcription 3 (STAT3) to bind with miR-4500. SNHG16 aggravated the development of HCC via sponging miR-4500 so as to upregulate STAT3. In other words, this study was the first to investigate the potential mechanism of SNHG16 in HCC and verified SNHG16 exerted its carcinogenesis by miR-4500/STAT3 axis, suggesting SNHG16 may be a new underlying therapeutic target for HCC treatment.

Characterization of a novel genus of jumbo phages and their application in wastewater treatment.

Phages widely exist in numerous environments from wastewater to deep ocean, representing a huge virus diversity, yet remain poorly characterized. Among them, jumbo phages are of particular interests due to their large genome (>200 kb) and unusual biology. To date, only six strains of jumbo phages infecting Klebsiella pneumoniae have been described. Here, we report the isolation and characterization of two jumbo phages from hospital wastewater representing the sixth genus: φKp5130 and φKp9438. Both phages showed lytic activity against broad range of clinical antibiotic-resistant K. pneumoniae strains and distinct physiology including long latent period, small burst size, and high resistance to thermal and pH stress. The treatment of sewage water with the phages cocktail resulted in dramatic decline in K. pneumoniae population. Overall, this study provides detailed molecular and genomics characterization of two novel jumbo phages, expands viral diversity, and provides novel candidate phages to facilitate environmental wastewater treatment.

Lestaurtinib Has the Potential to Inhibit the Proliferation of Hepatocellular Carcinoma Uncovered by Bioinformatics Analysis and Pharmacological Experiments.

Patients diagnosed with hepatocellular carcinoma (HCC) seek a satisfactory prognosis. However, most HCC patients present a risk of recurrence, thus highlighting the lack of effectiveness of current treatments and the urgent need for improved treatment options. The purpose of this study was to identify new candidate factors in the STAT family, which is involved in hepatocellular carcinogenesis, and new targets for the treatment of HCC. Bioinformatics web resources, including Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), The Human Protein Atlas (HPA), Tumor Immune Estimation Resource (TIMER), and GSCALite, were used to identify candidate genes among the STAT family in HCC. STAT1 was significantly overexpressed in hepatocellular carcinoma. More meaningfully, the high STAT1 expression was significantly associated with poor prognosis. Therefore, STAT1 is expected to be a therapeutic target. The JAK2 inhibitor lestaurtinib was screened by the Genomics of Cancer Drug Sensitivity Project (GDSC) analysis. Pharmacological experiments showed that lestaurtinib has the ability to prevent cell migration and colony formation from single cells. We also found that STAT1 is involved in inflammatory responses and immune cell infiltration. Immune infiltration analysis revealed a strong association between STAT1 levels and immune cell abundance, immune biomarker levels, and immune checkpoints. This study suggests that STAT1 may be a key oncogene in hepatocellular carcinoma and provides evidence that the JAK2 inhibitor lestaurtinib is a potent antiproliferative agent that warrants further investigation as a targeted therapy for HCC.

Downregulation of ZC3H13 by miR-362-3p/miR-425-5p is associated with a poor prognosis and adverse outcomes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is notorious for its poor prognosis. Previous studies identified several N6-methyladenosine (m6A)-related genes that play key roles in the initiation and progression of HCC patients. In particular, the N6-methyladenosine RNA methylation regulator ZC3H13 could be a candidate as a novel biomarker and therapeutic target for hepatocellular carcinoma. In HCC, low expression of ZC3H13 was reported, but the molecular reason is unclear. In this study, we performed pan cancer analysis for ZC3H13 expression and prognosis using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data and found that ZC3H13 might be a potential tumor suppressor gene in HCC. Subsequently, miRNAs contributing to ZC3H13 downregulation were identified by a series of in silico analyses, including expression analysis, correlation analysis, and survival analysis. Finally, the miR-362-3p/miR-425-5p-ZC3H13 axis was identified as the most likely upstream miRNA-related pathway of ZC3H13 in HCC. Additionally, miR-362-3p/miR-425-5p mimic and inhibitor results were detected by quantitative real-time PCR (qPCR) analysis and western blotting. We identified an upstream regulatory mechanism of ZC3H13 in HCC, namely, the miR-362-3p/miR-425-5p-ZC3H13 axis. Moreover, the ZC3H13 level was significantly positively associated with tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression. Collectively, our findings elucidated that ncRNA-mediated downregulation of ZC3H13 was correlated with a poor prognosis and tumor immune infiltration in HCC. In conclusion, this study demonstrates that ZC3H13 is a direct target of miR-362-3p/miR-425-5p in liver hepatocellular carcinoma (LIHC) that regulates immune modulation in the microenvironment of LIHC.

Dermatopontin inhibits WNT signaling pathway via CXXC finger protein 4 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of tumor associated deaths globally. Annually, the prevalence of HCC is increasing and the lack of early prognostic indicators manifests a dismal prognosis for HCC patients. A deep understanding of the molecular events that promote HCC progression are required for the design of new diagnostics and therapeutics. Dermatopontin (DPT) is an extracellular matrix protein that regulates the metastatic phenotypes of many cancers. However, the effects of DPT on HCC cell growth remain undefined. In this study, we demonstrate that the exogenous expression of DPT inhibits HCC cell growth both in vitro and in vivo. Furthermore, we show that DPT regulates CXXC4, which in turn targets c-Myc, EZH2, SOX2 and ß-catenin, through its ability to impact Wnt signaling pathway. These data suggest that DPT regulates CXXC4, c-Myc, EZH2, SOX2 and ß-catenin, through Wnt signaling to repress HCC proliferation. This highlights DPT as promising target for future HCC diagnostics and therapeutic targets.

TUG1 long non-coding RNA enlists the USF1 transcription factor to overexpress ROMO1 leading to hepatocellular carcinoma growth and metastasis.

Hepatocellular carcinoma (HCC) is a prevalent and highly aggressive cancer. Long non-coding RNAs (lncRNAs) are recognized as potential molecular targets for HCC and are currently under increased research focus. Here, we investigate the regulatory processes underlying the axis of the lncRNA taurine upregulated gene 1 (TUG1), Upstream Transcription Factor 1 (USF1), and reactive oxygen species modulator 1 (ROMO1) in the propagation and metastasis of HCC cells. Distribution of lncRNA TUG1 was found to be prominent in HCC cell cytoplasm and nuclei. LncRNA TUG1 conscripted the USF1 transcription factor to enhance the promoter function of ROMO1. Enlisting the USF1 transcription factor to increase ROMO1 expression following upregulation of TUG1 lncRNA enhanced HCC Huh7 cell proliferation, motility, and metastasis. Rapid tumor proliferation in nude mice provided in vivo verification. The importance of the lncRNA TUG1/USF1/ROMO1 complex as a target for HCC therapy is a key result of this investigation which is exemplified by its role in regulating the proliferation, motility, and metastasis of HCC cells.

LncRNA MALAT1 acts as a miR-125a-3p sponge to regulate FOXM1 expression and promote hepatocellular carcinoma progression.

Background: Hepatocellular carcinoma (HCC) is a prominent cancer type, with long non-coding RNAs (lncRNAs) being known to be relevant to its progression. We therefore investigated how a particular lncRNA known as the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was associated with HCC. Methods: Quantitative reverse transcriptase PCR (qPCR) was used to assess expression of MALAT1, Forkhead Box M1 (FOXM1) and miR-125a-3p in HCC tissue samples. How MALAT1 regulates HCC proliferation and metastasis was assessed through appropriate assays. FOXM1 was identified as a miR-125a-3p target using luciferase assays, and how MALAT1/miR-125a-3p alter FOXM1 expression was explored via qPCR and Western blotting. Results: HCC tissues exhibited MALAT1 upregulation. miR-125a-3p targeted FOXM1 and could be regulated by MALAT1. MALAT1 knockdown disrupted proliferation and invasion, whereas miR-125a-3p knockdown partially reversed this phenotype. Conclusions: These results indicate that MALAT1 modulates FOXM1 expression via being a miR-125a-3p sponge, thus promoting HCC progression.

Lentiviral RNA interference-mediated downregulation of Forkhead box M1 expression suppresses growth of oral squamous cell carcinoma in vitro.

Oral squamous cell carcinoma (OSCC) is one of the most fatal types of oral cancer worldwide. Forkhead box M1 (FOXM1) is associated with the occurrence and development of a number of types of human cancer, but its function in OSCC remains unclear. The present study aimed to explore the effect of FOXM1 downregulation using lentivirus-mediated short hairpin (sh)RNA against FOXM1 (LV-shFOXM1) in the cell line Tca8113 in vitro. Infection of Tca8113 cells with LV-shFOXM1 inhibited the mRNA and protein expression level of FOXM1. The downregulation of FOXM1 resulted in cell cycle arrest of Tca8113 cells, and the inhibition of proliferation, migration and invasion. The protein expression level of cyclins B1 and D1 were downregulated, whereas those of p27 and p21 were upregulated following infection with LV-shFOXM1, compared with the blank control and LV-shCON groups. In addition, FOXM1 downregulation decreased the expression of matrix metalloproteinase-2 and LV-shFOXM1 significantly suppressed OSCC cell viability. Therefore, FOXM1 may be a target for the treatment of OSCC.

[Preparation and characterization of monoclonal antibodies against RalB].

OBJECTIVE: In order to explore the expression of RalB (ras related; GTP binding protein B) in mammal eucaryotic cell, we prepared and characterized monoclonal antibodies against RalB. METHODS: Hybridomas were generated by the fusion with Sp2/0 myelomas and spleen cells, which were from mice immunized with RalB recombinant proteins. The monoclonal antibodies against RalB were then used to identify the expression of RalB in mammal eucaryotic cell, including normal hepatic cell and hepatoma carcinoma cells, by Western blot and Immunohistochemistry. RESULTS: Two hybridoma cell lines, F001, F002, had been produced, each of which stably secrets antibodies against RalB. Subclass of IgG are both belonged to IgG1. Immunohistochemistry demonstrated that RalB was presented in plasma membrane of hepatoma tissue. Western-blot showed that RalB was expressed in all concerned cell. CONCLUSION: The monoclonal antibodies against RalB protein have been successfully prepared, which should provide useful reagent for further investigation into the biological function of RalB.

Identification of microRNA-124 in regulation of Hepatocellular carcinoma through BIRC3 and the NF-κB pathway.

MicroRNAs (miRNAs) being proved to be involved in the carcinogenesis of numerous tumors. MicroRNA-124 (miR-124), identified as a tumor suppressor, has been demonstrated to exert pivotal roles in multiple processes of tumorigenesis. The present study demonstrated that miR-124 was low-expressed in human hepatocellular carcinoma (HCC) tissues and cell lines. In addition, overexpression of miR-124 through infected with miR-124 lentivirus inhibited the proliferation and migration of HCC in vitro and tumorigenesis in vivo, whereas inhibition of miR-124 expression can reverse the process. Moreover, Baculoviral IAP Repeat Containing 3 (BIRC3) was identified as a target gene of miR-124. The BIRC3 mRNA expression was increased in HCC tissues and negatively correlated with miR-124 expression. Knockdown of BIRC3 recovered the miR-124-induced inhibiting effect on HCC progression. Furthermore, we found that up-regulation of miR-124 significantly inhibited p-P65, p-IκBα and c-Myc proteins expression. However, the effect of miR-124 up-regulation on HCC development was partly reversed by BIRC3 restoration. In conclusion, our data proved that miR-124 inhibits the proliferation and migration of HCC at least partly through targeting BIRC3 and regulating NF-κB signaling pathway, and it may be a therapeutic target for HCC prognosis.

Autologous platelet-rich plasma induces bone formation of tissue-engineered bone with bone marrow mesenchymal stem cells on beta-tricalcium phosphate ceramics.

BACKGROUND: The purpose of the study is to investigate whether autologous platelet-rich plasma (PRP) can serve as bone-inducing factors to provide osteoinduction and improve bone regeneration for tissue-engineered bones fabricated with bone marrow mesenchymal stem cells (MSCs) and beta-tricalcium phosphate (ß-TCP) ceramics. The current study will give more insight into the contradictory osteogenic capacity of PRP. METHODS: The concentration of platelets, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-ß1 (TGF-ß1) were measured in PRP and whole blood. Tissue-engineered bones using MSCs on ß-TCP scaffolds in combination with autologous PRP were fabricated (PRP group). Controls were established without the use of autologous PRP (non-PRP group). In vitro, the proliferation and osteogenic differentiation of MSCs on fabricated constructs from six rabbits were evaluated with MTT assay, alkaline phosphatase (ALP) activity, and osteocalcin (OC) content measurement after 1, 7, and 14 days of culture. For in vivo study, the segmental defects of radial diaphyses of 12 rabbits from each group were repaired by fabricated constructs. Bone-forming capacity of the implanted constructs was determined by radiographic and histological analysis at 4 and 8 weeks postoperatively. RESULTS: PRP produced significantly higher concentration of platelets, PDGF-AB, and TGF-ß1 than whole blood. In vitro study, MTT assay demonstrated that the MSCs in the presence of autologous PRP exhibited excellent proliferation at each time point. The results of osteogenic capacity detection showed significantly higher levels of synthesis of ALP and OC by the MSCs in combination with autologous PRP after 7 and 14 days of culture. In vivo study, radiographic observation showed that the PRP group produced significantly higher score than the non-PRP group at each time point. For histological evaluation, significantly higher volume of regenerated bone was found in the PRP group when compared with the non-PRP group at each time point. CONCLUSIONS: Our study findings support the osteogenic capacity of autologous PRP. The results indicate that the use of autologous PRP is a simple and effective way to provide osteoinduction and improve bone regeneration for tissue-engineered bone reconstruction.

The Value of lncRNA NEAT1 as a Prognostic Factor for Survival of Cancer Outcome: A Meta-Analysis.

The present meta-analysis aimed to analyze available data to identify the prognostic role of NEAT1 in multiple carcinomas. A systematic search was performed by using several computerized databases from inception to June 7, 2017. The quantity of the publications was assessed according to MOOSE checklist. Pooled HRs with 95% CI was calculated to summarize the effect. A total of 12 studies with 3,262 cancer patients were pooled in the analysis to evaluate the prognostic value of NEAT1 in multiple tumors. High expression levels of NEAT1 were demonstrated to be associated with poor OS (HR = 1.71, 95%CI: 1.37-2.14, P < 0.001) and tumor progression (III/IV vs. I/II: HR 1.76, 95%CI: 1.40-2.21, P < 0.00001). Subgroup analysis showed that NEAT1 detection method (qRT-PCR) and sample size (more or less than 100) did not alter the predictive value of NEAT1 on OS in various cancers. According to the meta-regression results, the large heterogeneity of meta-analysis may be attributed to the differences of NEAT1 detection method. Furthermore, elevated NEAT1 expression significantly predicted lymph node metastasis (HR: 2.10, 95%CI: 1.32-3.33, P = 0.002) and distant metastasis (HR: 2.80, 95%CI: 1.60-4.91, P = 0.0003) respectively. The results indicate that NEAT1 expression level is a prognostic biomarker for OS and metastasis in general tumors.

Upregulated microRNA-429 inhibits the migration of HCC cells by targeting TRAF6 through the NF-κB pathway.

Increasing evidence indicates that miR-429 is involved in tumor suppression in various human cancers. however, its role in hepatocellular carcinoma (HCC) remains unclear. In the present study, we found that miR-429 was significantly downregulated in HCC tissue samples and cell lines. Upregulation of miR-429 markedly suppressed proliferation and migration of HCC cells. Moreover, we identified TRAF6 as a direct target of miR-429. Downregulation of TRAF6 partially attenuated the oncogenic effect of anti­miR-429 on HCC cells. Ectopic expression of miR-429 in HCC cells inhibited TCF-4 activity as well as nuclear accumulation of P65 and expression of the NF-κB targets c-Myc and phosphorylation of TAK1. In a nude xenograft model, miR-429 upregulation significantly decreased HCC growth. In conclusion, by targeting TRAF6, miR-429 is downregulated in HCC and inhibits HCC cell proliferation and motility. Our data suggest that miR-429 may serve as a potential anticancer target for the treatment of HCC.

Glycogen synthase kinase-3ß regulates tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis via the NF-κB pathway in hepatocellular carcinoma.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for its ability to selectively induce apoptosis in malignant cells. However, human hepatocellular carcinoma (HCC) cells display resistance to TRAIL-induced cell death. The present study investigated whether TRAIL-induced apoptosis in HCC cells was enhanced by the administration of an inhibitor of glycogen synthase kinase-3ß (GSK-3ß) or by short hairpin RNA-mediated inhibition of GSK-3ß. The results of the current study demonstrated that inhibition of GSK-3ß significantly impairs the expression of the nuclear factor-κB (NF-κB) target genes Bcl-xL and clAP2 in HCC cells (P<0.05). This indicates that GSK-3ß may regulate NF-κB target genes involved in cell survival. Furthermore, knockdown of Bcl-xL significantly enhanced the sensitizing effect of GSK-3ß inhibitor on TRAIL-induced apoptosis (P<0.05). Overall, the present study provides a rationale for further exploration of GSK-3ß inhibition combined with TRAIL as a novel treatment for HCC.

Downregulation of SNAIL sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by regulating the NF-κB pathway.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second most lethal cancer worldwide. Evidence has shown HCC cell resistance to TRAIL­mediated apoptosis. In a previous study, we verified that silencing SNAIL downregulated the growth of HCC cells. In addition, the mechanism of resistance to TRAIL in HCC cells was connected with the activation of nuclear factor-κB (NF-κB). Thus, it was hypothesized that the downregultaion of SNAIL sensitizes HCC cells to TRAIL-induced apoptosis by regulating the NF-κB pathway. In the present study, the most effective lentiviral vectors carrying shRNA against SNAIL were selected and adenoviral vectors harboring TRAIL were constructed. The expression of SNAIL and TRAIL was detected by quantitative PCR and western blotting. HCC cell viability and apoptosis were assessed using an MTT assay and the Hoechst test. To determine how to sensitize HCC cells to TRAIL-induced apoptosis after silencing SNAIL, p53 was assessed by western blot analysis. We also investigated the expression of Bcl-xL, cIAP2, survivin and Raf-1 protein using western blot analysis and the apoptotic degree of HuH-7 cells was detected using the Hoechst test following the suppression of each gene, which was a possible molecular mechanism to sensitive TRAIL-induced apoptosis through the downregulation of SNAIL in HCC cells. Silencing SNAIL resulted in increased apoptosis by enhancing sensitization to TRAIL in all the HCC cells. Additionally, p53 protein was upregulated in HuH-7 cells. Expression of Bcl-xL, cIAP2, survivin and Raf-1 was downregulated following silencing of SNAIL, while down-regulation of any of the proteins contributed to SNAIL suppression enhancing HCC cell sensitivity to TRAIL­induced apoptosis, with the exception of cIAP2. The results demonstrated that silencing SNAIL can sensitize TRAIL-induced apoptosis in HCC cells by upregulating p53 protein and by regulating related genes of the NF-κB pathway such as Bcl-xL, survivin and Raf-1.