AIM: To investigate the anti-tumor function of ginsenoside Rg3 on hepatocellular carcinoma (HCC) in vitro and in vivo, and its mechanism.
METHODS: Hep1-6 and HepG2 cells were treated by Rg3 in different concentrations (0, 50, 100 and 200 μg/mL) in vitro. After incubation for 0, 6, 12, 24 and 48 h, cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. Caspase-3 activity was measured by chromophore p-nitroanilide and flow cytometry. Bcl-2 family proteins were ascertained by Western-blotting. Mitochondria membrane potential was detected by 5, 5’, 6’ 6’ - tetrachloro-1, 1’, 3, 3’ - tetraethylbenzimidazolylcarbocyanine iodide. Forty liver tumor-bearing C57Bl6 mice were divided randomly into 4 groups for intra-tumor injection of saline, ginsenoside Rg3, cyclophosphamide (CTX) and ginsenoside Rg3 + CTX combination.
RESULTS: The survival time was followed up to 102 d. The mice in the Rg3 + CTX group showed significant increased survival time compared with those in the control group (P < 0.05). Rg3 could inhibit HCC cell proliferation and induce cell apoptosis in vitro in the concentration and time dependent manner. It also induced mitochondria membrane potential to decrease. Caspase-3 activation can be blocked by the inhibitor z-DEVD-FMK. Bax was up-regulated while Bcl-2 and Bcl-XL were down-regulated after Rg3 treatment.
CONCLUSION: Our data suggested that Rg3 alone or combined with CTX inhibited tumor growth in vivo and prolonged mouse survival time by inducing HCC cell apoptosis via intrinsic pathway by expression alterations of Bcl-2 family proteins.
Ginsenoside Rg3; Apoptosis; Hepatocellular Carcinoma; Bcl-2 family proteins; Cyclophosphamide
Human organic cation transporter 3 (OCT3, SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer and is markedly under-expressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency (MAF) 11.5% in African American) and g.-2G>A (rs555754) (MAF>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 mRNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.
SLC22A3; Polymorphism; Methylation and Prostate Cancer
At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We have explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of AKT and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This “side population” (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ABC drug transporter proteins that also contribute to chemoresistance in cancer. We found that the MDR1 transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1.
The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer.
Drug design is crucial for the effective discovery of anti-cancer drugs. The success or failure of drug design often depends on the leading compounds screened in pre-clinical studies. Many efforts, such as in vivo animal experiments and in vitro drug screening, have improved this process, but these methods are usually expensive and laborious. In the post-genomics era, it is possible to seek leading compounds for large-scale candidate small-molecule screening with multiple OMICS datasets. In the present study, we developed a computational method of prioritizing small molecules as leading compounds by integrating transcriptomics and toxicogenomics data. This method provides priority lists for the selection of leading compounds, thereby reducing the time required for drug design. We found 11 known therapeutic small molecules for breast cancer in the top 100 candidates in our list, 2 of which were in the top 10. Furthermore, another 3 of the top 10 small molecules were recorded as closely related to cancer treatment in the DrugBank database. A comparison of the results of our approach with permutation tests and shared gene methods demonstrated that our OMICS data-based method is quite competitive. In addition, we applied our method to a prostate cancer dataset. The results of this analysis indicated that our method surpasses both the shared gene method and random selection. These analyses suggest that our method may be a valuable tool for directing experimental studies in cancer drug design, and we believe this time- and cost-effective computational strategy will be helpful in future studies in cancer therapy.
TNF is a pleiotropic cytokine with intriguing biphasic pro-inflammatory and anti-inflammatory effects. Our previous studies demonstrated that TNF up-regulated FoxP3 expression and activated and expanded CD4+FoxP3+ regulatory T cells (Tregs) by utilizing TNFR2. Further, TNFR2-expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL-2, preferentially up-regulated mRNA and surface expression of TNFR2, 4-1BB and OX40 on Tregs. Agonistic antibodies against 4-1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti-TNF Ab blocked LPS-induced expansion of splenic Tregs and up-regulation of TNFR2, OX40 and 4-1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co-stimulatory TNFRSF members in response to LPS is mediated by TNF. Taken together, our novel data indicate that TNF preferentially up-regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4-1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.
TNF; regulatory T cells; co-stimulation; immune regulation
Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis, and can be targeted for cancer prevention. In order to develop potent topical agents for oral cancer chemoprevention, five known 5-Lox inhibitors from dietary and synthetic sources, Zileuton, ABT-761, Licofelone, Curcumin and Garcinol, were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis.
Oral cancer; Chemoprevention; Garcinol; 5-Lipoxygenase; Topical
Claudins are known as tight junction proteins, and their expression pattern in gastric cancer is still controversial. The relationship between the expression patterns of tight junction proteins and tumor proliferation in early gastric cancer is still far from clear.
To investigate the expression patterns of claudin-18 and Ki-67 in early gastric cancer at the invasive front and surrounding normal gastric mucosa and to investigate the biological function of claudin-18 in the proliferation and invasion of cancer cells.
Seventy-five early gastric cancer lesions removed via endoscopic mucosal resection or endoscopic submucosal resection were evaluated. All gastric cancer lesions were diagnosed as differentiated adenocarcinoma using the Japanese Classification of Gastric Carcinoma. To assess epithelial proliferation, immunostaining with Ki-67 was performed, and the labeling index was calculated. To assess the expression of epithelial tight junction proteins, immunofluorescent staining of claudin-18 was performed. The immunoreactivity of claudin-18 was graded according to the number of stained cells. Correlation analysis was performed by Spearman’s rank correlation coefficient. Transfection of claudin-18 small interfering RNA (siRNA) was accomplished in MKN74, a claudin-18-positive gastric cancer cell line, to investigate the effect of claudin-18 on proliferation and invasion of cancer cells.
Claudin-18 was significantly down-regulated in gastric cancer compared to surrounding gastric normal mucosa or intestinal metaplasia. The Ki-67 labeling index of gastric cancer at the invasive front was inversely correlated with the claudin-18 level, but that at the mucosal lesion was not correlated. Claudin-18 knockdown significantly promoted the proliferation of MKN74 compared with control siRNA-transfected cells. MKN74 invasion increased significantly with claudin-18 siRNA transfection compared with control siRNA transfection.
Down-regulation of claudin-18 is associated with the proliferative potential at the invasive front of gastric cancer, suggesting that it has a pivotal role in gastric cancer progression.
Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC), de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.
Background & Aims
Aberrant activation of the AKT oncogenic pathway and downregulation of the Sprouty 2 (Spry2) tumor suppressor gene are frequently observed molecular events in human hepatocarcinogenesis. The goal of the present study was to investigate the eventual biochemical and genetic crosstalk between activated AKT and inactivation of Spry2 during liver cancer development by using in vivo and in vitro approaches.
Activated AKT and/or Spry2Y55F, a dominant negative form of Spry2, were overexpressed in the mouse liver via hydrodynamic gene delivery. Histological and biochemical assays were applied to characterize the molecular features of AKT and AKT/Spry2Y55F liver tumors. The human HLE hepatocellular carcinoma (HCC) cell line, stably overexpressing AKT, was transfected with Spry2Y55F to study the molecular mechanisms underlying hepatocarcinogenesis driven by Spry2 loss.
Spry2Y55F overexpression significantly accelerated AKT induced hepatocarcinogenesis in the mouse. AKT/Spry2Y55F liver lesions had increased proliferation and glycolysis and decreased lipogenesis when compared with AKT corresponding lesions. At the molecular level, AKT/Spry2Y55F HCCs exhibited a significantly stronger induction of activated mitogen-activated protein kinase (MAPK) and Pyruvate Kinase M2 (PKM2) pathways than in AKT corresponding lesions. This phenotype was reproduced in HLE cells overexpressing AKT following transfection with Spry2Y55F. Furthermore, we found that concomitant suppression of the MAPK cascade and PKM2 strongly inhibited the growth induced by Spry2Y55F in AKT-overexpressing cells.
Inactivation of Spry2 accelerates AKT induced hepatocarcinogenesis via activation of MAPK and PKM2 pathways.
HCC; AKT; Spry2; MAPK; PKM2
The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow–derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.
myofibroblast; muscularis mucosae; stem cell niche; mucosal immunology; colorectal cancer
NADPH oxidase 4 (Nox4) is reported to be the major source of reactive oxygen species (ROS) in the kidneys during the early stages of diabetic nephropathy. It has been shown to mediate TGFβ1-induced differentiation of cardiac fibroblasts into myofibroblasts. Despite TGFβ1 being recognised as a mediator of renal fibrosis and functional decline role in diabetic nephropathy, the renal interaction between Nox 4 and TGFβ1 is not well characterised. The aim of this study was to investigate the role of Nox4 inhibition on TGFβ1-induced fibrotic responses in proximal tubular cells and in a mouse model of diabetic nephropathy. Immortalised human proximal tubular cells (HK2) were incubated with TGFβ1 ± plumbagin (an inhibitor of Nox4) or specific Nox4 siRNA. Collagen IV and fibronectin mRNA and protein expression were measured. Streptozotocin (STZ) induced diabetic C57BL/6J mice were administered plumbagin (2 mg/kg/day) or vehicle (DMSO; 50 µl/mouse) for 24 weeks. Metabolic, physiological and histological markers of nephropathy were determined. TGFβ1 increased Nox4 mRNA expression and plumbagin and Nox4 siRNA significantly inhibited TGF-β1 induced fibronectin and collagen IV expression in human HK2 cells. STZ-induced diabetic C57BL/6J mice developed physiological features of diabetic nephropathy at 24 weeks, which were reversed with concomitant plumbagin treatment. Histologically, plumbagin ameliorated diabetes induced upregulation of extracellular matrix protein expression compared to control. This study demonstrates that plumbagin ameliorates the development of diabetic nephropathy through pathways that include Nox4 signalling.
Functionally selective G protein-coupled receptor (GPCR) ligands, which differentially modulate canonical and non-canonical signaling, are extremely useful for elucidating key signal transduction pathways essential for both the therapeutic actions and side-effects of drugs. However, few such ligands have been created and very little purposeful attention has been devoted to studying what we term: ‘structure-functional selectivity relationships’ (SFSR). We recently disclosed the first β-arrestin-biased dopamine D2 receptor (D2R) agonists UNC9975 (44) and UNC9994 (36), which have robust in vivo antipsychotic drug-like activities. Here we report the first comprehensive SFSR studies focused on exploring four regions of the aripiprazole scaffold, which resulted in the discovery of these β-arrestin-biased D2R agonists. These studies provide a successful proof-of-concept for how functionally selective ligands can be discovered.
To investigate the prognostic value of intratumoral invariant natural killer T (iNKT) cells and interferon-gamma (IFN-γ) in hepatocellular carcinoma (HCC) after curative resection.
Expression of TRAV10, encoding the Vα24 domain of iNKT cells, and IFN-γ mRNA were assessed by quantitative real-time polymerase chain reaction in tumor from 224 HCC patients undergoing curative resection. The prognostic value of these two and other clinicopathologic factors was evaluated.
Either intratumoral iNKT cells and IFN-γ alone or their combination was an independent prognostic factor for OS (P = 0.001) and RFS (P = 0.001) by multivariate Cox proportional hazards analysis. Patients with concurrent low levels of iNKT cells and IFN-γ had a hazard ratio (HR) of 2.784 for OS and 2.673 for RFS. The areas under the curve of iNKT cells, IFN-γand their combination were 0.618 vs 0.608 vs 0.654 for death and 0.591 vs 0.604 vs 0.633 for recurrence respectively by receiver operating characteristic curve analysis. The prognosis was the worst for HCC patients with concurrent low levels of iNKT cells and IFN-γ, which might be related with more advanced pTNM stage and more vascular invasion.
Combination of intratumoral iNKT cells and IFN-γ is a promising independent predictor for recurrence and survival in HCC, which has a better power to predict HCC patients’ outcome compared with intratumoral iNKT cells or IFN-γ alone.
The aim of this study was to investigate the clinical value of serum cytokeratin 19 fragment (CYFRA21-1) and carcinoembryonic antigen (CEA) in the prediction of chemotherapy response and prognosis in patients with advanced non-small cell lung cancer (NSCLC). Serum CYFRA21-1 and CEA levels of 98 patients with advanced NSCLC were measured using immunoradiometric kits prior to and after 2 cycles of chemotherapy. After 2 cycles of chemotherapy, 45 patients achieved a radiological objective response (OR), 30 patients achieved stable disease (SD) and 23 patients had progressive disease (PD). Serum CYFRA21-1 and CEA were significantly decreased compared to baseline levels (P<0.001). By ROC curve analysis, a ≥60% reduction in CYFRA21-1 and a ≥25% reduction in CEA were the optimal cut-off levels with best sensitivity and specificity for the diagnosis of radiologic OR. The median survival of all patients was 10.2 months (range 2.6–26.3). Univariate survival analysis showed that the Eastern Cooperative Oncology Group (ECOG) performance status (PS) score, radiologic OR, a ≥60% reduction in CYFRA21-1 and a ≥25% reduction in CEA were significant prognostic factors for better overall survival. The median overall survival time in patients with a ≥60% reduction in CYFRA21-1 was significantly longer than in those with a <60% reduction (P<0.001). Similarly, the median overall survival time in patients with a ≥25% reduction in CEA was also significantly longer than in those with a <25% reduction (P<0.001). Multivariate analysis showed that ECOG PS score, a ≥60% reduction in CYFRA21-1 and a ≥25% reduction in CEA were independent prognostic factors of survival, while radiologic OR was not. In conclusion, a ≥60% reduction in CYFRA21-1 and a ≥25% reduction in CEA may be reliable surrogate markers for the prediction of chemothrapy response and prognosis, especially for the diagnosis of radiologic OR.
cytokeratin 19 fragment; carcinoembryonic antigen; non-small cell lung cancer; chemotherapy response; prognosis
Specific basic and aromatic amino acids in the C-terminal tail of Mgm101 play key roles in mediating ssDNA binding, stabilizing the oligomeric rings, and maintaining protein stability in vivo. The C-tail may couple ssDNA binding with ring disassembly, which facilitates the formation of nucleoprotein complexes competent for the repair of mtDNA.
Mgm101 is a Rad52-type single-stranded annealing protein (SSAP) required for mitochondrial DNA (mtDNA) repair and maintenance. Structurally, Mgm101 forms large oligomeric rings. Here we determine the function(s) of a 32–amino acid carboxyl-terminal tail (Mgm101238–269) conserved in the Mgm101 family of proteins. Mutagenic analysis shows that Lys-253, Trp-257, Arg-259, and Tyr-268 are essential for mtDNA maintenance. Mutations in Lys-251, Arg-252, Lys-260, and Tyr-266 affect mtDNA stability at 37°C and under oxidative stress. The Y268A mutation severely affects single-stranded DNA (ssDNA) binding without altering the ring structure. Mutations in the Lys-251–Arg-252–Lys-253 positive triad also affect ssDNA binding. Moreover, the C-tail alone is sufficient to mediate ssDNA binding. Finally, we find that the W257A and R259A mutations dramatically affect the conformation and oligomeric state of Mgm101. These structural alterations correlate with protein degradation in vivo. The data thus indicate that the C-tail of Mgm101, likely displayed on the ring surface, is required for ssDNA binding, higher-order structural organization, and protein stability. We speculate that an initial electrostatic and base-stacking interaction with ssDNA could remodel ring organization. This may facilitate the formation of nucleoprotein filaments competent for mtDNA repair. These findings could have broad implications for understanding how SSAPs promote DNA repair and genome maintenance.
To explore the utility of cytogenetic abnormalities in the distinction of hepatic adenoma (HA) and well-differentiated hepatocellular carcinoma (HCC).
Methods and results
Array-based comparative genomic hybridization (CGH) was used to determine chromosomal abnormalities in 39 hepatocellular neoplasms: 12 HA, 15 atypical hepatocellular neoplasms (AHN) and 12 well-differentiated HCC. The designation of AHN was used in two situations: (i) adenoma-like neoplasms (n = 8) in male patients (any age) and women >50 years and <15 years old; (ii) adenoma-like neoplasms with focal atypical features (n = 7). CGH abnormalities were seen in none of the HAs (0/12), eight (53%) AHNs and 11 (92%) HCCs. The number and nature of abnormalities in AHN was similar to HCC with gains in 1q, 8q and 7q being the most common. Although follow-up information was limited, recurrence and/or metastasis were observed in three AHNs (two with abnormal, one with normal CGH).
Adenoma-like neoplasms with focal atypical morphological features or unusual clinical settings such as male gender or women outside the 15–50 year age group can show chromosomal abnormalities similar to well-differentiated HCC. Even though these tumours morphologically mimic adenoma, they can recur and metastasize. Determination of chromosomal abnormalities can be useful in the diagnosis of AHN.
CGH; cytogenetic abnormalities; hepatic adenoma; hepatocellular carcinoma
Mitochondria are intracellular organelles involved in ATP synthesis, apoptosis, calcium signaling, metabolism, and the synthesis of critical metabolic cofactors. Mitochondrial dysfunction is associated with age-related degenerative diseases. How mitochondrial dysfunction causes cell degeneration is not well understood. Recent studies have shown that mutations in the adenine nucleotide translocase (Ant) cause aging-dependent degenerative cell death (DCD) in yeast, which is sequentially manifested by inner membrane stress, mitochondrial DNA (mtDNA) loss, and progressive loss of cell viability. Ant is an abundant protein primarily involved in ADP/ATP exchange across the mitochondrial inner membrane. It also mediates basal proton leak and regulates the mitochondrial permeability transition pore. Missense mutations in the human Ant1 cause several degenerative diseases which are commonly manifested by fractional mtDNA deletions. Multiple models have been proposed to explain the Ant1-induced pathogenesis. Studies from yeast have suggested that in addition to altered nucleotide transport properties, the mutant proteins cause a global stress on the inner membrane. The mutant proteins likely interfere with general mitochondrial biogenesis in a dominant-negative manner, which secondarily destabilizes mtDNA. More recent work revealed that the Ant-induced DCD is suppressed by reduced cytosolic protein synthesis. This finding suggests a proteostatic crosstalk between mitochondria and the cytosol, which may play an important role for cell survival during aging.
Rehmannia glutinosa, a traditional Chinese medicine herb, is unable to grow normally in a soil where the same species has recently been cultivated. The biological basis of this so called “replanting disease” is unknown, but it may involve the action of microRNAs (miRNAs), which are known to be important regulators of plant growth and development. High throughput Solexa/Illumina sequencing was used to generate a transcript library of the R. glutinosa transcriptome and degradome in order to identify possible miRNAs and their targets implicated in the replanting disease. A total of 87,665 unigenes and 589 miRNA families (17 of which have not been identified in plants to date) was identified from the libraries made from a first year (FP) and a second year (SP) crop. A comparison between the FP and SP miRNAs showed that the abundance of eight of the novel and 295 of the known miRNA families differed between the FP and SP plants. Sequencing of the degradome sampled from FP and SP plants led to the identification of 165 transcript targets of 85 of the differentially abundant miRNA families. The interaction of some of these miRNAs with their target(s) is likely to form an important part of the molecular basis of the replanting disease of R. glutinosa.
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Emerging evidence indicates that the host microRNAs (miRNAs) are involved in host-pathogen interactions. However, whether host miRNAs can target PRRSV and be used to inhibit PRRSV infection has not been reported. Recently, microRNA 181 (miR-181) has been identified as a positive regulator of immune response, and here we report that miR-181 can directly impair PRRSV infection. Our results showed that delivered miR-181 mimics can strongly inhibit PRRSV replication in vitro through specifically binding to a highly (over 96%) conserved region in the downstream of open reading frame 4 (ORF4) of the viral genomic RNA. The inhibition of PRRSV replication was specific and dose dependent. In PRRSV-infected Marc-145 cells, the viral mRNAs could compete with miR-181-targeted sequence in luciferase vector to interact with miR-181 and result in less inhibition of luciferase activity, further demonstrating the specific interactions between miR-181 and PRRSV RNAs. As expected, miR-181 and other potential PRRSV-targeting miRNAs (such as miR-206) are expressed much more abundantly in minimally permissive cells or tissues than in highly permissive cells or tissues. Importantly, highly pathogenic PRRSV (HP-PRRSV) strain-infected pigs treated with miR-181 mimics showed substantially decreased viral loads in blood and relief from PRRSV-induced fever compared to negative-control (NC)-treated controls. These results indicate the important role of host miRNAs in modulating PRRSV infection and viral pathogenesis and also support the idea that host miRNAs could be useful for RNA interference (RNAi)-mediated antiviral therapeutic strategies.
The objective of this study was to confirm the biological role of p38γ in human gliomas. The expression profiles of p38γ and hTERT in human glioma samples were detected by Western Blot and immunohistochemistry. RNA interference was performed in U251 cells by p38γ silencing. Cell proliferation and apoptosis were assayed by CCK-8 and flow cytometric analysis, and then RNA and protein expression levels were measured by real-time RT-PCR and Western Blot, respectively. Telomerase activity assays and Caspase-3,-9 activation assays were also conducted. The results showed p38γ had a positive correlation with the glioma's malignancy grade and that the treatment of U251 cells with p38γ-siRNA inhibited proliferation and induced apoptosis. Correspondingly, hTERT expression and telomerase activity were down regulated and Caspase-3 and -9 activities were elevated. In conclusion, p38γ may serve as an oncogenic factor promoting the growth and progression of gliomas and may become a useful therapeutic target.
Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients.
Laryngeal cancer; paclitaxel; gene expression profiles; miR expression profiles; cell lines
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, with limited treatment options. AKT/mTOR and Ras/MAPK pathways are frequently deregulated in human hepatocarcinogenesis. Recently, we generated an animal model characterized by the co-expression of activated forms of AKT and Ras in the mouse liver. We found that concomitant activation of AKT/mTOR and Ras/MAPK cascades leads to rapid liver tumor development in AKT/Ras mice, mainly through mTORC1 induction. To further define the role of mTORC1 cascade in AKT/Ras induced HCC development, the mTORC1 inhibitor Rapamycin was administered to AKT/Ras mice at the time when small tumors started to emerge in the liver. Of note, Rapamycin treatment significantly delayed hepatocarcinogenesis in AKT/Ras mice. However, some microscopic lesions persisted in the livers of AKT/Ras mice despite the treatment and rapidly gave rise to HCC following Rapamycin withdrawal. Mechanistically, Rapamycin inhibited mTORC1 and mTORC2 pathways, lipogenesis and glycolysis, resulting in inhibition of proliferation in the treated livers. However, activated ERK and its downstream effectors, Mnk1 and eIF4E, were strongly upregulated in the residual lesions. Concomitant suppression of AKT/mTOR and Ras/MAPK pathways was highly detrimental for the growth of AKT/Ras cells in vitro. The study indicates the existence of a complex interplay between AKT/mTOR and Ras/MAPK pathways during hepatocarcinogenesis, with important implications for the understanding of HCC pathogenesis as well as for its prevention and treatment.
AKT; Rapamycin; Ras; liver cancer; mTOR; mouse models
Objective: This study investigated the effect of proximal contact strength on the three-dimensional displacements of cantilever fixed partial denture (CFPD) under vertically concentrated loading with digital laser speckle (DLS) technique. Methods: Fresh mandible of beagle dog was used to establish the implant-supported CFPD for specimen. DLS technique was employed for measuring the three-dimensional displacement of the prosthesis under vertically concentrated loading ranging from 200 to 3 000 g. The effect of the contact tightness on the displacement of CFPD was investigated by means of changing the contact tightness. Results: When an axial concentrated loading was exerted on the pontic of the implant-supported CFPD, the displacement of the CFPD was the greatest. The displacement of the prosthesis decreased with the increase of contact strength. When the contact strength was 0, 0.95, and 3.25 N, the displacement of the buccolingual direction was smaller than that of the mesiodistal direction but greater than that of the occlusogingival direction. When the force on the contact area was 6.50 N, the mesiodistal displacement of the prosthesis was the biggest while the buccolingual displacement was the smallest. Conclusions: The implant-supported CFPD is an effective therapy for fully or partially edentulous patients. The restoration of the contact area and the selection of the appropriate contact strength can reduce the displacement of the CFPD, and get a better stress distribution. The most appropriate force value is 3.25 N in this study.
Cantilever fixed partial denture; Digital laser speckle technique; Contact strength; Three-dimensional displacement
Atherosclerosis is a public health concern affecting many worldwide, but its pathogenesis remains unclear. In this study we investigated the role of IKKε during the formation of atherosclerosis and its molecular mechanism in the mouse aortic vessel wall.
Methods and Results
C57BL/6 wild-type or IKKε knockout mice bred into the ApoE knockout genetic background were divided into 4 groups: (1) wild-type (WT), (2) ApoE knockout (AK), (3) IKKε knockout (IK), (4) or both ApoE and IKKε knockout (DK). Each group of mice were fed with a high fat diet (HFD) for 12 weeks from 8 weeks of age. Immunohistochemistry and Western blotting analysis demonstrated obvious increases in the expression of IKKε in the AK group compared with the WT group, especially in the intima. Serum lipid levels were significantly higher in the AK and DK groups than in the other two groups. Staining with hematoxylin-eosin and Oil Red, as well as scanning electron microscopy revealed less severe atherosclerotic lesions in the DK group than in the AK group. Immunofluorescence and Western blot analysis demonstrated obvious increases in the expression of NF-κB pathway components and downstream factors in the AK group, especially in the intima, while these increases were blocked in the DK group.
The knockout of IKKε prevented significant atherosclerosis lesions in the mouse aorta from in both wild-type and ApoE knockout mice fed a HFD, suggesting that IKKε may play a vital role in HFD-induced atherosclerosis and would be an important target for the treatment of atherosclerosis.