Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro.

AIM: Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells. METHODS: Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays. RESULTS: In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factor-dependent manner. CONCLUSION: This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis.

A functional p.82G>S polymorphism in the RAGE gene is associated with multiple sclerosis in the Chinese population.

BACKGROUND: The receptor for advanced glycation end products (RAGE) and its proinflammatory ligand, S100-calgranulins, are critically implicated in the pathological progression of multiple sclerosis (MS). A functional polymorphism within the V-type immunoglobulin domain of RAGE gene, p.82G>S (c.557G>A), has been shown to affect ligand binding affinity and thus may affect susceptibility to MS. METHODS: The RAGE p.82G>S polymorphism was genotyped in 144 MS patients and 156 healthy controls using polymerase chain reaction - restriction fragment length polymorphism. A replication study was performed on a second cohort comprising 138 patients and 150 controls. The relationship between the RAGE p.82G>S polymorphism and circulating levels of soluble RAGE (sRAGE), a secreted decoy receptor against RAGE signaling, was also investigated. RESULTS: In both initial and replication cohorts, an increased MS risk was detected in RAGE p.82G>S variant allele carriers (odds ratio [OR] = 1.786, p = 0.0134 and OR = 1.732, p = 0.0210, respectively). This association signal persisted in subgroups of women and patients with relapsing-remitting MS. Moreover, compared with the wild-type 82GG carriers, carriers of the variant allele presented a faster progression of disability and a reduced serum sRAGE level. CONCLUSIONS: The present study provides preliminary evidence that the gain-of-function p.82G>S polymorphism in the RAGE gene is associated with an increased risk of MS in the Chinese population.

[Association of combined polymorphisms in MIP-1α and ApoE genes with the susceptibility of inflammatory bowel disease].

OBJECTIVE: To investigate whether the macrophage inflammatory protein 1 alpha (MIP-1α) and apolipoprotein E (ApoE) gene polymorphisms, either alone or in combination, affect the susceptibility to inflammatory bowel disease (IBD). METHODS: Genomic DNA of IBD patients with Crohn's disease (CD, n = 41) and with ulcerative colitis (UC, n = 142) and healthy controls (n = 160) was extracted and genotyped for the MIP-1α and ApoE gene polymorphisms by restriction fragment length polymorphism assay. RESULTS: MIP-1α -906(TA)(6)/(TA)(6) homozygotes had a significantly elevated risk of UC (OR = 1.909, 95%CI = 1.204 - 3.028). The carriers of APOE4ε4 were at a significantly higher risk for UC with OR of 2.379 (95% CI = 1.451 - 3.896). And a combination of these two loci, MIP-1α -906(TA)(6)/(TA)(6)/APOE4ε4 were strongly associated with a higher risk of UC (OR = 3.288; 95%CI = 1.777 - 6.084). CONCLUSION: The polymorphisms of MIP-1α -906 (TA)(6)/(TA)(6) and ApoE are probably independent genetic risk factors for UC. And the coexistence of both may exert an additive effect on the UC risks.

[Association of IL-8 gene polymorphisms with inflammatory bowel disease in Chinese patients].

OBJECTIVE: To investigate the association of interleukin 8 (IL-8) gene polymorphisms with the risks of inflammatory bowel disease (IBD). METHODS: Single nucleotide polymorphisms (SNPs) of IL-8 gene at -845 T/C, -738 T/A, -353 A/T, -251 T/A and +678 T/C were analyzed in 183 IBD patients. They included Crohn's disease (CD, n = 41), ulcerative colitis (UC, n = 142) and healthy controls (n = 160). The methods of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction-sequence specific primers (PCR-SSP) were employed. RESULTS: No association was observed between any of these five SNPs in IL-8 gene with the occurrence of IBD. A specific haplotype AAT (-353 A/T, -251 T/A & +678 T/C) was over-represented in UC cases when compared with controls (31.0% vs 23.7%, P = 0.046). But the distributions of this haplotype did not show significant difference between CD cases and controls. CONCLUSION: Our data support a significant but modest association between the AAT haplotype of IL-8 gene and UC (OR = 1.441, 95%CI 1.007 - 2.063).

Silencing of R-Spondin1 increases radiosensitivity of glioma cells.

Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma. We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival. Rspo1 expression was also associated with reduced survival rates in glioma patients after treatment with radiotherapy and temozolomide (RT-TMZ). Importantly, Rspo1 was dramatically upregulated after radiation treatment in patients with glioma. Rspo1 silencing by shRNA potentiated glioma cell death upon radiation treatment. In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition. Thus, combining radiotherapy with silencing of Rspo1 is a potential therapeutic approach.

A functional polymorphism of the microRNA-146a gene is associated with susceptibility to drug-resistant epilepsy and seizures frequency.

PURPOSE: Epilepsy is the third most common chronic brain disorder and is characterized by an enduring predisposition for seizures. Recently, a growing body of evidence has suggested that microRNA-146a (miR-146a) is upregulated in the brains of epilepsy patients and of mouse models; furthermore, miR-146a may be involved in the development and progression of seizures through the regulation of inflammation and immune responses. In this report, we performed a case-control study to analyze the relationship between the two potentially functional single nucleotide polymorphisms (SNPs) of the miR-146a gene (rs2910464 and rs57095329) and the risk of epilepsy in a Chinese population comprising 249 cases and 249 healthy controls. METHOD: Our study comprised 249 epilepsy patients and 249 healthy controls in two regions of China. The DNA was genotyped using the ABI PRISM SNapShot method. The statistical analysis was estimated using the chi-square test or Fisher's exact test. RESULTS: Our results indicated a significant association between the rs57095329 SNP of the miR-146a gene and the risk of drug resistant epilepsy (DRE) (genotypes, p = 0.0258 and alleles, p = 0.0108). Moreover, the rs57095329 A allele was found to be associated with a reduced risk of seizures frequency in DRE patients (all p < 0.001). However, the rs2910164 variant was not associated with epilepsy. CONCLUSION: Our data indicate that the rs57095329 polymorphism in the promoter region of miR-146a is involved in the genetic susceptibility to DRE and the seizures frequency.

Protectin D1 promotes resolution of inflammation in a murine model of lipopolysaccharide-induced acute lung injury via enhancing neutrophil apoptosis.

BACKGROUND: Protectin D1 (PD1), derived from docosahexaenoic acid, has been shown to control and resolve inflammation in some experimental models of inflammatory disorders. We investigated the protective roles of protectin D1 in pulmonary inflammation and lung injury induced by lipopolysaccharide (LPS). METHODS: Mice were randomly assigned to six groups (n = 6 per group): sham-vehicle group, sham-PD1 group, sham-zVAD-fmk group, LPS-vehicle group, LPS-PD1 group, and LPS-PD1-zVAD-fmk group. Mice were injected intratracheally with 3 mg/kg LPS or saline, followed 24 hours later by intravenous injection of 200 µg/mouse PD1 or vehicle. At the same time, some mice were also injected intraperitoneally with the pan-caspase inhibitor zVAD-fmk. Seventy-two hours after LPS challenge, samples of pulmonary tissue and bronchoalveolar lavage fluid were collected. Optical microscopy was used to examine pathological changes in lungs. Cellularity and protein concentration in bronchoalveolar lavage fluid were analyzed. Lung wet/dry ratios and myeloperoxidase activity were measured. Apoptosis of neutrophils in bronchoalveolar lavage fluid (BALF) was also evaluated by flow cytometry. RESULTS: Intratracheal instillation of LPS increased neutrophil counts, protein concentration in bronchoalveolar lavage fluid and myeloperoxidase activity, it induced lung histological injury and edema, and also suppressed apoptosis of neutrophils in BALF. Posttreatment with PD1 inhibited LPS-evoked changes in BALF neutrophil counts and protein concentration and lung myeloperoxidase activity, with the outcome of decreased pulmonary edema and histological injury. In addition, PD1 promoted apoptosis of neutrophils in BALF. The beneficial effects of PD1 were blocked by zVAD-fmk. CONCLUSION: Posttreatment with PD1 enhances resolution of lung inflammation during LPS-induced acute lung injury by enhancing apoptosis in emigrated neutrophils, which is, at least in part, caspase-dependent.

TCTP promotes glioma cell proliferation in vitro and in vivo via enhanced ß-catenin/TCF-4 transcription.

Background The translationally controlled tumor protein (TCTP) is a multifunctional protein that plays important roles in immune responses, cell proliferation, tumorigenicity and cell apoptosis. Here, we examined the clinical value of TCTP in glioma patient survival and investigated the functional roles and mechanism of TCTP in glioma development. Methods TCTP expression was determined through immunohistochemical staining, immunoblotting, and quantitative real-time PCR (qRT-PCR). TCTP or TCF-4 expression was silenced using short hairpin (sh) RNA. In vitro cell proliferation was detected using MTT, BrdU and colony formation assays, and in vivo tumor growth was performed using the xenograft model. TCTP/TCF-4/ß-catenin association was detected using a co-immunoprecipitation (co-IP) assay. TCF-4 transcription activity was detected using a TOPflash/FOPflash report gene assay. Wnt/ß-catenin-targeted gene expression was detected through Western blotting. Results TCTP protein levels were significantly elevated in high-grade gliomas compared with low-grade gliomas and normal brain tissues. Importantly, the expression of TCTP was significantly associated with poorer overall survival and disease-free survival, and TCTP also reduced the survival rate after treatment with radiotherapy and temozolomide (RT-TMZ) for glioma patients. The ectopic expression of TCTP enhanced glioma cell proliferation both in vitro and in vivo, whereas the knockdown of TCTP inhibited this effect. Similarly, the overexpression of TCTP increased ß-catenin binding to TCF-4, TOPflash report gene transcription activity, and the expression of Wnt/ß-catenin signaling target genes including c-Myc and cyclin D1; notably, the knockdown of TCTP reduced these effects. The knockdown of TCF-4 using shRNA rescued the enhanced cell proliferation induced by the overexpression of TCTP. Conclusion TCTP is associated with reduced survival of glioma patients and induces glioma tumor growth through enhanced Wnt/ß-catenin signaling.

Nuclear export of VP19C is not essential for replication of herpes simplex virus type 1.

BACKGROUND: Herpes simplex virus (HSV) type 1 has a 152 kb double-stranded DNA genome that may encode more than 80 gene products, many of which remain uncharacterized. The HSV-1 triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. Previous studies have demonstrated that HSV-1 VP19C contains an atypical nuclear localization signal and a functional nuclear export signal (NES), which are both important for the nucleocytoplasmic shuttling of VP19C. However, whether the VP19C NES is required for efficient HSV-1 production is unknown. FINDINGS: In the present study, a VP19C NES-mutated recombinant virus was generated by using bacterial artificial chromosome recombineering technology to investigate the role of VP19C nuclear export in HSV-1 replication. Our results demonstrate that the growth curves, plaque areas, subcellular localization and viral gene expression are indistinguishable between the VP19C NES-mutated virus and the wild-type virus. CONCLUSIONS: Our findings reported herein indicate abrogation of the nuclear export of VP19C did not affect HSV-1 replication and viral gene expression.

Postoperative plasma copeptin levels independently predict delirium and cognitive dysfunction after coronary artery bypass graft surgery.

Copeptin can reflect individual's stress state and are correlated with poor outcome of critical illness. The occurrence of postoperative delirium (POD) and cognitive dysfunction (POCD) is associated with worse outcome after coronary artery bypass graft (CABG) surgery. The present study aimed to investigate the ability of postoperative plasma copeptin level to predict POD and POCD in patients undergoing CABG surgery. Postoperative plasma copeptin levels of 108 patients were measured by an enzyme-linked immunosorbent assay. It was demonstrated that plasma copeptin levels were substantially higher in patients with POD than without POD (1.8±0.6 ng/mL vs. 1.1±0.3 ng/mL; P<0.001) and in patients with POCD than without POCD (1.9±0.6 ng/mL vs. 1.1±0.4 ng/mL; P<0.001). Plasma copeptin level and age were identified as independent predictors for POD [odds ratio (OR), 67.386; 95% confidence interval (CI), 12.031-377.426; P<0.001 and OR, 1.202; 95% CI, 1.075-1.345; P=0.001] and POCD (OR, 28.814; 95% CI, 7.131-116.425; P<0.001 and OR, 1.151; 95% CI, 1.030-1.285; P=0.003) using a multivariate analysis. For prediction of POD, the area under receiver operating characteristic curve (AUC) of the copeptin concentration (AUC, 0.883; 95% CI, 0.807-0.937) was markedly higher than that of age (AUC, 0.746; 95% CI, 0.653-0.825; P=0.020). For prediction of POCD, the AUC of the copeptin concentration (AUC, 0.870; 95% CI, 0.792-0.927) was markedly higher than that of age (AUC, 0.735; 95% CI, 0.641-0.815; P=0.043). Thus, postoperative plasma copeptin level may be a useful, complementary tool to predict POD and POCD in patients undergoing CABG surgery.

Gene expression profiling of Clostridium botulinum under heat shock stress.

During growth, C. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and pH change; however, its corresponding global transcriptional profile is uncharacterized. This study is the first description of the genome-wide gene expression profile of C. botulinum in response to heat shock stress. Under heat stress (temperature shift from 37°C to 45°C over a period of 15 min), 176 C. botulinum ATCC 3502 genes were differentially expressed. The response included overexpression of heat shock protein genes (dnaK operon, groESL, hsp20, and htpG) and downregulation of aminoacyl-tRNA synthetase genes (valS, queA, tyrR, and gatAB) and ribosomal and cell division protein genes (ftsZ and ftsH). In parallel, several transcriptional regulators (marR, merR, and ompR families) were induced, suggesting their involvement in reshuffling of the gene expression profile. In addition, many ABC transporters (oligopeptide transport system), energy production and conversion related genes (glpA and hupL), cell wall and membrane biogenesis related genes (fabZ, fabF, and fabG), flagella-associated genes (flhA, flhM, flhJ, flhS, and motAB), and hypothetical genes also showed changed expression patterns, indicating that they may play important roles in survival under high temperatures.

Transcriptome analysis of adaptive heat shock response of Streptococcus thermophilus.

Streptococcus thermophilus, a gram-positive facultative anaerobe, is one of the most important lactic acid bacteria widely used in the dairy fermentation industry. In this study, we have analyzed the global transcriptional profiling of S. thermophilus upon temperature change. During a temperature shift from 42°C to 50°C, it is found that 196 (10.4%) genes show differential expression with 102 up-regulated and 94 down-regulated at 50°C. In particular, 1) Heat shock genes, such as DnaK, GroESL and clpL, are identified to be elevated at 50°C; 2) Transcriptional regulators, such as HrcA, CtsR, Fur, MarR and MerR family, are differentially expressed, indicating the complex molecular mechanisms of S. thermophilus adapting to heat shock; 3) Genes associated with signal transduction, cell wall genes, iron homeostasis, ABC transporters and restriction-modification system were induced; 4) A large number of the differentially expressed genes are hypothetical genes of unknown function, indicating that much remains to be investigated about the heat shock response of S. thermophilus. Experimental investigation of selected heat shock gene ClpL shows that it plays an important role in the physiology of S. thermophilus at high temperature and meanwhile we confirmed ClpL as a member of the CtsR regulon. Overall, this study has contributed to the underlying adaptive molecular mechanisms of S. thermophilus upon temperature change and provides a basis for future in-depth functional studies.