Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble.

Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, the in vitro preparation of dsRNA is costly and laborious. In this study, we have developed a novel and interesting method designated as pfRCT (promoter-free rolling-circle transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without directional preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced into the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just-transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of the dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

Effects of n-3PUFAs on autophagy and inflammation of hypothalamus and body weight in mice.

OBJECTIVE: Fat-1 transgenic mice were used as a model to study the effect of endogenous n-3 polyunsaturated fatty acids (n-3PUFAs) on the body weight, inflammatory factors and autophagy proteins in hypothalamus to explore the mechanism of n-3PUFAs inhibiting obesity. METHOD: The mice were divided into two groups after genotype identification: fat-1 transgenic mice and wild-type mice. The body weight and body length of mice were measured at 14th week, and calculated the Lee 's index. The autophagosome in arcuate nucleus neurons was observed through electron microscopy; the expression of autophagy protein P62, LC3 and ATG7 in hypothalamus were detected and analyzed quantitatively by immunofluorescence and Western blot techniques. The mRNAs of inflammatory factor TNF-α, IL-6, IL-1ß, NF-kB, chemokine MCP-1, CCL5, CXCL12, CX3CL1, microglia markers TMEM119, GFAP were detected by real-time fluorescence quantitative PCR. RESULT: The Lee's index of fat-1 transgenic mice was lower than that of wild-type mice(P < 0.05). The autophagosome of the arcuate nucleus in fat-1 transgenic mice were more than those in wild-type mice, and the expression of autophagy-related protein P62 was significantly decreased (P < 0.05) in hypothalamus of fat-1 transgenic mice, while the expression of autophagy related protein ATG7 was significantly up-regulated (P < 0.05), and the ratio of LC3 II/I was significantly increased (P < 0.05), The results of qPCR showed that the mRNAs of TNF-α, IL-6, IL-1ß, NF-kB, MCP-1, CCL5, CXCL12, and GFAP was significantly down regulated (P < 0.05), but CX3CL1 was significantly up-regulated (P < 0.05) in hypothalamus of fat-1 transgenic mice. CONCLUSION: Fat-1 gene or n-3 PUFAs possesses the function of reducing body weight, which involves the enhancement of autophagy and reduction of inflammatory factor in hypothalamus.

The suppressive effect of co-inhibiting PD-1 and CTLA-4 expression on H22 hepatomas in mice.

OBJECTIVE: We investigated the suppressive effect of siRNA-mediated co-inhibition of PD-1 and CTLA-4 expression on H22 hepatomas in mice. METHODS: Murine H22 cells were cultured in vivo in ICR mice. An allograft tumor model was also established in another ICR mouse group. The tumor-bearing mice were randomly divided into four groups: control, single PD-1 siRNA, single CTLA-4 siRNA, and double PD-1 + CTLA-4 siRNAs. The survival time and physiological condition of the mice were observed after the injection of the siRNAs and placebo. The volume and weight of the solid tumor were measured to assess the inhibition of the tumor. To assess the effects of siRNAs on mouse immune function, the protein levels of IFN-γ and IL-10 in the blood and PD-L1 in the tumor and liver were determined using ELISA, and the mRNA levels of IFN-γ, PD-L1, PD-1, CTLA-4, IL-6 and Survivin in the tumor, liver and spleen were determined using quantitative RT-PCR. The ratios of Bax and Bcl-2 protein were determined via western blot to analyze the effect of siRNAs on tumor cell apoptosis. RESULTS: The anti-tumor effect appeared in all groups with siRNA-mediated inhibition. The tumor growth suppression was stronger in the group with double inhibition. The weight and volume of the tumors were significantly lower and the survival rate improved in the three siRNA groups. IFN-γ levels increased but IL-10 levels decreased in the blood of the siRNA group mice compared with the results for the control group. In the tumor and spleen tissue, the IFN-γ levels significantly increased, but in the liver tissue they significantly decreased in the three siRNA groups. The results of quantitative RT-PCR showed that the mRNAs for PD-1 and CTLA-4 were downregulated in spleen tissue in the three siRNA groups, while the PD-L1 mRNA and protein levels increased significantly in the tumor, but decreased in the liver. Survivin and IL-6 mRNA levels decreased in the tumor. Western blot results showed that ratio of Bax and Bcl-2 had significantly increased. These results indicated that downregulating PD-1 and CTLA-4 could increase the body's immune response and promote apoptosis of tumor cells. CONCLUSION: Co-inhibiting the expressions of PD-1 and CTLA-4 can effectively suppress the growth of H22 hepatoma and promote the apoptosis of tumor cells in mice. Blocking PD-1 and CTLA-4 can improve the vitality of T cells, and improve the immune environment and response.

Family-based association study between monoamine oxidase A (MAOA) gene promoter VNTR polymorphism and Tourette's syndrome in Chinese Han population.

To clarify the association of monoamine oxidase A- variable number of tandem repeat (MAOA-pVNTR) with susceptibility to Tourette's syndrome (TS) in Chinese Han population we discuss the genetic contribution of MAOA-VNTR in 141 TS patients including all their parents in Chinese Han population using transmission disequilibrium test (TDT) design. Our results revealed that no significant association was found in the MAOA gene promoter VNTR polymorphism and TS in Chinese Han population (TDT = 1.515, df = 1, p > 0.05). The negative result may be mainly due to the small sample size, but we don't deny the role of gene coding serotonergic or monoaminergic structures in the etiology of TS.

[DUOX2 mutations in children with congenital hypothyroidism].

OBJECTIVE: To study the features of DUOX2 mutations and genotype-phenotype relationship in children with congenital hypothyroidism (CH), in order to provide evidence for gene diagnosis and gene treatment of CH. METHODS: Blood samples were collected from 10 CH children with thyromegaly. Genomic DNA was extracted from peripheral blood leukocytes. All exons of DUOX2 gene were analyzed using PCR and direct sequencing. RESULTS: G3632A mutation in the exon 28 of DUOX2 that may result in arginine to histidine substitution at codon 1211 was found in one patient. T2033C mutation in the exon 17 of DUOX2 that may result in histidine to arginine substitution at codon 678 was found in three patients. They were all heterozygous mutations. CONCLUSIONS: Heterozygous mutations in DUOX2 may affect protein function and cause CH. The relationship between DUOX2 genotypes and clinical phenotypes is unclear and needs further studies.

Downregulation of KDR expression induces apoptosis in breast cancer cells.

Angiogenesis plays a crucial role in the growth, invasion and metastasis of breast cancer. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the key regulators of tumor angiogenesis. VEGFR-2, known as the kinase insert domain receptor (KDR), is a key receptor involved in malignant angiogenesis. We previously showed that knocking down KDR with short interference RNA (KDR-siRNA) markedly decreased KDR expression and suppressed tumor growth in a xenograft model. However, the mechanisms underlying the anti-cancer effects of KDR-siRNA are not clearly understood. This study aimed to elucidate the molecular mechanisms that induce apoptosis in human breast cancer MCF-7 cells after transfection with KDR-siRNA. We studied the effects of KDR-siRNA on proliferation, apoptosis, antiapoptotic and pro-apoptotic proteins, mitochondrial membrane permeability, cytochrome c release and caspase-3 activity. The results indicated that KDR-siRNA treatment significantly inhibited the proliferation and induced the apoptosis of MCF-7 cells, reduced the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, and increased the level of the pro-apoptotic protein Bax, resulting in a decreased Bcl-2/Bax ratio. KDR-siRNA also enhanced the mitochondrial membrane permeability, induced cytochrome c release from the mitochondria, upregulated apoptotic protease-activating factor-1 (Apaf-1), cleaved caspase-3, and increased caspase-3 activity in MCF-7 cells. Furthermore, KDR-siRNA-induced apoptosis in MCF-7 cells was blocked by the caspase inhibitor Z-VAD-FMK, suggesting a role of caspase activation in the induction of apoptosis. These results indicate that the Bcl-2 family proteins and caspase-related mitochondrial pathways are primarily involved in KDR-siRNAinduced apoptosis in MCF-7 cells and that KDR might be a potential therapeutic target for human breast cancer treatments.

Aberrant NDRG1 methylation associated with its decreased expression and clinicopathological significance in breast cancer.

BACKGROUND: Cancer cell differentiation is an important characteristic of malignant tumor and has a great impact on prognosis and therapeutic decision for patients. The N-myc downstream regulated gene 1 (NDRG1), a putative tumor suppression gene, is involved in the regulation of human cell differentiation and metastasis in various cancers. Changes in the status of methylation of the NDRG1 gene have not been studied in detail in human breast cancer. RESULTS: The MDA-MB-231 breast tumor cell line could express NDRG1. However, it was only expressed after treatment with 5-Aza-2'-deoxycytidine (AZA) in T47D cell line, which revealed that NDRG1 expression could modulated by DNA methylation. Therefore, the fragment surrounding the transcript start site of NDRG1 gene promoter was cloned after sodium bisulfite DNA treatment. A high density (66%) of methylation for human NDRG1 gene promoter was detected in T47D; however, there was only 16% of methylated CpG dinucleotides in the NDRG1 gene promoter in MDA-MB-231. DNA methylation in the NDRG1 promoter was detected in 31.1% of primary breast cancer samples. Furthermore, the NDRG1 promoter methylation correlated with the Tumor Node Metastasis (TNM) at stage III/IV, metastasis, lymph invasion, moderate and poor histological grade in the breast cancer patients. CONCLUSION: These findings suggest that the DNA methylation status of NDRG1 gene may play an important role in the pathogenesis and/or development of breast cancer, and the expression could be regulated by aberrant DNA methylation.

Fucoidan inhibited 4T1 mouse breast cancer cell growth in vivo and in vitro via downregulation of Wnt/ß-catenin signaling.

Fucoidan is a sulfated polysaccharide derived from brown algae and is known to possess anticancer properties. However, the relationship between fucoidan and ß-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, mouse breast cancer cells (4T1) were exposed to fucoidan to investigate the relationship between fucoidan and the Wnt/ß-catenin signaling pathway in vivo and in vitro. We found that fucoidan significantly inhibited cell growth, increased cell death, and induced G1 cell cycle arrest in 4T1 cells. Fucoidan also reduced ß-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity. Furthermore, fucoidan downregulated the expression of downstream target genes such as c-myc, cyclin D1, and survivin. Intraperitoneal injection of fucoidan in tumor-bearing mice reduced the tumor volume and weight. Fucoidan induced aberrant downregulation of ß-catenin in tumor tissues with a significant increase in apoptosis. Thus, our data suggested that fucoidan exerts its anticancer activity through downregulation of Wnt/ß-catenin signaling. Fucoidan may be an effective therapy for the chemoprevention and treatment of mouse breast cancer.

Decreased expression of the DBC2 gene and its clinicopathological significance in breast cancer: correlation with aberrant DNA methylation.

Loss of DBC2 (deleted in breast cancer 2) gene expression is frequent in breast cancer tissues. This can be explained by homozygous deletions or other mutations in a minority of cases but alternative mechanisms need to be investigated. Here, DBC2 expression was significantly suppressed compared with normal breast tissues in breast cancer tissues when analyzed by RT-PCR. Furthermore, DNA methylation on DBC2 was more prevalent in breast tumors than in normal tissues. DBC2 mRNA levels correlated with the degree of DBC2 methylation in breast cancer tissues and in a breast cancer cell line (T47D). Clinico-pathological correlation analysis showed that DBC2 promoter methylation was associated with tumor-node-metastasis stages II and III/IV, lymph node metastasis, p53 mutation, and HER2-positive status. Thus loss of DBC2 expression is caused by abnormal methylation of DBC2 and might have a role in breast cancer development.

Selection and Characterization of a Novel DNA Aptamer, Apt-07S Specific to Hepatocellular Carcinoma Cells.

BACKGROUND: The efficacy of traditional therapeutic methods for liver cancer is unsatisfying because of the poor targeting, and inefficient drug delivery system. A recent study has proven that aptamers, developed through cell-SELEX, could specifically recognize cancer cells and show great potential in the development of a delivery system for anticancer drugs. PURPOSE: To develop a hepatocellular carcinoma specific aptamer using two kinds of hepatocellular carcinoma cell lines, HepG2 and SMMC-7721, as double targets and a normal hepatocyte, L02, as a negative control cell. METHODS: Hepatocellular carcinoma specific aptamer was developed via cell-SELEX. The enrichment of the library was monitored by flow cytometric analysis. The specificity, affinity, and distribution of the candidate aptamer were explored. Further study was carried to assess its potential in drug delivery. RESULTS: The library was enriched after 14 rounds of screening. Candidate aptamer Apt-07S can recognize four kinds of hepatocellular carcinoma cells and show little cell-binding ability to normal cells and four cell lines of different cancer types, revealing a high specificity of Apt-07S. Confocal imaging showed that Apt-07S distributed both on the surface and in the cytoplasm of the two target cells. Moreover, an anti-sense nucleotide to gene Plk1 (ASO-Plk1) was connected at the 3' end of Apt-07S to form an integrated molecule (Apt-07S-ASO-Plk1); the functional analysis indicated that the structure of Apt-07S may help ASO-Plk1 enter the cancer cells. CONCLUSION: The study indicates that Apt-07S can specifically target HCC and may have potential in the delivery of anticancer drugs.

Effects of neuregulin on expression of MMP-9 and NSE in brain of ischemia/reperfusion rat.

The aim is to investigate the effects of neuregulin-1beta (NRG-1beta) on expression of matrix metalloproteinase-9 (MMP-9) and neuron-specific enolase (NSE) in brain tissue in rats following cerebral ischemia/reperfusion. One hundred and fifty adult healthy male Wistar rats were used in the present study. Ten of them were randomized into a sham-operation group (n = 10) and the rest suffered surgery operation of middle cerebral artery occlusion/reperfusion with intraluminal monofilament suture from the left external-internal carotid artery. As a result, 100 rats of successful models were randomly divided into a control group (n = 50) and a treatment group (n = 50). Rats in the treatment group were injected 1.5% NRG-1beta at a dosage of 0.3 microg/kg from the stump of the left external carotid artery into the internal carotid artery. The expressions of MMP-9 and NSE proteins were determined by immunohistochemical, immunofluorescent double labeling, and Western blot assay. Ischemia/reperfusion induced morphological changes of brain tissue, including neurocyte shrinkage, chromatin condensation, nuclei fragment, and gliacyte and endothelial cell swelling. NRG-1beta obviously reduced and delayed the cerebral damage. With the duration of ischemia, the expression of MMP-9 gradually increased in the control group. NRG-1beta decreased the level of MMP-9 compared with that in the control group (P < 0.01). NSE immunoreaction transiently elevated at the early stage of cerebral ischemia insult, and then gradually decreased in the control group. The administration of NRG-1beta significantly increased the level of NSE, and thus delayed the time and the degree of neuron damage. There were statistical differences in contrast to the control group (P < 0.01). There was no relationship between the expressions of the two proteins. MMP-9 might aim at various target cells at different stages and contribute to the inflammatory reaction after cerebral ischemia-reperfusion insult. NRG-1beta inhibits the activation of MMP-9 and development of inflammation, enhances the activity of NSE, improves the microenvironment of neuron survivals, and delays the phase of irreversible neuron necrosis. Therefore, NRG-1beta may play a neuroprotective role in cerebral ischemia/reperfusion.

The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo.

We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.

Fucoidan prevent murine autoimmune diabetes via suppression TLR4-signaling pathways, regulation DC/Treg induced immune tolerance and improving gut microecology.

BACKGROUND: This study was to investigate the effect and its possible mechanism of fucoidan on the development of spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice. METHODS: 7-week-old NOD mice were randomly divided into three groups: control group, low-dose (300 mg/kg) and high-dose (600 mg/kg) fucoidan-treatment groups. After 5 weeks of treatment, 10 mice per group were randomly selected to be sacrificed after feces collection. The remaining 12 mice per group were fed until 26 weeks of age to assess the incidence of diabetes. RESULTS: Treatment with fucoidan increased serum insulin level, delayed the onset and decreased the development of diabetes in NOD mice. Fucoidan reduced the levels of strong Th1 proinflammatory cytokines, but induced Th2-bias ed. cytokine response. And dentridic cells (DCs) in fucoidan treatment group were characterized as low expression of MHC class II and CD86 molecules. TLR4 expressions and the downstream molecules in pancreas were down-regulated in fucoidan-treated groups. There were significant differences in the composition of gut flora between NOD control group and fucoidan group. Lactobacillus and Akkermansia were significantly enriched in fucoidan group. CONCLUSIONS: Fucoidan could prevent the development of autoimmune diabetes in NOD mice via regulating DC/Treg induced immune tolerance, improving gut microecology, down-regulating TLR4 signaling pathway, and maintaining pancreatic internal environment.

Identification of Two Missense Mutations in DUOX1 (p.R1307Q) and DUOXA1 (p.R56W) That Can Cause Congenital Hypothyroidism Through Impairing H2O2 Generation.

Context: The DUOX/DUOXA systems play a key role in H2O2 generation in thyroid cells, which is required for iodine organification and thyroid hormone synthesis. DUOX2/DUOXA2 defects can cause congenital hypothyroidism (CH), but it is unknown whether DUOX1/DUOXA1 mutations can also cause CH. Objective: We aimed to identify DUOX1/DUOXA1 mutations and explore their role in the development of CH by investigating their functional impacts on H2O2 generation. Patients and Methods: Forty-three children with CH with goiter were enrolled, in whom all exons and flanking intronic regions of DUOX1/DUOXA1 were directly sequenced. We characterized the functional effects of identified mutations on the expression of DUOX1 and DUOXA1 and H2O2 generation. Results: We identified a heterozygous DUOX1 missense mutation (G > A base substitution at nucleotide 3920 in exon 31) that changed a highly conserved arginine to glutamine at residual 1307 (p.R1307Q) in patient 1. A heterozygous-missense mutation (c.166 C>T; p.R56W) was identified in DUOXA1 in patient 2. Functional studies demonstrated that both p.R1307Q mutant or p.R56W mutant decreased the DUOX1 expression at mRNA and protein levels, with a corresponding impairment in H2O2 generation (P < 0.01). The results also showed that intact DUOXA1 was required for full activity of DUOX1 and H2O2 generation. Conclusions: We have identified two heterozygous missense mutations in DUOX1 and DUOXA1 in two patients that can cause CH through disrupting the coordination of DUOX1 and DUOXA1 in the generation of H2O2. This study for the first time demonstrates that the DUOX1/DUOXA1 system, if genetically defective, can cause CH.

Antitumor effects of Isatin on human neuroblastoma cell line (SH-SY5Y) and the related mechanism.

The purpose of the study was to investigate the antitumor effects of Isatin and the related mechanism. Human neuroblastoma cells (SH-SY5Y) were exposed to Isatin at different concentrations for 48 h. Apoptotic features were demonstrated by means of nuclei staining with Hoechst 33258 and flow cytometry with propidium iodide (PI). Expressions of Bcl-2, Bax and vascular endothelial growth factor (VEGF) mRNA were analyzed via RT-PCR. Expressions of Bcl-2, Bax proteins and phosphorylated extracellular signal regulated protein kinases (ERKs, p42/p44) were analyzed via Western blot. Activation of caspase-3 was assayed by flow cytometry with anti-active caspase-3-McAb-PE. VEGF protein was determined by ELISA kits. And the results showed that apoptosis of SH-SY5Y cells were induced by Isatin in a dose-dependent manner. Expressions of Bcl-2, VEGF mRNA and Bcl-2, VEGF proteins were down-regulated, while expressions of Bax mRNA and Bax protein were not changed obviously. Expression of phosphorylated ERKs decreased, but the level of activated caspase-3 increased after treatment of Isatin. These results suggest that Isatin promotes the apoptosis of neuroblastoma cells, therefore, it might be a potential candidate for the treatment of neuroblastoma.

RETRACTED: The function of long non-coding RNA MT1JP in the development and progression of gastric cancer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Executive Editor (Chairman) as panels from Figures 3A,B and 4D are similar to panels from Figures 4A,B and 5E of the article published by Mingjun Bi, Hongmei Yu, Bin Huang and Cuiyan Tang in Gene 626 (2017) 337­343 http://dx.doi.org/10.1016/j.gene.2017.05.049. Also, Figures 3C and 4A are similar to Figures 4C and 5A of the Gene article. One of the conditions of submission of a paper for publication is that authors declare explicitly that their work is original and has not appeared in a publication elsewhere. As such this article represents an abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Inhibitory effect of vascular endothelial growth factors-targeted small interfering RNA on proliferation of gastric cancer cells.

AIM: To examine the effects of vascular endothelial growth factor (VEGF)-targeted small interfering RNA (siRNA) on proliferation of gastric cancer cells in vitro. METHODS: Several siRNAs were transfected into human gastric cancer cell line SGC-7901 with Lipofectamine 2000. Cells not transfected with Lipofectamine 2000 or scrambled (SCR) siRNA served as controls. The inhibitory effect of siRNA on the expression of VEGF mRNA and protein was detected by RT-PCR and ELISA. MTT assay was used to examine the inhibition rate of cell growth. The change in cell cycling of siRNA-treated cells was detected by flow cytometry. RESULTS: siRNA targeting human VEGF effectively inhibited the proliferation of gastric cancer cell line SGC-7901 and the distribution of cell cycle. The percentage of G(0)/G(1) phase was significantly higher in siRNA(1)- and siRNA(2)-transfected cells than in control cells. The expression of VEGF mRNA was significantly inhibited in siRNA(1)- and siRNA(2)-transfected cells compared with that in control cells. VEGF protein notably decreased in siRNA-transfected cells, but had no effect on SCR siRNA. CONCLUSION: VEGF siRNA inhibits proliferation of gastric cancer cells in vitro.

Apoptosis is induced by docosahexaenoic acid in breast cancer cells via death receptor and mitochondria-mediated pathways.

In the present study, the antitumor effect of n­3 fatty acid was evaluated, and the effect of docosahexaenoic acid (DHA) on the induction of apoptosis and its underlying mechanism were examined. Flow cytometry and western blot analysis were performed to analyze apoptosis and the expression of protein factors in human breast cancer cells. The data revealed that DHA inhibited the viability of MCF­7 breast cancer cells in vitro, and promoted cell death by the induction of apoptosis. DHA decreased the expression of B­cell lymphoma 2 (Bcl­2), whereas the expression of Bcl­2­associated X protein was increased. DHA was also shown to promote the release of Smac/Diablo and cytochrome c from the mitochondria. DHA increased the levels of cleaved caspase­8, ­9 and ­3. Additionally, the protein expression of tumor necrosis factor­related apoptosis­inducing ligand, death receptor 4 and Fas were increased following DHA treatment. In conclusion, DHA caused apoptosis of the human breast cancer cells in vitro through the death receptor and mitochondria­mediated pathways. The results of this study encourage further investigation of the effect of fish oil on the prevention and treatment of human breast cancer.

Detection of Diverse N-Acyl-Homoserine Lactones in Vibrio alginolyticus and Regulation of Biofilm Formation by N-(3-Oxodecanoyl) Homoserine Lactone In vitro.

Quorum sensing (QS) is a cell-to-cell communication system based on the exchange of small intercellular signal molecules, such as N-Acyl homoserine lactones (AHLs), which act as cell-density mediators of QS gene expression, and are highly variable both in types and amounts in most Gram-negative Proteobacteria. Understanding the regulation of AHLs may contribute to the elucidation of cell density-dependent phenomena, such as biofilm formation. Vibrio alginolyticus is among the most frequently observed marine opportunistic Vibrio pathogens. However, AHL production of this species and its effects on biofilm formation remain to be understood. Here, our study reported the diverse AHL profiles of 47 marine-isolated V. alginolyticus strains and the effects of exogenous 3-oxo-C10-HSL on biofilm formation under different temperature conditions (16°C and 28°C). A total of 11 detected AHLs were produced by the isolates, of which 3-OH-C4-HSL, 3-oxo-C10-HSL and 3-oxo-C14-HSL comprised the largest proportions. We also observed that moderate levels of exogenous 3-oxo-C10-HSL (10 and 20 µM) could induce or enhance biofilm formation and alter its structure, while high levels (40 and 100 µM) did not significantly improve and even inhibited biofilm formation in V. alginolyticus. Further, regulation by exogenous 3-oxo-C10-HSL was both concentration- and temperature-dependent in V. alginolyticus.