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Gene co-expression network analysis has been widely used in gene function annotation, especially for long noncoding RNAs (lncRNAs). However, there is a lack of effective cross-platform analysis tools. For biologists to easily build a gene co-expression network and to predict gene function, we developed GCEN, a cross-platform command-line toolkit developed with C++. It is an efficient and easy-to-use solution that will allow everyone to perform gene co-expression network analysis without the requirement of sophisticated programming skills, especially in cases of RNA-Seq research and lncRNAs function annotation. Because of its modular design, GCEN can be easily integrated into other pipelines.

Oxidative stress-induced neuronal cell death contributes significantly to the physiological processes of a number of neurological disorders. Polydatin (PD) has been reported to protect against Alzheimer's disease (AD), ischemic stroke and traumatic brain injury. However, the underlying neuroprotective mechanisms remain to be elucidated. The current study suggested that PD activates AKT/cAMP response element-binding protein (CREB) signaling and induces neuroglobin (Ngb) to protect neuronal cells from hydrogen peroxide (H2O2) in vitro. PD inhibited the H2O2-induced neuronal cell death of primary mouse cortical neurons and N2a cells. Functional studies showed that PD attenuated H2O2-induced mitochondrial dysfunction and mitochondrial reactive oxygen species production. Mechanistically, PD was verified to induce the phosphorylation of AKT and CREB and increase the protein level of Ngb. The luciferase assay results showed that Ngb transcriptional activity was activated by CREB, especially after PD treatment. It was further indicated that PD increased the transcription of Ngb by enhancing the binding of CREB to the promoter region of Ngb. Finally, Ngb knockdown largely attenuated the neuroprotective role of PD against H2O2. The results indicated that PD protected neuronal cells from H2O2 by activating CREB/Ngb signaling in neuronal cells, indicating that PD has a neuroprotective effect against neurodegenerative diseases.

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.

The chemical and physical structure of cationic liposomes pays an important effect on their gene transfection efficiency. Investigation on the structure-function relationship of cationic liposomes will guide the design of novel cationic liposomes with high transfection efficiency and biosafety. In this paper, two novel series of lipids based on the backbone of pentaerythritol and trimethylolpropane were discovered, and their gene transfection efficiencies were assayed in vitro. The four lipids 8c, 9c, 14b, and 15b, exhibited much better transfection efficiency in the HEK293 cell lines compared with Lipo2000, lipid 9c also showed good transfection efficiency in the SW480 cell lines. And the structure-efficiency relationship revealed that a hydroxyethyl polar head group boosted transfer potency in trimethylolpropane-type lipids, but reduced in pentaerythritol-type lipids.

Transcription factor AP-2β mediates the transcription of a number of genes implicated in mammalian development, cell proliferation, and carcinogenesis. Although the expression pattern of AP-2β has been analyzed in cervical cancer cell lines, the functions and molecular mechanism of AP-2β are unknown. Here, we found that AP-2β significantly inhibits TCF/LEF reporter activity. Moreover, AP-2β and β-catenin interact both in vitro through GST pull-down assays and in vivo by co-immunoprecipitation. We further identified the interaction regions to the DNA-binding domain of AP-2β and the 1-9 Armadillo repeats of β-catenin. Moreover, AP-2β binds with β-TrCP and promotes the degradation of endogenous β-catenin via the proteasomal degradation pathway. Immunohistochemistry analysis revealed a negative correlation between the two proteins in cervical cancer tissues and cell lines. Finally, functional analysis showed that AP-2β suppresses cervical cancer cell growth in vitro and in vivo by inhibiting the expression of Wnt downstream genes. Taken together, these findings demonstrated that AP-2β functions as a novel inhibitor of the Wnt/β-catenin signaling pathway in cervical cancer.

MicroRNA-140-5p (miRNA-140-5p) is important for embryonic bone development. In this study, we found that miRNA-140-5p and its binding site in the 3'UTR of bone morphogenetic protein 2 (BMP-2) are highly conserved among vertebrates, and miRNA-140-5p negatively regulates both zebrafish and human BMP-2 genes. Microinjection of miRNA-140-5p or BMP-2b morpholino into zebrafish embryos led to a similar phenotype, including shortened tails, curved trunks, and defects in cranial cartilage. Moreover, miRNA-140-5p injection induced zebrafish embryo malformation that could be significantly rescued by microinjection of BMP-2 mRNA. In conclusion, our results indicated that miRNA-140-5p regulates zebrafish embryonic bone development via targeting BMP-2.

Neuroglobin is an endogenous neuroprotective protein, but the underlying neuroprotective mechanisms remain to be elucidated. Our previous yeast two-hybrid screening study identified that Dishevelled-1, a key hub protein of Wnt/&beta, Catenin signaling, is an interaction partner of Neuroglobin. In this study, we further examined the role of Neuroglobin in regulating Dishevelled-1 and the downstream Wnt/&beta, Catenin and NF&kappa, B signaling pathway. We found that Neuroglobin directly interacts with Dishevelled-1 by co-immunoprecipitation, and the two proteins are co-localized in both cytoplasma and nucleus of SK-N-SH cells. Moreover, the ectopic expression of Neuroglobin promotes the degradation of exogenous and endogenous Dishevelled-1 through the proteasomal degradation pathway. Furthermore, our results showed that Neuroglobin significantly inhibits the luciferase activity of Topflash reporter and the expression of &beta, Catenin mediated by Dishevelled-1 in SK-N-SH cells. In addition, we also documented that Neuroglobin enhances TNF-&alpha, induced NF&kappa, B activation via down-regulating Dishevelled-1. Finally, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays showed that Neuroglobin is an important neuroprotectant that protects SK-N-SH cells from TNF-&alpha, induced decrease in cell viability. Taken together, these findings demonstrated that Neuroglobin functions as an important modulator of the Wnt/&beta, B signaling pathway through regulating Dishevelled-1.

Tumor necrosis factor alpha (TNFα)-induced protein 1 (TNFAIP1) was originally identified as a protein involved in DNA replication, DNA damage repair, apoptosis and the progression of certain diseases, such as Alzheimer's disease. In the present study, forskolin, a stimulant of cyclic adenosine monophosphate (cAMP), was found to significantly reduce human TNFAIP1 mRNA levels and TNFAIP1 promoter activity in the SKNSH human neuroblastoma cell line as indicated by polymerase chain reaction analysis and a luciferase reporter assay. The association between transcription factor cAMP response element‑binding protein (CREB) and TNFAIP1 was further investigated using loss- and gain of function-studies with western blot analysis and luciferase reporter assays. The CREB-specific inhibitor KG‑501 significantly increased TNFAIP1 protein levels, while overexpression of wild‑type CREB, but not CREB mutated at ser133a or its DNA-binding site, significantly decreased human TNFAIP1 protein levels and TNFAIP1 promoter activity in SKNSH cells. Furthermore, two CRE sites located at ‑285 and ‑425 bp of the human TNFAIP1 promoter were identified to be responsible for CREB‑induced inhibition of human TNFAIP1 promoter activity. Chromatin immunoprecipitation assays confirmed that CREB bound to the TNFAIP1 promoter region harboring these two CRE sites. A further luciferase reporter assay demonstrated that CREB phosphorylation on ser133 was responsible for forskolin‑induced inhibition of TNFAIP1 expression. In conclusion, the present study suggested that CREB is a negative regulator of the TNFAIP1 gene.

Glycolipids might become a new type of promising non-viral gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, appropriate density and distribution of positive charges, high transfer efficiency and potential targeting function. In this study, four kinds of L-arabinose-based cationic glycolipids (Ara-DiC12MA, Ara-DiC14MA, Ara-DiC16MA and Ara-DiC18MA) containing quaternary ammonium as hydrophilic headgroup and two alkane chains as hydrophobic domain were synthesized and characterized. They were observed to have strong affinities for plasmid DNA (pDNA) and siRNA, the pDNA can be completely condensed at N/P ratio less than 2, and the siRNA can be completely retarded at N/P ratio less than 3. The dynamic light scattering (DLS) experiment and atomic force microscopy (AFM) experiment demonstrated that cationic lipids and their lipoplexes possessed suitable particle sizes with near-spherical shape and proper ζ-potentials for cell transfection. The Ara-DiC16MA liposome was found to have good transfection efficacy in HEK293, PC-3 and Mat cells compared with other three kinds of liposomes, and also maintain low cytotoxicity and better uptake capability in vitro. Furthermore, the gene silencing assay showed that Ara-DiC14MA and Ara-DiC16MA liposomes have demonstrated effective delivery and higher gene knockdown activity (>80%) in the above mentioned cells than Lipofectamine 2000. These results indicated Ara-DiC16MA can be developed for efficient and low toxic gene delivery.

Prediction of microRNA–mRNA interaction typically relies on bioinformatic methods, but these methods only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. A major obstacle to the miRNA research has been the lack of experimental procedures for the identification of miRNA–mRNA interactions. Recently, a few studies have attempted to explore experimental methods to isolate and identify miRNA targets or miRNAs targeting a single gene. Here, we developed an more convenient experimental approach for the isolation and identification of miRNAs targeting a single gene by applying short biotinylated DNA anti-sense oligonucleotides mix to enhanced green fluorescent protein (EGFP) mRNA which was fused to target gene mRNA. This method does not require a design of different anti-sense oligonucleotides to any mRNA. This is a simple and an efficient method to potentially identify miRNAs targeting specific gene mRNA combined with chip screen.

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10−8) level: 14q24.2 (rs227425, P-value 3.98 × 10−13, SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10−9, CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

To investigate the effects of curcumin on proliferation and apoptosis in testicular cancer cells in vitro and to investigate its molecular mechanisms of action. NTera-2 human malignant testicular germ cell line and F9 mouse teratocarcinoma stem cell line were used. The anti-proliferative effect was examined using MTT and colony formation assays. Hoechst 33258 staining, TUNEL and Annexin V-FITC/PI staining assays were used to analyze cell apoptosis. Protein expression was examined with Western blot analysis and immunocytochemical staining. Curcumin (5, 10 and 15 μmol/L) inhibited the viability of NTera-2 cells in dose- and time-dependent manners. Curcumin significantly inhibited the colony formation in both NTera-2 and F9 cells. Curcumin dose-dependently induced apoptosis of NTera-2 cells by reducing FasL expression and Bcl-2-to-Bax ratio, and activating caspase-9, -8 and -3. Furthermore, curcumin dose-dependently reduced the expression of AP transcription factor AP-2γ in NTera-2 cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Curcumin inhibited ErbB2 expression, and decreased the phosphorylation of Akt and ERK in NTera-2 cells. Curcumin induces apoptosis and inhibits proliferation in NTera-2 cells via the inhibition of AP-2γ-mediated downstream cell survival signaling pathways.

Aspirin has been reported to trigger apoptosis in various cancer cell lines. However, the detailed mechanisms involved remain elusive. The present study aimed to investigate whether aspirin plays a role in apoptosis of MDA-MB-453 cells. The effect of aspirin on the proliferation of human MDA-MB-453 cells breast cancer cells was evaluated using MTT assay, flow cytometry and western blotting. The present study reports that aspirin induces the apoptosis of MDA‑MB‑453 breast cancer cells which was attributed to the increased expression and activation of caspase‑3. Moreover, AP‑2α, a transcription factor highly expressed in MDA‑MB‑453 cells, was identified as a negative regulator of caspase‑3 transcription and AP‑2α was attenuated following aspirin treatment. Therefore, aspirin may increase the expression of caspase‑3 by inducing the degradation of AP‑2α, which increases activated caspase‑3 expression, thereby triggering apoptosis in MDA‑MB‑453 cells. Thus, aspirin may be used in breast cancer therapy.

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

Participates in actin remodeling through Rac and receptor endocytosisvia Rab5. Here, we used yeast two-hybrid systemwith Eps8 as bait to screen a human brain cDNA library.ITSN2 was identified as the novel binding factor of Eps8. Theinteraction between ITSN2 and Eps8 was demonstrated by thein vivo co-immunoprecipitation and colocalization assays andthe in vitro GST pull-down assays. Furthermore, we mappedthe interaction domains to the region between amino acids260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition,protein stability assays and immunofluorescence analysisshowed ITSN2 overexpression induced the degradation ofEps8 proteins, which was markedly alleviated with the lysosomeinhibitor NH4Cl treatment. Taken together, our resultssuggested ITSN2 interacts with Eps8 and stimulates the degradationof Eps8 proteins. [BMB reports 2012; 45(3): 183-188]

TNFAIP1 is a protein which can be induced by tumor necrosis factoralpha (TNFalpha) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2beta was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.

Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a co-activator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.

Intersectin-2Long (ITSN2L) is a multi-domain protein participating in endocytosis and exocytosis. In this study, RABEP1 was identified as a novel ITSN2L interacting protein using a yeast two-hybrid screen from a human brain cDNA library and this interaction, specifically involving the ITSN2L CC domain and RABEP1 CC3 regions, was further confirmed by in vitro GST (glutathione-S-transferase) pull-down and in vivo co-immunoprecipitation assays. Corroboratively, we observed that these two proteins co-localize in the cytoplasm of mammalian cells. Furthermore, over-expression of ITSN2L promotes RABEP1 degradation and represses RABEP1-enhanced endosome aggregation, indicating that ITSN2L acts as a negative regulator of RABEP1. Finally, we showed that ITSN2L and RABEP1 play opposite roles in regulating endocytosis. Taken together, our results indicate that ITSN2L interacts with RABEP1 and stimulates its degradation in regulation of endocytosis.

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNA target sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)- and femoral neck (FN)-bone mineral density (BMD). In stage I, 41 102 poly-miRTSs were meta-analyzed in seven cohorts with a genome-wide significance (GWS) α = 0.05/41 102 = 1.22 × 10(-6). By applying α = 5 × 10(-5) (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P = 7.67 × 10(-6) and 1.58 × 10(-5)) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P = 5.08 × 10(-3)) at α = 0.10/11 = 9.09 × 10(-3). PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P = 7.55 × 10(-6)) at α = 0.05/2 = 0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P = 8.87 × 10(-12)). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation.

Over the last few years, RNA Interference (RNAi), a naturally occurring mechanism of gene regulation conserved in plant and mammalian cells, has opened numerous novel opportunities for basic research across the field of biology. While RNAi has helped accelerate discovery and understanding of gene functions, it also has great potential as a therapeutic and potentially prophylactic modality. Challenging diseases failing conventional therapeutics could become treatable by specific silencing of key pathogenic genes. More specifically, therapeutic targets previously deemed "undruggable" by small molecules, are now coming within reach of RNAi based therapy. For RNAi to be effective and elicit gene silencing response, the double-stranded RNA molecules must be delivered to the target cell. Unfortunately, delivery of these RNA duplexes has been challenging, halting rapid development of RNAi-based therapies. In this review we present current advancements in the field of siRNA delivery methods, including the pros and cons of each method.

The etiology and significance of genomic instability (GIN), a hallmark of human cancers, remains controversial. The paradigm that inactivation of tumor suppressors [e.g. p53 or adenomatous polyposis coli (APC) genes] leads to GIN is largely based on experiments in vitro and in animal models. It remains unclear whether GIN is a cause or a result of cancer, particularly in patients. Precancerous Barrett's esophagus (BE) provides a clinical model to investigate GIN in cancer progression. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (ten cases, including five cases with multilayered epithelium (ME)), BE-associated esophageal adenocarcinoma (ten cases), or with normal gastro-esophageal junction (five cases). Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of p53 and APC were determined by immunohistochemistry. Increased p53 expression, a measurement of p53 mutations, was observed in BE with high grade dysplasia (HGD) and in BE-associated esophageal cancer (EC). The expression of wild type APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities were found in all EC samples. Numeric changes of chromosome 7, 11 and 12 were observed in BE in 14%, 64% and 43% of cases, respectively. Aneusomy of chromosome 11 and 12 were found in ME and in BE without dysplasia, in the presence of normal expression pattern of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stages prior to changes in tumor suppressor genes (p53 and APC) in BE-associated tumorigenesis in patients, suggesting that GIN may serve as a causative link between chronic inflammation and cancer.

As a member of the polymerase delta-interacting protein 1 (PDIP1) gene family, potassium channel tetramerisation domain-containing 10 (KCTD10) interacts with proliferating cell nuclear antigen (PCNA) and polymerase δ, participates in DNA repair, DNA replication and cell-cycle control. In order to further investigate the physiological functions of KCTD10, we generated the KCTD10 knockout mice. The heterozygous KCTD10(+/-) mice were viable and fertile, while the homozygous KCTD10(-/-) mice showed delayed growth from E9.0, and died at approximately E10.5, which displayed severe defects in angiogenesis and heart development. Further study showed that VEGF induced the expression of KCTD10 in a time- and dose-dependent manner. Quantitative real-time PCR and western blotting results revealed that several key members in Notch signaling were up-regulated either in KCTD10-deficient embryos or in KCTD10-silenced HUVECs. Meanwhile, the endogenous immunoprecipitation (IP) analysis showed that KCTD10 interacted with Cullin3 and Notch1 simultaneously, by which mediating Notch1 proteolytic degradation. Our studies suggest that KCTD10 plays crucial roles in embryonic angiogenesis and heart development in mammalians by negatively regulating the Notch signaling pathway.

Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both in vivo by co-immunoprecipitation as well as in vitro through GST pull-down assays. We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.

Neuroglobin (Ngb) has been demonstrated to be a novel neuroprotective protein that protects against hypoxia/ischemia and oxidative stress-induced injury in the nervous system. However, the regulation mechanisms of Ngb gene expression under both normal resting and stress conditions have not been fully elucidated. The cyclic AMP response element binding protein (CREB) is a key transcription factor that regulates a variety of pro-survival genes, but its role in regulating the neuroprotective gene Ngb has not been studied. In this study we investigated the transcriptional regulation of mouse Ngb gene by CREB in mouse neuroblastoma cell line N2a. Our results showed that CREB knockdown decreased Ngb gene expression, and overexpression of the wild-type CREB, but not the mutant CREB, significantly increased Ngb gene expression in N2a cells. Moreover, a cAMP response element (CRE) site located at −854 in the promoter region of mouse Ngb gene was found to be responsible for both basal and CREB-induced Ngb promoter activity. Using chromatin immunopreciptation (ChIP) assays, we found that CREB could bind to the Ngb promoter region spanning from −1016 to −793 that harbors the CRE site. Taken together, our results suggested that transcription factor CREB participates in the transcriptional regulation of mouse Ngb gene.

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.

The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger domain-containing protein tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) was first identified as a gene whose expression can be induced by the tumor necrosis factor alpha. Some studies showed that TNFAIP1 may function in DNA replication, apoptosis and human diseases. However, the definite functions and the mechanisms of TNFAIP1 are poorly known. In this study, we performed a yeast two-hybrid assay and used TNFAIP1 as the bait to screen human brain cDNA library. Potassium channel tetramerisation domain containing 10 (KCTD10) was identified as TNFAIP1-interacting partner. The KCTD10–TNFAIP1 interaction was then confirmed by the in vitro GST pull-down assays and the in vivo co-immunoprecipitation and colocalization assays. In addition, protein degradation and ubiquitin assays revealed TNFAIP1 overexpression resulted in ubiquitin-mediated degradation of KCTD10 proteins, which was significantly alleviated with the proteasome inhibitor MG132 treatment. Furthermore, transient transfection assays with two reporters showed that TNFAIP1 and KCTD10 inhibited the transcriptional activities of nuclear factor kappa B (NF-κB) and activating protein-1 reporters. Taken together, our results indicated the novel interaction and function between KCTD10 and TNFAIP1 in human PDIP1 family.

Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.

Signal regulatory protein α (SIRPα) is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. We hypothesized that interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. Here, we show that pig CD47 does not interact with mouse SIPRα. Similar to CD47−/− mouse cells, porcine red blood cells (RBCs) failed to induce SIRPα tyrosine phosphorylation in mouse macrophages. Blocking SIRPα with antimouse SIRPα mAb (P84) significantly enhanced the phagocytosis of CD47+/+ mouse cells, but did not affect the engulfment of porcine or CD47−/− mouse cells by mouse macrophages. CD47-deficient mice, whose macrophages do not phagocytose CD47−/− mouse cells, showed markedly delayed clearance of porcine RBCs compared with wild-type mouse recipients. Furthermore, mouse CD47 expression on porcine cells markedly reduced their phagocytosis by mouse macrophages both in vitro and in vivo. These results indicate that interspecies incompatibility of CD47 contributes significantly to phagocytosis of xenogeneic cells by macrophages and suggest that genetic manipulation of donor CD47 to improve its interaction with the recipient SIRPα may provide a novel approach to prevent phagocyte-mediated xenograft rejection.

RNA-interference (RNAi) is a potent mechanism, conserved from plants to humans for specific silencing of genes, which holds promise for functional genomics and gene-targeted therapies. Here we show that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo. Nonpathogenic Escherichia coli were engineered to transcribe shRNAs from a plasmid containing the invasin gene Inv and the listeriolysin O gene HlyA, which encode two bacterial factors needed for successful transfer of the shRNAs into mammalian cells. Upon oral or intravenous administration, E. coli encoding shRNA against CTNNB1 (catenin beta-1) induce significant gene silencing in the intestinal epithelium and in human colon cancer xenografts in mice. These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies.