WNK-SPAK/OSR1-NCC kinase signaling pathway as a novel target for the treatment of salt-sensitive hypertension.

Hypertension is the most prevalent health condition worldwide, affecting ~1 billion people. Gordon's syndrome is a form of secondary hypertension that can arise due to a number of possible mutations in key genes that encode proteins in a pathway containing the With No Lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1). This pathway regulates the activity of the thiazide-sensitive sodium chloride cotransporter (NCC), which is responsible for NaCl reabsorption in the distal nephron. Therefore, mutations in genes encoding proteins that regulate the NCC proteins disrupt ion homeostasis and cause hypertension by increasing NaCl reabsorption. Thiazide diuretics are currently the main treatment option for Gordon's syndrome. However, they have a number of side effects, and chronic usage can lead to compensatory adaptations in the nephron that counteract their action. Therefore, recent research has focused on developing novel inhibitory molecules that inhibit components of the WNK-SPAK/OSR1-NCC pathway, thereby reducing NaCl reabsorption and restoring normal blood pressure. In this review we provide an overview of the currently reported molecular inhibitors of the WNK-SPAK/OSR1-NCC pathway and discuss their potential as treatment options for Gordon's syndrome.

The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii.

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.

Targeting Cullin-RING Ubiquitin Ligases and the Applications in PROTACs.

Cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases, have become an attractive target for drug discovery, primarily due to their ability to regulate the degradation of numerous functionally and structurally diverse proteins, thereby controlling a myriad of biological processes. As the abnormal expressions of CRLs and their substrate proteins are associated with human diseases, elucidating their roles in these physiological and pathological processes will facilitate CRL-targeting drug development for the treatment of these diseases. Notably, these studies are also providing new concepts for the design of potential small-molecule therapeutics targeting CRLs and for the use of CRLs to degrade "undruggable" proteins. In this chapter, we systematically review the development of small molecules that target CRLs and especially emphasize the applications of CRLs in a chemical chimera for protein degradation, termed proteolysis-targeting chimeras (PROTACs).

The Bardet-Biedl syndrome protein complex regulates cell migration and tissue repair through a Cullin-3/RhoA pathway.

Cell motility and migration play critical roles in various physiological processes and disease states. Here, we show that the BBBsome, a macromolecule composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1, is a critical determinant of cell migration and wound healing. Fibroblast cells derived from mice or humans harboring a homozygous missense mutation (BBS1M390R/M390R) that disrupt the BBSome exhibit defects in migration and wound healing. Furthermore, we demonstrate that BBS1M390R/M390R mice have significantly delayed wound closure. In line with this, we provide data suggesting that BBS1M390R/M390R fibroblasts have impaired platelet-derived growth factor-AA (PDGF) receptor-α signaling, a key regulator of directional cell migration acting as a chemoattractant during postnatal migration responses such as wound healing. In addition, we show that BBS1M390R/M390R fibroblasts have upregulated RhoA expression and activity. The relevance of RhoA upregulation is demonstrated by the ability of RhoA-kinase inhibitor Y27632 to partially rescue the migration defect of BBS1M390R/M390R fibroblasts cells. We also show that accumulation of RhoA protein in BBS1M390R/M390R fibroblasts cells is associated with reduction and inactivation of the ubiquitin ligase Cullin-3. Consistent with this, Cullin-3 inhibition with MLN4924 is sufficient to reduce migration of normal fibroblasts. These data implicate the BBSome in cell motility and tissue repair through a mechanism that involves PDGF receptor signaling and Cullin-3-mediated control of RhoA.

Cullin 3 regulates ADAMs-mediated ectodomain shedding of amphiregulin.

A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.

Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics via Survivin.

The Cullin9 gene encodes a putative E3 ligase that serves a wide variety of biological functions in mitosis, whereas its roles in meiosis have not yet clearly defined. Here, we report that Cullin9 accumulates on the spindle apparatus and colocalizes with the microtubule fibers during mouse oocyte meiotic maturation. Depletion of Cullin9 by morpholino microinjection results in a remarkably higher rate of disorganized spindles and misaligned chromosomes in oocytes, which is coupled with the impaired kinetochore-microtubule attachments. Resultantly, the incidence of aneuploid eggs significantly increases in Cullin9-depleted oocytes. Moreover, we show that Cullin9 controls Survivin's protein level during meiotic maturation, and thus regulates microtubule stability in oocytes. Thus, our study assigns a new meiotic function to Cullin9 and reveals that it prevents mouse eggs from aneuploidy by regulating microtubule dynamics via Survivin.

Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.

Loss of CUL4A expression is underlying cisplatin hypersensitivity in colorectal carcinoma cells with acquired trabectedin resistance.

BACKGROUND: Colorectal carcinoma (CRC) is the third most common cancer worldwide. Platinum-based anticancer compounds still constitute one mainstay of systemic CRC treatment despite limitations due to adverse effects and resistance development. Trabectedin has shown promising antitumor effects in CRC, however, again resistance development may occur. In this study, we aimed to develop strategies to circumvent or even exploit acquired trabectedin resistance in novel CRC treatment regimens. METHODS: Human HCT116 CRC cells were selected for acquired trabectedin resistance in vitro and characterised by cell biological as well as bioinformatic approaches. In vivo xenograft experiments were conducted. RESULTS: Selection of HCT116 cells for trabectedin resistance resulted in p53-independent hypersensitivity of the selected subline against cisplatin. Bioinformatic analyses of mRNA microarray data suggested deregulation of nucleotide excision repair and particularly loss of the ubiquitin ligase CUL4A in trabectedin-selected cells. Indeed, transient knockdown of CUL4A sensitised parental HCT116 cells towards cisplatin. Trabectedin selected but not parental HCT116 xenografts were significantly responsive towards cisplatin treatment. CONCLUSIONS: Trabectedin selection-mediated CUL4A loss generates an Achilles heel in CRC cancer cells enabling effective cisplatin treatment. Hence, inclusion of trabectedin in cisplatin-containing cancer treatment regimens might cause profound synergism based on reciprocal resistance prevention.

A Human Long Non-Coding RNA ALT1 Controls the Cell Cycle of Vascular Endothelial Cells Via ACE2 and Cyclin D1 Pathway.

BACKGROUND/AIMS: ALT1 is a novel long non-coding RNA derived from the alternatively spliced transcript of the deleted in lymphocytic leukemia 2 (DLEU2). To date, ALT1 biological roles in human vascular endothelial cells have not been reported. METHODS: ALT1 was knocked down by siRNAs. Cell proliferation was analyzed by cck-8. The existence and sequence of human ALT1 were identified by 3' rapid amplification of cDNA ends. The interaction between lncRNA and proteins was analyzed by RNA-Protein pull down assay, RNA immunoprecipitation, and mass spectrometry analysis. RESULTS: ALT1 was expressed in human umbilical vein endothelial cells (HUVECs). The expression of ALT1 was significantly downregulated in contact-inhibited HUVECs and in hypoxia-induced, growth-arrested HUVECs. Knocking down of ALT1 inhibited the proliferation of HUVECs by G0/G1 cell cycle arrest. We observed that angiotensin converting enzyme Ⅱ(ACE2) was a direct target gene of ALT1. Knocking-down of ALT1 or its target gene ACE2 could efficiently decrease the expression of cyclin D1 via the enhanced ubiquitination and degradation, in which HIF-1α and protein von Hippel-Lindau (pVHL) might be involved. CONCLUSION: The results suggested the human long non-coding RNA ALT1 is a novel regulator for cell cycle of HUVECs via ACE2 and cyclin D1 pathway.

Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells.

The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition.

CUL4A expression in pediatric osteosarcoma tissues and its effect on cell growth in osteosarcoma cells.

Osteosarcoma (OS) is the most common bone malignancy in the pediatric population, and it comprises about 3 % of all pediatric tumors. Aberrant expression of the Cullin 4A (CUL4A) is found in many tumor types, but the role of CUL4A in OS progression remains largely unknown. The aim of this study was to investigate the expression and function of CUL4A in OS. CUL4A expression was detected in 30 samples of surgically resected OS and matched tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Cell proliferation was assessed by MTT, and migration and invasion were analyzed by Transwell and Matrigel assays after CUL4A knockdown in OS in vitro. Our result showed increased CUL4A expression in OS tissues. CUL4A knockdown inhibited the proliferation of MG63 cells. Furthermore, CUL4A siRNA ameliorated the migration and invasion of MG63 cell lines with altered expression of epithelial-mesenchymal transition (EMT)-associated molecules. Taken together, our findings indicate that CUL4A plays a pivotal role in OS progression and may serve as a potential marker for clinical diagnosis and target for therapy.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation.

In the last decade, the ubiquitin-proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin-RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

Downregulation of MicroRNA-145 Caused by Hepatitis B Virus X Protein Promotes Expression of CUL5 and Contributes to Pathogenesis of Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BACKGROUND: Hepatitis B viral infection-induced hepatocellular carcinoma (HCC) is a major threat to human health in China. Hepatitis B virus X protein (HBX), an HBV protein, has been reported to be involved in regulating the cellular activities of the host cells and is responsible for HCC oncogenesis. METHODS AND RESULTS: In this study, we performed real-time PCR in tumor tissue samples collected from 53 HCC patients (25 HBV-positive cases and 28 HBV-negative cases) to screen the candidate miRNAs that have previously been reported to be aberrantly expressed in HBV-associated HCC and found that miR-145 was significantly downregulated. The following computational analysis identified CUL5 and RAB5C as virtual targets of miR-145, whereas only CUL5 was verified as a validated target gene of miR-145 in liver cells via luciferase reporter assay. In line with this result, we found that both the mRNA and protein expression levels of CUL5 were significantly higher in HBV-positive than in HBV-negative HCC. An in vitro experiment demonstrated a significant decrease in the expression of miRNA-145, a substantial increase in the mRNA and protein expression of CUL5, and an enhanced proliferation of HBX over-expressing HepG2 cells compared with the control. In HepG2.2.15, we found significant decreases in both the expression of CUL5 and the cell growth rate of H cells transfected with 60 nM miR-145 mimics compared with the scramble controls. CONCLUSION: HBV infection promotes cell growth, at least partially, through the HBX-induced downregulation of miRNA-145 expression, which is responsible for the oncogenesis of HBV-associated HCC.

New strategies to inhibit KEAP1 and the Cul3-based E3 ubiquitin ligases.

E3 ubiquitin ligases that direct substrate proteins to the ubiquitin-proteasome system are promising, though largely unexplored drug targets both because of their function and their remarkable specificity. CRLs [Cullin-RING (really interesting new gene) ligases] are the largest group of E3 ligases and function as modular multisubunit complexes constructed around a Cullin-family scaffold protein. The Cul3-based CRLs uniquely assemble with BTB (broad complex/tramtrack/bric-à-brac) proteins that also homodimerize and perform the role of both the Cullin adapter and the substrate-recognition component of the E3. The most prominent member is the BTB-BACK (BTB and C-terminal Kelch)-Kelch protein KEAP1 (Kelch-like ECH-associated protein 1), a master regulator of the oxidative stress response and a potential drug target for common conditions such as diabetes, Alzheimer's disease and Parkinson's disease. Structural characterization of BTB-Cul3 complexes has revealed a number of critical assembly mechanisms, including the binding of an N-terminal Cullin extension to a bihelical '3-box' at the C-terminus of the BTB domain. Improved understanding of the structure of these complexes should contribute significantly to the effort to develop novel therapeutics targeted to CRL3-regulated pathways.

BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis.

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

Aberrant reduction of MiR-141 increased CD47/CUL3 in Hirschsprung's disease.

BACKGROUND: MiR-141 has been confirmed to be associated with various human diseases. However, whether miR-141 is involved in the pathogenesis of Hirschsprung's disease (HSCR) remains unknown. Here, we design the experiment to reveal the relationship between miR-141 and HSCR. METHODS: Quantitative real-time PCR and Western blot were used to detect the expression levels of miR-141 and its potential genes in 70 tissues of HSCR compared with 60 controls. Bisulfite sequencing PCR (BSP) assay was applied to explain the possible mechanism of the aberrant expression level of miR-141. We employed a dual-luciferase reporter assay to validate the regulation relation between miR-141 and CD47/CUL3. Cell migration, proliferation, apoptosis, and cell cycle progression were examined by transwell assay, MTT assay, and flow cytometry, respectively. RESULTS: MiR-141 was down-regulated whereas CD47 and CUL3 expression was increased in colon tissues from patients with HSCR compared with control group, The increased level of CD47 and CUL3 induced by miR-141 reduced proliferation and migration of 293T and SH-SY5Y cells. Furthermore, this suppression was reversed by reducing of CD47 and CUL3. Hypermethylation of a CpG Island in the promoter region of miR-141 gene was confirmed in HSCR tissues. CONCLUSION: Aberrant reduction of miR-141 may play an important role in the pathogenesis of HSCR with the inhibiting affection on cell migration and proliferation abilities. The present study demonstrates for the first time the role of miR-141 and its target genes in the occurrence of HSCR, and provides us a new direction for the study of the pathogenesis of Hirschsprung's disease.

Design, synthesis and biological evaluation of indolizine derivatives as HIV-1 VIF-ElonginC interaction inhibitors.

The HIV-1 viral infectivity factor (VIF) protein is essential for viral replication. VIF recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. Thus, the A3G-Vif-E3 complex represents an attractive target for the development of novel anti-HIV drugs. In this study, we describe the design and synthesis of indolizine derivatives as VIF inhibitors targeting the VIF-ElonginC interaction. Many of the synthesized compounds exhibited obvious inhibition activities of VIF-mediated A3G degradation, and 5 compounds showed improvement of activity compared to the known VIF inhibitor VEC-5 (1) with IC(50) values about 20 µM. The findings described here will be useful for the development of more potent VIF inhibitors.