Beyond identity: Understanding the contribution of the 5' nucleotide of the antisense strand to RNAi activity.

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene's mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5' nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5' nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5' nt identities and different pairing states between the 5' antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5' nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5' thermodynamic rules governing topical siRNA efficacy across plants and animals.

PIWIL1 governs the crosstalk of cancer cell metabolism and immunosuppressive microenvironment in hepatocellular carcinoma.

Altered energy metabolism of cancer cells shapes the immune cell response in the tumor microenvironment that facilitates tumor progression. Herein, we reported the novel of tumor cell-expressed Piwi Like RNA-Mediated Gene Silencing 1 (PIWIL1) in mediating the crosstalk of fatty acid metabolism and immune response of human hepatocellular carcinoma (HCC). PIWIL1 expression in HCC was increased compared to normal hepatic tissues and was positively correlated with the proliferation rate of HCC cell lines. PIWIL1 overexpression accelerated in vitro proliferation and in vivo growth of HCC tumors, while PIWIL1 knockdown showed opposite effects. PIWIL1 increased oxygen consumption and energy production via fatty acid metabolism without altering aerobic glycolysis. Inhibition of fatty acid metabolism abolished PIWIL1-induced HCC proliferation and growth. RNA-seq analysis revealed that immune system regulation might be involved, which was echoed by the experimental observation that PIWIL1-overexpressing HCC cells attracted myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment. MDSCs depletion reduced the proliferation and growth of PIWIL1-overexpressing HCC tumors. Complement C3, whose secretion was induced by PIWIL1 in HCC cells, mediates the interaction of HCC cells with MDSCs by activated p38 MAPK signaling in MDSCs, which in turn initiated expression of immunosuppressive cytokine IL10. Neutralizing IL10 secretion reduced the immunosuppressive activity of MDSCs in the microenvironment of PIWIL1-overexpressing HCC. Taken together, our study unraveled the critical role of PIWIL1 in initiating the interaction of cancer cell metabolism and immune cell response in HCC. Tumor cells-expressed PIWIL1 may be a potential target for the development of novel HCC treatment.

Structure-guided screening strategy combining surface plasmon resonance with nuclear magnetic resonance for identification of small-molecule Argonaute 2 inhibitors.

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.

Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila.

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.This article has an associated 'The people behind the papers' interview.

The people behind the papers - Olga Zaytseva and Leonie Quinn.

Unregulated cell proliferation can be disastrous for development and underlies the progression of cancers throughout the lifespan. A new paper in Development dissects the molecular regulation of a key cell proliferation promoter (and infamous oncogene) Myc, using Drosophila as a model system. We caught up with Olga Zaytseva, recent PhD graduate and one of the paper's first authors, and her supervisor Leonie Quinn, Associate Professor at the John Curtin School of Medical Research in Canberra, to find out more.

Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of siRNA Duplexes.

In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.

Re-Engineering RNA Molecules into Therapeutic Agents.

Efforts to chemically modify nucleic acids got underway merely a decade after the discovery of the DNA double helix and initially targeted nucleosides and nucleotides. The origins of three analogues that remain staples of modification strategies and figure prominently in FDA-approved nucleic acid therapeutics can be traced to the 1960s: 2'-deoxy-2'-fluoro-RNA (2'-F RNA), 2'- O-methyl-RNA (2'- OMe RNA), and the phosphorothioates (PS-DNA/RNA). Progress in nucleoside phosphoramidite-based solid phase oligonucleotide synthesis has gone hand in hand with the creation of second-generation (e.g., 2'- O-(2-methoxyethyl)-RNA, MOE-RNA) and third-generation (e.g., bicyclic nucleic acids, BNAs) analogues, giving rise to an expanding universe of modified nucleic acids. Thus, beyond site-specifically altered DNAs and RNAs with a modified base, sugar, and/or phosphate backbone moieties, nucleic acid chemists have created a host of conjugated oligonucleotides and artificial genetic polymers (XNAs). The search for oligonucleotides with therapeutic efficacy constitutes a significant driving force for these investigations. However, nanotechnology, diagnostics, synthetic biology and genetics, nucleic acid etiology, and basic research directed at the properties of native and artificial pairing systems have all stimulated the design of ever more diverse modifications. Modification of nucleic acids can affect pairing and chemical stability, conformation and interactions with a flurry of proteins and enzymes that play important roles in uptake, transport or processing of targets. Enhancement of metabolic stability is a central concern in the design of antisense, siRNA and aptamer oligonucleotides for therapeutic applications. In the antisense approach, uniformly modified oligonucleotides or so-called gapmers are used to target a specific RNA. The former may sterically block transcription or direct alternative splicing, whereas the latter feature a central PS window that elicits RNase H-mediated cleavage of the target. The key enzyme in RNA interference (RNAi) is Argonaute 2 (Ago2), a dynamic multidomain enzyme that binds multiple regions of the guide (antisense) and passenger (sense) siRNAs. The complexity of the individual interactions between Ago2 and the siRNA duplex provides significant challenges for chemical modification. Therefore, a uniform (the same modification throughout, e.g., antisense) or nearly uniform (e.g., aptamer) modification strategy is less useful in the pursuit of siRNA therapeutic leads. Instead, unique structural features and protein interactions of 5'-end (guide/Ago2MID domain), seed region, central region (cleavage site/Ago2 PIWI domain), and 3'-terminal nucleotides (guide/Ago2 PAZ domain) demand a more nuanced approach in the design of chemically modified siRNAs for therapeutic use. This Account summarizes current siRNA modification strategies with an emphasis on the regio-specific interactions between oligonucleotide and Ago2 and how these affect the choice of modification and optimization of siRNA efficacy. In addition to standard assays applied to measure the effects of modification on the stability of pairing and resistance against nuclease degradation, structural insights based on crystallographic data for modified RNAs alone and in complex with Ago2 from molecular modeling studies are a valuable guide in the design of siRNA therapeutics. Thus, this comprehensive approach is expected to result in accelerated generation of new siRNA-based therapies against various diseases, now that the first siRNA has obtained approval by the US FDA for treatment of hereditary hATTR amyloidosis.

PIWI proteins contribute to apoptosis during the UPR in human airway epithelial cells.

Small noncoding microRNAs (miRNAs) post-transcriptionally regulate a large portion of the human transcriptome. miRNAs have been shown to play an important role in the unfolded protein response (UPR), a cellular adaptive mechanism that is important in alleviating endoplasmic reticulum (ER) stress and promoting cell recovery. Another class of small noncoding RNAs, the Piwi-interacting RNAs (piRNAs) together with PIWI proteins, was originally shown to play a role as repressors of germline transposable elements. More recent studies, however, indicate that P-element induced WImpy proteins (PIWI proteins) and piRNAs also regulate mRNA levels in somatic tissues. Using genome-wide small RNA next generation sequencing, cell viability assays, and caspase activity assays in human airway epithelial cells, we demonstrate that ER stress specifically up-regulates total piRNA expression profiles, and these changes correlate with UPR-induced apoptosis as shown by up-regulation of two pro-apoptotic factor mRNAs, CHOP and NOXA. Furthermore, siRNA knockdown of PIWIL2 and PIWIL4, two proteins involved in piRNA function, attenuates UPR-related cell death, inhibits piRNA expression, and inhibits the up-regulation of CHOP and NOXA mRNA expression. Hence, we provide evidence that PIWIL2 and PIWIL4 proteins, and potentially the up-regulated piRNAs, constitute a novel epigenetic mechanism that control cellular fate during the UPR.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

PIWIL2 suppresses Siah2-mediated degradation of HDAC3 and facilitates CK2α-mediated HDAC3 phosphorylation.

HDAC3 is involved in deacetylation of histone and non-histone proteins, having a key role in the regulation of gene transcription and also in the process of tumorgenesis. However, how HDAC3 is regulated in cancer remains largely unclear. Here, we showed that PIWIL2 can interact with HDAC3, leading to stabilization of HDAC3 from ubiquitin-mediated degradation by competitive association with E3 ubiquitin ligase Siah2. Furthermore, we found that expression of PIWIL2 enhanced HDAC3 activity via CK2α. PIWIL2 facilitated the interaction between HDAC3 and CK2α, thus exhibiting a promotion on the HDAC3 phosphorylation by CK2α. Further work showed that PIWIL2 could promote cell proliferation and suppress cell apoptosis via regulating HDAC3. Our present study firstly revealed that PIWIL2 can play a role in HDAC3-mediated epigenetic regulation on cancer cell proliferation and apoptosis. These findings provide a novel insight into the roles of PIWIL2 in tumorigenesis.

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases.

BACKGROUND: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. RESULTS: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. CONCLUSIONS: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.

Click Quantitative Mass Spectrometry Identifies PIWIL3 as a Mechanistic Target of RNA Interference Activator Enoxacin in Cancer Cells.

Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting.

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival.

BACKGROUND: Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity. METHODS: Cereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis. RESULTS: We identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death. CONCLUSION: These results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.

The Role of PIWIL4, an Argonaute Family Protein, in Breast Cancer.

P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interacting RNAs and play key roles in the biogenesis and functions of PIWI-interacting RNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms and that they are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cells derived from breast cancer. Reducing PIWIL4 expression drastically impairs the migration ability of MDA-MB-231 cells, significantly increases their apoptosis, and mildly affects their proliferation. Our transcriptome and proteome analysis reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-ß and FGF signaling pathways and MHC class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer.

Non-equivalent Roles of AGO1 and AGO2 in mRNA Turnover and Translation of Cyclin D1 mRNA.

Mammalian Argonaute proteins (AGO1-4), in combination with microRNAs (miRs), bind to target mRNAs to initiate degradation and/or translation repression, but the relationships between these two effects is unclear. Although the AGO isoforms ofDrosophilaand plants perform different functions, mammalian AGO isoforms are considered to be functionally degenerate in terms of miR loading and downstream silencing effects. However, we found that, in quiescent (G0) rat myoblasts transiting to the G1 phase, cyclin D1 (Ccnd1) mRNA was associated with two functionally distinct AGO-miR complexes. While AGO1-miR-1 down-regulated the mRNA level, AGO2-let-7 delayed the timing of translation. Knockdown (KD) of AGO2, or antisense-mediated depletion of Let-7, caused Ccnd1 translation to occur earlier, but had no significant effect on mRNA abundance. Conversely, down-regulation of either AGO1 or miR-1, resulted in elevated Ccnd1 mRNA levels at early times, but failed to affect the timing of translation. These results show that the two miR-mediated silencing effects, viz. mRNA decay and translation repression, are independent processes induced by individual AGO isoforms in association with specific miRs.

KRAS Engages AGO2 to Enhance Cellular Transformation.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

Sodium orthovanadate induces the apoptosis of SH-SY5Y cells by inhibiting PIWIL2.

PIWIs have been shown to be abnormally expressed in a variety of cancers and may be important in the maintenance and invasion of cancer cells. The high expression of PIWIL2 contributed to the resistance effect of cisplatin in colon cancer cells, and the knockout of the PIWIL2 gene reduced the aggressive nature and malignant degree of colon cancer cells. Sodium orthovanadate (SOV) is a vanadium compound, and exhibited antineoplastic activity in certain types of human cancer cells, including lung, kidney and prostate cancer cells. However, its effects in human neuroblastoma (NB) cells have not yet been reported. The objective of this study was to investigate the effect of SOV on the apoptosis of NB cells and to explore how PIWIL2 is involved in the mechanism underlying this effect. In the present study, SH­SY5Y cells were treated with SOV and the optimal concentration was determined for further assays. Cell apoptosis, cell count, viability, the cell cycle, and the expression of PIWIL2 mRNA and protein were then determined. The results showed that SOV could induce cell apoptosis, reduce the percentage of viable cells, induce accumulation of SH­SY5Y cells at the G2/M and S phase of the cell cycle, and inhibit the expression of PIWIL2 and Bcl­2 mRNA and protein. The results suggested that the underlying mechanisms may be, at least in part, due to SOV inhibiting the expression of PIWIL2. These findings demonstrated the effect of SOV and supported its further evaluation as a treatment for human NB.

Distinct mechanisms underlie oral vs aboral regeneration in the cnidarian Hydractinia echinata.

Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1(+) stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology.