Towards a therapy for Angelman syndrome by targeting a long non-coding RNA.

Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.

Tripterine emerges as a potential anti-scarring agent in NIH/3T3 cells by repressing ANRIL.

Extensive scarring normally causes hypertrophic or keloid scars. This intuitively results in psychosocial distress and reduction in life quality. Tripterine is a bioactive pentacyclic triterpenoid compound while it is still poorly understood whether it possesses an anti-scarring function. NIH/3T3 cells were administrated with tripterine at increased concentrations (0-10 µM). Antisense RNA to INK4 locus (ANRIL) was transformed into NIH/3T3 cells, and the cells transfected with empty vector (mock transfection) were used as negative control. Then, cell viability and migration were profiled by cell counting kit-8 (CCK-8) and 24-Transwell assay. Protein expression was analyzed by Western blot assay. ANRIL was quantified by quantitative reverse transcription-PCR (qRT-PCR). Tripterine administration induced the growth inhibition of NIH/3T3 cells indicated by a trend toward the decreased expression of matrix metallopeptidase (MMPs), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). This process was accompanied by the decreased phosphorylation of p65, inhibitor of nuclear factor kappa-B alpha (IκBα) and the downregulation of ß-catenin. Moreover, ANRIL expression was notably repressed by tripterine. By contrast, in ANRIL-transfected cells, the effect of tripterine was abolished. Tripterine exhibited an anti-scarring bioactivity in NIH/3T3 cells by inhibiting ANRIL, and this process was accompanied by the blockade of nuclear factor-kappa B (NF-κB) and ß-catenin cascades.

Long noncoding RNA SOX21-AS1 promotes cervical cancer progression by competitively sponging miR-7/VDAC1.

Growing evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in cervical cancer. Dy000sregulation of lncRNA SOX21 antisense RNA 1 (SOX21-AS1) has been reported in several tumors. However, its expression pattern and potential biological function in cervical cancer (CC) have not been investigated. In this study, we first reported that SOX21-AS1 expression was significantly upregulated in both CC tissues and cell lines. High expression of SOX21-AS1 was found to be significantly correlated with Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis and depth of cervical invasion. Further clinical assay confirmed that high SOX21-AS1 expression was associated with shorter overall survival and could be used as a potential prognostic biomarker for CC patients. Functional investigation showed that knockdown of SOX21-AS1 suppressed CC cells proliferation, migration, and invasion, as well as epithelial to mesenchymal transition progress. Furthermore, our data showed that microRNA-7 (miR-7) interacted with SOX21-AS1 by directly targeting the miRNA-binding site in the SOX21-AS1 sequence, and quantitative real-time polymerase chain reaction results showed overexpression of SOX21-AS1 decreased the levels of miR-7 in CC cells. Moreover, we confirmed that miR-7 directly targeted the 3'-untranslated region of voltage dependent anion channel 1 (VDAC1). Final in vitro assay suggested that in CC cells with SOX21-AS1, VDAC1 overexpression resulted in an increase of cell proliferation, migration, and invasion. Overall, our findings illuminate how SOX21-AS1 formed a regulatory network to confer an oncogenic function in CC and SOX21-AS1 could be regarded as an efficient therapeutic target and potential biomarker for CC patients.

Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway.

AIM: To investigate whether long non-coding RNA (lncRNA) ZEB1 antisense 1 (ZEB1-AS1) affects cell migration and invasion of cervical cancer by regulating epithelial-mesenchymal transition (EMT) via the p38MAPK pathway. METHODS: Human cervical cancer cell line Hela was classified into Control, NC siRNA, ZEB1-AS1 siRNA, SB203580 (p38MAPK pathway inhibitor) and ZEB1-AS1 siRNA + Anisomycin (p38MAPK pathway activator) groups. Quantitative real-time polymerase chain reaction was performed for ZEB1-AS1 expression, Western blotting to measure p38MAPK signaling pathway-/EMT-related proteins, and Wound-healing and Transwell assays to evaluate cell migration and invasion respectively. RESULTS: ZEB1-AS1 was upregulated in cancer tissues and related to major clinicopathological features of cervical cancer. Besides, patients with lower-ZEB1-AS1-expression had a higher 5-year survival rate than those patients with higher-ZEB1-AS1-expression. High ZEB1-AS1 expression and advanced Federation of Gynecology and Obstetrics stage were independent risk factors for patients' prognosis. Both ZEB1-AS1 siRNA and SB203580 effectively reduced p-p38 expression and the migration and invasion of Hela cells, with elevation of E-cadherin and reduction of Vimentin and N-cadherin. However, inhibitory effects of ZEB1-AS1 siRNA on EMT as well as cell migration and invasion of the Hela cell were reversed by Anisomycin. CONCLUSION: Inhibition of ZEB1-AS1 can block the p38MAPK signaling pathway, ultimately restricting the EMT and suppressing cell migration and invasion of cervical cancer cells.

Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress antisense RNAs.

Eukaryotic transcriptomes are characterized by widespread transcription of noncoding and antisense RNAs, which is linked to key chromosomal processes, such as chromatin remodelling, gene regulation and heterochromatin assembly. However, these transcripts can be deleterious, and their accumulation is suppressed by several mechanisms including degradation by the nuclear exosome. The mechanisms by which cells differentiate coding RNAs from transcripts targeted for degradation are not clear. Here we show that the variant histone H2A.Z, which is loaded preferentially at the 5' ends of genes by the Swr1 complex containing a JmjC domain protein, mediates suppression of antisense transcripts in the fission yeast Schizosaccharomyces pombe genome. H2A.Z is partially redundant in this regard with the Clr4 (known as SUV39H in mammals)-containing heterochromatin silencing complex that is also distributed at euchromatic loci, and with RNA interference component Argonaute (Ago1). Loss of Clr4 or Ago1 alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that as well as performing other functions, H2A.Z is a component of a genome indexing mechanism that cooperates with heterochromatin and RNAi factors to suppress read-through antisense transcripts.

Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli.

Short regulatory RNAs are widespread in bacteria, and many function through antisense recognition of mRNA. Among the best studied antisense transcripts are RNA antitoxins that repress toxin mRNA translation. The hok/sok locus of plasmid R1 from Escherichia coli is an established model for RNA antitoxin action. Base-pairing between hok mRNA and Sok-antisense-RNA increases plasmid maintenance through post-segregational-killing of plasmid-free progeny cells. To test the model and the idea that sequestration of Sok-RNA activity could provide a novel antimicrobial strategy, we designed anti Sok peptide nucleic acid (PNA) oligomers that, according to the model, would act as competitive inhibitors of hok mRNA::Sok-RNA interactions. In hok/sok-carrying cells, anti Sok PNAs were more bactericidal than rifampicin. Also, anti Sok PNAs induced ghost cell morphology and an accumulation of mature hok mRNA, consistent with cell killing through synthesis of Hok protein. The results support the sense/antisense model for hok mRNA repression by Sok-RNA and demonstrate that antisense agents can be used to out-compete RNA::RNA interactions in bacteria. Finally, BLAST analyses of approximately 200 prokaryotic genomes revealed that many enteric bacteria have multiple hok/sok homologous and analogous RNA-regulated toxin-antitoxin loci. Therefore, it is possible to activate suicide in bacteria by targeting antitoxins.

In vivo knockdown of antisense non-coding mitochondrial RNAs by a lentiviral-encoded shRNA inhibits melanoma tumor growth and lung colonization.

The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy.

Long noncoding RNA FEZF1-AS1 indicates a poor prognosis of gastric cancer and promotes tumorigenesis via activation of Wnt signaling pathway.

Long non-coding RNAs (lncRNAs) have been demonstrated that it plays very important role in development and progression of carcinomas. LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1) has been proved to be implicated in tumor initiation and progression of various cancers, recently. Nevertheless, the biological function and clinical significance of lncRNA FEZF1-AS1 in gastric cancer (GC) are not clear enough. Here, we concentrated on the association of FEZF1-AS1 expression and clinicopathological factors in GC tissues and cells. Moreover, we explored the potential regulatory mechanisms. The results showed that lncRNA FEZF1-AS1 was observably upregulated in human GC tissues and GC cell lines, compared with the adjacent non-tumor tissues and human gastric epithelial cell line (GES-1). Moreover, high expression of lncRNA FEZF1-AS1 was significantly associated with later stage and higher grade. Furthermore, Kaplan-Meier survival analysis was conducted, indicating that lncRNA FEZF1-AS1 may be an independent prognostic factor in GC. Additionally, the area under the receiver operating characteristic (ROC) curve of lncRNA FEZF1-AS1 exhibited its diagnostic value in GC. Notably, whenever the lncRNA FEZF1-AS1 was silenced, the proliferation of GC cells were significantly inhibited and the cell cycle was arrested at a G0/G1 stage in GC cells. Furthermore, downregulation of lncRNA FEZF1-AS1 could suppress the activation of the Wnt/ß-catenin signaling pathway. Conclusively, our findings indicated that lncRNA FEZF1-AS1 could be considered as a novel biomarker for the treatment of GC.

Inhibition of long noncoding RNA BDNF-AS rescues cell death and apoptosis in hypoxia/reoxygenation damaged murine cardiomyocyte.

BACKGROUND: Hypoxia/reoxygenation (H/R)-induced cardiomyocyte damage in vitro bears many similarities to myocardial infraction in vivo. In this study, we investigated whether long noncoding RNA (lncRNA) BDNF-AS has functional role in rescuing H/R-induced damage in cardiomyocyte in vitro. METHOD: Murine neonatal cardiomyocytes were cultured in vitro, and treated with either long (Hypoxia (L)) or short (Hypoxia (S)) H/R conditions. Viability and TUNEL assays were conducted to assess cardiomyocyte death or apoptosis. QRT-PCR was used to examine BDNF-AS mRNA expression. Endogenous BDNF-AS was downregulated by siRNA in cardiomyocyte. Its effect on H/R-induced cardiomyocyte damage was then examined. In addition, survival-associated proteins, including BDNF, VEGF and Akt were examined by western blot in siRNA-transfected cardiomyocytes. RESULTS: Hypoxia (S) induced apoptosis whereas Hypoxia (L) induced cell death in murine neonatal cardiomyocyte in vitro. Under both conditions, endogenous cardiomyocyte BDNF-AS was significantly upregulated. SiRNA-mediated BDNF-AS downregulation rescued cardiomyocyte death under Hypoxia (L) condition, and reduced cardiomyocyte apoptosis under Hypoxia (L) condition. Moreover, BDNF-AS downregulation activated BDNF and VEGF through upregulation, and activated Akt through phosphorylation in H/R-damaged cardiomyocyte. CONCLUSION: Inhibition of lncRNA BDNF-AS may be an efficient way to rescue cardiomyocyte from H/R-induced apoptosis or cell death.

AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription.

Interleukin-6 (IL-6) is widely expressed in a variety of malignant tumors; thus, targeting the IL-6/STAT3 pathway represents a promising therapeutic strategy for malignant cancers. In this study, we identified a noncoding RNA, AS-IL6, which is transcribed antisense to IL6 and induces IL6 expression in glioma cells. Knockdown of AS-IL6 attenuates LPS-induced IL6 transcription. Interestingly, AS-IL6 does not change IL6 mRNA stability, but induces the enrichment of histone H3 acetylated at lysine 27 (H3K27Ac) at the IL6 promoter. In addition, we found that depletion of AS-IL6 inhibits the invasive ability of glioblastoma cells, while treatment of cells with recombinant IL6 reverses this effect. Our results reveal a novel mechanism of IL6 regulation and demonstrate an oncogenic role for AS-IL6 in glioma cells.

Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition.

BACKGROUND: The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells. RESULTS: The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis. CONCLUSIONS: The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.

Treatment efficacy of the lead RNAIII-inhibiting peptide YSPWTNF-NH2 in acquired Staphylococcus aureus sepsis: a histopathological assessment.

The quorum-sensing interfering RNAIII-inhibiting peptide (RIP) YSPXTNF and its synthetic analogues YSPWTNF and YSPITNF have been shown to prevent and suppress diseases caused by Staphylococcus aureus at different body sites in different animal models. This study was designed to investigate histopathologically the therapeutic efficacy of lead peptide RIP YSPWTNF-NH(2) in the subcutaneous air sac murine model of acquired S. aureus sepsis. Two experimental protocols were evaluated: an infection/therapy protocol, for which twenty BALB/c mice per group were infected with a subcutaneous inoculum of S. aureus strain ATCC 25923 ( [Formula: see text] colony forming units) that were either pretreated or not with 150microg of peptide RIP, and a safety protocol, for which three uninfected mice per group received treatment with either 150microg of peptide RIP or saline. Therapeutic efficacy was assessed by clinical examination for a period of 20 days and histopathology at 12, 24, 36, 48, 96 and 168h after inoculation. Treatment safety was assessed histopathologically at 24, 48 and 264h after inoculation. Subcutaneous administration of uninfected control mice with a single dose of peptide RIP YSPWTNF caused no significant histopathology in most organs examined, except for slight to moderate lung and liver congestions. In contrast to the situation with the untreated infected control group mice that presented with histopathological alterations consistent with the diagnosis of rapidly progressive and highly erosive disease (100% mortality by day 3), treatment of infected animals with peptide RIP YSPWTNF had a profound therapeutic effect on survival rate (67% by day 20) and on disease progression. The histopathological examination confirmed the clinical findings showing that extensive tissue damage at the site of the infection and in organs were greatly suppressed in the peptide RIP-treated animals.

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6nt from its 5' end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok-6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok-6 for further rapid degradation by PNPase from its 3' end. We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase.

Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase zeta.

To examine a role of DNA polymerase zeta in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the V(H) genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase zeta, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.