Sh RNAs Research Articles

Gartanin is a novel NEDDylation inhibitor for induction of Skp2 degradation, FBXW2 expression, and autophagy.

Gartanin, a 4-prenylated xanthone, has been identified from the purple mangosteen fruit as a potent growth inhibitor of various cancer cell lines, including prostate cancer. However, much of Gartanin's anticancer mechanism remains unknown. We have discovered that Gartanin docked onto the regulatory subunit of the precursor cell-expressed developmentally downregulated 8 (NEDD8)-activating enzyme (NAE) complex and next to the NEDD8 binding complex, which leads to inhibit NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 NEDDylation in an in vitro assay. The S phase kinase-associated protein (Skp2) and F-box and WD-repeat domain-containing 2 (FBXW2), the NEDD8 family members of E3 ubiqutin ligases, were also downregulated and upregulated by Gartainin, respectively. Knock-down of NEDD8 expression by short harpin (Sh) RNAs blocked or attenuated these effects of Gartainin. Finally, Gartanin demonstrated its ability to inhibit growth of prostate cancer lines via autophagy initiation. Our data support that Gartanin is a naturally occurring NEDDylation inhibitor and deserves further investigation for prostate cancer prevention and treatment.

Urate transport capacity of glucose transporter 9 and urate transporter 1 in cartilage chondrocytes.

Chronic gouty arthritis, caused by a persistent increase in, and the deposition of, soluble uric acid (sUA), can induce pathological chondrocyte destruction; however, the effects of urate transport and intracellular sUA on chondrocyte functionality and viability are yet to be fully determined. Thus, the aim of the present study was to investigate the presence and functionality of a urate transport system in chondrocytes. The expression profiles of two primary urate reabsorptive transporters, glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), in human articular cartilage and chondrocyte cell lines were examined via western blotting, reverse transcription-quantitative PCR, immunohistochemistry and immunofluorescence. Then, chondrocytes were incubated with exogenous sUA at increasing concentrations. Negative control assays were conducted via the specific knockdown of GLUT9 and URAT1 with lentiviral short hairpin (sh)RNAs, and by pretreatment with benzbromarone, a known inhibitor of the two transporters. Intracellular UA concentrations were measured using colorimetric assays. The expression levels of GLUT9 and URAT1 were determined in cartilage tissues and chondrocyte cell lines. Incubation of chondrocytes with sUA led to a concentration-dependent increase in intracellular urate concentrations, which was inhibited by GLUT9 or URAT1 knockdown, or by benzbromarone pretreatment (27.13±2.70, 44.22±2.34 and 58.46±2.32% reduction, respectively). In particular, benzbromarone further decreased the already-reduced intracellular UA concentrations in HC-shGLUT9 and HC-shURAT1 cells by 46.79±2.46 and 39.79±2.22%, respectively. Cells overexpressing GLUT9 and URAT1 were used as the positive cell control, which showed increased intracellular UA concentrations that could be reversed by treatment with benzbromarone. In conclusion, chondrocytes may possess an active UA transport system. GLUT9 and URAT1 functioned synergistically to transport UA into the chondrocyte cytoplasm, which was inhibited by specific gene knockdowns and drug-induced inhibition. These results may be fundamental in the further investigation of the pathological changes to chondrocytes induced by sUA during gouty arthritis, and identified UA transport processes as potential targets for the early control of chronic gouty arthritis.

PF230 THE RNA HELICASE DDX17 PROTECTS ACUTE MYELOID LEUKEMIA CELLS AGAINST DNA DAMAGE

Background:Alternative splicing (AS) is essentially regulated by RNA‐binding proteins (RBPs) and is one of many deregulated cellular processes in Acute Myeloid Leukemia (AML). The alternative usage of exons substantially increases the coding potential of mRNAs and frequently leads to the expression of tumor‐promoting mRNA variants in cancer modulating apoptosis, proliferation, invasion and migration of cancer cells. This underlines the great plasticity of AS in cancer and justifies the need to elucidate the function of nuclear RBPs in specific cancer types. We found that the RNA helicase DDX17 is significantly more highly expressed in primary human AML specimens with high versus low Leukemia Stem Cell (LSC) frequency.Aims:In this study, we aimed to understand the functional role of DDX17 in normal hematopoietic stem and progenitor cells (HSPCs) and in AML cells. In particular, we sought to determine whether DDX17 exerts its function in the hematopoietic system primarily via AS or AS‐unrelated mechanisms.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether DDX17 was essential for primary human cord blood CD34+ HSPCs and leukemia cells. Furthermore, mRFP tagged DDX17 was overexpressed through lentiviral transduction to track protein localization by immunofluorescence (IF) imaging. Lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) was used to determine how DDX17 KD affected gene expression and alternative exon usage. Pull down experiments combined with mass spectrometry revealed binding partners in presence and absence of chemotherapeutic agents. Finally, we performed homologous recombination (HR) and drug sensitivity assays to determine the role of DDX17 in DNA damage.Results:Knockdown of DDX17 induced DNA damage and severely impaired the function of healthy HSPCs and TP53 wild type AML cells. TP53 null cells maintained their proliferative capacity during short term in vitro expansion in absence of DDX17, but were significantly sensitized towards DNA damaging agents. In line, overexpression of DDX17 protected the cells against double strand break (DSB) inducing drugs. IF imaging revealed direct recruitment of DDX17 to DNA damage sites, while RNA‐Seq showed suppression of multiple DNA repair genes.Summary/Conclusion:RNA‐Seq, IF imaging, proteomics, and HR assays suggest an essential role of DDX17 in DNA repair via different mechanisms including direct recruitment to DNA damage sites, alternative splicing, and regulation of DNA repair genes on the transcriptional level.

PS980 GRIK5 DRIVES SELF RENEWAL PATHWAYS IN HUMAN ACUTE MYELOID LEUKEMIA CELLS

Background:Acute myeloid leukemia (AML) is characterized by a block in differentiation and uncontrolled proliferation of immature hematopoietic precursor cells. Self‐renewing leukemia stem cells (LSCs) are supposed to initiate and promote the disease. In our previous study, we had identified a set of genes associated with high LSC frequency in normal karyotype AML. Among these, we identified the Glutamate receptor ionotropic, kainate 5 (GRIK5) coding for a transmembrane protein, which is known to be expressed in the central nervous system as a ligand‐gated ion channel receptor.Aims:In this study, we aimed to understand the functional role of GRIK5 in normal HSPCs and AML cells. In particular, we investigated how GRIK5 drives self‐renewal pathways in AML and whether GRIK5 might represent a novel cell surface target for anti‐leukemic therapy.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether GRIK5 was essential for proliferation, differentiation, and survival of primary human cord blood CD34+ HSPCs, leukemic cell lines, and primary human‐derived AML xenograft cells. Furthermore, lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) and whole cell mass spectrometry followed by enrichment analysis were used to identify key signaling pathways affected by GRIK5 KD.Results:We found that knockdown of GRIK5 expression strongly impaired leukemic cell proliferation and totally abrogated human leukemic engraftment in immunocompromised mice. In the healthy system, however, GRIK5 KD predominantly reduced the proliferative output of more committed progenitors, while more immature HSPCs were less affected. RNA‐Seq data points towards a major involvement in several key self‐renewal pathways.Summary/Conclusion:Here we found that the surface receptor GRIK5 is overexpressed in the majority of AML samples compared to healthy HSPCs. GRIK5 plays a pivotal role in proliferation, differentiation, and engraftment capacity of primary human leukemia cells in xenotransplantation assays and might, therefore, be used as a novel therapeutic target for anti‐leukemic therapies.

RNA Interference Therapy for Machado–Joseph Disease: Long-Term Safety Profile of Lentiviral Vectors Encoding Short Hairpin RNAs Targeting Mutant Ataxin-3

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by an abnormal repetition of a CAG codon in the MJD1 gene. This expansion translates into a long polyglutamine tract, leading to the misfolding of the mutant protein ataxin-3, which abnormally accumulates in the nucleus, thus leading to neurodegeneration in specific brain regions. No treatment able to modify the progression of the disease is available. However, it has previously been shown that specific silencing of mutant ataxin-3 by RNA interference with viral vectors is a promising therapeutic strategy for MJD. Nevertheless, reports of cytotoxic effects of this technology led to the safety profile of the previously tested lentiviral vectors encoding short hairpin (sh)RNAs (LV-shmutatx3) targeting mutant ataxin-3 upon brain injection being investigated. For this purpose, the vectors were injected in the mouse striata, and neuronal dysfunction, degeneration, gliosis, off-target effects, and saturation of the RNA interference machinery were evaluated. It was found that: (1) LV-shmutatx3 mediated stable and long-term expression of the shRNA in neurons of the mouse striatum; (2) neuronal dysfunction evaluated by darpp-32, NeuN, and cresyl violet staining, initially more pronounced, became indistinguishable from the phosphate-buffered saline group at 8 weeks and resolved within 20 weeks; (3) astrocytic activation was present, which resolved within 8 weeks; (4) microglial activity and proinflammatory cytokines release were present, which resolved and normalized within 20 weeks; and (5) there were no off-target effects or saturation of the endogenous RNA interference processing machinery in the mouse striatum. The data show that injection of lentiviral vectors encoding a shRNA targeting mutant ataxin-3 in the mouse brain induce transient dysfunctions, which resolve within 20 weeks. Importantly, long-term expression (up to 20 weeks post injection) of this shRNA (driven by H1 promoter) led to no toxic effect in vivo. This study thus constitutes an additional step in a future translation of gene silencing as a therapy for MJD.

PTEN inhibits non-small cell lung cancer cell growth by promoting G0/G1 arrest and cell apoptosis.

Non-small cell lung cancer (NSCLC) is a major type of human lung cancer and the primary cause of cancer-associated cases of mortality worldwide. Phosphatase and tensin homolog (PTEN) is a potent tumor suppressor gene in various human cancer types. The aim of the current study was to explore the role of PTEN and its associated regulatory mechanisms in NSCLC. Firstly, the expression of PTEN was detected using western blotting in a variety of NSCLC cell lines. The results revealed that compared with normal control cells, PTEN levels were significantly decreased in NSCLC cell lines (P<0.01). Short hairpin (sh)RNAs specific to PTEN were also used to knockdown endogenous PTEN in NSCLC cells. The results indicated that cell viability was significantly increased in PTEN-knockdown cells compared with those transfected with negative control shRNA (P<0.01). Conversely, overexpression of PTEN in A549 and SK-MES-1 cells significantly decreased the optical density of NSCLC cells (P<0.01). Flow cytometry was used to investigate the cell cycle; the results revealed that PTEN knockdown significantly increased the percentage of cells at G0/G1 phase (P<0.01) and decreased the number of cells at S phase (P<0.01). The molecular mechanism was further explored using western blotting and the results demonstrated that PTEN overexpression increased the levels of cleaved caspase-3 (P<0.01). These results suggest that PTEN may be a potential target gene for gene therapy in patients with NSCLCs.

Abstract 2001: Transcriptional integrator p300 in human prostate tumor-initiating cells

Abstract Introduction p300 is a well-known coactivator of the androgen receptor (AR) and has been shown to play an important role in prostate cancer (PCa). The expression of p300 has been correlated to tumor volume and PCa progression. Therefore, we hypothesize that p300 may play an important role in tumor-initiating cells and therapy resistance. Due to its multifunctional role p300 may serve as a promising new therapeutic target for treatment of PCa. Ultimately, it is our aim to find new innovative therapeutic targets for castration resistant PCa. Material &amp; Methods The tumor-initiating capacity of the AR-negative PCa cell lines PC3 and Du145, as well as primary prostate epithelial cells was characterized by seeding a limiting number of cells per flask. Colony formation efficiency, colony type and detailed morphological information about each colony were determined by an automated pipeline that we have developed. Stable cell lines with doxycycline inducible p300 sh RNAs were generated by lentiviral transduction. Knockdown of p300 was verified by Western Blot and reduced p300 activity was confirmed by measuring histone h3 acetylation. Results In concordance with the literature we identified three colony types (holo-, mero- and paraclones) in PC3 and Du145 cell lines as well as primary PCa cells. Serial clonogenic assays confirmed in both cell lines that holoclones show the highest colony formation potential and maintain their tumor-initiating capacity over numerous rounds. In PC3 we detected 10% and in Du145 30% holoclones. Under optimal growth conditions p300 was detected in all colony types, however no differences in protein expression of p300 as well as on mRNA level were observed. Knockdown of p300 did not alter colony formation efficiency or the relative distribution of colony types. Conclusion Taken together, we confirmed the presence of tumor-initiating holoclones in PCa cell lines and primary prostate epithelial cells and established stable cell lines with inducible knockdown of p300. In contrast to several other types of malignancies, p300 has no specific role in regulation of stemness in our PCa models at the tested conditions. As a next step we will determine the effects of p300 knockdown during chemotherapy and radiation therapy to evaluate if p300 depletion may enhance the treatment efficacy and which role p300 plays in therapy resistance. Citation Format: Martina Gruber, Florian Handle, Zoran Culig. Transcriptional integrator p300 in human prostate tumor-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2001.

PSMD4 is a novel therapeutic target in chemoresistant colorectal cancer activated by cytoplasmic localization of Nrf2.

Nuclear Nrf2 (nNrf2) binding to the antioxidant response element may promote chemoresistance in colorectal cancer. However, the shuttling of Nrf2 between cytoplasm and nucleus in colon cancer cells has revealed the possibility that cytoplasmic location of Nrf2 (cNrf2) may play a specific role in chemoresistance. Transfection of a nuclear location sequence (NLS)-wild-type or NLS-mutated Nrf2 expression vector into a stable shNrf2 HCT116 clone using the MTT assay to examine whether chemoresistance induced by cNrf2 may be greater than nNrf2. Different specific inhibitors and small hairpin (sh)RNAs of targeting genes were used to verify the mechanistic action of cNrf2 in chemoresistance and further confirmed by an animal model. The association of cNrf2 with chemotherapeutic response in patients with colorectal cancer was statistically analyzed. The MTT assay indicated that cNrf2 may play a more important role than nNrf2 in conferring 5-fluorouracil (5-FU) and oxaliplatin resistance in HCT116 cells. Mechanistically, cNrf2-induced PSMD4 expression was responsible for chemoresistance in the NLS-mutated Nrf2-tranfected shNrf2HCT116 clone via the NF-κB/AKT/β-catenin/ZEB1 cascades. The tumor burden induced by the NLS-mutated Nrf2-transfected shNrf2HCT116 clone was completely suppressed by treatment with 5-FU in combination with carfilzomib. A higher prevalence of unfavorable chemotherapeutic response in colorectal cancer patients with cNrf2, PSMD4-positive, p-p65-positive, and nuclear β-catenin tumors was observed when compared to their counterparts. cNrf2 may play a more important role than nNrf2 in the chemoresistance of colorectal cancer. Activation of the NF-κB/AKT/β-catenin/ZEB1 cascade by PSMD4 may be responsible for cNrf2-mediated chemoresistance.Condensed abstractCNrf2 may play a more important role than nNrf2 in conferring 5-FU and oxaliplatin resistance. This observation in patients seemed to support the findings of the cell and animal models and suggested that PSMD4 may be responsible cNrf2-mediated chemoresistance via the NF-κB/AKT/β-catenin /ZEB1 cascades.

Evaluation of anti-insulin receptor antibodies as potential novel therapies for human insulin receptoropathy using cell culture models

Aims/hypothesisBi-allelic loss-of-function mutations in the INSR gene (encoding the insulin receptor [INSR]) commonly cause extreme insulin resistance and early mortality. Therapeutic options are limited, but anti-INSR antibodies have been shown to activate two mutant receptors, S323L and F382V. This study evaluates four well-characterised murine anti-INSR monoclonal antibodies recognising distinct epitopes (83-7, 83-14, 18-44, 18-146) as surrogate agonists for potential targeted treatment of severe insulin resistance arising from insulin receptoropathies.MethodsTen naturally occurring mutant human INSRs with defects affecting different aspects of receptor function were modelled and assessed for response to insulin and anti-INSR antibodies. A novel 3T3-L1 adipocyte model of insulin receptoropathy was generated, permitting conditional knockdown of endogenous mouse Insr by lentiviral expression of species-specific short hairpin (sh)RNAs with simultaneous expression of human mutant INSR transgenes.ResultsAll expressed mutant INSR bound to all antibodies tested. Eight mutants showed antibody-induced autophosphorylation, while co-treatment with antibody and insulin increased maximal phosphorylation compared with insulin alone. After knockdown of mouse Insr and expression of mutant INSR in 3T3-L1 adipocytes, two antibodies (83-7 and 83-14) activated signalling via protein kinase B (Akt) preferentially over signalling via extracellular signal-regulated kinase 1/2 (ERK1/2) for seven mutants. These antibodies stimulated glucose uptake via P193L, S323L, F382V and D707A mutant INSRs, with antibody response greater than insulin response for D707A.Conclusions/interpretationAnti-INSR monoclonal antibodies can activate selected naturally occurring mutant human insulin receptors, bringing closer the prospect of novel therapy for severe insulin resistance caused by recessive mutations.

The contribution of SP100 to cottontail rabbit papillomavirus transcription and replication.

SP100 proteins are components of nuclear domain 10 structures and have been implicated as inhibitors of human papillomavirus (HPV) replication. In this study, we have addressed the role of SP100 in tumour formation by the cottontail rabbit (Sylvilagus floridanus) papillomavirus (CRPV or SfPV1) in a rabbit model. Tissue culture studies using rabbit keratinocyte lines indicated that rabbit SP100 is an interferon-beta-inducible gene similar to its human counterpart. Stable knockdown of SP100 by shRNA in a cell line harbouring CRPV genomes resulted in a decrease of viral early transcripts. In contrast, infection of domestic rabbits with recombinant CRPV genomes expressing short hairpin (sh)RNAs directed against SP100 did not reveal changes in tumour formation rate, tumour size or early viral transcript levels. However, late viral transcript levels and viral genome copies were consistently lower in CRPV/shSP100-induced tumours than in the control, but these differences did not reach statistical significance. In summary, this study suggests that rabbit SP100 is not an inhibitor but an activator of CRPV replication and transcription.

Mesenchymal stem cell deficiency influences megakaryocytopoiesis through the TNFAIP3/NF-κB/SMAD pathway in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease. Mesenchymal stem cells (MSCs) play important roles in the physiology and homeostasis of the haematopoietic system, including supporting megakaryocytic differentiation from CD34+ haematopoietic progenitor cells. Tumour necrosis factor alpha-induced protein 3 (TNFAIP3, also termed A20) plays a key role in terminating NF-κB signalling. Human genetic studies showed that the polymorphisms of the TNFAIP3 gene may contribute to ITP susceptibility. In this study, we showed a significant decrease in TNFAIP3 and increase in NF-κB/SMAD7 in ITP-MSCs. In co-cultures with CD34+ cells, NF-κB was overexpressed in MSCs from healthy controls (HC-MSCs) after transfection with NFKBIA (IκB)-specific short hairpin (sh)RNAs, resulting in MSC deficiency and a reduction in megakaryocytic differentiation and thrombopoiesis. Knockdown of TNFAIP3 expression using TNFAIP3-specific shRNAs in HC-MSCs affected megakaryocytopoiesis. However, IKBKB knockdown corrected megakaryocytopoiesis inhibition in the ITP-MSCs by decreasing NF-κB expression. Amplified TNFAIP3 expression in ITP-MSCs by TNFAIP3 cDNA can facilitate megakaryocyte differentiation. shRNA-mediated knockdown of SMAD7 expression rescued the impaired MSC function in ITP patients. Therefore, we demonstrate that a pathological reduction in TNFAIP3 levels induced NF-κB/SMAD7 pathway activation, causing a deficiency in MSCs in ITP patients. The ability of ITP-MSCs to support megakaryocytic differentiation and thrombopoiesis of CD34+ cells was impaired.

Mortalin deficiency suppresses fibrosis and induces apoptosis in keloid spheroids

Mortalin (Mot) is a mitochondrial chaperone of the heat shock protein 70 family and it’s pro-proliferative and anti-apoptosis functions could be associated with keloid pathogenesis, and blocking of mortalin and its interaction with p53 might be a potential novel target for the treatment of keloid. Therefore, we generated mortalin-specific small hairpin (sh) RNAs (dE1-RGD/GFP/shMot) and introduced into keloid spheroids for examination of its apoptotic and anti-fibrotic effect. On keloid tissues, mortalin expression was higher than adjacent normal tissues and it’s protein expressions were activated keloid fibroblasts (KFs). After primary keloid spheroid were transduced with dE1-RGD/GFP/shMot for knockdown of mortalin, expression of type I, III collagen, fibronectin, and elastin was significantly reduced and transforming growth factor-β1, epidermal growth factor receptor (EGFR), Extracellular Signal-Regulated Kinases 1 and 2 (Erk 1/2), and Smad 2/3 complex protein expression were decreased. In addition, increased TUNEL activities and cytochrome C were observed. Further, for examine of mortalin and p53 interaction, we performed immunofluorescence analysis. Knockdown of mortalin relocated p53 to the cell nucleus in primary keloid spheroids by dE1-RGD/GFP/shMot transduction. These results support the utility of knockdown of mortalin to induce apoptosis and reduce ECMs expression in keloid spheroid, which may be highly beneficial in treating keloids.

AKAP-Lbc mediates protection against doxorubicin-induced cardiomyocyte toxicity

Doxorubicin (DOX) is a chemotherapic agent that is widely used to treat hematological and solid tumors. Despite its efficacy, DOX displays significant cardiac toxicity associated with cardiomyocytes death and heart failure. Cardiac toxicity is mainly associated with the ability of DOX to alter mitochondrial function. The current lack of treatments to efficiently prevent DOX cardiotoxicity underscores the need of new therapeutic approaches. Our current findings show that stimulation of cardiomyocytes with the α1-adrenergic receptor (AR) agonist phenylephrine (PE) significantly inhibits the apoptotic effect of DOX. Importantly, our results indicate that AKAP-Lbc is critical for transducing protective signals downstream of α1-ARs. In particular, we could show that suppression of AKAP-Lbc expression by infecting primary cultures of ventricular myocytes with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly impairs the ability of PE to reduce DOX-induced apoptosis. AKAP-Lbc-mediated cardiomyocyte protection requires the activation of anchored protein kinase D1 (PKD1)-dependent prosurvival pathways that promote the expression of the anti-apoptotic protein Bcl2 and inhibit the translocation of the pro-apoptotic protein Bax to mitochondria. In conclusion, AKAP-Lbc emerges as a coordinator of signals that protect cardiomyocytes against the toxic effects of DOX.

Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function invivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy.

Cytochrome P450 1B1 inhibition suppresses tumorigenicity of prostate cancer via caspase-1 activation.

Cytochrome P450 1B1 (CYP1B1) is recognized as a universal tumor biomarker and a feasible therapeutic target due to its specific overexpression in cancer tissues. Despite its up-regulation in prostate cancer (PCa), biological significance and clinicopathological features of CYP1B1 are still elusive. Here, we show that overexpression or hyperactivation of CYP1B1 stimulated proliferative, migratory and invasive potential of non-tumorigenic PCa cells. Attenuation of CYP1B1 with its specific small hairpin (sh) RNAs greatly reduced proliferation through apoptotic cell death and impaired migration and invasion in PCa cells. Intratumoral injection of CYP1B1 shRNA attenuated growth of pre-existing tumors. The antitumor effect of CYP1B1 shRNA was also observed in prostate tumor xenograft mouse models. Among the genes altered by CYP1B1 knockdown, reduction of caspase-1 (CASP1) activity attenuated the antitumor effect of CYP1B1 inhibition. Indeed, CYP1B1 regulates CASP1 expression or activity. Finally, CYP1B1 expression was increased in higher grades of PCa and overall survival was significantly reduced in patients with high levels of CYP1B1 protein. CYP1B1 expression was reversely associated with CASP1 expression in clinical tissue samples. Together, our results demonstrate that CYP1B1 regulates PCa tumorigenesis by inhibiting CASP1 activation. Thus, the CYP1B1-CASP1 axis may be useful as a potential biomarker and a therapeutic target for PCa.

4.5SI and 4.5SH RNAs: Expression in various rodent organs and abundance and distribution in the cell

Studying the structure, functions, and cell physiology of small RNAs remains important. The 4.5SI and 4.5SH small RNAs, which were among the first to be discovered and sequenced, share several features, i.e., they are both approximately 100 nt in size, are synthesized by RNA polymerase III, and are found only in rodents of several related families. Genes coding for these RNAs are evolutionarily related to short interspersed elements (SINEs). However, the two RNAs differ in nucleotide sequence, half-life in the cell, and the organization of their genes in the genome. Although the 4.5SI and 4.5SH RNAs have been identified more than three decades ago, several aspects of their metabolism in the cell are still poorly understood. The 4.5SI and 4.5SH RNA levels were measured in various organs of three rodent species (mouse, rat, and hamster). Both of the RNAs were found to occur at high levels, which were much the same in different organs in the case of the 4.5SI RNA and varied among organs in the case of the 4.5SH RNA. Both 4.5SI and 4.5SH RNAs demonstrated a predominantly nuclear localization with a detectable presence in the cytoplasm. The copy number per cell for the RNAs was estimated at 0.4-2.4 × 10^(6). A quantitative study for the 4.5SI and 4.5SH RNAs was performed for the first time and resolved a number of contradictions in data from other studies.

4.5SI and 4.5SH RNAs: Expression in various rodent organs and abundance and distribution in the cell

4.5SI and 4.5SH RNAs: Expression in various rodent organs and abundance and distribution in the cell

Silencing Genes in the Heart.

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

Downregulation of Annexin A1 is correlated with radioresistance in nasopharyngeal carcinoma.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance often remains an obstacle to successful treatment. In our previous study, it was demonstrated that Annexin A1 (ANXA1) was involved in the p53-mediated radioresponse in NPC cells, which suggested that it may be associated with radioresistance in NPC; however, the role of ANXA1 in NPC radioresistance is unknown. In the present study, CNE2 cells were stably transfected with pLKO.1-ANXA1-small hairpin (sh)RNAs to investigate the effects of ANXA1 on the radiosensitivity of NPC. CNE2 cells transfected with pLKO.1 were used as the control. The radiosensitivities of the cells in vitro were analyzed using the clonogenic survival assay, cell growth analysis, flow cytometry and Hoechst 33258 staining. ANXA1 downregulation significantly enhanced clonogenic survival and cell growth following treatment of CNE2 cells with ionizing radiation (IR), increased the number of cells in the S phase and decreased IR-induced apoptosis. These results suggested that the radiosensitivity of CNE2 cells transfected with ANXA1-specific shRNA was significantly lower compared with the control cells. Therefore, ANXA1 downregulation may be involved in the radioresistance of NPC, and ANXA1 may be considered a novel biomarker for predicting NPC response to radiotherapy.

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.