Inhibition Of Target Gene Expression Research Articles

Antisense yycG Regulation of Antibiotic Sensitivity of Methicillin-Resistant Staphylococcus aureus in Chronic Osteomyelitis.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an urgent medical problem in osteomyelitis. The YycFG two-component regulatory system (TCS) allows bacteria to adapt rapidly to physical, chemical, and biological stresses. The recombinant plasmid shuttle vector was used to overexpress an antisense RNA (asRNA) to inhibit target gene expression by sequence-specific double-stranded RNA complex degradation. In the current study, antisense yycG RNA (ASyycG)-overexpression MRSA clinical isolates were constructed. Methods: Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Quantitative reverse transcription polymerase chain reaction analysis was used to identify expression of yycF/G/H and icaA/D in MRSA and ASyycG strains. The expression of YycG protein was quantified by Western blot assays. The antibiotic resistance of ASyycG strains was compared with that of the MRSA strains. Results: The ASyycG strains showed a decrease in growth rate compared with the MRSA strains. Of note, overexpression of ASyycG led to a reduction in biofilm formation and adhesion force. ASyycG strains had decreased expressions of the yycF/G/H and icaA/D. Furthermore, Western blot data showed that expression of the YycG protein decreased by 40% in ASyycG strains compared with MRSA strains. In addition, the effect of yycG asRNA improved the susceptibility of ASyycG strains to cefoxitin. Conclusions: The ASyycG strains inhibited biofilm organization and increased antibiotic sensitivity, which may be attributed to altered intracellular polysaccharide construction.

SLN124, a Galnac-siRNA Conjugate Targeting TMPRSS6, for the Treatment of Iron Overload and Ineffective Erythropoiesis Such As in Beta-Thalassemia

Accumulation of excess iron in tissues causes organ damage and dysfunction and may lead to serious clinical consequences including liver cirrhosis, diabetes, growth retardation and heart failure. Iron overload is a major health threat in iron loading anemias, like beta-thalassemia, myelodysplastic syndrome and in hereditary hemochromatosis. In patients with beta-thalassemia major, iron overload develops due to frequent blood transfusions to control the severe anemia. In addition, iron overload also occurs in patients with beta thalassemia intermedia (non-transfusion dependent beta-thalassemia). In the later cases, iron overload develops through gastrointestinal iron hyperabsorption due to stressed and ineffective erythropoiesis. Importantly, expression of the peptide hormone hepcidin, which is the key modulator in iron homeostasis, is abnormally low and unable to block ferroportin-mediated intestinal iron absorption. In hereditary hemochromatosis, gene defects in the hepcidin-ferroportin axis controlling iron homeostasis, lead to hepatic iron overload. Therefore, in these indications, iron overload is caused by dysregulation or dysfunction of the hepcidin-ferroportin axis. Hepcidin is predominantly produced by the liver and is induced by activation of the BMP/SMAD signaling pathway. Furthermore, hepcidin is under the negative control of the transmembrane protease matriptase-2, encoded by the TMPRSS6 gene.RNA interference is a natural mechanism and a powerful approach for inhibiting the expression of disease-associated genes. Silence Therapeutics has developed short interfering RNA (siRNA) conjugate technology for the selective inhibition of target gene expression in the liver. GalNAc-conjugated siRNAs bind efficiently to the asialoglycoprotein (ASGP) receptor expressed predominantly by hepatocytes thereby providing a highly specific, safe and efficient delivery technology to enable a new class of therapeutic use.Here we present the pharmacological characterization of SLN124, our GalNAc-siRNA conjugate targeting TMPRSS6 expression, in preclinical models. A single subcutaneous administration is sufficient to achieve significant modulation of target gene expression in mice and in non-human primates over several weeks. SLN124 treatment reduces systemic iron levels, transferrin saturation and tissue iron levels in a rodent model for hereditary hemochromatosis type 1. In addition, we report for the first time the therapeutic efficacy of iron restriction by SLN124 in mice with established iron overload both as monotherapy and in combination with an oral iron chelator - current standard of care- over an extended treatment period. The effects of these treatments on red blood cell parameters and tissue iron levels will be presented. In addition, we assessed the therapeutic effects of SLN124 in an animal model for beta-thalassemia intermedia, showing dose-dependent and long-lasting effects on target gene expression as well as on modulation of iron stores and normalization of erythropoiesis and anemia. Safety and tolerability studies in relevant preclinical models confirmed that SLN124 is well tolerated and shows promise as an effective and safe treatment for unmet medical need in iron loading anemias, such as beta-thalassemia. SLN124 is currently in preclinical development. The first in human study is planned to commence in 2019 in patients with beta-thalassemia and in patients with myelodysplastic syndrome. DisclosuresMuckenthaler:Novartis: Research Funding. Dames:Silence Therapeutics GmbH: Employment. Frauendorf:Silence Therapeutics GmbH: Employment. Schubert:Silence Therapeutics GmbH: Employment. Aleku:Silence Therapeutics GmbH: Employment. Zügel:Silence Therapeutics GmbH: Employment.

Quantitative fluorescence imaging determines the absolute number of locked nucleic acid oligonucleotides needed for suppression of target gene expression.

Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.

S-nitrosylation of the zinc finger protein SRG1 regulates plant immunity

Nitric oxide (NO) orchestrates a plethora of incongruent plant immune responses, including the reprograming of global gene expression. However, the cognate molecular mechanisms remain largely unknown. Here we show a zinc finger transcription factor (ZF-TF), SRG1, is a central target of NO bioactivity during plant immunity, where it functions as a positive regulator. NO accumulation promotes SRG1 expression and subsequently SRG1 occupies a repeated canonical sequence within target promoters. An EAR domain enables SRG1 to recruit the corepressor TOPLESS, suppressing target gene expression. Sustained NO synthesis drives SRG1 S-nitrosylation predominantly at Cys87, relieving both SRG1 DNA binding and transcriptional repression activity. Accordingly, mutation of Cys87 compromises NO-mediated control of SRG1-dependent transcriptional suppression. Thus, the SRG1-SNO formation may contribute to a negative feedback loop that attenuates the plant immune response. SRG1 Cys87 is evolutionary conserved and thus may be a target for redox regulation of ZF-TF function across phylogenetic kingdoms.

Abstract LB-385: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Abstract Bromodomain Containing 4 (BRD4) mediates transcriptional elongation of the oncogene MYC by binding to acetylated histones. BRD4 has been shown to play a critical role in tumorigenesis in several cancers, and the BRD4-NUT fusion gene is a driver of NUT midline carcinoma (NMC), a rare but highly lethal cancer. microRNAs (miRNAs) are endogenous small non-coding RNAs that suppress target gene expression by binding to complementary mRNA sequences. Here, we show that miR-3140, which was identified as a novel tumor suppressive miRNA by function-based screening of a library containing 1090 miRNA mimics, directly suppressed BRD4 by binding to its coding sequence (CDS). miR-3140 concurrently downregulated BRD3 by bind to its CDS as well as CDK2 and EGFR by binding to their 3' untranslated regions. miR-3140 inhibited tumor cell growth in vitro in various cancer cell lines, including EGFR tyrosine kinase inhibitor-resistant cells. Interestingly, we found that miR-3140 downregulated the BRD4-NUT fusion protein and suppressed in vitro tumor cell growth in a NMC cell line, Ty-82 cells. Furthermore, administration of miR-3140 suppressed in vivo tumor growth in a xenograft mouse model. Our results suggest that miR-3140 is a candidate for the development of miRNA-based cancer therapeutics. Citation Format: Tomoki Muramatsu, Erina Tonouchi, Yasuyuki Gen, Hidekazu Hiramoto, Kousuke Tanimoto, Jun Inoue, Johji Inazawa. miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein [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 LB-385.

MiR-132 couples the circadian clock to daily rhythms of neuronal plasticity and cognition.

The microRNA miR-132 serves as a key regulator of a wide range of plasticity-associated processes in the central nervous system. Interestingly, miR-132 expression has also been shown to be under the control of the circadian timing system. This finding, coupled with work showing that miR-132 is expressed in the hippocampus, where it influences neuronal morphology and memory, led us to test the idea that daily rhythms in miR-132 within the forebrain modulate cognition as a function of circadian time. Here, we show that hippocampal miR-132 expression is gated by the time-of-day, with peak levels occurring during the circadian night. Further, in miR-132 knockout mice and in transgenic mice, where miR-132 is constitutively expressed under the control of the tetracycline regulator system, we found that time-of-day dependent memory recall (as assessed via novel object location and contextual fear conditioning paradigms) was suppressed. Given that miRNAs exert their functional effects via the suppression of target gene expression, we examined the effects that transgenic miR-132 manipulations have on MeCP2 and Sirt1—two miR-132 targets that are associated with neuronal plasticity and cognition. In mice where miR-132 was either knocked out, or transgenically expressed, rhythmic expression of MeCP2 and Sirt1 was suppressed. Taken together, these results raise the prospect that miR-132 serves as a key route through which the circadian timing system imparts a daily rhythm on cognitive capacity.

MiR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Bromodomain Containing 4 (BRD4) mediates transcriptional elongation of the oncogene MYC by binding to acetylated histones. BRD4 has been shown to play a critical role in tumorigenesis in several cancers, and the BRD4-NUT fusion gene is a driver of NUT midline carcinoma (NMC), a rare but highly lethal cancer. microRNAs (miRNAs) are endogenous small non-coding RNAs that suppress target gene expression by binding to complementary mRNA sequences. Here, we show that miR-3140, which was identified as a novel tumor suppressive miRNA by function-based screening of a library containing 1090 miRNA mimics, directly suppressed BRD4 by binding to its coding sequence (CDS). miR-3140 concurrently downregulated BRD3 by bind to its CDS as well as CDK2 and EGFR by binding to their 3’ untranslated regions. miR-3140 inhibited tumor cell growth in vitro in various cancer cell lines, including EGFR tyrosine kinase inhibitor-resistant cells. Interestingly, we found that miR-3140 downregulated the BRD4-NUT fusion protein and suppressed in vitro tumor cell growth in a NMC cell line, Ty-82 cells. Furthermore, administration of miR-3140 suppressed in vivo tumor growth in a xenograft mouse model. Our results suggest that miR-3140 is a candidate for the development of miRNA-based cancer therapeutics.

Biomass production and salinity response in plants: role of MicroRNAs

Small non-coding RNAs are one of the major contributors for diverse cellular functions in plants. MicroRNAs (miRNAs) are one such class of small non-coding RNAs playing crucial role in normal plant growth and development as well as under environmental stresses. miRNA modulates the transcripts level by directly binding to the transcript generated from the target gene and mediate either the cleavage of the target mRNA transcript or the inhibitition of translation of the target transcript or inhibition of target gene expression through epigenetic modification. In the current scenario, understanding the link between the diverse miRNAs and their orchestrated functioning for regulation of stress signaling as well as biomass production is a major challenge. In the present review, we have explored the current knowledge about the plant miRNA families and their functional aspects, particularly in the context of salinity stress tolerance and higher biomass production. We conclude that miRNA families such as miR156, 159, 164, 166, 319, 393, 396 and 414 are possibly mediating the cross talk between biomass production and salinity stress response. The present review may improve the current knowledge about the plant miRNAs and thus may help in generating crop plants for marginal lands.

Rational design of biosafe crop resistance to a range of nematodes using RNA interference.

SummaryDouble‐stranded RNA (dsRNA) molecules targeting two genes have been identified that suppress economically important parasitic nematode species of banana. Proteasomal alpha subunit 4 (pas‐4) and Actin‐4 (act‐4) were identified from a survey of sequence databases and cloned sequences for genes conserved across four pests of banana, Radopholus similis, Pratylenchus coffeae, Meloidogyne incognita and Helicotylenchus multicinctus. These four species were targeted with dsRNAs containing exact 21 nucleotide matches to the conserved regions. Potential off‐target effects were limited by comparison with Caenorhabditis, Drosophila, rat, rice and Arabidopsis genomes. In vitro act‐4 dsRNA treatment of R. similis suppressed target gene expression by 2.3‐fold, nematode locomotion by 66 ± 4% and nematode multiplication on carrot discs by 49 ± 5%. The best transgenic carrot hairy root lines expressing act‐4 or pas‐4 dsRNA reduced transcript message abundance of target genes in R. similis by 7.9‐fold and fourfold and nematode multiplication by 94 ± 2% and 69 ± 3%, respectively. The same act‐4 and pas‐4 lines reduced P. coffeae target transcripts by 1.7‐ and twofold and multiplication by 50 ± 6% and 73 ± 8%. Multiplication of M. incognita on the pas‐4 lines was reduced by 97 ± 1% and 99 ± 1% while target transcript abundance was suppressed 4.9‐ and 5.6‐fold. There was no detectable RNAi effect on nontarget nematodes exposed to dsRNAs targeting parasitic nematodes. This work defines a framework for development of a range of nonprotein defences to provide broad resistance to pests and pathogens of crops.

Abstract 2080: Polycomb-mediated disruption of an AR-feedback loop drives castration-resistant prostate cancer

Abstract Androgen receptor (AR) is an androgen-stimulated transcription factor that critically promotes prostate cancer development and progression. In addition to its conventional role in transcriptional activation, AR also acts as a transcriptional repressor to directly inhibit target gene expression. Through meta-analysis of androgen-regulated expression microarray data, we nominated CCN3/NOV, a CCN family protein, as one of the top AR-repressed genes in prostate cancer, implicating that CCN3 may play a tumor suppressive role in prostate cancer development. Therefore, we sought to investigate the molecular functions of CCN3 and its roles in prostate cancer progression especially in castration-resistant prostate cancer (CRPC). We first showed that CCN3 protein physically interacts with AR. The CCN3 interaction domain was mapped in the N-terminal domain of AR which is also present in constitutively active AR variants. Using immunofluorescence microscopy as well as subcellular fractionation technique, we demonstrated that CCN3 sequesters AR or AR variants into the cytoplasm to inhibit AR nuclear translocation, which in turn suppresses AR chromatin targeting and transcriptional activation. However, this negative feedback loop between AR and AR-repressed gene CCN3is disrupted in CRPC through CCN3 persistent epigenetic silencing by the Polycomb group protein EZH2, rendering AR transcriptional activation and CRPC progression. Taking advantage of this repressive mechanism, we restored CCN3 in CRPC cell model, either through ectopic overexpression or treatment with EZH2 inhibitor, which results in strong suppression of CRPC cell proliferation and anchorage-independent cell growth. Consistently, application of EZH2 inhibitor remarkably reduced tumor size in a castrated mice model. Taken together, our data highlight CCN3 as a novel regulator of AR signaling and support the promise of EZH2-targeting agents in treating CRPC. Citation Format: Ka-Wing Fong, Jonathan Zhao, Jung Kim, Shangze Li, Angela Yang, Bing Song, Laure Rittié, Ming Hu, Ximing Yang, Bernard Perbal, Jindan Yu. Polycomb-mediated disruption of an AR-feedback loop drives castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2080. doi:10.1158/1538-7445.AM2017-2080

New insights on microRNAs in diabetic nephropathy: potential biomarkers for diagnosis and therapeutic targets

Diabetic nephropathy (DN) is a severe complication of diabetes mellitus associated with the progressive deterioration of renal function. Although microalbuminuria is considered as a gold standard for DN diagnosis, it has limited predictive powers and specificity as a diagnostic tool for the early stage of DN. Therefore, new biomarkers are required for the early detection of DN. Studies using in vitro and in vivo models of DN have revealed an important role of microRNAs (miRNAs), short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression, in DN development. Recent studies have shown that the dysregulation of miRNAs, which is associated with the key features of DN, such as the mesangial expansion and accumulation of extracellular matrix proteins, is related to fibrosis and glomerular dysfunction. Thus, the up- and downregulation of miRNA expression in the renal tissue or biological fluids, including urine, may represent new biomarkers for the diagnosis and monitoring of DN progression. In this review, we highlight the significance of miRNAs as biomarkers for the early detection of DN and emphasise their potential role as a therapeutic target.

Multi-step encapsulation of chemotherapy and gene silencing agents in functionalized mesoporous silica nanoparticles

Drug to carrier ratio is an important consideration in designing drug platforms, since a low loading capacity necessitates the use of high doses of carriers, which can result in side effects. Here, we have engineered a platform to co-deliver small molecule drugs and small interfering RNA (siRNA). This platform consists of cyclodextrin-grafted polyethylenimine (CP) functionalized mesoporous silica nanoparticles (MSNP). A unique multi-step encapsulation procedure was used to obtain a high loading capacity for doxorubicin (DOX) and siRNA oligos specific for the PKM2 gene that encodes pyruvate kinase M2, an enzyme catalyzing the final rate-limiting step in glycolysis. We systematically characterized this platform (CP-MSNP@DOX/PKM2) in vitro and evaluated its therapeutic efficacy in vivo with a mouse model of triple negative breast cancer (TNBC). Exposure of TNBC cells to CP-MSNP@DOX/PKM2 resulted in suppressed target gene expression, reduced cell proliferation, and enhanced apoptosis. Intravenous administration of the drug substantially decreased the tumor burden in comparison to DOX or siRNA monotherapy. In conclusion, we have developed a platform for efficient co-delivery of small molecule drugs and therapeutic siRNA.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Efficient in vivo delivery of antisense oligonucleotide to choroid plexus.

The choroid plexus (CP) is present on the ventricular walls of the brain, produces cerebrospinal fluid (CSF), contains many blood vessels, and is a major functional component of the blood-CSF barrier. The CP is an important site in the pathophysiology of various neurological diseases, including Alzheimer's disease and meningeal amyloidosis. We performed gene silencing in the CP in vivo by using an antisense oligonucleotide (ASO). A short ASO of length 12 nucleotides was intravenously injected into rats. The ASO was not delivered to neurons or glia in the central nervous system, but was successfully delivered into the CP, and resulted in a significant reduction of endogenous target gene expression in epithelial cells within the CP. Although the mechanism of uptake of the ASO by the CP was not elucidated, the ASO bound to albumin in vivo, and the distribution of ASO delivery was similar to that of albumin delivery. These findings suggest that we inhibited target gene expression in the epithelial cells of the CP via albumin-ASO conjugates. This strategy should be useful for investigations of the function of CP, and for the development of new gene-silencing therapies for diseases with pathophysiology related to the CP.

MiR-1271 upregulated by saturated fatty acid palmitate provokes impaired insulin signaling by repressing INSR and IRS-1 expression in HepG2 cells

Dietary saturated fatty acids (SFA) in excess not only induce hepatic insulin resistance, but also result in type 2 diabetes (T2DM). Although microRNAs (miRNAs) participate widely in the pathogenesis of a range of diseases through the suppression of target gene expression at the post-transcriptional level, the implications of SFA-induced miRNAs in the dysregulation of metabolism, particularly in the development of insulin resistance, are largely unclear. SFA palmitate provoked an impairment of insulin signaling in HepG2 cells via a reduction in the expression of INSR and IRS-1 protein. The significant upregulation of miR-1271, which was presumed to target INSR and IRS-1 3′UTRs, was observed in the palmitate-treated HepG2 cells. Using a reporter gene assay, miR-1271 authentically targeted the 3′UTRs of INSR and IRS-1. Furthermore, the overexpression of miR-1271 caused a substantial decrease in INSR and IRS-1 expression, which led to an impairment in insulin signaling and glycogen metabolism. Therefore, these findings suggest that the induction of miR-1271 by SFA palmitate promotes the development of insulin resistance by targeting INSR and IRS-1 in hepatocytes.

Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication

RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors.

Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation

Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.

Protection of rats spinal cord ischemia-reperfusion injury by inhibition of MiR-497 on inflammation and apoptosis: Possible role in pediatrics.

MicroRNAs are extensively included in the pathogenesis and progression of many diseases by inhibiting target gene expression. Recently, studies have demonstrated that microRNA-497 (miR-497) may be implicated in human breast cancer that miR-497 predicts the prognosis of breast cancer patients from the posttranscriptional level. However, the specific function of miR-497 in spinal cord ischemia-reperfusion (IR) injury is far from clear nowadays. The present study was designed to determine the role of miR-497 in spinal cord IR injury and investigate the underlying spinal cord protective mechanism. The rat spinal cord IR injury model was performed by occluding the left anterior descending coronary artery for 30 min, which is then followed by 12h reperfusion. As predicted, miR-497 over-expression markedly decreased the expression of IL-1 receptor associated kinase (IRAK1) and Cyclic AMP response element binding protein (CREB). Moreover, Toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB) and Caspase-3, as miR-497 potential targets were significantly suppressed after miR-497 transfection, then preventing inflammatory cytokines and factors regulating apoptosis. We also found that tumor necrosis factor-a (TNF-α) and interleukin-1beta (IL-1β) activity, pro-apoptotic related genes, such as extracellular regulated protein kinases (ERK), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL levels were all decreased associated with the down-regulation of IRAK1 and CREB. In conclusion, our data demonstrate that miR-497 could inhibit inflammation and apoptosis of spinal cord IR through its targets, IRAK1 of TLR4 and CREB signaling pathway. Thus, miR-497 may constitute a new therapeutic target for the prevention of spinal cord IR injury.

MicroRNA-154 functions as a tumor suppressor in osteosarcoma by targeting Wnt5a.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumor initiation and development by suppressing target gene expression. miRNA-154 has been shown to be important in tumorigenesis in many types of cancers. However, its role in osteosarcoma (OS) remains unknown. In the present study, we focused on the roles and mechanisms of miR‑154 in OS development. The results of quantitative RT‑PCR showed that miR‑154 expression was decreased in primary OS tumor samples and cell lines compared to levels in the matched adjacent normal tissues and human normal osteoblast cells (NHOst). Restoration of expression in U2OS cells inhibited cell proliferation, colony formation, migration and invasion, as well as induced cell cycle arrest at the G1 stage. Bioinformatic prediction suggested that Wnt5a is a target gene of miR‑154. It was further verified that Wnt5a is a target gene of miR‑150 in OS cells using luciferase assay, mRNA and protein expression analysis. Wnt5a was upregulated in OS cell lines and primary tumor samples, and its mRNA expression level was negatively correlated with the miR‑154 level in the OS tissues. Restored expression of Wnt5a weakened miR‑154‑mediated suppression of tumor progression. Taken together, these findings suggest that miR‑154 functions as a tumor suppressor in OS by partially suppressing Wnt5a expression.

Transfection of microRNA Mimics Should Be Used with Caution

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.