Antisense LNA Research Articles

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signalling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CAMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CAMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function.

Knockdown of SIRLNT by antisense LNA GapmeRs and evaluation of its inhibitory effect on proliferation and apoptosis in MCF7 cell line

Knockdown of SIRLNT by antisense LNA GapmeRs and evaluation of its inhibitory effect on proliferation and apoptosis in MCF7 cell line

Acetyl Co-A Carboxylase Inhibition Halts Hyperglycemia Induced Upregulation of De Novo Lipogenesis in Podocytes and Proximal Tubular Cells.

The effect of glycemic stress on de novo lipogenesis (DNL) in podocytes and tubular epithelial cells is understudied. This study is aimed (A) to show the effect of glycemic stress on DNL, and (B) to assess the effect of acetyl-Co A (ACC) inhibition on halting upregulation of DNL, on the expression of other lipid regulatory genes in the DNL pathway, and on markers of fibrosis and apoptosis in podocytes and tubular epithelial cells. We used cultured mouse primary tubular epithelial cells, mouse proximal tubular (BUMPT) cells, and immortal mouse podocytes and measured their percentage of labeled 13C2-palmitate as a marker of DNL after incubation with 13C2 acetate in response to high glucose concentration (25 mM). We then tested the effect of ACC inhibition by complimentary strategies utilizing CRISPR/cas9 deletion or incubation with Acaca and Acacb GapmeRs or using a small molecule inhibitor on DNL under hyperglycemic concentration. Exposure to high glucose concentration (25 mM) compared to osmotic controlled low glucose concentration (5.5 mM) significantly increased labeled palmitate after 24 h up to 72 h in podocytes and primary tubular cells. Knocking out of the ACC coding Acaca and Acacb genes by CRISPR/cas9, downregulation of Acaca and Acacb by specific antisense LNA GapmeRs and inhibition of ACC by firsocostat similarly halted/mitigated upregulation of DNL and decreased markers of fibrosis and programmed cell death in podocytes and various tubular cells. ACC inhibition is a potential therapeutic target to mitigate or halt hyperglycemia-induced upregulation of DNL in podocytes and tubular cells.

428. CHARACTERIZATION OF CD82/KAI1 IN ESOPHAGEAL ADENOCARCINOMA

Abstract The incidence of esophageal adencocarcinoma (EAC) has increased in last decades. Regardless to improved perioperative chemotherapies and surgical approaches, the prognosis for EAC in advanced stages is poor. CD82 is a tumor suppressor gene, and its downregulation correlates with invasive growth, metastases formation and advanced clinical stages. CD82 might influence the Wnt/ß-catenin signaling pathway. We used an in vitro Barrett’s esophagus cell culture model, represented by esophageal squamous cell epithelium (EPC1, EPC2), metaplasia (CP-A), dysplasia (CP-B) and esophageal adenocarcinoma (OE33, OE19, SKGT4, FLO1). Antisense LNA Oligonucleotides (GapmeRs, Qiagen) were used to achieve a transient knockdown of CD82 in OE33 and OE19 cells. mRNA and proteins were analyzed by q-RT-PCR and western blot analyses, respectively. A fluorescence antibody specific to CD82 was used for flow cytometry analysis. Proliferation assay, colony formation assay and boyden chamber assay were performed after CD82-knockdown to further analyze cell proliferation, cell growth and survival, colony forming and cell migration. CD82 was expressed in all investigated cell lines by flow cytometry with a significant overexpression in OE19 and FLO1 cells. A stable downregulation of CD82 was achieved in OE33 and OE19 cells. Proliferation assays showed a significantly higher proliferation rate at 72 hours after CD82-knockdown in OE33 cells. A significantly higher mRNA expression of vimentin was observed after CD82-knockdown in OE33. Colony formation assay showed a significantly higher colony count after CD82-knockdown in both cell lines. Cell migration was increased after CD82-downregulated in OE33 and OE19 cells. CD82 might influence the expression of mesenchymal marker proteins, cell migration abilities and the process of colony forming and thus enhance the metastatic potential of OE33 and OE19 cells. Low expression of CD82 could alter metastasis and cancer progression but further molecular characterizations are needed to elucidate the impact on carcinogenesis of EAC.

Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function.Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation.Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively.Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation.Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

ZCTs knockdown using antisense LNA GapmeR in specialized photomixotrophic cell suspensions of Catharanthus roseus: Rerouting the flux towards mono and dimeric indole alkaloids.

The online version contains supplementary material available at 10.1007/s12298-021-01017-y.

Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B12 is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B12 by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B12 but also both B12-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6–9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B12-ASO conjugates is strongly affected by the low uptake rate of the B12 in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.

A chromatin-associated splicing isoform of OIP5-AS1 acts in cis to regulate the OIP5 oncogene

ABSTRACT A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Knockdown of SOX12 Expression Induced Apoptotic Factors is Associated with TWIST1 and CTNNB1 Expression in Human Acute Myeloid Leukemia Cells.

Recent improvements in molecular treatment and gene therapy led to discovering novel cancer remedies. Antisense LNA GapmeRs is a state-of-the-art molecular research field for diagnosing and treating various cancer types. Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy defined by the rapid accumulation and malignant proliferation of immature myeloid progenitors. SOX12 is a new potential target for acute myeloid leukemia. In this study, SOX12 was blocked by antisense LNA GapmeRs (ALG) in human AML cell lines (KG1 and M07e). Cells were transfected with Gapmer anti-SOX12 at 24, 48, and 72 h post-transfection. Transfection efficiency was assessed by a fluorescent microscope. Furthermore, evaluation of SOX12, TWIST1, CTNNB1, CASP3, and CASP9 expression was performed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell viability was determined by MTT assay. SOX12 expression was decreased remarkably in the ALG group. Moreover, SOX12 knockdown was associated with a decrease in TWIST1 and CTNNB1 expression. Besides, downregulation of SOX12 in both cell lines could induce apoptosis, probably through upregulation of CASP3 and CASP9. The findings reveal that SOX12 knockdown could be a new target for reducing AML cells proliferation through antisense therapy approach.

Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.

LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.

Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Downregulation of MIR100HG Induces Apoptosis in Human Megakaryoblastic Leukemia Cells.

Long noncoding ribonucleic acids (lncRNAs) are ribonucleic acid (RNA) molecules longer than 200 nucleotides without protein-coding capacity. Several studies have shown that lncRNAs play a pivotal role in the initiation, maintenance, and progression of acute myeloid leukemia (AML), which could make them a promising candidate in the diagnosis and treatment of leukemia. Acute Megakaryoblastic leukemia (AMKL) is a rare form of AML with a poor prognosis and low survival. It has been reported that lncRNA MIR100HG is involved several types of malignancies. In the present study, MIR100HG was downregulated in a human acute megakaryoblastic leukemia cell line (M-07e) using Antisense LNA GapmeRs. In order to assess the expression level of MIR100HG, cell viability, apoptosis, and necrosis (late apoptosis), quantitative reverse transcription polymerase chain reaction (qRT-PCR), Methyl-thiazol Tetrazolium assay, AnnexinV, and propidium iodide staining was performed at different time points after the transfection. In addition, the expression level of TGFβ was evaluated by qRT-PCR. Our results revealed that inhibition of MIR100HG might serve as a new method for inhibition of the proliferation of AMKL cells and therefore, could be a promising approach in medicine for targeted therapy in AMKL.

Degradation of HOX Transcript Antisense RNA Provoked Apoptosis and Necrosis in Human Ovarian Cancer Cells

Ovarian cancer (OC) is one of the common malignancies with poor survival rates in women. Long noncoding RNAs (lncRNAs) are a class of regulatory RNA via gene expression regulation and dysregulation results in several disorders such as cancer. LncRNA HOX transcript antisense RNA (HOTAIR) is reported to be upregulated in OC. It plays a major role in cell proliferation and metastasis. The aim of this study is to investigate the effect of HOTAIR blockage in OC cells proliferation and death. We blocked HOTAIR in OC cell line SKOV3 by Antisense LNA GapmeRs. The qPCR and Annexin V/propidium iodide staining assay were performed at three time points after transfection. Moreover, we examined STAT3 and MAPK8 expression levels as cancer proliferation-associated agents under the influence of GapmeRs using qPCR at 24, 48 and 72 h after transfection. The rate of apoptotic cells increased in Antisense LNA GapmeRs-transfected group compared with the two other groups at all three time points. The ratio of the necrotic cell was also higher in Antisense LNA GapmeRs-treated group in contrary to the other groups in a time-dependent manner. However, among all selected genes only MAPK8 was significantly downregulated at 24 h, followed by upregulation at 48 and 72 h. STAT3 was remarkably increased in all three time points. These data demonstrated that HOTAIR can act as an onco-lncRNA and its inhibition significantly boosted apoptosis and necrosis in the SKOV3 cell line. Our findings can pave the way for antisense-based therapy as a potentially effective approach in the treatment of OC.

Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA

ObjectiveA widely recognized metabolic side effect of glucocorticoid (GC) therapy is steroid-induced diabetes mellitus (DM). However, studies on the molecular basis of GC-induced pancreatic beta cell dysfunction in human beta cells are lacking. The significance of non-coding RNAs in various cellular processes is emerging. In this study, we aimed to show the direct negative impact of GC on beta cell function and elucidate the role of riborepressor GAS5 lincRNA in the GC signaling pathway in human pancreatic beta cells. MethodsPatients undergoing two weeks of high-dose prednisolone therapy were monitored for C-peptide levels. Human pancreatic islets and the human beta cell line EndoC-βH1 were incubated in pharmacological concentrations of dexamethasone. The GAS5 level was modulated using anti-sense LNA gapmeR or short oligonucleotides with GAS5 HREM (hormone response element motif). Immunoblotting and/or real-time PCR were used to assess changes in protein and RNA expression, respectively. Functional characterization included glucose-stimulated insulin secretion and apoptosis assays. Correlation analysis was performed on RNAseq data of human pancreatic islets. ResultsWe found reduced C-peptide levels in patients undergoing high-dose GC therapy. Human islets and the human beta cell line EndoC-βH1 exposed to GC exhibited reduced insulin secretion and increased apoptosis. Concomitantly, reduced expression of important beta cell transcription factors, PDX1 and NKX6-1, as well as exocytotic protein SYT13 were observed. The expression of the glucocorticoid receptor was decreased, while that of serum and glucocorticoid-regulated kinase 1 (SGK1) was elevated. The expression of these genes was found to significantly correlate with GAS5 in human islet transcriptomics data. Increasing GAS5 levels using GAS5 HREM alleviated the inhibitory effects of dexamethasone on insulin secretion. ConclusionsThe direct adverse effect of glucocorticoid in human beta cell function is mediated via important beta cell proteins and components of the GC signaling pathway in an intricate interplay with GAS5 lincRNA, a potentially novel therapeutic target to counter GC-mediated beta cell dysfunction.

Long Non-Coding RNAs As Novel Therapeutic Targets in Juvenile Myelomonocytic Leukemia: Proof of Concept Study

BACKGROUND Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative disorder of early childhood. Hematopoietic stem cell transplantation results in long-term overall survival of only 50-60% and is fraught with frequent relapse and toxicity. Consequently, there is need to develop novel treatments. Evidence is growing that non-coding RNAs can serve as alternative therapeutic targets. Long non-coding RNAs (lncRNAs) are a recently discovered class of RNAs, with a minimum length of 200 nucleotides, of which perturbation can impact the cancer phenotype in vivo. Recently, we documented for the first time the lncRNA landscape in 44 JMML patients using microarrays and have associated lncRNA expression with clinical and molecular characteristics. Also, we demonstrated that JMML patients exhibit a distinct lncRNA expression profile compared to healthy controls and we identified 15 lncRNAs overexpressed in JMML patients. AIM The aim of this study is to identify overexpressed lncRNAs in JMML and to provide proof-of-concept for lncRNA perturbation as novel therapeutic strategy in this disease. METHODS To further identify overexpressed lncRNAs in JMML, total RNA from isolated mononuclear cell preparations from 19 previously untreated JMML patients (median age: 2.02 years) and 3 normal pediatric bone marrow (NPBM) samples from healthy controls (siblings screened for transplantation) was sequenced (HiSeq, Illumina). Differential gene expression (adjusted P-value < 0.05 and FDR < 0.10) was performed with DESEQ2 and EdgeR (R Bioconductor). Functional analysis of differentially expressed lncRNAs was performed through a lncRNA-mRNA interaction network (lncPath, R Bioconductor). A subset of overexpressed lncRNAs, based on fold change ≥2 and low to absent expression in NPBM, were subsequently validated by quantitative reverse-transcriptase PCR (qPCR). Antisense LNA™ GapmeRs (Qiagen), potent antisense oligonucleotides used for highly efficient inhibition of mRNA and lncRNA function, were designed for a subset of significantly overexpressed lncRNAs. As no JMML cell lines are available, cell lines from other pediatric and adult haematopoietic malignancies were selected based on publically available RNA-seq data and lncRNA expression was verified on qPCR. The effect of GapmeR treatment on lncRNA expression and cell viability was tested with qPCR and flow cytometry viability assays (7-AAD and annexin V staining). RESULTS Total RNA sequencing additionally revealed presence of 122 upregulated and 47 downregulated lncRNAs, most of which not present on the microarray. JMML patients clustered together and divergent from healthy controls on principal component analysis using only lncRNAs. Different pathways associated with RAS-MAPK signaling, cancer development, DNA metabolism and cell cycle were identified as being synergistically regulated by these differentially expressed lncRNAs through a lncRNA-mRNA interaction network. Forty-two lncRNAs (55 different transcripts) showing overexpression in JMML were validated with qPCR and in 13 transcripts from 11 lncRNAs a significantly higher expression could be observed in JMML patients compared to NPBM (Figure 1A), thus providing potential therapeutic targets. A minimum of four different antisense LNA GapmeRs were subsequently designed for each of these validated lncRNAs, overexpressed in JMML. For each lncRNA, one or more hematopoietic cell lines, showing expression of that lncRNA, could be identified and validated with qPCR. In vitro incubation of these cell lines with GapmeRs against lncRNA with expression in these cell lines, revealed concentration-dependent molecular knockdown in ≥ 2 GapmeRs for 4/5 lncRNAs (Figure 1B). For two targets, lnc-THADA4-1 and lnc-ACOT9, additional cell viability assays were performed and an effect on cell viability could be observed in GapmeR treated wells (Figure 1C). CONCLUSION Using microarray and total RNA sequencing, we documented the lncRNA landscape in JMML, and identified deregulated lncRNAs associated with key processes in JMML pathogenesis. We further provided proof-of-concept that knockdown of these lncRNAs using LNA GapmeRs can be a feasible therapeutic strategy in vitro in hematopoietic cell lines. Subsequently, safety and efficacy of this novel therapeutic strategy needs to be validated in JMML xenograft models in vivo. Figure 1 Disclosures Niemeyer: Celgene: Consultancy.

Blockage of HOTAIR Reduced Cell Proliferation in Human Ovarian Cancer Cells Through Upregulation of AKT2

Ovarian cancer (OC) is the most leading cause of cancer-related death in women. lncRNA HOTAIR is emerging as an onco-lncRNA that its overexpression provokes progression and metastasis in OC. In this study, we focused on evaluating the efficiency of antisense-based HOTAIR inhibition in OC tumor cells death. We transfected antisense LNA GapmeR into OC cell line (SKOV3). Then, the survival rates of SKOV3 were measured by MTT at all three time points after transfection. Moreover, we examined the expression level of AKT2 in SKOV3 under the influence of GapmeRs using qPCR at 24, 48 and 72 h after transfection. The viability of SKOV3 was significantly reduced over time under the suppression of lncRNA HOTAIR. The qPCR results disclosed the remarkable increase in AKT2 expression level via inhibition of HOTAIR in SKOV3 cell line. Our data imply that inhibition of HOTAIR by antisense LNA GapmeR induced OC cell death. Hence, we concluded that antisense-based strategies might be considered as a potential therapeutic approach in OC treatment.

THE EFFECT OF INHIBITION OF LNCRNA MIR100HG ON THE PROLIFERATION OF HUMAN PROMYELOCYTIC LEUKEMIA CELLS

lncRNAs are one of the main classes of non-coding RNAs, responsible for RNA regulation in a variety of cellular processes, and their effect on human cancer remains largely unexplored; Abnormal expression of lncRNAs has been shown to be associated with many human diseases and cancers, such as leukemia. Acute promyelocytic leukemia (APL) is the M3 subtype of acute myeloid leukemia (AML), with the aberrant accumulation of promyelocytes. One of the lncRNAs that showed upregulation in AML is lncRNA MIR100HG, involved in different cancers. The aim of our study is to investigate the functional role of MIR100HG antisense LNA GapmeRs, on APL cells. In this experimental study, we have used an Antisense LNA GapmeRs, in order to block MIR100HG in APL Cells. HL60 (APL cell line) cells were transfected with MIR100HG antisense LNA GapmeRs and at three different time points (24, 48 and 72 h) and were investigated apoptosis, necrosis, MIR100HG and TGFβ expression. MIR100HG inhibition could reduce the viability of HL- 60 cells, through induction of apoptosis; because of the TGFβ upregulation. qRT-PCR was performed to determine the MIR100HG expression by antisense LNA GapmeRs. Our results suggest that degradation of MIR100HG could serve as a novel approach for controlling the proliferation of APL cells and therefore, can be used in translational medicine for targeted therapy in APL

Functional Role of Linc-RNAs in Multiple Myeloma: Linc-MIR17HG Affects Fatty Acid Biosynthesis Via transcriptional Regulation of ACC1 with Potential Therapeutic Implications

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. DisclosuresAnderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Induction of apoptosis and necrosis in human acute erythroleukemia cells by inhibition of long non-coding RNA PVT1.

Recent advances in molecular medicine have proposed new therapeutic strategies for cancer. One of the molecular research lines for the diagnosis and treatment of cancer is the use of long non-coding RNAs (LncRNAs) which are a class of non-coding RNA molecules longer than 200 base pairs in length that act as the key regulator of gene expression. Different aspects of cellular activities like cell growth, proliferation, differentiation, apoptosis and migration are regulated by lncRNAs. In various cancers, aberrant expression of lncRNAs has been reported. One of the lncRNAs that showed upregulation in human acute myeloid leukemia (AML) is lncRNA plasmacytoma variant translocation 1 (PVT1). Here, we performed blockage of lncRNA PVT1 in human acute erythroleukemia (AEL) cell line (KG1) using antisense LNA GapmeRs. Then, at different time points (24, 48 and 72 hours) after transfection, qRT‑real‑time PCR and AnnexinV/Propidium Iodide staining assay were performed. The data were processed using the ANOVA test. At all three time points, the ratio of apoptotic cells in the PVT1 antisense LNA GapmeRs treated group was higher than the other groups. The ratio of necrotic cells in the antisense LNA GapmeRs group was also higher than the other groups. These assessments show that inhibition of lncRNA PVT1 could significantly induce apoptosis and necrosis in KG1 cells. Our findings can be used in translational medicine for future investigation in acute erythroleukemia and treatment approach based on antisense therapy.

Abstract 677: Genetic Depletion of the Long Non-coding RNA H19 in Mice Protects from Elastase-induced Abdominal Aortic Aneurysms

Long noncoding RNAs (lncRNAs) have been shown as crucial molecular regulators in various biological processes and diseases. Recently we demonstrated that lncRNA H19 is highly upregulated during abdominal aortic aneurysm (AAA) development and progression in murine models (Angiotensin II in ApoE-/- mice; porcine pancreatic elastase model (PPE) in C57BL/6 mice). Experimental H19 knock-down using specific antisense LNA oligonucleotides showed a significant reduction in AAA growth in both models. Aim of this current study was to utilize genetically mutated H19-depleted mice (H19-/-) vs. wildtype littermate controls, to assess their behavior upon experimental AAA induction using PPE. In addition, we studied the proliferation rates of smooth muscle cells, originating from either H19-/- or H19+/+ mice in a kinetic live-cell imaging system. H19-/- on a C57BL/6J background were exposed to PPE. The aortic diameter in H19-/- mice was compared to WT littermate controls (upon PPE-AAA induction) at baseline, and then consecutively at days 7, 14, and 28. Primary mouse aortic smooth muscle cells were isolated from wild type or H19-depleted aortas, and cultured and monitored in the IncuCyte live cell imaging system for 48 hours, in an effort to study their proliferation rate. H19-/- mice upon PPE-AAA induction displayed significantly lower diameters throughout the study compared to WT controls. Primary aortic smooth muscle cells from H19-depleted mice showed greatly increased proliferation rates (based on cell confluency detection) in our kinetic live-cell imaging system in comparison to WT control cells. In conclusion, our study in H19-depleted mice supports our previously presented efforts, that H19 is an important contributor to experimental AAA development and progression. Further mechanistic studies will have to reveal the molecular properties of this long non-coding RNA in smooth muscle cell survival and proliferation.