Malat1 Knockout Research Articles

Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and the β-catenin-OPG/Jagged1 pathway.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

PXR Activation Relieves Deoxynivalenol-Induced Liver Oxidative Stress Via Malat1 LncRNA m6A Demethylation.

Deoxynivalenol (DON) is a prevalent toxin causing severe liver damage through hepatocellular oxidative stress. However, the underlying mechanisms and effective therapeutic approaches remain unknown. Here, the unique role of the xenobiotic metabolism factor pregnane X receptor (PXR) in mediating DON-induced hepatocellular oxidative stress is investigated. Treatment with the PXR agonist 3-indole-propionic acid (IPA) alleviates DON-induced oxidative stress and liver injury both in vitro and in vivo. Mechanistically, it is discovered for the first time that PXR agonist IPA directly transactivates the m6A demethylase FTO expression, leading to site-specific demethylation and decreased abundance of YTHDC1-bound Malat1 lncRNA at single-nucleotide resolution. The diminished m6A modification of Malat1 lncRNA reduces its stability and augments antioxidant pathways governed by NRF2, consequently mitigating DON-induced liver injury. Furthermore, Malat1 knockout mice exhibit decreased DON-induced liver injury, emphasizing the role of Malat1 lncRNA in oxidative stress. Collectively, the findings establish that PXR-mediated m6A-dependent Malat1 lncRNA expression determines hepatocyte oxidative stress via m6A demethylase FTO, providing valuable insights into the potential mechanisms underlying DON-induced liver injury and offers potential therapeutic strategies for its treatment.

Dental Pulp Stem Cell-Derived Exosomes Inhibit Senescence-Induced Chronic Obstructive Pulmonary Disease Through the Nuclear Factor Kappa B Signaling Pathway

The respiratory condition known as chronic obstructive pulmonary disease (COPD) is widespread, but its pathogenesis remains unclear. To investigate the mechanism by which dental pulp stem cells (DPSCs) and their exosomes inhibit cellular senescence, senescence was first induced in bronchial epithelial cells by treatment with 5% cigarette smoke extract (5% CSE). Our results revealed that the senescence of bronchial epithelial cells induced by 5% CSE was decreased when co-cultured with dental pulp stem cells or their exosomes. Furthermore, this study identified that 5% CSE promoted cell senescence through the Nuclear factor kappa B (NF-kB) pathway. In addition, 5% CSE-induced cell senescence was limited when IKKβ was knocked out in bronchial epithelial cells. Meanwhile, DPSCs inhibited cell senescence through exosomal-MALAT1. Contrastingly, this effect was reversed by MALAT1 knockout. In the mouse COPD model, it was found that DPSCs could effectively inhibit COPD progression via reducing cell senescence-related proteins in mouse lung tissues, such as p21 and GLB1, and upregulating the MALAT1 expression. TNF-α and p21 expression levels were considerably reduced after treatment with dental pulp stem cells, according to IHC staining. Finally, we validated that DPSCs and their exosomes inhibit cell senescence by regulating MALAT1 and the NF-kB pathway in vitro as well as in vivo, thereby exerting a therapeutic effect in COPD.

Targeting the MALAT1 gene with the CRISPR/Cas9 technique in prostate cancer.

BackgroundThe MALAT1 lncRNA acts as an oncogene in Prostate cancer (PC); thus, it can be severe as a cancer biomarker.MethodsUsing bioinformatics datasets including (HTSeq-Counts, GDC, and TCGA) 5501 gene expression profiling specimens were gathered. Then, expression profiles and sample survival of lncRNA were investigated using COX regression analyses, ROC curve analysis. The Database for Annotation, Visualization, and Integrated Discovery was used to conduct GO and KEGG studies on the lncRNA-related PCGs. After MALAT1 Knockout via CRISPR/Cas9 technique, the MALAT1 expression was assessed in DU-145 cells. The deletion of the target fragment was examined by polymerase chain reaction (PCR). Also, the expression of apoptosis genes was investigated by qRT-PCR. The viability and cell proliferation were measured using the MTT assay. Cell migration capability was determined using the cell scratch assay. The results of qRT-PCR were assessed by the ΔΔCt method, and finally, statistical analysis was performed in SPSS software.ResultsA maximum of 451 lncRNAs were discovered to reflect different expressions between PC and non-carcinoma tissue samples, with 307 being upregulated and 144 being down-regulated. Thirty-six lncRNAs related to OS were carefully selected, which were then subjected to stepwise multivariate Cox regression analysis, with 2 lncRNAs (MALAT1, HOXB-AS3). MALAT1 is highly expressed in PC cells. MALAT1 Knockout in DU-145 cells increases apoptosis and prevents proliferation and migration, and DU-145 transfected cells were unable to migrate based on the scratch recovery test. Overall, data suggest that MALAT1 overexpression in PC helps metastasis and tumorigenesis. Also, MALAT1 knockout can be considered a therapeutic and diagnostic target in PC.ConclusionTargeting MALAT1 by CRISPR/Cas9 technique inhibit the cell proliferation and migration, and in addition induce apoptosis. Thus, MALAT1 can act as a tumor biomarker and therapeutic target.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41021-022-00252-3.

Abstract LB025: Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation

Abstract Metastatic relapse is the major causes of mortality in patients with cancer and occur due to metastatic reactivation of dormant tumor cells. Early dissemination of tumor cells undergoing a protected period of dormancy in the target organs potentially explains this prevalent clinical behavior. Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 is one of the most abundant and highly conserved nuclear lncRNAs and have shown the associated with metastasis and serving as a predictive marker for various tumor progression. However, the correlation of tumor intrinsic lncRNAs in regulation of tumor dormancy and immune evasion are largely unknown. Using an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To dissect the functional role of Malat1 in metastatic reactivation, we developed a clean Malat1 knockout (KO) model using paired gRNA CRISPR-Cas9 in metastatic murine syngeneic breast cancer. As a proof of concept, we also used inducible knockdown system under in vivo models. To delineate the immune microenvironment, we used single cell RNA-seq, ChIRP-seq, multicolor flowcytometry, RNA-FISH, and coculture experiments. Our data revealed that deletion of Malat1 induces dormancy and attenuated the metastatic colonization resulting in long-term survival of syngeneic mice model. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Interestingly, 4T1-Malat1 KO dormant breast cancer cells exhibit metastatic outgrowth in T cells defective mice. Our single-cell RNA-seq and multicolor flowcytometry evaluation reveal enhanced T cells and reduced neutrophils proportions in mice with Malat1 KO cells. This indicates a critical role of immune microenvironment via Malat1-dependent immune evasion. Additionally, Malat1 KO inhibits cancer stemness properties. Similarly, RNA-seq and ChIRP-seq data suggest that KO of Malat1 hampers immune evasion and downregulates metastasis associated genes including Serpins and Wnts. Additionally, our data strongly suggests that Malat1 KO cells persists as non-proliferative dormant cells in lung due to CD8+ T cell-umpired immune activity. Our mechanistic studies showed that Malat1 regulated Sepinb6b blocks the CstG enzymatic activity and suppress cleavage of GsdmD and T cells induced pyroptosis. Interestingly, rescue experiments suggest that Malat1 or Serpinb6b protects T cell-induced cell death and induces dormancy re-awakening thereby rescue the metastatic potential of 4T1 Malat1 KO cells. Combination of Malat1 ASO with double immune checkpoint inhibitors greatly affects the metastatic outgrowth in breast cancer. Taken together, our studies demonstrate that tumor intrinsic Malat1 regulates Serpinb6b that suppresses CstG-GsdmD mediated pyroptosis which eventually promotes immune evasion and breast cancer dormancy metastatic reactivation. Citation Format: Dhiraj Kumar, Filippo Giancotti. Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB025.

An intersegmental single-cell profile reveals aortic heterogeneity and identifies a novel Malat1+ vascular smooth muscle subtype involved in abdominal aortic aneurysm formation

The developmental origin, anatomical location, and other factors contribute to aortic heterogeneity in a physiological state. On this basis, vascular diseases occur at different ratios based on position specificity, even with the same risk factor. However, the continuous intersegmental aortic profile has been rarely reported at the single-cell level. To reveal aortic heterogeneity, we identified 15 cell subtypes from five continuous aortic segments by marker genes and functional definitions. The EC1 subtype highly expressed Vcam1 and Scarb2 genes in the aortic arch, which were reported to be associated with atherosclerosis. The newly identified Fbn1+ fibroblasts were found highly expressed in thoracic segments. More importantly, vascular smooth muscle cells (VSMCs) demonstrated a novel composition in which VSMC 4 marked with the gene Malat1 were mainly distributed in the abdominal segment. Malat1 knockout reduced MMPs and inflammatory factor production induced by Ang II in smooth muscle cells, and the Malat1 inhibitor exerted preventive, inhibitory, and reversing effects on AngII-induced abdominal aortic aneurysm (AAA) in vivo revealed by a series of animal experiments. Single-cell analysis of AngII-induced AAA tissues treated with or without the inhibitor further clarified the key role of Malat1+VSMC in the occurrence and progression of AAA. In summary, segmental gene expression and cell subtype features in normal aorta associated with different vascular diseases might provide potential therapeutic targets.

MALAT1 Maintains the Intestinal Mucosal Homeostasis in Crohn’s Disease via the miR-146b-5p-CLDN11/NUMB Pathway

Intestinal homeostasis disorder is critical for developing Crohn's disease [CD]. Maintaining mucosal barrier integrity is essential for intestinal homeostasis, preventing intestinal injury and complications. Among the remarkably altered long non-coding RNAs [lncRNAs] in CD, we aimed to investigate whether metastasis-associated lung adenocarcinoma transcript 1 [MALAT1] modulated CD and consequent disruption of intestinal homeostasis. Microarray analyses on intestinal mucosa of CD patients and controls were performed to identify dysregulated lncRNAs. MALAT1 expression was investigated via qRT-PCR and its distribution in intestinal tissues was detected using BaseScope. Intestines from MALAT1 knockout mice with colitis were investigated using histological, molecular, and biochemical approaches. Effects of intestinal epithelial cells, transfected with MALAT1 lentiviruses and Smart Silencer, on monolayer permeability and apical junction complex [AJC] proteins were analysed. MiR-146b-5p was confirmed as a critical MALAT1 mediator in cells transfected with miR-146b-5p mimic/inhibitor and in colitis mice administered agomir-146b-5p/antagomir-146b-5p. Interaction between MALAT1 and miR-146b-5p was predicted via bioinformatics and validated using Dual-luciferase reporter assay and Ago2-RIP. MALAT1 was aberrantly downregulated in the intestine mucosa of CD patients and mice with experimental colitis. MALAT1 knockout mice were hypersensitive to DSS-induced experimental colitis. MALAT1 regulated the intestinal mucosal barrier and regained intestinal homeostasis by sequestering miR-146b-5p and maintaining the expression of the AJC proteins NUMB and CLDN11. Downregulation of MALAT1 contributed to the pathogenesis of CD by disrupting AJC. Thus, a specific MALAT1-miR-146b-5p-NUMB/CLDN11 pathway that plays a vital role in maintaining intestinal mucosal homeostasis may serve as a novel target for CD treatment.

Long non‑coding RNA MALAT1 promotes high glucose‑induced rat cartilage endplate cell apoptosis via the p38/MAPK signalling pathway.

Diabetes mellitus (DM) contributes to intervertebral disc degeneration (IDD). The long non-coding RNA MALAT1 has been revealed to play an important role in diabetes-associated complications. However, the specific role of MALAT1 in diabetes-associated IDD has not been determined. The aim of the present study was to evaluate the roles of MALAT1 in the apoptosis of cartilage endplate (CEP) cells induced by high glucose and to explore the mechanisms underlying this effect. Rat CEP cells were cultured in high-glucose medium (25 mM glucose) for 24 or 72 h. Cells cultured in medium containing 5 mM glucose were used as a control. Flow cytometry was used to detect the degree of apoptosis. Reverse transcription-quantitative PCR was used to measure the expression of MALAT1 mRNA. In addition, CEP cells were treated with different conditions (high glucose, high glucose + MALAT1 negative control, high glucose + MALAT1 RNAi, normal control) for 72 h. Flow cytometry was subsequently used to detect apoptosis and western blotting was used to measure the expression levels of total and phosphorylated p38. The results revealed that high glucose concentration promoted apoptosis and enhanced expression of MALAT1 in CEP cells. Furthermore, MALAT1 knockout decreased the expression levels of total and phosphorylated p38 and reduced the apoptosis of rat CEP cells. The results obtained in the present study indicated that MALAT1 may serve as an important therapeutic target for curing or delaying IDD in patients with diabetes.

New Insights into Long Non-Coding RNA MALAT1 in Cancer and Metastasis.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is one of the most abundant, long non-coding RNAs (lncRNAs) in normal tissues. This lncRNA is highly conserved among mammalian species, and based on in vitro results, has been reported to regulate alternative pre-mRNA splicing and gene expression. However, Malat1 knockout mice develop and grow normally, and do not show alterations in alternative splicing. While MALAT1 was originally described as a prognostic marker of lung cancer metastasis, emerging evidence has linked this lncRNA to other cancers, such as breast cancer, prostate cancer, pancreatic cancer, glioma, and leukemia. The role described for MALAT1 is dependent on the cancer types and the experimental model systems. Notably, different or opposite phenotypes resulting from different strategies for inactivating MALAT1 have been observed, which led to distinct models for MALAT1’s functions and mechanisms of action in cancer and metastasis. In this review, we reflect on different experimental strategies used to study MALAT1’s functions, and discuss the current mechanistic models of this highly abundant and conserved lncRNA.

MALAT1 and HOTAIR—Key Epigenetic Regulators in Diabetic Retinopathy

Introduction: With the increasing incidence of diabetic retinopathy (DR), the need for novel and specific therapy is essential. Genomic studies have identified several long non-coding RNAs (lncRNAs). However, majority of lncRNAs have not been characterized in DR. We examined the roles of lncRNAs MALAT1 and HOTAIR in DR. Methods: Human retinal endothelial cells (HRECs) were cultured in 5 mM/L (NG) or in 25 mM/L (HG) glucose. RNA expressions of MALAT1, HOTAIR, and inflammatory and angiogenic cytokines were tested. HRECs were similarly examined following siRNA-mediated MALAT1 or HOTAIR knockdown, or treatment with histone (DZNep) and DNA methylation (52019-aza-dC) blockers. DNA methylation patterns and RNA-protein interactions were analyzed. Retinal tissues from Malat1 knockout (KO) and wild type (WT) mice with or without diabetes were examined. We further assessed MALAT1 a’nd HOTAIR in human diabetic and nondiabetic vitreous. Results: HG caused upregulations of MALAT1, HOTAIR, IL-6, TNF-α, and VEGF-A transcripts in HRECs. HG increased binding of EZH2 (a PRC2 component) with MALAT1 and HOTAIR and evoked unique DNA methylation patterns in the MALAT1 and HOTAIR CpG regions. DZNep, 5-aza-dC and knockdown of MALAT1 or HOTAIR reduced mRNA expressions of IL-6, TNF-α, and VEGF-A. Similarly, diabetes-induced elevations of these inflammatory and angiogenic cytokines in the retina were prevented in the Malat1 KO mice. Furthermore, MALAT1 and HOTAIR levels were elevated in the diabetic vitreous. Conclusions: Our findings allude to the importance of lncRNAs in influencing DR through epigenetic mediators. Understanding the role of lncRNAs may allow the development of better-targeted therapies in DR. Disclosure S. Biswas: None. A.A. Thomas: None. B. Feng: None. S. Chen: None. E. Aref-Eshghi: None. J. Gonder: None. B. Sadikovic: None. S. Chakrabarti: None.

MALAT1: An Epigenetic Regulator of Inflammation in Diabetic Retinopathy

Despite possessing limited protein-coding potential, long non-coding RNAs (lncRNAs) have been implicated in a myriad of pathologic conditions. Most well documented in cancer, one prominent intergenic lncRNA known as MALAT1 is notorious for its role in impacting epigenetic mechanisms. In this study, we established a novel epigenetic paradigm for MALAT in diabetic retinopathy (DR) by employing siRNA-mediated MALAT1 knockdown in human retinal endothelial cells (HRECs), a Malat1 knockout animal model, vitreous humor from diabetic patients, pharmacological inhibitors for histone and DNA methylation, RNA immunoprecipitation, western blotting, and a unique DNA methylation array to determine glucose-related alterations in MALAT1. Our findings indicated that MALAT1 is capable of impacting the expressions of inflammatory transcripts through its association with components of the PRC2 complex in diabetes. Furthermore, the vitreous humors from diabetic patients revealed increased expressions of MALAT1, TNF-α, and IL-6. Intriguingly, our DNA methylation array demonstrated that transient high glucose exposure in HRECs does not contribute to significant methylation alterations at CpG sites across the MALAT1 gene. However, global inhibition of DNA methyltransferases induced significant increases in MALAT1 and associated inflammatory transcripts in HRECs. Our findings collectively demonstrate the importance of MALAT1 in inflammation and epigenetic regulation in DR.

Hypoxia-induced long non-coding RNA Malat1 is dispensable for renal ischemia/reperfusion-injury

Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Non-coding RNAs are crucially involved in its pathophysiology. We identified hypoxia-induced long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) to be upregulated in renal I/R injury. We here elucidated the functional role of Malat1 in vitro and its potential contribution to kidney injury in vivo. Malat1 was upregulated in kidney biopsies and plasma of patients with AKI, in murine hypoxic kidney tissue as well as in cultured and ex vivo sorted hypoxic endothelial cells and tubular epithelial cells. Malat1 was transcriptionally activated by hypoxia-inducible factor 1-α. In vitro, Malat1 inhibition reduced proliferation and the number of endothelial cells in the S-phase of the cell cycle. In vivo, Malat1 knockout and wildtype mice showed similar degrees of outer medullary tubular epithelial injury, proliferation, capillary rarefaction, inflammation and fibrosis, survival and kidney function. Small-RNA sequencing and whole genome expression analysis revealed only minor changes between ischemic Malat1 knockout and wildtype mice. Contrary to previous studies, which suggested a prominent role of Malat1 in the induction of disease, we did not confirm an in vivo role of Malat1 concerning renal I/R-injury.

MALAT1: A regulator of inflammatory cytokines in diabetic complications.

SummaryObjectives and DesignIn this study, we examined the role of MALAT1, a highly conserved nuclear long non‐coding RNA molecule, in chronic diabetic complications affecting the heart and kidneys using both in vitro and in vivo models: human endothelial cell culture and a Malat1 knockout mice model.ResultsFindings from our in vitro experiments demonstrated that MALAT1 was predominantly localized to nuclear speckles in endothelial cells and MALAT1 expression was significantly increased following incubation with high glucose in association with increased expression of inflammatory cytokines. As for our in vivo experiments, we used Malat1 knockout mice and wild‐type controls with or without streptozotocin‐induced diabetes over 2 months of follow‐up, where all of our diabetic animals showed hyperglycaemia and polyuria. Examination of cardiac and renal tissues demonstrated altered MALAT1 RNA expression in wild‐type diabetic animals. Such changes were associated with augmented production of downstream inflammatory molecules at the mRNA and protein levels. Diabetes‐induced elevations of inflammatory markers were significantly decreased in Malat1 knockout diabetic animals. In addition to transcript and protein analyses, we examined functional changes in the heart and kidneys. Organ functions were affected in the wild‐type diabetic mice but were rescued in Malat1 knockout mice.ConclusionsTaken together, findings from this study will provide direct evidence and insight into the importance of MALAT1 in the pathogenesis of chronic diabetic complications involving the heart and kidneys.

Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1β/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.

Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke.

The study was designed to determine the role of long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (Malat1), in ischemic stroke outcome. Primary mouse brain microvascular endothelial cells (BMECs) were cultured and treated with Malat1 GapmeR before 16 h oxygen and glucose depravation (OGD). Cell death was assayed by LDH and MTT methods. Malat1 knock-out and wild-type mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) and 24-72 h of reperfusion. To explore the underlying mechanism, apoptotic and inflammatory factors were measured by qPCR, ELISA, and Western blotting. The physical interaction between Malat1 and apoptotic or inflammatory factors was measured by RNA immunoprecipitation. Increased Malat1 levels were found in cultured mouse BMECs after OGD as well as in isolated cerebral microvessels in mice after MCAO. Silencing of Malat1 by Malat1 GapmeR significantly increased OGD-induced cell death and Caspase 3 activity in BMECs. Silencing of Malat1 also significantly aggravated OGD-induced expression of the proapoptotic factor Bim and proinflammatory cytokines MCP-1, IL-6, and E-selectin. Moreover, Malat1 KO mice presented larger brain infarct size, worsened neurological scores, and reduced sensorimotor functions. Consistent with in vitro findings, significantly increased expression of proapoptotic and proinflammatory factors was also found in the cerebral cortex of Malat1 KO mice after ischemic stroke compared with WT controls. Finally, we demonstrated that Malat1 binds to Bim and E-selectin both in vitro and in vivo Our study suggests that Malat1 plays critical protective roles in ischemic stroke.SIGNIFICANCE STATEMENT Accumulative studies have demonstrated the important regulatory roles of microRNAs in vascular and neural damage after ischemic stroke. However, the functional significance and mechanisms of other classes of noncoding RNAs in cerebrovascular pathophysiology after stroke are less studied. Here we demonstrate a novel role of Malat1, a long noncoding RNA that has been originally identified as a prognostic marker for non-small cell lung cancer, in cerebrovascular pathogenesis of ischemic stroke. Our experiments have provided the first evidence that Malat1 plays anti-apoptotic and anti-inflammatory roles in brain microvasculature to reduce ischemic cerebral vascular and parenchymal damages. Our studies also suggest that lncRNAs can be therapeutically targeted to minimize poststroke brain damage.

Abstract IA08: Functional long noncoding RNAs in lung cancer

Abstract The majority of the human genome is transcribed into non-protein-coding RNA. Hence, RNA is also the primary product of the cancer genome. We have defined the ncRNA expression landscape of lung, breast and liver cancer providing a comprehensive expression map of over 17000 long ncRNAs and discovering new lncRNAs associated with cancer whose molecular and cellular functions we are currently elucidating exploiting our custom siRNA library targeting 638 tumor-associated lncRNAs. The nuclear lncRNA MALAT1 was one of the first lncRNAs associated with cancer: it is associated with metastasis development in lung cancer. However, its high abundance and nuclear localization have hampered its functional analysis. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements site-specifically into the MALAT1 locus. This approach yielded a 1000-fold silencing of MALAT1 providing a unique loss-of-function model. Proposed mechanisms of MALAT1 action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but regulates gene expression inducing a signature of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft model. Encouraged by this discovery of the essential function of MALAT1 in lung cancer metastasis, we analyzed whether MALAT1 could serve as therapeutic target for an Antisense oligonucleotide (ASO). Notably, MALAT1-ASO treatment prevented metastasis formation after tumor implantation. Thus, targeting MALAT1 with ASOs provided a therapeutic approach to prevent lung cancer metastasis with MALAT1 serving as both, predictive marker and therapeutic target. In summary, ten years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unraveled the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis. Citation Format: Sven Diederichs. Functional long noncoding RNAs in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA08.

Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice

BackgroundLong non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice.ResultsAbsence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97±0.26, TAC 1.57±0.40; p<0.0001) and KO mice (sham: 3.64±0.37; TAC: 2.24±0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each).ConclusionThese findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133.

Abstract 1121: The long non-coding RNA MALAT1 is an essential gene regulator for lung cancer metastasis in a novel human knockout model .

Abstract The long non-coding RNA MALAT1 was one of the first lncRNAs associated with cancer: it is a highly conserved nuclear ncRNA and a predictive marker for metastasis development in lung cancer. However, its high abundance and nuclear localization have greatly hampered its functional analysis since it is only inefficiently knocked down by RNA interference (RNAi). To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements site-specifically into the MALAT1 locus using Zinc Finger Nucleases (ZFN).This approach yielded a more than 1000-fold silencing of MALAT1 providing a unique loss-of-function model. Proposed mechanisms of action of MALAT1 include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but actively regulates gene expression inducing a signature of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft model. Encouraged by this discovery of the essential function of MALAT1 in lung cancer metastasis, we wanted to analyze whether MALAT1 could also be therapeutically targeted: We developed Antisense oligonucleotides (ASOs) effectively blocking MALAT1 expression in the cell culture and in the animal. Notably, MALAT1-ASO treatment prevents metastasis formation after tumor implantation. Thus, targeting MALAT1 with antisense oligonucleotides provides a potential therapeutic approach to prevent lung cancer metastasis with MALAT1 serving as both, predictive marker and therapeutic target. Lastly, regulating gene expression, but not alternative splicing is the critical function of MALAT1 in lung cancer metastasis. In summary, ten years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unravels the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis. Citation Format: Tony Gutschner, Monika Hämmerle, Moritz Eißmann, Jeff Hsu, Youngsoo Kim, Gene Hung, Alexey Revenko, Gayatri Arun, Marion Stentrup, Matthias Groß, Martin Zörnig, A. Robert MacLeod, David L. Spector, Sven Diederichs. The long non-coding RNA MALAT1 is an essential gene regulator for lung cancer metastasis in a novel human knockout model . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1121. doi:10.1158/1538-7445.AM2013-1121

The lncRNA Malat1 Is Dispensable for Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult

Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3'-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

A survey of the sensitivity of fresh clinical isolates to cefuroxime and other antibiotics.

The sensitivities to cefuroxime and cephradine of potentially pathogenic bacteria isolated in two British general hospitals comprising 900 beds have been assessed. In a three-month period 2537 strains were studied; 30 microgram cefuroxime discs were used with 2511 strains, and cephradine discs of the same strength were used with 2525 strains. The organisms were also examined routinely for sensitivity to other antibiotics. Overall, 91.7% of the isolates were sensitive to cefuroxime and 85.8% were inhibited by cephradine, the differences in percentage strain susceptibility to cefuroxime and cephradine being mainly a result of the greater activity of cefuroxime against the Gram-negative bacteria. The wide antibacterial effectiveness of cefuroxime should make it a useful antibiotic for the treatment of serious infections including those conditions in which the causative organism has not been identified.