Endogenous lncRNA Research Articles

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

MiRNA let-7 family regulated by NEAT1 and ARID3A/NF-κB inhibits PRRSV-2 replication in vitro and in vivo.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry worldwide. To investigate the role of miRNAs in the infection and susceptibility of PRRS virus (PRRSV), twenty-four miRNA libraries were constructed and sequenced from PRRSV-infected and mock-infected Porcine alveolar macrophages (PAMs) of Meishan, Landrace, Pietrain and Qingping pigs at 9 hours post infection (hpi), 36 hpi, and 60 hpi. The let-7 family miRNAs were significantly differentially expressed between PRRSV-infected and mock-infected PAMs from 4 pig breeds. The let-7 family miRNAs could significantly inhibit PRRSV-2 replication by directly targeting the 3’UTR of the PRRSV-2 genome and porcine IL6, which plays an important role in PRRSV replication and lung injury. NEAT1 acts as a competing endogenous lncRNA (ceRNA) to upregulate IL6 by attaching let-7 in PAMs. EMSA and ChIP results confirmed that ARID3A could bind to the promoter region of pri-let-7a/let-7f/let-7d gene cluster and inhibit the expression of the let-7 family. Moreover, the NF-κB signaling pathway inhibits the expression of the let-7 family by affecting the nuclear import of ARID3A. The pEGFP-N1-let-7 significantly reduced viral infections and pathological changes in PRRSV-infected piglets. Taken together, NEAT1/ARID3A/let-7/IL6 play significant roles in PRRSV-2 infection and may be promising therapeutic targets for PRRS.

LncRNA FTX promotes the malignant progression of colorectal cancer by regulating the miR-214-5p-JAG1 axis.

BackgroundLong non-coding RNAs (lncRNAs) have recently been found to be vital regulators of various cancers, including colorectal cancer (CRC). It has been previously reported that the dysregulated expression of lncRNA Five prime to Xist (FTX) is involved in carcinogenesis. However, the role of lncRNA FTX in the progression of CRC is still unclear.MethodsFluorescence in situ hybridization (FISH) was used to detect the expression of lncRNA FTX and miR-214-5p in CRC tissues. Cell Counting Kit-8 assay, transwell assay, wound-healing assay, and proliferation assay were used to explore the function of lncRNA FTX in CRC cells. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and luciferase reporter assay were used to confirm the relationship between lncRNA FTX and miR-214-5p–jagged canonical Notch ligand 1 (JAG1). We further explored the role of lncRNA FTX in vivo using xenograft tumor assay.ResultslncRNA FTX was found to be upregulated in CRC tissues by FISH. The downregulation of endogenous lncRNA FTX expression inhibited CRC cell proliferation, migration, and invasion. Mechanistically, lncRNA FTX sequestered miR-214-5p and thus released its repression on JAG1, driving the malignant progression of CRC.ConclusionsThese findings give rise to a new perspective, the lncRNA FTX–miR-214-5p–JAG1 regulatory axis, in exploring the cancer-promoting mechanism of lncRNA FTX in CRC.

Long noncoding RNA lncRNA354 functions as a competing endogenous RNA of miR160b to regulate ARF genes in response to salt stress in upland cotton.

Long non-coding RNAs (lncRNAs) play important roles in response to biotic and abiotic stress through acting as competing endogenous RNAs (ceRNAs) to decoy mature miRNAs. However, whether this mechanism is involved in cotton salt stress response remains unknown. We report the characterization of an endogenous lncRNA, lncRNA354, whose expression was reduced in salt-treated cotton and was localized at the nucleus and cytoplasm. Using endogenous target mimic (eTM) analysis, we predicted that lncRNA354 had a potential binding site for miR160b. Transient expression in tobacco demonstrated that lncRNA354 was a miR160b eTM and attenuated miR160b suppression of its target genes, including auxin response factors (ARFs). Silencing or overexpressing lncRNA354 affected the expression of miR160b and target ARFs. Silencing lncRNA354 and targets GhARF17/18 resulted in taller cotton plants and enhanced the resistant to salt stress. Overexpression of lncRNA354 and targets GhARF17/18 in Arabidopsis led to dwarf plants, decreased root dry weight and reduced salt tolerance. Our results show that the lncRNA354-miR160b effect on GhARF17/18 expression may modulate auxin signalling and thus affect growth. These results also shed new light on a mechanism of lncRNA-associated responses to salt stress.

LncRNA H19 Mitigates Oxidized Low-Density Lipoprotein Induced Pyroptosis via Caspase-1 in Raw 264.7 Cells

Atherosclerosis (AS) is mainly characterized by the activation of inflammatory cells and chronic inflammatory responses after cell injury. Pyroptosis is a form of programmed cell death (PCD) accompanied by the release of inflammatory factors. Many studies have shown that pyroptosis plays an important role in AS. Increasing evidence also indicates that long non-coding RNA H19 (lncRNA H19) involved in AS. However, whether the role of lncRNA H19 in AS is related to pyroptosis and the underlying mechanisms are largely unknown. In this study, we found that oxidized low-density lipoprotein (ox-LDL) induced pyroptosis and decreased the expression of lncRNA H19 in Raw 264.7 cells. Besides, silencing endogenous lncRNA H19 increased inflammatory responses and pyroptosis while exogenous overexpression of lncRNA H19 reversed this effect. Notably, we identified that the inhibitor of caspase-1 (XV-765) completely abrogated the silencing endogenous lncRNA H19 mediated pyroptosis. In addition, we found that lncRNA H19 inhibited ox-LDL-induced activation of nuclear factor-kappa B (NF-κB), mitochondrial dysfunction, and reduced the production of reactive oxygen species (ROS). Moreover, VX-765 impaired the silencing endogenous lncRNA H19 mediated pyroptosis. Overall, these findings indicated that lncRNA H19 may play an important role in pyroptosis and may serve as a potential therapeutic target for AS.

LncRNA MALAT1/miR‑26a/26b/ST8SIA4 axis mediates cell invasion and migration in breast cancer cell lines.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA that is overexpressed in various human cancers, including breast cancer. Evidence has associated the function of the α-2,8-sialyltransferase (ST8SIA) family with breast cancer. The present study aimed to investigate the potential roles of MALAT1 in breast cancer development and progression using analyses of both breast cancer tissues and cell lines. The mRNA levels of MALAT1, microRNA (miR)-26a/26b and ST8SIA4 were detected by reverse transcription-quantitative PCR (RT-qPCR) and the protein level of ST8SIA4 was assessed by western blot analysis. Cell proliferation, invasion and migration were detected by CCK-8, wound healing and Transwell assays, respectively. Interactions between MALAT1 and miR-26a/26b were assessed using fluorescence in situ hybridization, RNA immunoprecipitation and luciferase reporter assays. Herein, different levels of MALAT1 were primarily observed in human breast cancer samples and cells. Upregulated MALAT1 was a crucial predictor of poor breast cancer prognosis. Altered MALAT1 modulated cell progression in breast cancer. Moreover, miR-26a/26b was confirmed as a direct regulator of MALAT1, and ST8SIA4 was predicted as a target of miR-26a/26b. Functional analysis in human breast cancer cell lines demonstrated that MALAT1 modulated breast cancer cell tumorigenicity by acting as a competing endogenous lncRNA (ceRNA) to regulate ST8SIA4 levels by sponging miR-26a/26b. The identification of the MALAT1/miR-26a/26b/ST8SIA4 axis which contributes to breast cancer progression may constitute a potential new therapeutic target.

TCONS_00230836 silencing restores stearic acid-induced \u03b2 cell dysfunction through alleviating endoplasmic reticulum stress rather than apoptosis

BackgroundChronic exposure of pancreatic β cells to high levels of stearic acid (C18:0) leads to impaired insulin secretion, which accelerates the progression of type 2 diabetes mellitus (T2DM). Recently, long noncoding RNAs (lncRNAs) were found to participate in saturated fatty acid-induced metabolism dysfunction. However, their contribution to stearic acid-induced β-cell dysfunction remains largely unknown. This study evaluated the possible role of the lncRNA TCONS_00230836 in stearic acid-stimulated lipotoxicity to β cells.MethodUsing high-throughput RNA-sequencing, TCONS_00230836 was screened out as being exclusively differentially expressed in stearic acid-treated mouse β-TC6 cells. Co-expression network was constructed to reveal the potential mRNAs targeted for lncRNA TCONS_00230836. Changes in this lncRNA’s and candidate mRNAs’ levels were further assessed by real-time PCR in stearic acid-treated β-TC6 cells and islets of mice fed a high-stearic-acid diet (HSD). The localization of TCONS_00230836 was detected by fluorescent in situ hybridization. The endogenous lncRNA TCONS_00230836 in β-TC6 cells was abrogated by its Smart Silencer.ResultsTCONS_00230836 was enriched in mouse islets and mainly localized in the cytoplasm. Its expression was significantly increased in stearic acid-treated β-TC6 cells and HSD-fed mouse islets. Knockdown of TCONS_00230836 significantly restored stearic acid-impaired glucose-stimulated insulin secretion through alleviating endoplasmic reticulum stress. However, stearic acid-induced β cell apoptosis was not obviously recovered.ConclusionOur findings suggest the involvement of TCONS_00230836 in stearic acid-induced β-cell dysfunction, which provides novel insight into stearic acid-induced lipotoxicity to β cells. Anti-lncRNA TCONS_00230836 might be a new therapeutic strategy for alleviating stearic acid-induced β-cell dysfunction in the progression of T2DM.

IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

BackgroundInterleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE.MethodsIn vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student’s t test, as appropriate.ResultsKnockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process.ConclusionsThese findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.

LncRNA NORAD regulates scar hypertrophy via miRNA-26a mediating the regulation of TGFβR1/2.

Transforming growth factor-β (TGF-β) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation. The study aims to analyze the potential mechanism of long non-coding RNA NORAD (LncRNA NORAD) and microRNA (miR-26a) regulation of the TGF-β pathway in hypertrophic scar fibroblasts (HSFs). Hypertrophic scar tissues were collected and assayed for LncRNA NORAD, miR-26a, transforming growth factor β receptor I (TGF-βR1) and TGF-βR2, with enzyme-linked immunosorbent assay (ELISA) or qualitative polymerase chain reaction (qPCR). LncRNA NORAD interfering plasmids were transfected into HSFs and induced with TGF-β1. Cell Counting Kit-8 (CCK-8) assays were performed to assess HSF proliferation, and flow cytometry to analyze apoptosis and the cell cycle. TGF-βR1, TGF-βR2, Smad2, and p-Smad2 levels were detected using western blot (WB). The related proteins (p21, cyclin D1 and cyclin-dependent kinase 4 (CDK4)) regulating the cell cycle, and apoptosis-related proteins (caspase-3 and Bcl-2) were also detected using WB. The binding sites of miRNA-26a and LncRNA NORAD, TGF-βR2, or UBE3A were predicted using Starbase and confirmed with dual luciferase reporter assay. RNA immunoprecipitation (RIP) was utilized to explore the interplay of miR-26a with its target genes. LncRNA NORAD is decreased, miR-26a is increased and TGF-β receptors show abnormal expression in scar tissue. LncRNA NORAD knockdown inhibits proliferation of HSF cells induced by TGF-β1 treatment. In addition, cell apoptotic levels are markedly increased and cell numbers in G0/G1 phase are increased. Moreover, the TGF-β/Smad pathway is regulated by decreasing endogenous LncRNA NORAD levels, possibly by affecting the relative levels of TGF-βR1. p21 is notably upregulated, while cyclin D1 and CDK4 are downregulated. Apoptosis-related proteins are significantly affected. LncRNA NORAD may act as a sponge, binding miR-26a and changing its expression. Finally, RIP shows that miR-26a targets the 3'UTRs of TGF-βR2 and UBE3A. LncRNA NORAD regulates HSF proliferation via miR-26a mediating the regulation of TGF-βR2/R1. LncRNA NORAD/miR-26a could be a potential target for treating HS.

The lncRNA XIST promotes proliferation, migration and invasion of gastric cancer cells by targeting miR-337

Background and study aims: Gastric cancer (GC) is one of the most common malignant tumours worldwide. Long non-coding RNAs (lncRNAs) and microRNAs regulate the occurrence and development of various cancers and play an important role in GC progression. X-inactive specific transcript (XIST), a carcinogenic lncRNA, is involved in human tumourigenesis and is altered in GC. Janus kinase 2 (JAK2), a transcription factor, is involved in cancer cell metastasis and differentiation. However, the exact mechanism underlying the biological roles of XIST and JAK2 in cancer cells remains unclear. Material and methodsThis study was conducted using GES-1, HGC-27, AGS and HEK-293 T cells. Quantitative polymerase chain reaction and western blotting were performed to detect XIST, microRNA-337 (miR-337) and JAK2 expressions. GC cell invasion was investigated by using the Transwell assay. Fluorescein reporter gene detection was used to determine the relationship between JAK2 and XIST. ResultsCompared with that in GES-1 cells, XIST expression was significantly up-regulated in AGS and HGC-27 cells. miR-337 expression in GC cell lines was decreased. The proliferation, invasion and migration of GC cells were simultaneously inhibited by XIST knockdown, and the relationship between XIST and miR-337 was confirmed by bioinformatics analysis. JAK2 is expected to be the target gene of miR-337. MiR-337 can negatively regulate JAK2 expression in vitro. In addition, si-XIST decreased JAK2 expression by up-regulating miR-337 in vitro, thereby inhibiting GC cell proliferation and migration. Therefore, we speculated that XIST regulates JAK2 by competing with miR-337 as a competitive endogenous lncRNA in GC. ConclusionWe elucidated the effects of migration and invasion after XIST inhibition, at least in part, by inhibiting miR-337 expression in GC cells to regulate JAK2. These data indicate that a positive feedback loop exists between XIST and JAK2 and suggest that JAK2 and XIST play a vital role in cancer cell migration and invasion.

Long Non-coding RNA Depletion Using Self-Cleaving Ribozymes.

Some long non-coding RNA (lncRNA) genes encode a functional RNA product, whereas others act as DNA elements or via the act of transcription . We describe here a ribozyme-based approach to deplete an endogenous lncRNA in mouse embryonic stem cells, with minimal disruption of its gene. This enables the role of the lncRNA product to be tested.

225-LB: A High-Throughput CRISPR-Activation Screen Defines Human LncRNA Metabolic Regulators

In the past decade, long noncoding RNAs (lncRNAs) have emerged as important regulators of cellular and systemic metabolism. Deciphering the physiological functions of lncRNA by gain- or loss-of-function analyses in key metabolic organs, such as the liver, is essential to understand their disease relevance. Conventional transgenic or knockout approaches, however, have limitations in defining lncRNA function as lncRNAs transgenically expressed from ectopic genomic loci could be non-functional and partial deletion of lncRNAs. Domain negative Cas9 (dCas9) coupled with transcriptional activators can trigger endogenous lncRNA expression by targeting guide RNA (gRNA) to transcription start sites of lncRNAs, thus increasing their transcription efficiency and targeting multiple different lncRNA isoforms simultaneously. Adenovirus vector is the most commonly used tool for specifically targeting liver-enriched genes both in vitro and in animal models. A CRISPR-activation (CRISPR-a) adenovirus system thus constitutes a powerful means to active liver lncRNAs transcriptionally. In this study, we established a CRISPR-a adenovirus platform by transferring CRISPR-a units into the adenovirus vector, and our system allows one digestion-ligation step to construct the gRNA adenovirus vector seamlessly. Efficacy and versatility of this pipeline has been confirmed by the successful construction of a CRISPR-a adenovirus library containing 96 gRNAs targeting 20 liver-enriched, metabolic sensitive lncRNAs, which shows high CRISPR activation efficiency after primary human hepatocytes infection. Moreover, the expression levels of several lncRNA target genes predicted by co-relation analysis are significantly changed. This method thus provides a fast and efficient way to activate the endogenous liver lncRNAs and simplifies the procedure of lncRNA functional screening using CRISPR-a adenovirus system, which can be scaled up to a genome level to quickly define lncRNA metabolic regulators. Disclosure Y. Ma: None. H. Cao: None.

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in

In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

Visualization of Long Noncoding RNA MEG3 in Living Cells by a Triple-Helix-Powered 3D Catcher.

Visual imaging of long noncoding RNA (lncRNA) MEG3, a newfound regulator of transactivation and tumor growth suppression, is conducive to unlock the secrets of MEG3 in some important biological processes. Here, for the first time, we designed a DNA tetrahedron-based three-dimensional (3D) catcher for imaging cytoplasmic lncRNA MEG3 in living cells. The 3D catcher is composed of a triple-helix-forming dsDNA with capacity to bind the 5'-end GA-rich domain of the lncRNA MEG3 and four hairpin-shaped antisense sequences toward contiguous domain on MEG3. Once ingested by the cell, the 3D catcher quickly captures lncRNA MEG3 via forming a DNA-RNA triple-helix structure and triggering the hybridization-based string disassembly of the catcher. Concomitantly, the quenched hairpin is opened and the fluorescent signal undergoes lighting on conversion. Ascribed to the triple-helix-induced "domino effect," the disassembly reaction time is greatly shorter than the reaction with the inability to form a triple helix. The 3D catcher allows detection of long-chain targets as long as 129 nucleotide (129 nt) with a detection limit of 0.36 nM and distinguishes endogenous lncRNA MEG3 fragments in living cells between hepatoma cells and normal hepatocytes, which provides a reliable strategy for monitoring endogenous long fragment nucleic acid biomarkers in early clinical lesion diagnoses.

The Long Noncoding RNA Hotair Regulates Oxidative Stress and Cardiac Myocyte Apoptosis during Ischemia-Reperfusion Injury.

Oxidative stress and subsequent cardiac myocyte apoptosis play central roles in the initiation and progression of myocardial ischemia-reperfusion (I/R) injury. Homeobox transcript antisense intergenic RNA (Hotair) was previously implicated in various heart diseases, yet its role in myocardial I/R injury has not been clearly demonstrated. Mice with cardiac-restricted knockdown or overexpression of Hotair were exposed to I/R surgery. H9c2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) stimulation to further verify the role and underlying mechanisms of Hotair in vitro. Histological examination, molecular detection, and functional parameters were determined in vivo and in vitro. In response to I/R or H/R treatment, Hotair expression was increased in a bromodomain-containing protein 4-dependent manner. Cardiac-restricted knockdown of Hotair exacerbated, whereas Hotair overexpression prevented I/R-induced oxidative stress, cardiac myocyte apoptosis, and cardiac dysfunction. Mechanistically, we observed that Hotair exerted its beneficial effects via activating AMP-activated protein kinase alpha (AMPKα). Further detection revealed that Hotair activated AMPKα through regulating the enhancer of zeste homolog 2/microRNA-451/calcium-binding protein 39 (EZH2/miR-451/Cab39) axis. We provide the evidence that endogenous lncRNA Hotair is an essential negative regulator for oxidative stress and cardiac myocyte apoptosis in myocardial I/R injury, which is dependent on AMPKα activation via the EZH2/miR-451/Cab39 axis.

Development and functional characterization of a lncRNA-HIT conditional loss of function allele.

Analysis of the human and murine transcriptomes has identified long noncoding RNAs (lncRNAs) as major functional components in both species. Transcriptional profiling of the murine limb led to our discovery of lncRNA-HIT, which our previous in vitro analyses suggested a potential role for this lncRNA in the development of limb, craniofacial, and genitourinary tissues (Carlson et al., 2015). To test this hypothesis, we developed a conditional lncRNA-HIT loss of function allele which uses Cre recombinase to activate an shRNA specific for lncRNA-HIT. Activation of the lncRNA-HIT shRNA allele resulted in a robust knock-down of lncRNA-HIT as well as co-activation of a mCherry reporter, confirming the efficacy of the shRNA allele to reduce endogenous lncRNA levels in a tissue- and cell-type specific manner. Developmental analyses of embryos expressing the activated shRNA and mCherry co-reporter revealed multiple malformations corresponding to the sites of shRNA activation, affecting craniofacial, limb, and genitourinary tissue development. These results confirm the efficacy of lncRNA-HIT shRNA allele to knock-down endogenous transcripts in tissue- and cell type specific manner and indicate a requirement for lncRNA-HIT in the development of these tissues.

Knockdown of the lncRNA MALAT1 alleviates lipopolysaccharide‑induced A549 cell injury by targeting the miR‑17‑5p/FOXA1 axis.

Long‑noncoding RNAs (lncRNAs) are crucial for the pathophysiology of acute lung injury (ALI). Metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) suppresses inflammatory responses via microRNA (miR)‑146a in lipopolysaccharide (LPS)‑induced ALI. However, the molecular mechanisms underlying the MALAT1‑mediated regulation of cell proliferation and apoptosis in LPS‑induced ALI remain unclear. In the present study, it was found that LPS treatment upregulated MALAT1 expression and suppressed the proliferation of A549 cells. MALAT1 knockdown significantly promoted the proliferation and G1/S phase transition and inhibited apoptosis in LPS‑treated A549 cells. In addition, miR‑17‑5p was a direct target of MALAT1. miR‑17‑5p expression was downregulated and FOXA1 expression was upregulated in LPS‑treated A549 cells. Further, MALAT1 knockdown promoted miR‑17‑5p expression and inhibited FOXA1 expression, whereas the combined suppression of MALAT1 and miR‑17‑5p induced FOXA1 expression. Moreover, miR‑17‑5p knockdown reversed the effects of MALAT1 suppression on LPS‑induced A549 cell proliferation. These results indicated that MALAT1 serves as a competing endogenous lncRNA that, by sequestering miR‑17‑5p, stimulates FOXA1 expression and mediates LPS‑induced A549 cell injury. In conclusion, the present study demonstrated that MALAT1 knockdown alleviates LPS‑induced A549 cell injury by targeting the miR‑17‑5p/FOXA1 axis.

The lncRNA TDRG1 promotes cell proliferation, migration and invasion by targeting miR-326 to regulate MAPK1 expression in cervical cancer

BackgroundRecently, lncRNA-Testis developmental related gene 1 (TDRG1) was proved to be a key modulator in reproductive organ-related cancers. The biological role of TDRG1 in cervical cancer (CC) progression remains largely unknown.MethodReal-time PCR (qRT-PCR) examined the expression level of TDRG1, microRNA (miR)-326 and MAPK1 mRNA. OS tissues and corresponding relative normal tissues, as well as CC cell lines and normal cell line Ect1/E6E7 were collected to determine the expression of TDRG1 in CC. MTT, colony formation, wound-healing, transwell and flow cytometer assay detected the influence of TDRG1 and miR-326 on CC cells growth, metastasis and apoptosis. Western blot examined proteins level. Bioinformatics, RNA pull-down assay, RNA immunoprecipitation and dual-luciferase reporter assays detected the molecular mechanism of TDRG1 in CC. Xenograft tumour model was established to determine the role of TDRG1 in vivo.ResultsThe expression of TDRG1 was significantly increased in CC tissues and cell lines compared with normal tissue and normal cell line respectively and its expression was associated with clinicopathological characteristics of CC patients. Knockdown of TDRG1 inhibited the cell proliferation, migration and invasion in Hela and SIHA cells. Moreover, TDRG1 directly interacted with miR-326, and the inhibition effect on cell growth and metastasis induced by TDRG1 siRNA can be abrogated by miR-326 silencing by its inhibitor in Hela and SIHA cells. Further, MAPK1 was proved to be a direct target of miR-326, and its expression was negatively regulated by miR-326 while positively modulated by TDRG1.ConclusionTDRG1 acts as a competing endogenous lncRNA (ceRNA) to modulate MAPK1 by sponging miR-326 in CC, shedding new light on TDRG1-directed diagnostics and therapeutics in CC.

NEAT1/hsa-mir-98-5p/MAPK6 axis is involved in non-small-cell lung cancer development.

Long noncoding RNAs (lncRNAs) or microRNAs belong to the two most important noncoding RNAs and they are involved in a lot of cancers, including non-small-cell lung cancer (NSCLC). Therefore, currently, we focused on the biological and clinical significance of lncRNA nuclear enriched abundant transcript 1 (NEAT1) and hsa-mir-98-5p in NSCLC. It was observed that NEAT1 was upregulated while hsa-mir-98-5p was downregulated respectively in NSCLC cell lines compared to human normal lung epithelial BES-2B cells. Inhibition of NEAT1 can suppress the progression of NSCLC cells and hsa-mir-98-5p can reverse this phenomenon. Bioinformatics search was used to elucidate the correlation between NEAT1 and hsa-mir-98-5p. Additionally, a novel messenger RNA target of hsa-mir-98-5p,mitogen-activated protein kinase 6 (MAPK6), was predicted. Overexpression and knockdown studies were conducted to verify whether NEAT1 exhibits its biological functions through regulating hsa-mir-98-5p and MAPK6 in vitro. NEAT1 was able to increase MAPK6 expression and hsa-mir-98-5p mimics can inhibit MAPK6 via downregulating NEAT1 levels. We speculated that NEAT1 may act as a competing endogenous lncRNA to upregulate MAPK6 by attaching hsa-mir-98-5p in lung cancers. Taken these together, NEAT1/hsa-mir-98-5p/MAPK6 is involved in the development and progress in NSCLC. NEAT1 could be recommended as a prognostic biomarker and therapeutic indicator in NSCLC diagnosis and treatment.

MALAT1\u2013miR663a negative feedback loop in colon cancer cell functions through direct miRNA\u2013lncRNA binding

The lncRNA MALAT1 has multiple biological functions, including influencing RNA processing, miRNA sponging, and cancer development. It is acknowledged that miR663a and its targets are inflammation-related genes frequently deregulated in many cancers. The associations between MALAT1 and miR663a and their target genes remain unknown. In this study, it was found that in colon cancer (CC) cells, MALAT1 and miR663a were reciprocally repressed in cDNA array screening and qRT-PCR analysis. However, MALAT1 was significantly upregulated in CC tissues, and miR663a was significantly downregulated relative to the corresponding surgical margin (SM) tissues. An inverse relationship between MALAT1 and miR663a expression was detected among CC tissue samples (n = 172, r = −0.333, p < 0.0001). The RNA-pulldown results showed MALAT1 lncRNA–miR663a binding. The results of luciferase-reporter analysis further revealed that the MALAT1 7038–7059 nt fragment was the miR663a seed sequence. Both miR663a knockdown and MALAT1 activation alone significantly upregulated the expression levels of miR663a targets, including TGFB1, PIK3CD, P53, P21, and JUND, in the CC cell lines HCT116 and SW480. A positive relationship was also observed between the expression levels of MALAT1 and these miR663a targets in the above 172 CC samples and 160 CC samples in publicly available databases. In addition, reciprocal abolishment of the effects of miR663a overexpression and MALAT1 activation on the proliferation, migration, and invasion of cancer cells was also observed, while miR663a upregulation and MALAT1 activation alone inhibited and promoted the behaviors of these CC cell lines, respectively. All these suggested that, as a competing endogenous lncRNA, MALAT1 maybe a dominant protector for the degradation of miR663a targets. miR663a and MALAT1 may consist of a negative feedback loop to determine their roles in CC development.