Probes for LONG

Non-coding RNAs (ncRNAs) play a critical role in the entire body, and their mis-regulation is often associated with disease. In parallel with the advances in high-throughput sequencing technologies, there is a great deal of focus on this broad class of RNAs. Although these molecules are not translated into proteins, they are now well established as significant regulatory components in many biological pathways and pathological conditions. ncRNAs can be roughly divided into two main sub-groups based on the length of the transcript, with both the small and long non-coding RNAs having diverse regulatory functions. The smaller length group includes ribosomal RNAs (rRNA), transfer RNAs (tRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), microRNAs (miRNA), small interfering RNAs (siRNA), and PIWI-associated RNAs (piRNA). The longer length group includes linear long non-coding RNAs (lncRNA) and circular RNAs (circRNA). This review is designed to present the different classes of small and long ncRNA molecules and describe some of their known roles in physiological and pathological conditions, as well as methods used to assess the validity and function of miRNAs and lncRNAs, with a focus on their role and functions in the inner ear, hearing and deafness.

An increasing body of evidence suggests that long noncoding RNAs play critical roles in human cancer. Breast cancer is a heterogeneous disease and the potential involvement of long noncoding RNAs in breast cancer remains poorly understood. Herein, researchers identified a long noncoding RNA, DANCR, which promotes cisplatin chemoresistance in triple-negative breast cancer cells. Mechanistically, DANCR could bind to Krüppel-like factor 5 (KLF5) and induce acetylation of KLF5 at lysine 369 (K369), and DANCR knockdown resulted in down-regulation of KLF5 protein levels. Furthermore, researchers found that the DANCR/KLF5 signaling pathway induced hypersensitivity to cisplatin in chemoresistant patients by inhibiting p27 transcription. In summary, researcher's study reinforces the potential presence of a growth regulatory network found in triple-negative breast cancer cells, and a DANCR/KLF5/p27 signaling pathway was documented in the present study that mediates cisplatin chemoresistance in triple-negative breast cancer.

Psoriasis is a common immune-mediated skin disease characterized by epidermal hyperproliferation and chronic skin inflammation. Long non-coding RNAs (lncRNAs) are >200 nucleotide long transcripts, which possess important regulatory functions. To date, little is known about the contribution of lncRNAs to psoriasis. Here, we identify LINC00958 as a lncRNA overexpressed in keratinocytes from psoriasis skin lesions, in a transcriptomic screen performed on keratinocytes sorted from psoriasis and healthy skin. Increased levels of LINC00958 in psoriasis keratinocytes were confirmed by RT-qPCR and single molecule in situ hybridization. Confocal microscopy and analysis of subcellular fractions showed that LINC00958 is mainly localized in the cytoplasm of keratinocytes. IL-17A, a key psoriasis cytokine, induced LINC00958 in keratinocytes through C/EBP-β and the p38 pathway. Inhibition of LINC00958 led to decreased proliferation as measured by Ki67 expression, IncuCyte imaging and EdU assays. Transcriptomic analysis of LINC00958-depleted keratinocytes revealed enrichment of proliferation and cell cycle-related genes among differentially expressed transcripts. Moreover, LINC00958-depletion led to decreased basal and IL-17A-induced phosphorylation of p38. Furthermore, IL-17A-induced keratinocyte proliferation was counteracted by the inhibition of LINC00958. In summary, our data support a role for the IL-17A-induced lncRNA, LINC00958, in the pathological circuits in psoriasis by reinforcing IL-17A-induced epidermal hyperproliferation.

The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN, which is known to dictate specification towards the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms.The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific function in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced.These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development.

Metastasis is the predominant cause of mortality in patients with breast cancer. Long noncoding RNAs (lncRNAs) have been shown to drive important phenotypes in tumors, including invasion and metastasis. However, the lncRNAs involved in metastasis and their molecular and cellular mechanisms are still largely unknown.The transcriptional and posttranscriptional processing of LINC00478-associated cytoplasmic RNA (LacRNA) was determined by RT-qPCR, semiquantitative PCR and 5'/3' RACE. Paired-guide CRISPR/cas9 and CRISPR/dead-Cas9 systems was used to knock out or activate the expression of LacRNA. Cell migration and invasion assay was performed to confirm the phenotype of LacRNA. Tail vein model and mammary fat pad model were used for in vivo study. The LacRNA-PHB2-cMyc axis were screened and validated by RNA pulldown, mass spectrometry, RNA immunoprecipitation and RNA-seq assays.Here, we identified a novel cytoplasmic lncRNA, LacRNA (LINC00478-associated cytoplasmic RNA), derived from nucleus-located lncRNA LINC00478. The nascent transcript of LINC00478 full-length (LINC00478_FL) was cleaved and polyadenylated, simultaneously yielding 5' ends stable expressing LacRNA, which is released into the cytoplasm, and long 3' ends of nuclear-retained lncRNA. LINC00478_3'RNA was rapidly degraded. LacRNA significantly inhibited breast cancer invasion and metastasis in vitro and in vivo. Mechanistically, LacRNA physically interacted with the PHB domain of PHB2 through its 61-140-nt region. This specific binding affected the formation of the autophagy degradation complex of PHB2 and LC3, delaying the degradation of the PHB2 protein. Unexpectedly, LacRNA specifically interacted with PHB2, recruited c-Myc and promoted c-Myc ubiquitination and degradation. The negatively regulation of Myc signaling ultimately inhibited breast cancer metastasis. Furthermore, LacRNA and LacRNA-mediated c-Myc signaling downregulation are significantly associated with good clinical outcomes, take advantage of these factors we constructed a prognostic predict model.Therefore, our findings propose LacRNA as a potential prognostic biomarker and a new therapeutic strategy.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.

Here we describe a conserved motor neuron specific long non-coding RNA, Lhx1os, whose knock-out in mice produces motor impairment and post-natal reduction of mature motor neurons (MNs). The endoplasmic reticulum (ER)-stress response pathway resulted specifically altered with the downregulation of factors involved in the Unfolded Protein Response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function.

Based on high-throughput transcriptomic sequencing, SNHG3 was among the most highly expressed long noncoding RNAs in calcific aortic valve disease. SNHG3 upregulation was verified in human and mouse calcified aortic valves. Moreover, in vivo and in vitro studies showed SNHG3 silencing markedly ameliorated aortic valve calcification. In-depth functional assays showed SNHG3 physically interacted with polycomb repressive complex 2 to suppress the H3K27 trimethylation BMP2 locus, which in turn activated BMP2 expression and signaling pathways. Taken together, SNHG3 promoted aortic valve calcification by upregulating BMP2, which might be a novel therapeutic target in human calcific aortic valve disease.

Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR. However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV-host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA, with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Epigenetic reprogramming plays a critical role in chondrocyte senescence during osteoarthritis (OA) pathology, but the underlying molecular mechanisms remain to be elucidated. Here, using large-scale individual datasets and genetically engineered (Col2a1-CreERT2;Eldrflox/flox and Col2a1-CreERT2;ROSA26-LSL-Eldr+/+ knockin) mouse models, we show that a novel transcript of long noncoding RNA ELDR is essential for the development of chondrocyte senescence. ELDR is highly expressed in chondrocytes and cartilage tissues of OA. Mechanistically, exon 4 of ELDR physically mediates a complex consisting of hnRNPL and KAT6A to regulate histone modifications of the promoter region of IHH, thereby activating hedgehog signaling and promoting chondrocyte senescence. Therapeutically, GapmeR-mediated silencing of ELDR in the OA model substantially attenuates chondrocyte senescence and cartilage degradation. Clinically, ELDR knockdown in cartilage explants from OA-affected individuals decreased the expression of senescence markers and catabolic mediators. Taken together, these findings uncover an lncRNA-dependent epigenetic driver in chondrocyte senescence, highlighting that ELDR could be a promising therapeutic avenue for OA.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked downregulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared to wild type control. Meg3 was also induced by LPS in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

Renal interstitial fibrosis (RIF) is a common pathological manifestation of chronic kidney diseases. Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is considered a major cause of RIF. Although long non-coding RNAs (lncRNAs) are reportedly involved in various pathophysiological processes, the roles and underlying molecular mechanisms of lncRNAs in the progression of RIF are poorly understood. In this study, we investigated the function of lncRNAs in RIF. Microarray assays showed that expression of the lncRNA lnc-CHAF1B-3 (also called claudin 14 antisense RNA 1) was significantly upregulated in human renal proximal tubular cells by both transforming growth factor-β1 (TGF-β1) and hypoxic stimulation, accompanied with increased expression of EMT-related genes. Knockdown of lnc-CHAF1B-3 significantly suppressed TGF-β1-induced upregulated expression of collagen type I alpha 1, cadherin-2, plasminogen activator inhibitor-1, snail family transcriptional repressor I (SNAI1) and SNAI2. Quantitative reverse transcriptase PCR analyses of paraffin-embedded kidney biopsy samples from IgA nephropathy patients revealed lnc-CHAF1B-3 expression was correlated positively with urinary protein levels and correlated negatively with estimated glomerular filtration rate. In situ hybridization demonstrated that lnc-CHAF1B-3 is expressed only in proximal tubules. These findings suggest lnc-CHAF1B-3 affects the progression of RIF by regulating EMT-related signaling. Thus, lnc-CHAF1B-3 is a potential target in the treatment of RIF.