Long non-coding RNA chromogenic in situ hybridisation signal pattern correlation with breast tumour pathology.

J Clin Pathol.

2015 Aug 31

Zhang Z, Weaver DL, Olsen D, deKay J, Peng Z, Ashikaga T, Evans MF.
PMID: 26323944 | DOI: 10.1136/jclinpath-2015-203275

Abstract

AIM:
Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH).

METHODS:
Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring.

RESULTS:
HOTAIR, H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p<0.05), and HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p<0.05). HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens.

CONCLUSION:
These data corroborate the involvement of three lncRNAs (HOTAIR, H19 and KCNQ1OT1) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.

Long Non-Coding RNA H19 Prevents Lens Fibrosis through Maintaining Lens Epithelial Cell Phenotypes

Cells

2022 Aug 17

Xiong, L;Sun, Y;Huang, J;Ma, P;Wang, X;Wang, J;Chen, B;Chen, J;Huang, M;Huang, S;Liu, Y;
PMID: 36010635 | DOI: 10.3390/cells11162559

The integrity of lens epithelial cells (LECs) lays the foundation for lens function and transparency. By contrast, epithelial-mesenchymal transition (EMT) of LECs leads to lens fibrosis, such as anterior subcapsular cataracts (ASC) and fibrotic forms of posterior capsule opacification (PCO). However, the underlying mechanisms remain unclear. Here, we aimed to explore the role of long non-coding RNA (lncRNA) H19 in regulating TGF-β2-induced EMT during lens fibrosis, revealing a novel lncRNA-based regulatory mechanism. In this work, we identified that lncRNA H19 was highly expressed in LECs, but downregulated by exposure to TGF-β2. In both human lens epithelial explants and SRA01/04 cells, knockdown of H19 aggravated TGF-β2-induced EMT, while overexpressing H19 partially reversed EMT and restored lens epithelial phenotypes. Semi-in vivo whole lens culture and H19 knockout mice demonstrated the indispensable role of H19 in sustaining lens clarity through maintaining LEC features. Bioinformatic analyses further implied a potential H19-centered regulatory mechanism via Smad-dependent pathways, confirmed by in vitro experiments. In conclusion, we uncovered a novel role of H19 in inhibiting TGF-β2-induced EMT of the lens by suppressing Smad-dependent signaling, providing potential therapeutic targets for treating lens fibrosis.

In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration

Gastroenterology.

2018 Apr 03

Geng H, Bu HF, Liu F, Wu L, Pfeifer K, Chou PM, Wang X, Sun J, Lu L, Pandey A, Bartolomei MS, De Plaen IG, Wang P, Yu J, Qian J, Tan XD.
PMID: 29621481 | DOI: 10.1053/j.gastro.2018.03.058

Abstract

BACKGROUND & AIMS:

Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation.

METHODS:

We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs.

RESULTS:

In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, micewith LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19ΔEx1/+ mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19ΔEx1/+ mice vs control mice. Crypt epithelial cells from H19ΔEx1/+ mice proliferated more slowly than those from control miceafter exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium.

CONCLUSIONS:

The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration.

H19 Is Expressed in Hybrid Hepatocyte Nuclear Factor 4α + Periportal Hepatocytes but Not Cytokeratin 19+ Cholangiocytes in Cholestatic Livers

Hepatology Communications

2018 Sep 24

Jiang YC, Huang Y, Cai SY, Song YF, Boyer JL, Zhang KZ, Gao L, Zhao JJ, Huang WD, Liang G, Liangpunsakul S, Wang L.
PMID: - | DOI: 10.1002/hep4.1252

Abstract

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re‐induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situhybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell‐specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5‐diethoxycarbony1‐1,4‐dihydrocollidine (DDC), and multidrug‐resistant gene 2 knockout (Mdr2–/–) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double‐positive H19RNA+/sex‐determining region Y (SRY)‐box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple‐positive H19RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

Combined Single-Cell Profiling of lncRNAs and Functional Screening Reveals that H19 Is Pivotal for Embryonic Hematopoietic Stem Cell Development.

Cell Stem Cell (2019)

2019 Jan 10

Zhou J, Xu J, Zhang L, Liu S, Ma Y, Wen X, Hao J, Li Z, Ni Y, Li X, Zhou F, Li Q, Wang F, Wang X, Si Y, Zhang P, Liu C, Bartolomei M, Tang F, Liu B, Yu J, Lan Y.
| DOI: 10.1016/j.stem.2018.11.023

The generation of hematopoietic stem cells (HSCs) from embryonic endothelial precursors and pre-HSCs is precisely regulated by signaling pathways and transcription factors. Nevertheless, regulatory roles of non-coding RNAs remain unknown. Taking advantage of our ability to capture rare pre-HSCs and HSCs in vivo, we generated a single-cell landscape of long non-coding RNAs (lncRNAs) during HSC development. Combining bioinformatics and functional screening, we identified 6 lncRNAs influencing hematopoiesis in vitro. We further revealed that H19 lncRNA is pivotal for in vivo HSC emergence in aorta-gonads-mesonephros region. Early H19 lncRNA deficiency blocked endothelial-to-hematopoietic transition, which was independent of the H19-derived miR, miR-675. Moreover, H19-deficient pre-HSCs displayed promoter hypermethylation and concomitant downregulation of several master hematopoietic transcription factors, including Runx1 and Spi1. H19 deficiency increased the activity of S-adenosylhomocysteine hydrolase, a regulator of DNA methylation, which partially contributed to the observed hematopoietic defect. Our findings provide a resource for further analysis of lncRNAs in embryonic HSC development.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration

Experimental neurology

2022 Jun 20

Ribeiro, M;Ayupe, AC;Beckedorff, FC;Levay, K;Rodriguez, S;Tsoulfas, P;Lee, JK;Nascimento-Dos-Santos, G;Park, KK;
PMID: 35738417 | DOI: 10.1016/j.expneurol.2022.114147

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects

Genome medicine

2021 Aug 28

Li, Y;Zhang, Y;Hu, Q;Egranov, SD;Xing, Z;Zhang, Z;Liang, K;Ye, Y;Pan, Y;Chatterjee, SS;Mistretta, B;Nguyen, TK;Hawke, DH;Gunaratne, PH;Hung, MC;Han, L;Yang, L;Lin, C;
PMID: 34454586 | DOI: 10.1186/s13073-021-00937-4

Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy.Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models.We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity.Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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