A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

Molecular cancer

2022 Mar 22

Fletcher, CE;Deng, L;Orafidiya, F;Yuan, W;Lorentzen, MPGS;Cyran, OW;Varela-Carver, A;Constantin, TA;Leach, DA;Dobbs, FM;Figueiredo, I;Gurel, B;Parkes, E;Bogdan, D;Pereira, RR;Zhao, SG;Neeb, A;Issa, F;Hester, J;Kudo, H;Liu, Y;Philippou, Y;Bristow, R;Knudsen, K;Bryant, RJ;Feng, FY;Reed, SH;Mills, IG;de Bono, J;Bevan, CL;
PMID: 35317841 | DOI: 10.1038/s44161-022-00042-8

miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

Antisense oligonucleotides selectively suppress target RNA in nociceptive neurons of the pain system and can ameliorate mechanical pain

Pain.

2018 Jan 01

Mohan A, Fitzsimmons B, Zhao HT, Jiang Y, Mazur C, Swayze EE, Kordasiewicz HB.
PMID: 28976422 | DOI: 10.1097/j.pain.0000000000001074

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several compounds based on ASOs are currently being tested in clinical trials for various neurological disorders. Here we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that the activity of ASOs lasts up to 2 months after a single intrathecal bolus dose in the spinal cord. Interestingly, comparison of subcutaneous, central intracerebroventricular and intrathecal administration shows DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.

LncRNA4667 is dispensable for spermatogenesis and fertility in mice.

Reproductive and Developmental Medicine

2019 Apr 11

Dai YB, Lin Y, Song N, Sun F.
PMID: - | DOI: 10.4103/2096-2924.255985

Objective: Spermatogenesis is a complex process which is of vital importance for sexual reproduction. In many studies of spermatogenesis, the mRNAs, protein-coding genes, as well as small noncoding RNAs (ncRNAs) have been well characterized. However, there remain numerous questions despite previously characterized molecular mechanisms. Long ncRNAs (lncRNAs) are a relatively new addition to our knowledge of ncRNAs. Limited studies have examined the function of lncRNAs in spermatogenesis. Therefore, we selected a testis-specific lncRNA, lncRNA4667, to analyze its potential role in spermatogenesis and male fertility.

Methods: In situ hybridization and quantitative reverse transcription polymerase chain reaction analyses were used to confirm testis-specific expression of lncRNA4667. LncRNA4667 knockout mice were generated using CRISPR/Cas9 technology. Histology, sperm counts, sperm motility, body parameters, and fertility were compared between wild-type and knockout mice (n = 8/group).

Results: Expression analysis showed that lncRNA4667 was testis specific and localized to round spermatids in seminiferous tubules of adult mouse testes. Mice homozygous for a null mutation of lncRNA4667 displayed normal spermatogenesis and fertility compared with wild-type mice.

Conclusions: These data indicate that lncRNA4667 is dispensable for spermatogenesis and fertility in mice, and the localization of lncRNA4667 makes it a useful marker for the identification of round spermatids in mice.

LncRNA PAINT is Associated with Aggressive Prostate Cancer and Dysregulation of Cancer Hallmark Genes

International journal of cancer

2021 Mar 17

Hasan, MF;Ganapathy, K;Sun, J;Khatib, A;Andl, T;Soulakova, JN;Coppola, D;Zhang, W;Chakrabarti, R;
PMID: 33729568 | DOI: 10.1002/ijc.33569

Long non-coding RNAs (lncRNAs) play regulatory role in cellular processes and their aberrant expression may drive cancer progression. Here we report the function of a lncRNA PAINT (Prostate Cancer Associated Intergenic Non-Coding Transcript) in promoting prostate cancer (PCa) progression. Upregulation of PAINT was noted in advanced stage and metastatic PCa. Inhibition of PAINT decreased cell proliferation, S-phase progression, increased expression of apoptotic markers, and improved sensitivity to docetaxel and Aurora kinase inhibitor VX-680. Inhibition of PAINT decreased cell migration and reduced expression of Slug and Vimentin. Ectopic expression of PAINT suppressed E-cadherin, increased S-phase progression and cell migration. PAINT expression in PCa cells induced larger colony formation, increased tumor growth and higher expression of mesenchymal markers. Transcriptome analysis followed by qRT-PCR validation showed differentially expressed genes involved in epithelial mesenchymal transition (EMT), apoptosis and drug resistance in PAINT-expressing cells. Our study establishes an oncogenic function of PAINT in PCa. This article is protected by

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

Science Translational Medicine

2019 Mar 27

Smith EL, Harrington K, Staehr M, Masakayan R, Jones J, Long TJ, Ng KY, Ghoddusi M, Purdon TJ, Wang X, Do T, Pham MT, Brown JM, De Larrea CF, Olson E, Peguero E, Wang P, Liu H, Xu Y, Garrett-Thomson SC, Almo SC, Wendel H-G, Riviere I, Liu C, Sather B and Brentjens RJ
| DOI: 10.1126/scitranslmed.aau7746

Chimeric antigen receptor (CAR) T cells, a type of cell-based immunotherapy, have shown some promising results in multiple myeloma, a bone marrow cancer. These earlier CAR T cells targeted a protein called B cell maturation antigen, but some patients’ cancer cells expressed little to none of this antigen and were therefore resistant to the CAR T cells. Smith et al. identified another target antigen for multiple myeloma, called GPRC5D. The authors demonstrated that CAR T cells against GPRC5D are effective in mouse models, even those with tumors that are resistant to the earlier CARs, and they are safe in mice and primates.Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

	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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