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Nature Medicine May 2024
PubMed: 38332041
DOI: 10.1038/s41591-024-02842-w -
Epigenetics Dec 2024Long noncoding RNAs (lncRNAs) regulate the progression of type 2 diabetes mellitus complicated with obstructive sleep apnoea (T2DM-OSA). However, the role of the lncRNA...
LncRNA NEAT1 aggravates human microvascular endothelial cell injury by inhibiting the Apelin/Nrf2/HO-1 signalling pathway in type 2 diabetes mellitus with obstructive sleep apnoea.
Long noncoding RNAs (lncRNAs) regulate the progression of type 2 diabetes mellitus complicated with obstructive sleep apnoea (T2DM-OSA). However, the role of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in T2DM-OSA remains unknown. This study aimed to reveal the function of NEAT1 in T2DM-OSA and the underlying mechanism. KKAy mice were exposed to intermittent hypoxia (IH) or intermittent normoxia to generate a T2DM-OSA mouse model. HMEC-1 cells were treated with high glucose (HG) and IH to construct a T2DM-OSA cell model. RNA expression was detected by qRT-PCR. The protein expression of Apelin, NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and up-frameshift suppressor 1 (UPF1) was assessed using western blot. Cell injury was evaluated using flow cytometry, enzyme-linked immunosorbent assay, and oxidative stress kit assays. RIP, RNA pull-down, and actinomycin D assays were performed to determine the associations between NEAT1, UPF1, and Apelin. NEAT1 expression was upregulated in the aortic vascular tissues of mice with T2DM exposed to IH and HMEC-1 cells stimulated with HG and IH, whereas Apelin expression was downregulated. The absence of NEAT1 protected HMEC-1 cells from HG- and IH-induced damage. Furthermore, NEAT1 destabilized Apelin mRNA by recruiting UPF1. Apelin overexpression decreased HG- and IH-induced injury to HMEC-1 cells by activating the Nrf2/HO-1 pathway. Moreover, NEAT1 knockdown reduced HG- and IH-induced injury to HMEC-1 cells through Apelin. NEAT1 silencing reduced HMEC-1 cell injury through the Apelin/Nrf2/HO-1 signalling pathway in T2DM-OSA. LncRNAs, long non-coding RNAs; T2DM, type 2 diabetes mellitus; OSA, obstructive sleep apnoea; NEAT1, nuclear paraspeckle assembly transcript 1; IH, intermittent hypoxia; HMEC-1, human microvascular endothelial cells; HG, high glucose; Nrf2, NF-E2-related factor 2; UPF1, up-frameshift suppressor 1; HO-1, haem oxygenase-1; qRT-PCR, quantitative real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-α, tumour necrosis factor-α; CCK-8, Cell Counting Kit-8; IL-1β, interleukin-1β; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; RIP, RNA immunoprecipitation; SD, standard deviations; GSH, glutathione; AIS, acute ischaemic stroke; HMGB1, high mobility group box-1 protein; TLR4, toll-like receptor 4.
Topics: Animals; Humans; Mice; Apelin; Brain Ischemia; Diabetes Mellitus, Type 2; DNA Methylation; Endothelial Cells; Glucose; Heme Oxygenase (Decyclizing); Hypoxia; NF-E2-Related Factor 2; RNA Helicases; RNA, Long Noncoding; Sleep Apnea, Obstructive; Stroke; Trans-Activators; Tumor Necrosis Factor-alpha
PubMed: 38232183
DOI: 10.1080/15592294.2023.2293409 -
International Journal of Molecular... Dec 2023Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against -mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired...
Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against -mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired resistance. Therefore, enhancing PARP inhibitor sensitivity and preventing resistance in those cells are an unmet clinical need. Here, we investigated the ability of paraspeckle component 1 (), as an additional synthetic lethal partner with , to enhance olaparib sensitivity in preclinical models of -mutated breast and ovarian cancers. In vitro, the combined olaparib and small interfering RNA (siRNA) exhibited synergistic anti-proliferative activity in -mutated breast and ovarian cancer cells. The combination therapy also demonstrated synergistic tumor inhibition in a xenograft mouse model. Mechanistically, olaparib monotherapy increased the expressions of p-ATM and DNA-PKcs, suggesting the activation of a DNA repair pathway, whereas combining siRNA with olaparib decreased the expressions of p-ATM and DNA-PKcs again. As such, the combination increased the formation of γH2AX foci, indicating stronger DNA double-strand breaks. Subsequently, these DNA-damaged cells escaped G2/M checkpoint activation, as indicated by the suppression of p-cdc25C (Ser216) and p-cdc2 (Tyr15) after combination treatment. Finally, these cells entered mitosis, which induced increased apoptosis. Thus, this proves that inhibition enhances olaparib sensitivity by targeting DNA damage response in our preclinical model. The combination of olaparib and inhibition merits further clinical investigation to enhance PARP inhibitor efficacy.
Topics: Animals; Antineoplastic Agents; Poly(ADP-ribose) Polymerase Inhibitors; Ovarian Neoplasms; Humans; Female; Mice; Breast Neoplasms; Cell Line, Tumor; RNA-Binding Proteins; BRCA1 Protein; BRCA2 Protein; RNA, Small Interfering
PubMed: 38069409
DOI: 10.3390/ijms242317086 -
Nucleic Acids Research Apr 2024RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks...
RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.
Topics: Humans; DNA Breaks, Double-Stranded; DNA-Binding Proteins; Etoposide; RNA Precursors; Transcription Factors; DNA; RNA-Binding Proteins
PubMed: 38224452
DOI: 10.1093/nar/gkae022 -
Theriogenology Jul 2024Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of...
Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of blastocysts, the low ratio of the number of inner cell mass cells to the number of trophoblastic cells, and the high apoptosis rate of blastocysts, resulting in low embryo implantation rate. Therefore, optimizing in vitro culture conditions has become a key technology to im-prove the quality of preimplantation embryos. Oviduct Epithelial cells exosomes (OEVs) can be absorbed and internalized by embryos to improve the blastocyst rate and blastocyst quality of embryos in vitro. As a special nuclear structure, Paraspeckles are involved in the fate determination of mammalian early embryonic mammalian cells. However, the regulation of embryonic cell differentiation by OEVs remains unknown. We aimed to investigate the effects of OEVs on paraspeckle formation and cell fate determination in yak in vitro fertilization (IVF) of em-bryos. To simulate the in vivo oviduct environment after ovulation, we used follicular fluid exosomes (FEVs) to stimulate yak oviduct epithelial cells and collect OEVs. OEVs were added to the yak IVF embryo culture system. Paraspeckle formation, cell differentiation, and blastocyst quality in yak embryos were determined. Our results show that, development of yak embryos is unique compared to other bovine species, and OEVs can be used as a supplement to the in vitro culture system of yak embryos to improve embryonic development and blas-tocyst quality. And also Paraspeckles/CARM1 mediated the regulation of OEVs on cell differentiation during in vitro yak embryo production. These results provide new insights into the study of yak embryonic development and the role of OEVs in embryonic development.
Topics: Animals; Cell Differentiation; Female; Embryonic Development; Cattle; Epithelial Cells; Embryo Culture Techniques; Exosomes; Fertilization in Vitro; Fallopian Tubes; Blastocyst; Oviducts
PubMed: 38692037
DOI: 10.1016/j.theriogenology.2024.04.013 -
Drug Metabolism and Disposition: the... Oct 2023Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was...
Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.
Topics: Humans; Cytochrome P-450 CYP3A; Ligands; Paraspeckles; Pregnane X Receptor; Receptors, Steroid; Rifampin; RNA-Binding Proteins
PubMed: 37349114
DOI: 10.1124/dmd.122.001065 -
Naunyn-Schmiedeberg's Archives of... Oct 2023Polyphyllin B (PPB) is a compound with anti-tumor effects. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long-stranded noncoding RNA that induces...
Polyphyllin B (PPB) is a compound with anti-tumor effects. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long-stranded noncoding RNA that induces epithelial-mesenchymal transition (EMT) of tumor cells and promotes tumor growth and metastasis. However, the role and mechanism of PPB on melanoma and the correlation between them remain unclear. In this study we screened NEAT1 by using LncRNA transcriptomic sequencing, and then transfected B16F10 cells using OVER-NEAT1 lentivirus. Next, we found that PPB had significant proliferation inhibition of melanoma and B16F10 cells through MTT assay and establishment of mouse subcutaneous transplantation tumor model; in addition, through wound healing assay, transwell assay and establishment of mouse melanoma lung metastasis model, we found that PPB significantly inhibited the invasion and migration of B16F10 cells in vitro, and inhibited the metastasis of melanoma to lung, bone and liver in vivo. Finally, changes in the expression levels of EMT-related proteins were assessed by western blot (WB) and immunohistochemistry, and PPB significantly downregulated the expression levels of MMP-9, N-cadherin, etc., and upregulated E-cadherin. While overexpressed NEAT1 showed the ability to promote melanoma proliferation, migration and invasion, in addition to partially reversed the inhibition of proliferation, migration and invasion of melanoma by PPB mentioned above.
Topics: Animals; Mice; RNA, Long Noncoding; Epithelial-Mesenchymal Transition; Neoplasm Invasiveness; Cell Line, Tumor; Melanoma; Cell Proliferation; Gene Expression Regulation, Neoplastic; Cell Movement; MicroRNAs
PubMed: 37004552
DOI: 10.1007/s00210-023-02474-w -
Pathology, Research and Practice Jan 2024Cancer is a complicated illness that spreads indefinitely owing to epigenetic, genetic, and genomic alterations. Cancer cell multidrug susceptibility represents a severe... (Review)
Review
Cancer is a complicated illness that spreads indefinitely owing to epigenetic, genetic, and genomic alterations. Cancer cell multidrug susceptibility represents a severe barrier in cancer therapy. As a result, creating effective therapies requires a better knowledge of the mechanisms driving cancer development, progress, and resistance to medications. The human genome is predominantly made up of long non coding RNAs (lncRNAs), which are currently identified as critical moderators in a variety of biological functions. Recent research has found that changes in lncRNAs are closely related to cancer biology. The vascular endothelial growth factor (VEGF) signalling system is necessary for angiogenesis and vascular growth and has been related to an array of health illnesses, such as cancer. LncRNAs have been identified to alter a variety of cancer-related processes, notably the division of cells, movement, angiogenesis, and treatment sensitivity. Furthermore, lncRNAs may modulate immune suppression and are being investigated as possible indicators for early identification of cancer. Various lncRNAs have been associated with cancer development and advancement, serving as cancer-causing or suppressing genes. Several lncRNAs have been demonstrated through research to impact the VEGF cascade, resulting in changes in angiogenesis and tumor severity. For example, the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to foster the formation of oral squamous cell carcinoma and the epithelial-mesenchymal transition by stimulating the VEGF-A and Notch systems. Plasmacytoma variant translocation 1 (PVT1) promotes angiogenesis in non-small-cell lung cancer by affecting miR-29c and boosting the VEGF cascade. Furthermore, lncRNAs regulate VEGF production and angiogenesis by interacting with multiple downstream signalling networks, including Wnt, p53, and AKT systems. Identifying how lncRNAs engage with the VEGF cascade in cancer gives beneficial insights into tumor biology and possible treatment strategies. Exploring the complicated interaction between lncRNAs and the VEGF pathway certainly paves avenues for novel ways to detect better accurately, prognosis, and cure cancers. Future studies in this area could open avenues toward the creation of innovative cancer therapy regimens that enhance the lives of patients.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; RNA, Long Noncoding; Lung Neoplasms; Vascular Endothelial Growth Factor A; Carcinoma, Squamous Cell; Mouth Neoplasms; Gene Expression Regulation, Neoplastic
PubMed: 38056133
DOI: 10.1016/j.prp.2023.154998 -
Journal of Molecular Biology Dec 2023Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of...
Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, we find that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Our data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.
Topics: Biomolecular Condensates; RNA-Binding Proteins; DNA-Binding Proteins; RNA; Prions
PubMed: 37952770
DOI: 10.1016/j.jmb.2023.168364 -
Scientific Reports Jul 2023The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into...
The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.
Topics: Humans; Biomolecular Condensates; Capsid; Capsid Proteins; Cell Nucleus; HIV Infections; HIV Seropositivity; HIV-1; mRNA Cleavage and Polyadenylation Factors; Virus Replication
PubMed: 37414787
DOI: 10.1038/s41598-023-37364-x