Daling Zhu

Ribosomal RNA-derived small RNAs (rsRNAs) are nucleic acid fragments originating from rRNA, the functions and roles of which in diseases remain incompletely understood. We identified 28S rsRNA-CTC_L20, an rsRNA whose expression is upregulated in hypoxic pulmonary artery smooth muscle cells (PASMCs) and in lung tissues of pulmonary hypertension (PH) models. In hypoxic cells, 28S rsRNA-CTC_L20 promotes PASMC proliferation, and its knockdown mitigates SuHx-induced pulmonary hypertension and vascular remodeling. Mechanistically, 28S rsRNA-CTC_L20 modulates β-hydroxybutyrate (BHB) production by maintaining the stability of ketogenic enzyme mRNAs through its "CTGNNGG" motif. Elevated levels of BHB enhance the β-hydroxybutyrylation modification at K166 of pyruvate kinase M2 (PKM2), increasing its activity and driving glycolysis to sustain proliferation. These results link ketone body metabolism to glycolysis in hypoxic cells and define an rsRNA-BHB-PKM2 β-hydroxybutyrylation axis in PH. They also point to 28S rsRNA-CTC_L20 as a potential target for treating vascular remodeling.

BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Although noncoding RNAs, such as circular RNAs, have been implicated in PH, the role of tRNA-derived small RNA remains poorly understood. Among them, i-tRF (internal tRNA-derived fragment)-Asp represents a novel tRNA-derived small RNA whose function in hypoxic PH is unknown. This study aims to investigate whether i-tRF-Asp contributes to PASMC proliferation and vascular remodeling in hypoxic PH. METHODS: To elucidate the functional role of i-tRF-Asp, loss-of-function experiments were conducted in vitro using hypoxic mouse PASMCs, alongside in vivo studies using both SuHx (Sugen5416+hypoxia) induced PH models in both adult male C57BL/6 mice and Sprague-Dawley rats. These models employed antisense oligonucleotide inhibitors and adeno-associated virus-mediated silencing. The underlying mechanisms were further explored via RNA immunoprecipitation, liquid-liquid phase separation assays, cell proliferation assessment, Western blot, alternative splicing profiling, and molecular dynamics simulation. RESULTS: Inhibition of i-tRF-Asp ameliorated hypoxia-induced pulmonary vascular remodeling, primarily through suppressing excessive proliferation of mouse PASMCs. Further analysis indicated that i-tRF-Asp facilitates liquid–liquid phase separation of hnRNPU (heterogeneous nuclear ribonucleoprotein U). This process mediates selective recruitment of Tcf7l2 (transcription factor 7-like 2) pre-mRNA and promotes alternative splicing, resulting in a pro-proliferative Tcf7l2 transcript variant that drives mouse PASMC proliferation. Furthermore, we have identified that the endonuclease ERN1 (endoribonuclease IRE1) can upregulate the expression of i-tRF-Asp. CONCLUSIONS: The endoribonuclease ERN1 upregulates the expression of i-tRF-Asp, which in turn facilitates mouse PASMC proliferation and pulmonary vascular remodeling. This process is driven by hnRNPU liquid–liquid phase separation-dependent alternative splicing of the long Tcf7l2 transcript isoform.

BACKGROUND: Pulmonary hypertension (PH) represents a significant cardiovascular disorder marked by both functional and structural alterations within the pulmonary vasculature. Long noncoding RNAs have been closely associated with PH pathogenesis and progression, particularly in vascular remodeling and cell proliferation. Nonetheless, how long noncoding RNAs interact with downstream targets to modulate PH remains unclear. METHODS: The expression levels of LINC00599 were quantified in the mouse lung tissues and pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions. The involvement of LINC00599 in PH progression and vascular remodeling was evaluated through in vivo studies. To investigate its role in human PASMC proliferation, small interfering RNA and overexpression plasmids were used. RESULTS: The expression of LINC00599 is upregulated in the medial layer of pulmonary arteries in experimental PH models and hypoxic PASMCs. Administration of lentivirus-mediated shRNA targeting LINC00599 reverses hypoxic PH in murine models. Mechanistically, LINC00599 promotes PASMC proliferation by modulating stress granule formation through m6A (N6-methyladenosine) modification and facilitating liquid-liquid phase separation with MYH9 (myosin heavy chain 9), a process previously implicated in cell-cycle regulation. Furthermore, its expression is driven by a super-enhancer mediated by the transcription factor ZNF263. CONCLUSIONS: This study demonstrates that LINC00599 promotes PH progression by promoting PASMC proliferation via liquid-liquid phase separation—distinct from classical long noncoding RNA mechanisms. The identification of LINC00599 as a modulator of both m6A-dependent stress granule dynamics and MYH9-mediated phase separation expands our understanding of long noncoding RNA functionality in vascular diseases. As a target in a druggable pathway, LINC00599 holds promise for PH precision medicine.

BACKGROUND: Ferroptosis has been implicated in pulmonary hypertension (PH), and chromatin-associated RNAs are increasingly recognized as key regulators of this process. However, the detailed mechanism remains unexplored. METHODS: Bioinformatics, Sanger sequencing, and RNase R digestion were used to identify the upregulation of ca-circSCN8A. Functional gain and loss assays were used to unveil the role of ca-circSCN8A in hypoxic redox–dependent ferroptosis in human pulmonary arterial smooth muscle cells and a PH mice model. Interaction between ca-circSCN8A and FUS was detected via RNA immunoprecipitation and pull-down assays. Fluorescence recovery after photobleaching, ChIRP-qPCR (Chromatin Isolation by RNA Purification followed by Quatitative PCR), malondialdehyde, reduced glutathione, and glutathione were conducted to explore the potential molecular mechanism. RESULTS: ca-circSCN8A was identified and confirmed to be upregulated in PH. Its overexpression promoted hypoxia-induced ferroptosis in human pulmonary arterial smooth muscle cells. Under hypoxic conditions, ca-circSCN8A recruited EP300 to facilitate the lactylation of FUS (Fused in Sarcoma), triggering the formation of a ca-circSCN8A/FUS/EP300 complex via liquid-liquid phase separation. Liquid-liquid phase separation maintained the stability of the R-loop formed by ca-circSCN8A and ferroptosis-related gene SLC7A11 (solute carrier family 7 member 11) promoter that inhibits its transcription, further result in the disruption of the redox homeostasis and causing ferroptosis in human pulmonary arterial smooth muscle cells. CONCLUSIONS: ca-circSCN8A recruits EP300 to promote the lactylation of FUS, thereby driving liquid-liquid phase separation–mediated complex formation with FUS and EP300. This process enables ca-circSCN8A to form an R-loop with the nonhost SLC7A11 promoter, contributing to the regulation of hypoxia-induced ferroptosis in human pulmonary arterial smooth muscle cells. This study provides the first evidence that circRNAs can form R-loops with nonhost genes in a liquid-liquid phase separation–dependent manner. Our findings highlight ca-circSCN8A as a crucial regulator of ferroptosis in hypoxic PH and a potential therapeutic target for PH.

The intrinsic property of MXenes to adsorb dyes with high Raman scattering cross‐sections makes them promising candidates for surface‐enhanced Raman scattering (SERS) biosensors. In the study, we report a vanadium carbide MXene (V ₂ CT _x )‐based SERS biosensor tag, V ₂ CT _x @Thi (thionine)@Au NPs (gold nanoparticles)‐Ab (antibody), owing to its large interlayer spacing and superior dye adsorption capacity. The tag V ₂ CT _x @Thi@Au NPs‐Ab was fully characterized and validated, demonstrating a significantly enhanced Raman signal through both electromagnetic and chemical enhancement mechanisms. Using a handheld Raman spectrometer as a readout tool, the developed handheld SERS biosensor was successfully applied for the detection of viral antigens. The biosensor exhibited excellent linearity (1.562–100 nM) and achieved a low limit of detection (LOD) 1.562 nM. Moreover, the biosensor demonstrated good selectivity and stability for detecting the target S protein in saliva samples. Our study highlights the potential of V ₂ CT _x MXene as a powerful material for handheld SERS biosensors, paving the way for portable and efficient viral diagnostics.

BACKGROUND: Pulmonary artery endothelial cell (PAEC) dysfunction is a pathological hallmark of pulmonary hypertension (PH). Yet, the roles of long noncoding RNAs (lncRNAs) driven by super-enhancers (SEs) in PAECs are not well understood. In this study, we focused on the PAEC-specific SE-associated lncRNA HCG20 (HLA complex group 20) and to elucidate its role and underlying mechanisms in the progression of PH. METHODS: ChIP-qPCR, chromosome conformation capture followed by PCR, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and dual-luciferase reporter assays were used to identify dysregulated SE-associated lncRNAs in PAECs and to investigate the pathological role of HCG20. The role of HCG20 in pathological processes was validated in rodent models of PH induced by SU5416/hypoxia, monocrotaline, or hypoxia alone, through adeno-associated virus–mediated endothelial-specific HCG20 overexpression or knockdown of HCG20. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and RNA sequencing were used to elucidate the underlying mechanisms of HCG20-mediated PAEC dysfunction. RESULTS: We identified the SE-associated lncRNA HCG20 from histone H3 lysine-27 acetylation (H3K27ac) and histone H3 lysine-4 monomethylation (H3K4me1) ChIP-seq data derived from PAECs of patients with PH. A significant upregulation of HCG20 was found in hypoxia-induced human PAECs, lung tissues, and the plasma of patients with PH. Antisense oligonucleotide and CRISPR/Cas9, which, respectively, target HCG20 and its SE, alleviate hypoxia-induced pyroptosis and subsequent endothelial-to-mesenchymal transition. Human pulmonary artery smooth muscle cells internalize human PAEC–derived exosomes containing HCG20, inducing their excessive proliferation. Targeted delivery of HCG20 into the pulmonary vascular endothelium induced pulmonary vasculature remodeling and increased pulmonary artery systolic blood pressure in rodents. Mechanistically, HCG20 directly bound and stabilized the U2AF2 (U2 small nuclear RNA auxiliary factor 2) protein, thereby facilitating its impact on the alternative splicing of EIF2AK2 (eukaryotic translation initiation factor 2 alpha kinase 2). Furthermore, we identified a novel mouse ortholog gene, 4833427F10Rik (named Hcg20), of HCG20 for the first time. Our study demonstrated that specific interference with Hcg20 in the pulmonary vascular intima has been shown to ameliorate hypoxia-induced PH. CONCLUSIONS: Collectively, our data suggest that HCG20, driven by SE, contributes to PAEC dysfunction through U2AF2-mediated alternative splicing of EIF2AK2. Our work underscores the potential of using HCG20 as a novel biomarker and a promising target for the treatment of PH.

Long noncoding RNAs (lncRNAs) are implicated in pulmonary hypertension (PH) progression. However, the underlying mechanisms remain largely unknown. Although mitophagy plays a crucial role in hypoxia‐induced PH pathogenesis, the role of lncRNAs in mitophagy remains unclear. Especially, the mechanism of lncRNA encoded by the mitochondrial genome in regulating mitophagy needs to be elucidated. We explored the role of lncND5 in human pulmonary artery smooth muscle cells (PASMCs) and Sugen5416 plus hypoxia (SuHx)‐induced PH mouse model in vitro and in vivo. LncND5 expression and localization were detected using real‐time quantitative polymerase chain reaction (RT‐qPCR) and fluorescence in situ hybridization (FISH). We investigated the molecular mechanism of lncND5 using western blotting, flow cytometry, RNA immunoprecipitation, RNA pulldown, transmission electron microscopy (TEM), immunofluorescence (IF), and echocardiography. Mitochondrial lncND5 expression was decreased under hypoxia in human PASMCs. Mechanistically, in the mitochondria, lncND5 maintains complex I activity by binding with mitochondrial ADH‐ubiquinone oxidoreductase chain 5 (MT‐ND5) at nucleotides 1086–1159 bp, thereby regulating mitochondrial reactive oxygen species (mROS) release and alleviating mitophagy. Additionally, lncND5 regulates mitophagy via cardiolipin (CL), which regulates complex I activity, inhibiting ROS release then relieving mitophagy. In the cytoplasm, lncND5 inhibits mitophagy by directly interacting with hydroxymethylglutaryl‐CoA synthase 1 (HMGCS1). Notably, lncND5 is transported from the mitochondria to the cytoplasm and is mediated by TAR DNA‐binding protein 43 (TDP‐43). Our findings, for the first time, reveal that lncND5 may be a potential therapeutic approach for PH.