N6-methyladenosine-driven miR-143/145-KLF4 circuit orchestrates the phenotypic switch of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is characterized by vascular remodeling predominantly driven by a phenotypic switching in pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanisms for this phenotypic alteration remain incompletely understood. Here, we identified that RNA methyltransferase METTL3 is significantly elevated in the lungs of hypoxic PH (HPH) mice and rats, as well as in the pulmonary arteries (PAs) of HPH rats. Targeted deletion of Mettl3 in smooth muscle cells exacerbated hemodynamic consequences of hypoxia-induced PH and accelerated pulmonary vascular remodeling in vivo. Additionally, the absence of METTL3 markedly induced phenotypic switching in PASMCs in vitro. Mechanistically, METTL3 depletion attenuated m6A modification and hindered the processing of pri-miR-143/145, leading to a downregulation of miR-143-3p and miR-145-5p. Inhibition of hnRNPA2B1, an m6A mediator involved in miRNA maturation, similarly resulted in a significant reduction of miR-143-3p and miR-145-5p. We demonstrated that miR-145-5p targets Krüppel-like factor 4 (KLF4) and miR-143-3p targets fascin actin-bundling protein 1 (FSCN1) in PASMCs. The decrease of miR-145-5p subsequently induced an upregulation of KLF4, which in turn suppressed miR-143/145 transcription, establishing a positive feedback circuit between KLF4 and miR-143/145. This regulatory circuit facilitates the persistent suppression of contractile marker genes, thereby sustaining PASMC phenotypic switch. Collectively, hypoxia-induced upregulation of METTL3, along with m6A mediated regulation of miR-143/145, might serve as a protective mechanism against phenotypic switch of PASMCs. Our results highlight a potential therapeutic strategy targeting m6A modified miR-143/145-KLF4 loop in the treatment of PH.

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

Comprehensive characterization of small noncoding RNA profiles in hypoxia-induced pulmonary hypertension (HPH) rat tissues.

Hypoxia-induced pulmonary hypertension (HPH) is a fatal cardiovascular disease characterized by an elevation in pulmonary artery pressure, resulting in right ventricular dysfunction and eventual heart failure. Exploring the pathogenesis of HPH is crucial, and small noncoding RNAs (sncRNAs) are gaining recognition as potential regulators of cellular responses to hypoxia. In this study, we conducted a comprehensive analysis of sncRNA profiles in eight tissues of male HPH rats using high-throughput sequencing. Our study unveiled several sncRNAs, with the brain, kidney, and spleen exhibiting the highest abundance of microRNA (miRNA), tRNA-derived small RNA (tDR), and small nucleolar RNA (snoRNA), respectively. Moreover, we identified numerous tissue-specific and hypoxia-responsive sncRNAs, particularly miRNAs and tDRs. Interestingly, we observed arm switching in miRNAs under hypoxic conditions and a significant increase in the abundance of 5' tRNA-halves among the total tDRs during hypoxia. Overall, our study provides a comprehensive characterization of the sncRNA profiles in HPH rats.

Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification.

Cell-free RNAs (cfRNAs) offer an opportunity to detect diseases from a transcriptomic perspective, however, existing techniques have fallen short in generating a comprehensive cell-free transcriptome profile. We develop a sensitive library preparation method that is robust down to 100 µl input plasma to analyze cfRNAs independent of their 5'-end modifications. We show that it outperforms adapter ligation-based method in detecting a greater number of cfRNA species. We perform transcriptome-wide characterizations in 165 lung cancer, 30 breast cancer, 37 colorectal cancer, 55 gastric cancer, 15 liver cancer, and 133 cancer-free participants and demonstrate its ability to identify transcriptomic changes occurring in early-stage tumors. We also leverage machine learning analyses on the differentially expressed cfRNA signatures and reveal their robust performance in cancer detection and classification. Our work sets the stage for in-depth study of the cfRNA repertoire and highlights the value of cfRNAs as cancer biomarkers in clinical applications.

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.

AIMS: Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. METHODS AND RESULTS: SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. CONCLUSION: The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.

Critical view on oligo(dT)-based RNA-seq: bias arising, modeling, and mitigating.

The precise biological interpretation of oligo(dT)-based RNA sequencing (RNA-seq) datasets, particularly in single-cell RNA-seq (scRNA-seq), is invaluable for understanding complex biological systems. However, the presence of biases can lead to misleading results in downstream analysis. This study has now identified two additional biases that are not accounted for in established bias models: poly(A)-tail length bias and fixed-position GC-content bias. These biases have a significant negative impact on the overall quality of oligo(dT)-based RNA-seq data. To address these biases, we have developed a universal bias-mitigating method based on the lower-affinity binding of short and nonanchored oligo(dT) primers to poly(A) tails. This method significantly reduces poly(A) length bias and completely eliminates fixed-position GC bias. Furthermore, the use of short oligo(dT) with impartial binding behavior toward the diverse poly(A) tails renders RNA-seq with more reliable measurements. The findings of this study are particularly beneficial for scRNA-seq datasets, where accurate benchmarking is critical.

Identification and Validation of Genes Exhibiting Dynamic Alterations in Response to Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) carries a high mortality rate and has a poor prognosis. The pathogenesis of pulmonary fibrosis (PF) is highly related to dysregulation of multiple RNAs. This study aims to identify and validate dysregulated RNAs that exhibited dynamic alterations in response to bleomycin (BLM)-induced PF. The results will provide therapeutic targets for patients suffering from IPF. Whole transcriptomic profiles of BLM-induced PF were obtained through high-throughput RNA sequencing. miRNA profiling was downloaded from GSE45789 database in the Gene Expression Omnibus (GEO). We identified the differentially expressed RNAs (DERNAs) that exhibited dynamic alterations in response to BLM-induced PF. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis were conducted to discovery regulatory processes of PF. Weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) analysis, and co-expression analysis were performed to identify key genes and pathogenic pattern during the progression of PF. MiRanda, miRcode, and TargetScan were utilized to predict target relationships in the potential competing endogenous RNA (ceRNA) network. The results were verified by qRT-PCR analysis. In the context of BLM-induced PF, this study identified a total of 167 differentially expressed messenger RNAs (DEmRNAs), 115 differentially expressed long non-coding RNAs (DElncRNAs), 45 differentially expressed circular RNAs (DEcircRNAs), and 87 differentially expressed microRNAs (DEmiRNAs). These RNA molecules showed dynamic alterations in response to BLM-induced PF. These DEmRNAs exhibited a predominant association with the biological processes pertaining to the organization of extracellular matrix. A regulatory network was built in PF, encompassing 31 DEmRNAs, 18 DE lncRNAs, 13 DEcircRNAs, and 13 DEmiRNAs. Several DERNA molecules were subjected to validate using additional BLM-induced PF model. The outcomes of this validation process shown a strong correlation with the results obtained from RNA sequencing analysis. The GSE213001 dataset was utilized to validate the expression levels and diagnostic efficacy of four specific hub mRNAs (CCDC80, CLU, COL5A1, and COL6A3) in individuals diagnosed with PF. In this study, we identified and validated several RNA molecules that exhibited dynamic alternations in response to BLM-induced PF. These dysregulated RNAs participated in the pathogenesis of PF and can be used as therapeutic targets for early-stage IPF. Although more work must be done to confirm the results, our study may provide directions for future studies.

CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation.

Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.

Altered cfDNA fragmentation profile in hypomethylated regions as diagnostic markers in breast cancer.

BACKGROUND: Breast cancer, the most common malignancy in women worldwide, has been proven to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profiles. Nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear. METHODS: Improved cfMeDIP-seq were utilized to characterize both cfDNA methylation and fragmentation profiles in 49 plasma samples from both healthy individuals and patients with breast cancer. The feasibility of using cfDNA fragmentation profile in hypo- and hypermethylated regions as diagnostic markers for breast cancer was evaluated. RESULTS: Mean size of cfDNA fragments (100-220 bp) mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 to 167.73 bp) than in healthy individuals (2.87 bp, 174.54 to 171.67 bp). Furthermore, proportion of short cfDNA fragments (100-150 bp) in hypomethylated regions when compared with it in hypermethylated regions was found to increase more in patients with breast cancer in two independent discovery cohort. The feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic regions for breast cancer diagnosis in validation cohort was evaluated. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity). CONCLUSION: We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis.

The protective role of EP300 in monocrotaline-induced pulmonary hypertension.

Background: Pulmonary hypertension (PH) is a lethal disease characterized by pulmonary vascular remodeling, which is mediated by the abnormal proliferation/migration of pulmonary arterial smooth muscle cells (PASMCs). Recent reports suggest the involvement of histone acetylation in PAH development and that histone deacetylase (HDAC) inhibitors have therapeutic potential for the treatment of PAH. EP300 is an acetyltransferase that plays diverse roles in cell proliferation, differentiation, and apoptosis. However, the functions of EP3000 in PH are rarely studied. Results: In this work, we found that the expression of EP300 was increased in the pulmonary arteries of monocrotaline (MCT)-induced PH rats. Knockdown of EP300 by AAV-mediated shRNA exacerbated the PH, with a higher right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness in the pulmonary artery of MCT-induced PH rat. On the cellular level, the proliferation of PASMCs was promoted by EP300 knockdown. In addition, the expression of EP300 was increased in PASMCs by the overexpression of EGR1, while the deletion of EGR1 binding sites in the EP300 promoter region decreased the activity of EP300 promoter. Moreover, deleting the EP300 promoter region containing EGR1 binding sites using CRISPR/Cas9 abolished the upregulation of EP300 in MCT-induced rats and exacerbated MCT-induced PH. To summarize, our data indicate that EP300 upregulation mediated by EGR1 has a protective effect on MCT-induced PH. Conclusion: These findings showed EP300 expression was increased in the MCT-induced PH model in rats, which could be mediated by EGR1; the EP300 also displayed the potential to provide protection from PH.

Gut Microbiota and Metabolome Changes in Three Pulmonary Hypertension Rat Models.

Dysbiosis of the gut microbiota and metabolites is found in both pulmonary hypertension patients and pulmonary hypertension rodent models. However, the exact changes in gut microbiota during the development of pulmonary hypertension is unclear. The function of the gut microbiota is also ambiguous. Here, this study showed that the gut microbiota was disrupted in rats with hypoxia (Hyp)-, hypoxia/Sugen5416 (HySu)-, and monocrotaline (MCT)-induced pulmonary hypertension. The gut microbiota is dynamically changed during the development of Hyp-, HySu-, and MCT-induced rat pulmonary hypertension. The variation in the α diversity of the gut microbiota in Hyp-induced pulmonary hypertension rats was similar to that in rats with MCT-induced pulmonary hypertension and different from that in rats with HySu-induced pulmonary hypertension. In addition, six plasma biomarkers, His, Ala, Ser, ADMA, 2-hydroxybutyric acid, and cystathionine, were identified in Hyp-induced pulmonary hypertension rats. Furthermore, a disease-associated network connecting Streptococcus with Hyp-induced pulmonary hypertension-associated metabolites was described here, including trimethylamine N-oxide, Asp, Asn, Lys, His, Ser, Pro, and Ile.

2-Hydroxypropyl Group Linked Derivatives of Indole Azoles as Potential Multifunctional Antibacterial Candidates for Effectively Inhibiting the Activity of MRSA and Responding Inflammatory Factors.

The increasing resistance of methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics has led to escalating efforts to design and synthesize new structural agents with significant antimicrobial potential. A novel class of 2-hydroxypropyl group linked derivatives of indole azoles was developed as potential antibacterial agents. Bioactivity screening results demonstrated that metronidazole-modified indole derivative 4 a had excellent antibacterial capacity against MRSA (MIC=6 µM), which was about 4 times that of norfloxacin (MIC=25 µM). Highly active hybrid 4 a did not cause obvious drug-resistance in MRSA after multiple generations (15 passage operations). Compound 4 a showed low toxicity to normal mammalian cells (RAW 264.7). Molecular docking and molecular electrostatic potential (MEP) surface studies were used to map hydrogen bond interactions and the electron distribution in the highly active compounds. In addition, the preliminary exploration of the antibacterial mechanism revealed that the active molecule 4 a could infiltrate the membrane of MRSA and insert into MRSA DNA to prevent its replication, thus activating strong inhibition of the bacteria. Furthermore, highly active derivative 4 a could better respond to inflammatory factors (IL-6, IL-10, TNF-α and PGE-2), and it is less likely to cause inflammatory complications, hence diversifying the functions of antibacterial candidate molecules. These findings effectively indicate the potential of the bioactive hybrid 4 a as a multifunctional anti-MRSA agent. Further exploration of the development of antimicrobials combining these kinds of 2-hydroxypropyl group linked indole derivatives is of great value.

Circular RNA circFARSA promotes the tumorigenesis of non-small cell lung cancer by elevating B7H3 via sponging miR-15a-5p.

Non-small cell lung cancer (NSCLC) is currently one of the malignant tumors with the highest incidence and mortality rate in China. Circular RNA hsa_circ_0000896 (circFARSA) has been reported as being an oncogene and a potential biomarker for NSCL. However, the functional role and action mechanism of circFARSA in NSCLC progression have not been fully elucidated. The present study demonstrated that circFRASA was upregulated in NSCLC tissues and cell lines, and its expression was positively correlated with poor prognosis of patients with NSCLC. Further experiments revealed that circFARSA knockdown inhibited cell proliferation, migration, and invasion in vitro experiments, but overexpression of circFARSA exhibited opposite results. Mechanistically, circFARSA facilitated the malignant phenotype of NSCLC cells by enhancing B7H3 expression through sponging miR-15a-5p. In vivo experiments, knockdown of circFARSA restricted tumor growth and metastasis. In conclusion, circFARSA served as a sponge of miR-15a-5p to promote tumorigenesis and development of NSCLC by upregulation of B7H3 expression, which provided evidence of circFARSA maybe act as a novel therapeutic target for NSCLC.

Methylation-mediated silencing of PTPRD induces pulmonary hypertension by promoting pulmonary arterial smooth muscle cell migration via the PDGFRB/PLCγ1 axis.

OBJECTIVE: Pulmonary hypertension is a lethal disease characterized by pulmonary vascular remodeling and is mediated by abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Platelet-derived growth factor BB (PDGF-BB) is the most potent mitogen for PASMCs and is involved in vascular remodeling in pulmonary hypertension development. Therefore, the objective of our study is to identify novel mechanisms underlying vascular remodeling in pulmonary hypertension. METHODS: We explored the effects and mechanisms of PTPRD downregulation in PASMCs and PTPRD knockdown rats in pulmonary hypertension induced by hypoxia. RESULTS: We demonstrated that PTPRD is dramatically downregulated in PDGF-BB-treated PASMCs, pulmonary arteries from pulmonary hypertension rats, and blood and pulmonary arteries from lung specimens of patients with hypoxic pulmonary arterial hypertension (HPAH) and idiopathic PAH (iPAH). Subsequently, we found that PTPRD was downregulated by promoter methylation via DNMT1. Moreover, we found that PTPRD knockdown altered cell morphology and migration in PASMCs via modulating focal adhesion and cell cytoskeleton. We have demonstrated that the increase in cell migration is mediated by the PDGFRB/PLCγ1 pathway. Furthermore, under hypoxic condition, we observed significant pulmonary arterial remodeling and exacerbation of pulmonary hypertension in heterozygous PTPRD knock-out rats compared with the wild-type group. We also demonstrated that HET group treated with chronic hypoxia have higher expression and activity of PLCγ1 in the pulmonary arteries compared with wild-type group. CONCLUSION: We propose that PTPRD likely plays an important role in the process of pulmonary vascular remodeling and development of pulmonary hypertension in vivo .

MicroRNAs in Pulmonary Hypertension, from Pathogenesis to Diagnosis and Treatment.

Pulmonary hypertension (PH) is a fatal and untreatable disease, ultimately leading to right heart failure and eventually death. microRNAs are small, non-coding endogenous RNA molecules that can regulate gene expression and influence various biological processes. Changes in microRNA expression levels contribute to various cardiovascular disorders, and microRNAs have been shown to play a critical role in PH pathogenesis. In recent years, numerous studies have explored the role of microRNAs in PH, focusing on the expression profiles of microRNAs and their signaling pathways in pulmonary artery smooth muscle cells (PASMCs) or pulmonary artery endothelial cells (PAECs), PH models, and PH patients. Moreover, certain microRNAs, such as miR-150 and miR-26a, have been identified as good candidates of diagnosis biomarkers for PH. However, there are still several challenges for microRNAs as biomarkers, including difficulty in normalization, specificity in PH, and a lack of longitudinal and big sample-sized studies. Furthermore, microRNA target drugs are potential therapeutic agents for PH treatment, which have been demonstrated in PH models and in humans. Nonetheless, synthetic microRNA mimics or antagonists are susceptible to several common defects, such as low drug efficacy, inefficient drug delivery, potential toxicity and especially, off-target effects. Therefore, finding clinically safe and effective microRNA drugs remains a great challenge, and further breakthrough is urgently needed.

Decoding ceRNA regulatory network in the pulmonary artery of hypoxia-induced pulmonary hypertension (HPH) rat model.

BACKGROUND: Hypoxia-induced pulmonary hypertension (HPH) is a lethal cardiovascular disease with the characteristic of severe remodeling of pulmonary vascular. Although a large number of dysregulated mRNAs, lncRNAs, circRNAs, and miRNAs related to HPH have been identified from extensive studies, the competitive endogenous RNA (ceRNA) regulatory network in the pulmonary artery that responds to hypoxia remains largely unknown. RESULTS: Transcriptomic profiles in the pulmonary arteries of HPH rats were characterized through high-throughput RNA sequencing in this study. Through relatively strict screening, a set of differentially expressed RNAs (DERNAs) including 19 DEmRNAs, 8 DElncRNAs, 19 DEcircRNAs, and 23 DEmiRNAs were identified between HPH and normal rats. The DEmRNAs were further found to be involved in cell adhesion, axon guidance, PPAR signaling pathway, and calcium signaling pathway, suggesting their crucial role in HPH. Moreover, a hypoxia-induced ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed according to the ceRNA hypothesis. More specifically, the ceRNA network was composed of 10 miRNAs as hub nodes, which might be sponged by 6 circRNAs and 7 lncRNAs, and directed the expression of 18 downstream target genes that might play important role in the progression of HPH. The expression patterns of selected DERNAs in the ceRNA network were then validated to be consistent with sequencing results in another three independent batches of HPH and normal control rats. The diagnostic effectiveness of several hub mRNAs in ceRNA network was further evaluated through investigating their expression profiles in patients with pulmonary artery hypertension (PAH) recorded in the Gene Expression Omnibus (GEO) dataset GSE117261. Dysregulated POSTN, LTBP2, SPP1, and LSAMP were observed in both the pulmonary arteries of HPH rats and lung tissues of PAH patients. CONCLUSIONS: A ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed, 10 hub miRNAs and their corresponding interacting lncRNAs, circRNAs, and mRNAs were identified. The expression patterns of selected DERNAs were further validated to be consistent with the sequencing result. POSTN, LTBP2, SPP1, and LSAMP were suggested to be potential diagnostic biomarkers and therapeutic targets for PAH.

LncPTSR Triggers Vascular Remodeling in Pulmonary Hypertension by Regulating [Ca2+]i in Pulmonary Arterial Smooth Muscle Cells.

Pulmonary hypertension (PH) is characterized by vascular remodeling and sustained increase in right ventricular systolic pressure. The molecular mechanisms behind PH development remain unclear. Here, a long noncoding RNA (lncRNA) attenuated by platelet-derived growth factor BB (PDGF-BB) was identified, and its functional roles were investigated in vitro and in vivo. Using RNA-sequencing data and rapid amplification of cDNA ends, an lncRNA neighboring the locus of ATPase plasma membrane Ca2+ transporting 4 (PMCA4) was identified and named lncPTSR. It is a highly conserved nuclear lncRNA and was downregulated in pulmonary arterial smooth muscle cells (PASMCs) with PDGF-BB stimulation or hypoxia induction. Gene interruption or overexpression assays revealed that lncPTSR negatively regulates rat PASMC proliferation, apoptosis, and migration. LncPTSR interruption in Sprague Dawley rats using adeno-associated virus type 9-mediated shRNA resulted in a significant increase in right ventricular systolic pressure and vascular remodeling in normoxic condition. LncPTSR knockdown also suppressed PMCA4 expression and attenuated the intracellular Ca2 + efflux of PASMCs in vitro and in vivo. Further studies suggest a complex crosstalk between lncPTSR and mitogen-activated protein kinase pathway: inhibition of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase abolishes the PDGF-BB-mediated lncPTSR downregulation, and lncPTSR plays a feedback regulation for mitogen-activated protein kinase-signaling molecules. The present study suggests that lncPTSR participates in pulmonary artery remodeling via modulating the expression of PMCA4 and intracellular Ca2 + homeostasis downstream of PDGF-BB-driven mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling. These results suggest that lncPTSR may be a promising therapeutic target in PH treatment.

Integrative Modeling of Multiomics Data for Predicting Tumor Mutation Burden in Patients with Lung Cancer.

Immunotherapy has been widely used in the treatment of lung cancer, and one of the most effective biomarkers for the prognosis of immunotherapy currently is tumor mutation burden (TMB). Although whole-exome sequencing (WES) could be utilized to assess TMB, several problems prevent its routine clinical application. To develop a simplified TMB prediction model, patients with lung adenocarcinoma (LUAD) in The Cancer Genome Atlas (TCGA) were randomly split into training and validation cohorts and categorized into the TMB-high (TMB-H) and TMB-low (TMB-L) groups, respectively. Based on the 610 differentially expressed genes, 50 differentially expressed miRNAs and 58 differentially methylated CpG sites between TMB-H and TMB-L patients, we constructed 4 predictive signatures and established TMB prediction model through machine learning methods that integrating the expression or methylation profiles of 7 genes, 7 miRNAs, and 6 CpG sites. The multiomics model exhibited excellent performance in predicting TMB with the area under curve (AUC) of 0.911 in the training cohort and 0.859 in the validation cohort. Besides, the significant correlation between the multiomics model score and TMB was observed. In summary, we developed a prognostic TMB prediction model by integrating multiomics data in patients with LUAD, which might facilitate the further development of quantitative real time-polymerase chain reaction- (qRT-PCR-) based TMB prediction assay.

MSCs Therapy Reverse the Gut Microbiota in Hypoxia-Induced Pulmonary Hypertension Mice.

Mesenchymal stem cell (MSC) therapy is a promising therapeutic approach based on its strong effect on pulmonary hypertension (PH) in rats. However, the detailed mechanism of MSC therapy remains unknown. Alterations in the gut microbiota were found in both type 1 pulmonary arterial hypertension patients and hypoxia/SU5416- or monocrotaline (MCT)-induced PH rats. However, whether the therapeutic mechanism of MSCs is associated with the gut microbiota is poorly understood. Here, we found that gut microbiota homeostasis was disrupted in hypoxia-induced PH mice due to the increased Firmicutes-to-Bacteroidetes (F/B) ratio; enhanced abundances of harmful Marinifilaceae, Helicobacteraceae, and Lactobacillaceae; and decreased abundances of beneficial Bacteroidaceae, Prevotellaceae, Tannerellaceae, and Lachnospiraceae. Unexpectedly, reverses of the increase in disease-associated microbiota and decrease in anti-inflammatory and immunomodulatory functional microbiota were observed in the MSC-treated group. We also identified harmful Erysipelotrichaceae, Alphaproteobacteria, Christensenella timonensis, Coriobacteriales, and Rhodospirillales that may serve as gut microbiota biomarkers of hypoxia-induced PH mice. Micrococcaales, Nesterenkonia, Anaerotruncus, and Tyzzerella may serve as gut microbiota biomarkers of MSC-treated mice. In summary, MSC treatment suppresses hypoxia-induced pulmonary hypertension in mice, and alterated gut microbiota may play a role in the development and progression of PH. The mechanism of MSC therapy is associated with various metabolic pathways of the gut microbiota in hypoxia model PH mice.

Elevated plasma miR-133b and miR-221-3p as biomarkers for early Parkinson's disease.

Blood circulating microRNAs (miRNAs) are proposed to be promising biomarkers for many neurodegenerative disorders, including Parkinson's disease (PD). However, there is a lack of identified differentially expressed miRNAs in PD from different studies. The aim of this study was to evaluate miRNAs expression in PD. We measured plasma circulating miRNA expression in three independent sets with a total of 151 PD patients, 21 multiple system atrophy (MSA) patients and 138 healthy controls using high-throughput RT-PCR. We identified that elevated miR-133b and miR-221-3p discriminated early-stage PD from controls with 94.4% sensitivity and 91.1% specificity. Elevated miR-133b and miR-221-3p distinguished PD from controls with 84.8% sensitivity and 88.9% specificity. In addition, miR-4454 distinguished PD from MSA with 57.1% sensitivity and 82.6% specificity. Hence, elevated miR-133b and miR-221-3p potentially represent good biomarkers for early PD, and a combination of miR-133b, miR-221-3p and miR-4454 has the potential to serve as a non-invasive biomarker for PD diagnosis.