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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Advances in microRNAs (miRNAs) biomarkers have generated disease markers with potential clinical values. However, none of these published results have been applied in clinic until today. The main reason could be the lack of simple but robust miRNA measurements.Methods: We built up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting miRNAs without RNA purification. In this study, the method was optimized and compared with other RNA purification-based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, seven potential miRNA biomarkers of colorectal cancer were validated.Results: It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20 μl) and time (~ 140 min) with this approach. The sensitivity of this method was 2.7-343-fold higher than that of the stem-loop method, and comparable with S-Poly(T) plus method. 7 validated miRNA biomarkers of colorectal cancer by Direct S-Poly(T) plus assay could discriminate colorectal cancer stage I from healthy individuals, and promised satisfactory discrimination with the area under receiver operating characteristic (ROC) curve ranging from 0.79 to 0.94 (p value < 0.001).Conclusions: This simple and robust protocol may have strong impact on the development of specific miRNAs as biomarkers in clinic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]