Hemodynamic shear stress is a frictional force that acts on vascular endothelial cells and is essential for endothelial homeostasis. Physiological laminar shear stress (LSS) suppresses endothelial inflammation and protects arteries from atherosclerosis. Herein, we screened differentially expressed circular RNAs (circRNAs) that were significantly altered in LSS-stimulated endothelial cells and found that circRNA-LONP2 was involved in modulating the flow-dependent inflammatory response. Furthermore, endothelial circRNA-LONP2 overexpression promoted endothelial inflammation and atherosclerosis in vitro and in vivo. Mechanistically, circRNA-LONP2 competitively sponged miR-200a-3p and subsequently promoted Kelch-like ECH-associated protein 1, Yes-associated protein 1, and enhancer of zeste homolog 2 expression, thereby inactivating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling, promoting oxidative stress and endothelial inflammation, and accelerating atherosclerosis. LSS-induced down-regulation of circRNA-LONP2 suppresses endothelial inflammation, at least in part, by activating the miR-200a-3p-mediated nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway. CircRNA-LONP2 may serve as a new therapeutic target for atherosclerosis.

Background: N6-methyladenosine (m6A) plays a critical role in various biological processes. However, no study has addressed the role of m6A modification in the statin-induced protection of endothelial cells (ECs). Methods: Quantitative real-time polymerase chain reaction and Western blotting analyses were used to study the expression of m6A regulatory genes in atorvastatin-treated ECs. Gain- and loss-of-function assays, methylated RNA immunoprecipitation analysis, and dual-luciferase reporter assays were performed to clarify the function of FTO (fat mass and obesity-associated protein) in ECs. Results: Atorvastatin decreased FTO protein expression in ECs. The knockdown of FTO enhanced the mRNA and protein expression of KLF2 (Kruppel-like factor 2) and eNOS (endothelial NO synthase) but attenuated TNFα (tumor necrosis factor alpha)-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression, as well as the adhesion of monocytes to ECs. Conversely, FTO overexpression significantly upregulated the mRNA and protein levels of VCAM-1 and ICAM-1, downregulated those of KLF2 and eNOS, and strongly attenuated the atorvastatin-mediated induction of KLF2 and eNOS expression. Subsequent investigations demonstrated that KLF2 and eNOS are functionally critical targets of FTO. Mechanistically, FTO interacted with KLF2 and eNOS transcripts and regulated their expression in an m6A-dependent manner. After FTO silencing, KLF2 and eNOS transcripts with higher levels of m6A modification in their 3′ untranslated regions were captured by YTHDF3 (YT521-B homology m6A RNA-binding protein 3), resulting in mRNA stabilization and the induction of KLF2 and eNOS protein expression. Conclusions: FTO might serve as a novel molecular target to modulate endothelial function in vascular diseases.

The roles of micropeptides in cell cycle regulation and cancer development remain largely unknown. Here we found that a micropeptide STMP1 (small transmembrane protein 1) was up-regulated in multiple malignancies including hepatocellular carcinoma (HCC), and its high level was associated with short recurrence-free survival of HCC patients. Gain- and loss-of-function analyses revealed that STMP1 accelerated cell proliferation and clonogenicity in vitro and tumor growth in vivo, and silencing STMP1 blocked G1/S transition. Mechanistically, STMP1 promoted the mRNA and protein levels of CCNE2, CDK2, and E2F1. STMP1 was localized in the inner membrane of mitochondria and interacted with mitochondrial complex IV and then enhanced its activity. Moreover, treatment with the mitochondrial complex IV inhibitor tetrathiomolybdate dramatically abrogated the promoting effect of STMP1 on cell proliferation and the expression of cyclin E2, CDK2, and E2F1. These results suggest that STMP1 may promote Gl/S transition and cell proliferation by enhancing mitochondrial complex IV activity, which highlights STMP1 as a new regulator of the cell cycle and a potential target for anti-cancer therapy.

Extracellular vesicles (EVs) are a promising new therapeutic platform. However, the low cargo-loading efficiency limits their clinical translation. In this study, we developed a high-yield EV cargo-loading device and explored its ability to encapsulate gene editing proteins. A series of fusion protein-based systems were constructed and their cargo loading efficiencies were compared by a NanoGlo luciferase assay. A myristoylated (Myr) peptide tag cloned from the N-terminal region of charged multivesicular body protein 6 (CHMP6), termed Myr(CHMP6), outcompeted CD9, ARRDC1, and other short polypeptides as an active packaging device. As determined by nanoparticle tracking analysis and transmission electron microscopy, the overexpression of Myr(CHMP6) increased small EV (sEV) production in Lenti-X 293T cells without altering sEV morphology. The high passive packaging efficiency of Myr(CHMP6) was also elucidated for unmodified cargo loading. Western blotting revealed that Myr(CHMP6) facilitated the loading of Cre and Cas9 into sEVs without the generation of packaging device-cargo fusion proteins. Furthermore, Myr(CHMP6)-modified sEVs loaded with Cre or Cas9 promoted gene-editing in recipient cells, as observed using a fluorescence reporter system. Subsequent investigation demonstrated a dose-dependent effect of Myr(CHMP6) tag-induced cargo-loading. Mechanistically, N-myristoylation alone was necessary but not sufficient for the effective packaging of proteins into EVs. Thus, our results indicated that Myr(CHMP6) induces sEV production and may be effective in loading gene editing proteins into sEVs for therapeutic purposes.

Abstract Background Influenza A virus infection due to drug resistance and side effects of the conventional antiviral drugs yet remains a serious public health threat for humans and animals. Forsythiaside A is an effective ingredient isolated from the Chinese herbal medicine forsythia. It has various pharmacological effects and has a good therapeutic effect against a variety of infectious diseases. This study aimed to further explore the immunological mechanism of Forsythiaside A in the treatment of influenza virus-infected mice and its effect on the Toll-like receptor 7 (TLR7) signaling pathway in the lungs of these mice. Methods C57/BL6J mice and TLR7−/− mice were infected with the FM1 strains (H1N1 and A/FM/1/4) of the Influenza A virus. Each group of experimental mice were divided into the mock, virus, oseltamivir, and Forsythiaside A groups. Weight change, lung index change, and the mRNA and protein expression levels of key factors in the TLR7 signaling pathway were detected. Flow cytometry was used to detect the changes in the Th1/Th2 and Th17/Treg ratios. Results After infection with the Influenza A virus, the weight loss of C57/BL6J mice treated with forsythoside A and oseltamivir decreased, and the pathological tissue sections showed that the inflammatory damage was reduced. The expression levels of the key factors, TLR7, myeloid differentiation factor 88(Myd88), and nuclear factor-kappa B (NF-κB) in the TLR7 signaling pathway were significantly reduced. Flow cytometry showed that Th1/Th2 and Th17/Treg ratios decreased after Forsythiaside A treatment. In the TLR7−/− mice, there was no significant change after Forsythiaside A treatment in the virus group. Conclusions Forsythiaside A affects the TLR7 signaling pathway in mouse lung immune cells and reduces the inflammatory response caused by the Influenza A virus FM1 strain in mouse lungs.

Background The expression profile of lncRNAs in coronary artery disease (CAD) patients has not yet been fully explored. Therefore, the current study aimed to investigate lncRNA-based prognostic biomarkers for CAD. Methods The expression profiles of lncRNA and messenger RNA (mRNA) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed lncRNA (DElncRNAs) and DEmRNAs were identified from CAD and normal samples, and weighted gene co-expression network analysis (WGCNA) was conducted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to investigate the principal functions of significantly dysregulated genes. The potential drugs of new CAD-specific genes were identified by network distance method. Receiver operating characteristic (ROC) was used to verify the classification performance of genes. Results A total of 512 differentially expressed genes (DEGs) and 308 DElncRNAs were identified from GSE113079 dataset to classify CAD samples. Through WGCNA co-expression analysis, 24 co-expression modules were obtained. A total of 187 DElncRNAs and 253 DEGs were determined from 7 modules correlated with CAD. Functional enrichment analysis showed that these DEGs were mainly related to inflammatory and immune-related pathways. Furthermore, 36 regulatory pairs of significantly shared micro RNAs (miRNAs) were identified as dysregulated lncRNA-mRNA (LRM-CAD), which contained 11 lncRNAs and 33 genes. Compared with a single lncRNA or gene, LRM-CAD showed stronger classification performance [average area under the curve (AUC) =0.958]. We screened 3 potential therapeutic drugs, DB09105, DB12371, and DB12612, a by binding drug-target gene interaction network. Molecular docking verified that the S1PR1 gene bound relatively closely to DB12371 and DB12612. The ROC analysis on external data sets showed that S1PR1, AC012640.4, and S1PR1-AC012640.4 could effectively distinguish CAD samples from control samples. Conclusions We provided a transcriptome overview of abnormally expressed lncRNAs in CAD patients and identified novel biomarkers for diagnosing CAD.

Purpose: Enhanced external counterpulsation is a non-invasive treatment that increases coronary flow in patients with coronary artery disease (CAD). However, the acute responses of vascular and blood flow characteristics in the conduit arteries during and immediately after enhanced external counterpulsation (EECP) need to be verified. Methods: Forty-two patients with CAD and 21 healthy controls were recruited into this study to receive 45 min-EECP. Both common carotid arteries (CCAs), namely, the left carotid (LC) and right carotid (RC), the right brachial (RB), and right femoral (RF) artery were imaged using a Color Doppler ultrasound. The peak systolic velocity (PSV), end-diastolic velocity (EDV), mean inner diameter (ID), resistance index (RI), and mean flow rate (FR) were measured and calculated before, during, and after the 45 min-EECP treatment. Results: During EECP, in the CCAs, the EDV was significantly decreased, while the RI was markedly increased in the two groups (both P < 0.01). However, immediately after EECP, the RI in the RC was significantly lower than that at the baseline in the patients with CAD (P = 0.039). The FR of the LC was markedly increased during EECP only in the CAD patients (P = 0.004). The PSV of the patients with CAD was also significantly reduced during EECP (P = 0.015) and immediately after EECP (P = 0.005) compared with the baseline. Moreover, the ID of the LC, RB, and RF was significantly higher immediately after EECP than that at the baseline (all P < 0.05) in the patients with CAD. In addition, they were also higher than that in the control groups (all P < 0.05). Furthermore, by the subgroup analysis, there were significant differences in the FR, PSV, and RI between females and males during and immediately after EECP (all P < 0.05). Conclusions: Enhanced external counterpulsation creates different responses of vascular and blood flow characteristics in carotid and peripheral arteries, with more significant effects in both the carotid arteries. Additionally, the beneficial effects in ID, blood flow velocity, RI, and FR after 45 min-EECP were shown only in the patients with CAD. More importantly, acute improvement of EECP in the FR of the brachial artery was showed in males, while the FR and RI of the carotid arteries changed in females.

Currently, the role of lncRNA in myocardial infarction (MI) is poorly understood. 17 co-expression modules were determined, specifically, the greenyellow, saddlebrown, grey60, royalblue, lightgreen, white, and pink modules were specifically expressed in the acute phase of MI, and brown, darkred, and royalblue, while greenyellow modules were specifically expressed in MI compared with CAD. 12 time-dependent of lncRNA/mRNA clusters with consistent expression trends were also identified. MI-associated modules were mainly enriched to immune, cell cycle, and metabolic pathways. We further obtained a network of 1816 lncRNA-mRNAs with higher expression correlations among these lncRNAs by analyzing the topological properties of the network. Herein, lncRNA RP11-847H18.2 and KLHL28, SPRTN, and EPM2AIP1 were determined as gene markers specifically expressed in MI, and they demonstrated a high predictive performance for MI diagnosis and prognosis. Three drugs, namely, Calcium citrate, Calcium Phosphate, and Calcium phosphate dihydrate, were identified as potential precursors of MI. Finally, gene and lncRNA diagnostic models were developed based on these genes and lncRNAs, with their AUCs averaged above 0.89 in both training and validation datasets. The findings of this study improve the diagnosis and prognosis of MI and personalized treatment of MI.