The Functions and Mechanisms of Translatable Circular RNAs.

Circular RNAs (circRNAs) are covalently closed RNA produced by back-splicing. CircRNAs have been considered as a type of noncoding RNAs for a long time. However, recent studies have shown that circRNAs can be translated into functional proteins. Proteins specifically encoded by circRNAs have been proved to play important roles in cancer pathology. In this review, we introduce the methods commonly used to identify and validate circRNA translation in detail. We also describe the major mechanisms driving the translation of these circRNAs. In addition, we summarize the main functions of the circRNA-encoded proteins in both physiologic and pathologic conditions. Finally, we discuss the therapeutic potential and challenges in the usage of synthetic translatable circRNAs. This brief review highlights recent discoveries made in this field and the progress of therapy based on translatable circRNAs. SIGNIFICANCE STATEMENT: Understanding the translation of circRNA could facilitate the identification of novel drug targets in various diseases. Moreover, some circRNA encoded proteins were demonstrated to have therapeutic functions in cancer. The application of synthetic circRNAs as carriers to achieve stable protein expression in vitro and in vivo has tremendous therapeutic potential.

Exercise Training-Induced MicroRNA Alterations with Protective Effects in Cardiovascular Diseases.

Exercise training (ET) is an important non-drug adjuvant therapy against many human diseases, including cardiovascular diseases. The appropriate ET intensity induces beneficial adaptions and improves physiological function and cardiopulmonary fitness. The mechanisms of exercise-induced cardioprotective effects are still not fully understood. However, mounting evidence suggest that microRNAs (miRNAs) play crucial role in this process and are essential in responding to exercise-stress and mediating exercise-protective effects. Thus, this review summarizes the biogenesis of miRNAs, the mechanism of miRNA action, and specifically the miRNAs involved in exercise-induced cardio-protection used as therapeutic targets for treating cardiovascular diseases.

Genome Editing to Abrogate Muscle Atrophy.

Muscle atrophy is a multifactor syndrome, which not only decreases the patients' quality of life significantly but also increases the morbidity and mortality of patients with chronic diseases. At present, no effective clinical treatments for muscle atrophy except for exercise are available. The emerging field of genome editing is gaining momentum as it has shown great advantage in the treatment of various diseases, including muscle atrophy. In our current review, we systematically evaluate the etiology and related signaling pathways of muscle atrophy and discuss the application of genome editing in the treatment of muscle atrophy.

Exercise Training after Myocardial Infarction Attenuates Dysfunctional Ventricular Remodeling and Promotes Cardiac Recovery.

Recent evidences have shown that exercise training not only plays a necessary role in maintaining cardiac homeostasis, but also promotes cardiac repair after myocardial infarction. Post-myocardial infarction, exercise training has been observed to effectively increase the maximum cardiac output, and protect myocardial cells against necrosis and apoptosis, thus leading to an improved quality of life of myocardial infarction patients. In fact, exercise training has received more attention as an adjunct therapeutic strategy for both treatment and prevention of myocardial infarction. This review summarizes the experimental evidence of the effects of exercise training in ventricular remodeling after myocardial infarction, and tries to provide theoretical basis along with suitable references for the exercise prescription aimed at prevention and therapy of myocardial infarction.

Delivery of engineered extracellular vesicles with miR-29b editing system for muscle atrophy therapy.

Muscle atrophy is a frequently observed complication, characterized by the loss of muscle mass and strength, which diminishes the quality of life and survival. No effective therapy except exercise is currently available. In our previous study, repressing miR-29b has been shown to reduce muscle atrophy. In our current study, we have constructed artificially engineered extracellular vesicles for the delivery of CRISPR/Cas9 to target miR-29b (EVs-Cas9-29b). EVs-Cas9-29b has shown a favorable functional effect with respect to miR-29b repression in a specific and rapid manner by gene editing. In in vitro conditions, EVs-Cas9-29b could protect against muscle atrophy induced by dexamethasone (Dex), angiotensin II (AngII), and tumor necrosis factor-alpha (TNF-α). And EVs-Cas9-29b introduced in vivo preserved muscle function in the well-established immobilization and denervation-induced muscle atrophy mice model. Our work demonstrates an engineered extracellular vesicles delivery of the miR-29b editing system, which could be potentially used for muscle atrophy therapy.

PAX8, an Emerging Player in Ovarian Cancer.

Ovarian Cancer is one of the most lethal and widespread gynecological malignancies. It is the seventh leading cause of all cancer deaths worldwide. High-Grade Serous Cancer (HGSC), the most commonly occurring subtype, alone contributes to 70% of all ovarian cancer deaths. This is mainly attributed to the complete lack of symptoms during the early stages of the disease and absence of an early diagnostic marker.PAX8 is emerging as an important histological marker for most of the epithelial ovarian cancers, as it is expressed in about 90% of malignant ovarian cancers, specifically in HGSC. PAX8 is a member of the Paired-Box gene family (PAX1-9) of transcription factors whose expression is tightly controlled temporally and spatially. The PAX genes are well known for their role in embryonic development and their expression continues to persist in some adult tissues. PAX8 is required for the normal development of Müllerian duct that includes Fallopian tube, uterus, cervix, and upper part of vagina. In adults, it is expressed in the Fallopian tube and uterine epithelium and not in the ovarian epithelium. Considering the recent studies that predict the events preceding the tumorigenesis of HGSC from the Fallopian tube, PAX8 appears to have an important role in the development of ovarian cancer.In this chapter, we review some of the published findings to highlight the significance of PAX8 as an important marker and an emerging player in the pathogenesis of ovarian cancer. We also discuss regarding the future perspectives of PAX8 wherein it could contribute to the betterment of ovarian cancer diagnosis and treatment.

Long Non-Coding RNA HAND2-AS1 Acts as a Tumor Suppressor in High-Grade Serous Ovarian Carcinoma.

Long non-coding RNAs (lncRNAs) are increasingly being identified as crucial regulators in pathologies like cancer. High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer (OC), one of the most lethal gynecological malignancies. LncRNAs, especially in cancers such as HGSC, could play a valuable role in diagnosis and even therapy. From RNA-sequencing analysis performed between an OC cell line, SKOV3, and a Fallopian Tube (FT) cell line, FT194, an important long non-coding RNA, HAND2 Anti sense RNA 1 (HAND2-AS1), was observed to be significantly downregulated in OCs when compared to FT. Its downregulation in HGSC was validated in different datasets and in a panel of HGSC cell lines. Furthermore, this study shows that the downregulation of HAND2-AS1 is caused by promoter hypermethylation in HGSC and behaves as a tumor suppressor in HGSC cell lines. Since therapeutic relevance is of key importance in HGSC research, for the first time, HAND2-AS1 upregulation was demonstrated to be one of the mechanisms through which HDAC inhibitor Panobinostat could be used in a strategy to increase HGSC cells' sensitivity to chemotherapeutic agents currently used in clinical trials. To unravel the mechanism by which HAND2-AS1 exerts its role, an in silico mRNA network was constructed using mRNAs whose expressions were positively and negatively correlated with this lncRNA in HGSC. Finally, a putative ceRNA network with possible miRNA targets of HAND2-AS1 and their mRNA targets was constructed, and the enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified.

PAX8 expression in high-grade serous ovarian cancer positively regulates attachment to ECM via Integrin ß3.

BACKGROUND: Ovarian cancer is the third most common cause of death among gynecologic malignancies worldwide. Understanding the biology and molecular pathogenesis of ovarian epithelial tumors is key to developing improved prognostic indicators and effective therapies. We aimed to determine the effects of PAX8 expression on the migrative, adhesive and survival capabilities of high-grade serous carcinoma cells. METHODS: PAX8 depleted Fallopian tube secretory cells and ovarian cancer cells were generated using short interfering siRNA. Anoikis resistance, cell migration and adhesion properties of PAX8 silenced cells were analyzed by means of specific assays. Chromatin immunoprecipitation (ChIP) was carried out using a PAX8 polyclonal antibody to demonstrate that PAX8 is able to bind to the 5'-flanking region of the ITGB3 gene positively regulating its expression. RESULTS: Here, we report that RNAi silencing of PAX8 sensitizes non-adherent cancer cells to anoikis and affects their tumorigenic properties. We show that PAX8 plays a critical role in migration and adhesion of both Fallopian tube secretory epithelial cells and ovarian cancer cells. Inhibition of PAX8 gene expression reduces the ability of ovarian cancer cells to migrate and adhere to the ECM and specifically to fibronectin and/or collagen substrates. Moreover, loss of PAX8 strongly reduces ITGB3 expression and consequently the correct expression of the αvß3 heterodimer on the plasma membrane. CONCLUSIONS: Our results demonstrate that PAX8 modulates the interaction of tumor cells with the extracellular matrix (ECM). Notably, we also highlight a novel pathway downstream this transcription factor. Overall, PAX8 could be a potential therapeutic target for high-grade serous carcinoma.

The landscape of epigenetic regulation and therapeutic application of N6-methyladenosine modifications in non-coding RNAs.

RNA N6-methyladenosine (m6A) methylation is the most abundant and conserved RNA modification in eukaryotes. It participates in the regulation of RNA metabolism and various pathophysiological processes. Non-coding RNAs (ncRNAs) are defined as small or long transcripts which do not encode proteins and display numerous biological regulatory functions. Similar to mRNAs, m6A deposition is observed in ncRNAs. Studying RNA m6A modifications on ncRNAs is of great importance specifically to deepen our understanding of their biological roles and clinical implications. In this review, we summarized the recent research findings regarding the mutual regulation between RNA m6A modification and ncRNAs (with a specific focus on microRNAs, long non-coding RNAs, and circular RNAs) and their functions. We also discussed the challenges of m6A-containing ncRNAs and RNA m6A as therapeutic targets in human diseases and their future perspective in translational roles.

Nanoparticle-Patch System for Localized, Effective, and Sustained miRNA Administration into Infarcted Myocardium to Alleviate Myocardial Ischemia-Reperfusion Injury.

Timely blood reperfusion after myocardial infarction (MI) paradoxically triggers ischemia-reperfusion injury (I/RI), which currently has not been conquered by clinical treatments. Among innovative repair strategies for myocardial I/RI, microRNAs (miRNAs) are expected as genetic tools to rescue damaged myocardium. Our previous study identified that miR-30d can provide protection against myocardial apoptosis and fibrosis to alleviate myocardial injury. Although common methods such as liposomes and viral vectors have been used for miRNA transfection, their therapeutic efficiencies have struggled with inefficient in vivo delivery, susceptible inactivation, and immunogenicity. Here, we establish a nanoparticle-patch system for miR-30d delivery in a murine myocardial I/RI model, which contains ZIF-8 nanoparticles and a conductive microneedle patch. Loaded with miR-30d, ZIF-8 nanoparticles leveraging the proton sponge effect enable miR-30d to escape the endocytic pathway, thus avoiding premature degradation in lysosomes. Meanwhile, the conductive microneedle patch offers a distinct advantage by intramyocardial administration for localized, effective, and sustained miR-30d delivery, and it simultaneously releases Au nanoparticles to reconstruct electrical impulses within the infarcted myocardium. Consequently, the nanoparticle-patch system supports the consistent and robust expression of miR-30d in cardiomyocytes. Results from echocardiography and electrocardiogram (ECG) revealed improved heart functions and standard ECG wave patterns in myocardial I/RI mice after implantation of a nanoparticle-patch system for 3 and 6 weeks. In summary, our work incorporated conductive microneedle patch and miR-30d nanodelivery systems to synergistically transcend the limitations of common RNA transfection methods, thus mitigating myocardial I/RI.

The EV antibody database: An interactive database of curated antibodies for extracellular vesicle and nanoparticle research.

Antibodies are critical tools for research into extracellular vesicles (EVs) and other extracellular nanoparticles (ENPs), where they can be used for their identification, characterization, and isolation. However, the lack of a centralized antibody platform where researchers can share validation results thus minimizing wasted personnel time and reagents, has been a significant obstacle. Moreover, because the performance of antibodies varies among assay types and conditions, detailed information on assay variables and protocols is also of value. To facilitate sharing of results on antibodies that are relevant to EV/ENP research, the EV Antibody Database has been developed by the investigators of the Extracellular RNA Communication Consortium (ERCC). Hosted by the ExRNA Portal (https://exrna.org/resources/evabdb/), this interactive database aggregates and shares results from antibodies that have been tested by research groups in the EV/ENP field. Currently, the EV Antibody Database includes modules for antibodies tested for western Blot, EV Flow Cytometry, and EV Sandwich Assays, and holds 110 records contributed by 6 laboratories from the ERCC. Detailed information on antibody sources, assay conditions, and results is provided, including negative results. We encourage ongoing expert input and community feedback to enhance the database's utility, making it a valuable resource for comprehensive validation data on antibodies and protocols in EV biology.

Cardiomyocyte-derived circulating extracellular vesicles allow a non-invasive liquid biopsy of myocardium in health and disease.

The ability to track disease without tissue biopsy in patients is a major goal in biology and medicine. Here, we identify and characterize cardiomyocyte-derived extracellular vesicles in circulation (EVs; "cardiovesicles") through comprehensive studies of induced pluripotent stem cell-derived cardiomyocytes, genetic mouse models, and state-of-the-art mass spectrometry and low-input transcriptomics. These studies identified two markers (POPDC2, CHRNE) enriched on cardiovesicles for biotinylated antibody-based immunocapture. Captured cardiovesicles were enriched in canonical cardiomyocyte transcripts/pathways with distinct profiles based on human disease type (heart failure, myocardial infarction). In paired myocardial tissue-plasma from patients, highly expressed genes in cardiovesicles were largely cardiac-enriched (vs. "bulk" EVs, which were more organ non-specific) with high expression in myocardial tissue by single nuclear RNA-seq, largely in cardiomyocytes. These results demonstrate the first "liquid" biopsy discovery platform to interrogate cardiomyocyte states noninvasively in model systems and in human disease, allowing non-invasive characterization of cardiomyocyte biology for discovery and therapeutic applications.

Exercise Promotes Tissue Regeneration: Mechanisms Involved and Therapeutic Scope.

Exercise has well-recognized beneficial effects on the whole body. Previous studies suggest that exercise could promote tissue regeneration and repair in various organs. In this review, we have summarized the major effects of exercise on tissue regeneration primarily mediated by stem cells and progenitor cells in skeletal muscle, nervous system, and vascular system. The protective function of exercise-induced stem cell activation under pathological conditions and aging in different organs have also been discussed in detail. Moreover, we have described the primary molecular mechanisms involved in exercise-induced tissue regeneration, including the roles of growth factors, signaling pathways, oxidative stress, metabolic factors, and non-coding RNAs. We have also summarized therapeutic approaches that target crucial signaling pathways and molecules responsible for exercise-induced tissue regeneration, such as IGF1, PI3K, and microRNAs. Collectively, the comprehensive understanding of exercise-induced tissue regeneration will facilitate the discovery of novel drug targets and therapeutic strategies.

Expanding the horizon of EV-RNAs: LncRNAs in EVs as biomarkers for disease pathways.

Extracellular vesicles (EVs) are membrane-bound nanoparticles with different types of cargo released by cells and postulated to mediate functions such as intercellular communications. Recent studies have shown that long non-coding RNAs (lncRNAs) or their fragments are present as cargo within EVs. LncRNAs are a heterogeneous group of RNA species with a length exceeding 200 nucleotides with diverse functions in cells based on their localization. While lncRNAs are known for their important functions in cellular regulation, their presence and role in EVs have only recently been explored. While certain studies have observed EV-lncRNAs to be tissue-and disease-specific, it remains to be determined whether or not this is a global observation. Nonetheless, these molecules have demonstrated promising potential to serve as new diagnostic and prognostic biomarkers. In this review, we critically evaluate the role of EV-derived lncRNAs in several prevalent diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases, with a specific focus on their role as biomarkers.

Antioxidants Supplementation During Exercise: Friends or Enemies for Cardiovascular Homeostasis?

Exercise is a preferred strategy for improving cardiac function, especially for patients with cardiovascular diseases. Increasing evidence indicates that oxidative stress is involved in exercise-induced cardioprotection, while the underlying mechanism remains unclear. Furthermore, the effect of antioxidant supplementation during or post-exercise still exists despite divergences. To explore the effect of oxidative stress and antioxidant supplementation on cardiovascular homeostasis during or post-exercise, we take insights into the progress of exercise-induced oxidative stress, antioxidant supplementation, and cardiovascular homeostasis. In particular, antioxidants such as vitamin C or E, gamma-oryzanol, and other natural antioxidants are discussed concerning regulating exercise-associated oxidative stress. Additionally, our present study reviewed and discussed a meta-analysis of antioxidant supplementation during exercise. Overall, we take an insight into the essential biological adaptations in response to exercise and the effects of antioxidant supplementation on cardiac function, which aid us in giving recommendations on antioxidant supplementation for exercisers and exercised people. A better understanding of these issues will broaden our knowledge of exercise physiology.

Moderate-Intensity Exercise Maintains Redox Homeostasis for Cardiovascular Health.

It is well known that exercise is beneficial for cardiovascular health. Oxidative stress is the common pathological basis of many cardiovascular diseases. The overproduction of free radicals, both reactive oxygen species and reactive nitrogen species, can lead to redox imbalance and exacerbate oxidative damage to the cardiovascular system. Maintaining redox homeostasis and enhancing anti-oxidative capacity are critical mechanisms by which exercise protects against cardiovascular diseases. Moderate-intensity exercise is an effective means to maintain cardiovascular redox homeostasis. Moderate-intensity exercise reduces the risk of cardiovascular disease by improving mitochondrial function and anti-oxidative capacity. It also attenuates adverse cardiac remodeling and enhances cardiac function. This paper reviews the primary mechanisms of moderate-intensity exercise-mediated redox homeostasis in the cardiovascular system. Exploring the role of exercise-mediated redox homeostasis in the cardiovascular system is of great significance to the prevention and treatment of cardiovascular diseases.

Identification of a novel small-molecule inhibitor of miR-29b attenuates muscle atrophy.

Muscle atrophy is debilitating and can be induced by several stressors. Unfortunately, there are no effective pharmacological treatment until now. MicroRNA (miR)-29b is an important target that we identified to be commonly involved in multiple types of muscle atrophy. Although sequence-specific inhibition of miR-29b has been developed, in this study, we report a novel small-molecule miR-29b inhibitor that targets miR-29b hairpin precursor (pre-miR-29b) (Targapremir-29b-066 [TGP-29b-066]) considering both its three-dimensional structure and the thermodynamics of interaction between pre-miR-29b and the small molecule. This novel small-molecule inhibitor has been demonstrated to attenuate muscle atrophy induced by angiotensin II (Ang II), dexamethasone (Dex), and tumor necrosis factor α (TNF-α) in C2C12 myotubes, as evidenced by increase in the diameter of myotube and decrease in the expression of Atrogin-1 and MuRF-1. Moreover, it can also attenuate Ang II-induced muscle atrophy in mice, as evidenced by a similar increase in the diameter of myotube, reduced Atrogin-1 and MuRF-1 expression, AKT-FOXO3A-mTOR signaling activation, and decreased apoptosis and autophagy. In summary, we experimentally identified and demonstrated a novel small-molecule inhibitor of miR-29b that could act as a potential therapeutic agent for muscle atrophy.

Plasma extracellular vesicle transcriptome as a dynamic liquid biopsy in acute heart failure.

Background: Acute decompensation is associated with increased mortality in heart failure (HF) patients, though the underlying etiology remains unclear. Extracellular vesicles (EVs) and their cargo may mark specific cardiovascular physiologic states. We hypothesized that EV transcriptomic cargo, including long non-coding RNAs (lncRNAs) and mRNAs, is dynamic from the decompensated to recompensated HF state, reflecting molecular pathways relevant to adverse remodeling. Methods: We examined differential RNA expression from circulating plasma extracellular RNA in acute HF patients at hospital admission and discharge alongside healthy controls. We leveraged different exRNA carrier isolation methods, publicly available tissue banks, and single nuclear deconvolution of human cardiac tissue to identify cell and compartment specificity of the topmost significantly differentially expressed targets. EV-derived transcript fragments were prioritized by fold change (-1.5 to + 1.5) and significance (<5% false discovery rate), and their expression in EVs was subsequently validated in 182 additional patients (24 control; 86 HFpEF; 72 HFrEF) by qRT-PCR. We finally examined the regulation of EV-derived lncRNA transcripts in human cardiac cellular stress models. Results: We identified 138 lncRNAs and 147 mRNAs (present mostly as fragments in EVs) differentially expressed between HF and control. Differentially expressed transcripts between HFrEF vs. control were primarily cardiomyocyte derived, while those between HFpEF vs. control originated from multiple organs and different (non-cardiomyocyte) cell types within the myocardium. We validated 5 lncRNAs and 6 mRNAs to differentiate between HF and control. Of those, 4 lncRNAs (AC092656.1, lnc-CALML5-7, LINC00989, RMRP) were altered by decongestion, with their levels independent of weight changes during hospitalization. Further, these 4 lncRNAs dynamically responded to stress in cardiomyocytes and pericytes in vitro , with a directionality mirroring the acute congested state. Conclusion: Circulating EV transcriptome is significantly altered during acute HF, with distinct cell and organ specificity in HFpEF vs. HFrEF consistent with a multi-organ vs. cardiac origin, respectively. Plasma EV-derived lncRNA fragments were more dynamically regulated with acute HF therapy independent of weight change (relative to mRNAs). This dynamicity was further demonstrated with cellular stress in vitro . Prioritizing transcriptional changes in plasma circulating EVs with HF therapy may be a fruitful approach to HF subtype-specific mechanistic discovery. CLINICAL PERSPECTIVE: What is new?: We performed extracellular transcriptomic analysis on the plasma of patients with acute decompensated heart failure (HFrEF and HFpEF) before and after decongestive efforts.Long non-coding RNAs (lncRNAs) within extracellular vesicles (EVs) changed dynamically upon decongestion in concordance with changes within human iPSC-derived cardiomyocytes under stress.In acute decompensated HFrEF, EV RNAs are mainly derived from cardiomyocytes, whereas in HFpEF, EV RNAs appear to have broader, non-cardiomyocyte origins.What are the clinical implications?: Given their concordance between human expression profiles and dynamic in vitro responses, lncRNAs within EVs during acute HF may provide insight into potential therapeutic targets and mechanistically relevant pathways. These findings provide a "liquid biopsy" support for the burgeoning concept of HFpEF as a systemic disorder extending beyond the heart, as opposed to a more cardiac-focused physiology in HFrEF.

Circulating extracellular vesicles in human cardiorenal syndrome promote renal injury in a kidney-on-chip system.

BACKGROUNDCardiorenal syndrome (CRS) - renal injury during heart failure (HF) - is linked to high morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.METHODSWe investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip (KOC) model. The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV-miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.RESULTSRenal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from patients with HF. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. A total of 15 EV-miRNAs were associated with creatinine, targeting 1,143 gene targets specifying pathways relevant to renal injury, including TGF-ß and AMPK signaling. We observed directionally consistent changes in the expression of TGF-ß pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function.CONCLUSIONPlasma EVs in patients with CRS elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.TRIAL REGISTRATIONClinicalTrials.gov NCT03345446.FUNDINGAmerican Heart Association (AHA) (SFRN16SFRN31280008); National Heart, Lung, and Blood Institute (1R35HL150807-01); National Center for Advancing Translational Sciences (UH3 TR002878); and AHA (23CDA1045944).