miRNA Therapeutics Research Articles

Inhaled Gold Nano-star Carriers for Targeted Delivery of Triple Suicide Gene Therapy and Therapeutic MicroRNAs to Lung Metastases: Development and Validation in a Small Animal Model.

Pulmonary metastases pose significant treatment challenges for many cancers, including triple-negative breast cancer (TNBC). We developed and tested a novel suicide gene and therapeutic microRNAs (miRs) combination therapy against lung metastases in vivo in mouse models after intranasal delivery using nontoxic gold nanoparticles (AuNPs) formulated to carry these molecular therapeutics. We used AuNPs coated with chitosan-β-cyclodextrin (CS-CD) and functionalized with a urokinase plasminogen activator (uPA) peptide to carry triple cancer suicide genes (thymidine kinase-p53-nitroreductase: TK-p53-NTR) plus therapeutic miRNAs (antimiR-21, antimiR-10b and miR-100). We synthesized three AuNPs: 20nm nanodots (AuND), and 20nm or 50nm nanostars (AuNS), then surface coated these with CS-CD using a microfluidic-optimized method. We sequentially coated the resulting positively charged AuNP-CS-CD core with synthetic miRNAs followed by TK-p53-NTR via electrostatic interactions, and added uPA peptide through CD-adamantane host-guest chemistry. A comparison of transfection efficiencies for different AuNPs showed that the 50nm AuNS allowed ∼4.16-fold higher gene transfection than other NPs. The intranasal delivery of uPA-AuNS-TK-p53-NTR-microRNAs NPs (pAuNS@TK-p53-NTR-miRs) in mice predominantly accumulated in lungs and facilitated ganciclovir and CB1954 prodrug-mediated gene therapy against TNBC lung metastases. This new nanosystem may serve as an adaptable-across-cancer-type, facile, and clinically scalable platform to allow future inhalational suicide gene-miR combination therapy for patients harboring pulmonary metastases.

Intravenously transplanted mesenchymal stromal cells: a new endocrine reservoir for cardioprotection

BackgroundIntravenous administration of mesenchymal stromal cells (MSCs) has an acknowledged competence of cardiac repair, despite a lack of systematic description of the underlying biological mechanisms. The lung, but not the heart, is the main trapped site for intravenously transplanted MSCs, which leaves a spatial gap between intravenously transplanted MSCs and the injured myocardium. How lung-trapped MSCs after intravenous transplantation rejuvenate the injured myocardium remains unknown.MethodsMSCs were isolated from human placenta tissue, and DF-MSCs or Gluc-MSCs were generated by transduced with firefly luciferase (Fluc)/enhanced green fluorescence protein (eGFP) or Gaussia luciferase (Gluc) lactadherin fusion protein. The therapeutic efficiency of intravenously transplanted MSCs was investigated in a murine model of doxorubicin (Dox)-induced cardiotoxicity. Trans-organ communication from the lung to the heart with the delivery of blood was investigated by testing the release of MSC-derived extracellular vesicles (MSC-EVs), and the potential miRNA inner MSC-EVs were screened out and verified. The potential therapeutic miRNA inner MSC-EVs were then upregulated or downregulated to assess the further therapeutic efficiencyResultsDox-induced cardiotoxicity, characterized by cardiac atrophy, left ventricular dysfunction, and injured myocardium, was alleviated by consecutive doses of MSCs. These cardioprotective effects might be attributed to suppressing GRP78 triggering endoplasmic reticulum (ER) stress-induced apoptosis in cardiomyocytes. Our results confirmed that miR-181a-5p from MSCs-derived EVs (MSC-EVs) inhibited GRP78. Intravenous DF-MSCs were trapped in lung vasculature, secreted a certain number of EVs into serum, which could be confirmed by the detection of eGFP+ EVs. GLuc activity was increased in serum EVs from mice administrated with GLuc-MSCs. MiR-181a-5p, inhibiting GRP78 with high efficacy, was highly expressed in serum EVs and myocardium after injecting consecutive doses of MSCs into mice treated with Dox. Finally, upregulation or downregulation of miR-181a-5p levels in MSC-EVs enhanced or weakened therapeutic effects on Dox-induced cardiotoxicity through modulating ER stress-induced apoptosis.ConclusionsThis study identifies intravenously transplanted MSCs, as an endocrine reservoir, to secrete cardioprotective EVs into blood continuously and gradually to confer the trans-organ communication that relieves Dox-induced cardiotoxicity.

Abstract 1494: 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that regulate a wide array of genes. Because miRNA downregulate translation proteins associated with cancer progression, miRNA-based therapeutics have great potential as anticancer drugs. Nonetheless, the therapeutic potential of miRNA replacement is limited due to lack of safe and efficient delivery vehicles, and inability to target miRNAs to the intended cells/tissues. Thus, identifying molecules that are able to deliver therapeutic miRNA is of critical need. This could be achieved by targeting receptors that are overexpressed on target tumor cells. For example, the folate receptor (FR) is overexpressed on various tumor types including breast, lung, ovarian and brain tumors. Our laboratory has previously developed a unique strategy that directly links a miRNA mimic to the high affinity FR ligand, folate (FolamiRs). In the present work, to further improve the potency of FolamiRs, we introduce the reduced folate metabolite, 5-methyltetrahydrofolate (5-MTHF) that maintains nanomolar affinity for the FR at pH 7, but disengages from the FR at a significantly higher pH than that of folate, which is important when including an endosomal escape agent that typically disrupts endosomal acidification. We synthesize and conjugate 5-MTHF ligand to the potent tumor suppressor miRNA-34a (5-MTHF-miR-34a). We show that 5-MTHF-miR-34a uptake is specific to the cells overexpressing FR and represses target gene expression. Our data supports the use of 5-MTHF in combination with the endosomal escape agent, nigericin, to achieve robust cytosolic localization of therapeutically relevant miRNAs such as miR-34a. This work provides the foundation for future studies using 5-MTHF- conjugated therapeutic agents for targeting additional FR expressing cells, including myeloid derived suppressor cells (MDSCs) and macrophages. Collectively these studies will pave the way for developing 5-MTHF-based small RNA conjugates for treating FR-expressing cancers. The details of these intriguing results will be discussed in the poster. Citation Format: Sudarsan R. Kasireddy, Ahmed M. Abdelaal, Philip S. Low, Andrea L. Kasinski. 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1494.

Abstract 1493: Development of 5-FU-modified tumor suppressor microRNAs as a platform for novel microRNA-based cancer therapeutics

Abstract MicroRNA (miRNAs) are small non-coding RNAs that regulate gene expression and are frequently dysregulated, affecting many key oncogenic pathways. Drug resistance is one of the key barriers in the treatment of cancer, contributing to the poor prognoses in patients. To overcome this hurdle, we sought to exploit the inherent multi-targeted nature of miRNAs for the development of cancer therapeutics. Despite its potential, it remains a challenge to deliver nucleic acid-based therapies, including miRNAs, to cancer cells. We recently developed a novel approach to modify miRNAs by replacing its uracil bases with 5-fluorouracil (5-FU) in the guide strand of tumor suppressor miRNAs. This modification combines the therapeutic effects of a traditional chemotherapeutic with the effect of a tumor suppressor miRNA to create a potent, multi-targeted therapeutic molecule. We have previously demonstrated that 5-FU-modified miR-15a (where the prefix 5-FU- refers to the modified miRNA mimetic, i.e., 5-FU-miR-15a) and 5-FU-miR-129 have several key features such as the retention of target gene specificity, vehicle-free delivery, enhanced potency, and stability in colon and pancreatic cancer. To demonstrate the general applicability of this approach for other tumor suppressor miRNAs we selected 12 well-studied tumor suppressor miRNAs and modified them with 5-FU. The efficacy of these 12 different miRNA mimetics were screened in several cancer types including gastric, lung, breast, pancreatic, and blood cancer. Our results show that 5-FU-modified miRNAs are more effective at inhibiting cancer cell proliferation than its unmodified counterparts. In gastric cancer, there is an 8.4-fold increase in efficacy after modifying let-7a (let-7a IC50 = 59.5 nM vs. 5-FU-let-7a IC50 = 7.1 nM) and a 4.3-fold increase for miR-145 (miR-145 IC50 = 20.9 nM vs. 5-FU-miR-145 IC50 = 4.9 nM). Similarly in lung cancer, there is a 6.3-fold increase in efficacy after modifying let-7a (let-7a IC50 = 143.5 nM vs. 5-FU-let-7a IC50 = 22.8 nM) and a 13.8-fold increase (miR-145 IC50 = 51.7 nM vs. 5-FU-miR-145 IC50 = 11.0 nM). We also show that these miRNA mimetics can be delivered into cancer cells without the aid of a delivery vehicle. Additionally, this modification preserves miRNA target gene suppression activity and has 5-FU activity from its breakdown products. Lastly, in a CT26 syngeneic mouse colon cancer model, we demonstrate that 5-FU-miR-15a inhibits tumor growth by 58.2% without vehicle and is enhanced to 97.2% with a low concentration of vehicle. Notably, there was no observable toxicity in the animal studies. This work demonstrates the potential of fluoropyrimidine-modification that can be applicable to a broad spectrum of tumor suppressor miRNAs in different cancer types and represents an advancement in the development of therapeutic miRNAs for cancer treatment. Citation Format: John G. Yuen, Andrew Fesler, Ga-Ram Hwang, Thomas Dahl, Lan-Bo Chen, Henry Chen, Jingfang Ju. Development of 5-FU-modified tumor suppressor microRNAs as a platform for novel microRNA-based cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1493.

Deregulation of ceRNA Networks in Frontal Cortex and Choroid Plexus of Brain during SARS-CoV-2 Infection Aggravates Neurological Manifestations: An Insight from Bulk and Single-Cell Transcriptomic Analyses.

Although transcriptomic studies of SARS‐CoV‐2‐infected brains have depicted variability in gene expression, the landscape of deregulated cell‐specific regulatory circuits has not been elucidated yet. Hence, bulk and single‐cell RNA‐seq data are analyzed to gain detailed insights. Initially, two ceRNA networks with 19 and 3 differentially expressed (DE) hub lncRNAs are reconstructed in SARS‐CoV‐2 infected Frontal Cortex (FC) and Choroid Plexus (CP), respectively. Functional and pathway enrichment analyses of downstream mRNAs of deregulated ceRNA axes demonstrate impairment of neurological processes. Mapping of hub lncRNA‐mRNA pairs from bulk RNA‐seq with snRNA‐seq data has indicated that NORAD, NEAT1, and STXBP5‐AS1 are downregulated across 4, 4, and 2 FC cell types, respectively. At the same time, MIRLET7BHG and MALAT1 are upregulated in excitatory neurons of FC and neurons of CP, respectively. Here, it is hypothesized that downregulation of NORAD, NEAT1, and STXBP5‐AS1, and upregulation of MIRLET7BHG and MALAT1 might deregulate respectively 51, 6, and 37, and 31 and 19 mRNAs in cell types of FC and CP. Afterward, 13 therapeutic miRNAs are traced that might safeguard against deregulated lncRNA‐mRNA pairs of NORAD, NEAT1, and MIRLET7BHG in FC. This study helps to explain the plausible mechanism of post‐COVID neurological manifestation and also to devise therapeutics against it.

Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression.

miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Col1a2, Fn1, etc) and tissue inhibitor of metalloproteinase 3 (Timp3) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.

Targeting specificity protein 1 with miR-128-3p overcomes TGF-β1 mediated epithelial-mesenchymal transition in breast cancer: An in vitro study.

Specificity protein 1 (SP1) was found to play a critical role in the regulation of TGF-β1 driven epithelial-mesenchymal transition (EMT). Recent clinical findings demonstrated a significant drop in the expression of miR-128-3p with the cancer progression in breast cancer patients. However, the impact of miR-128-3p on the SP1 expression in breast cancer remains unknown. Herein, we evaluated the role of miR-128-3p mimics in suppressing EMT of breast cancer cell lines by regulating the TGF-β1/SP1 axis. miR-128-3p interaction with SP1 was detected by in silico tools and dual-luciferase reporter assay. qPCR, western blot, and immunocytochemistry experiments were conducted for determining the expression levels of miR-128-3p and EMT markers with and without the treatment of miR-128-3p mimics. Further, to understand the effect of miR-128-3p mimics on cancer progression, experiments such as wound healing assay, transwell assay, adhesion assay, and cell cycle analysis were performed. A significant inverse relation between SP1 and miR-128-3p levels was found in MCF-7 and MDA-MB-231 cell lines. miR-128-3p overexpression impeded the SP1 mediated EMT markers in TGF-β1 stimulated cells by inhibiting the SP1 nuclear function. Further, treatment with miR-128-3p mimics significantly reduced the migration, invasion and spreading capability of TGF-β1 stimulated cells. Flow cytometry results showed the impeding role of miR-128-3p on the cell cycle progression. Upregulated miR-128-3p inhibited SP1, thereby limiting the TGF-β1 induced EMT in MCF-7 and MDA-MB-231 cell lines for the first time. This study may pave the path to explore novel miRNA therapeutics for eradicating advanced breast cancer cases.

Clinical Applications of Short Non-Coding RNA-Based Therapies in the Era of Precision Medicine.

Simple SummaryRNA-based drugs are an attractive approach for personalized treatment of cancer and other diseases. This review focuses on two related classes of short non-coding RNA: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are endogenous short RNAs that bind multiple messenger RNAs (mRNAs) and prevent the production of their gene-products, whereas siRNAs are exogenous RNAs that target a single and specific mRNA for degradation. This review describes the development, challenges, and clinical successes of short RNA-based drugs. We provide several examples of how these RNA drugs are designed, chemically modified and delivered for treatment of different cancer types, cardiovascular disease, and rare genetic disorders. We highlight the similarities, differences, and considerations to maximize the treatment efficacy of miRNA-based vs. siRNA-based drugs.Traditional targeted therapeutic agents have relied on small synthetic molecules or large proteins, such as monoclonal antibodies. These agents leave a lot of therapeutic targets undruggable because of the lack or inaccessibility of active sites and/or pockets in their three-dimensional structure that can be chemically engaged. RNA presents an attractive, transformative opportunity to reach any genetic target with therapeutic intent. RNA therapeutic design is amenable to modularity and tunability and is based on a computational blueprint presented by the genetic code. Here, we will focus on short non-coding RNAs (sncRNAs) as a promising therapeutic modality because of their potency and versatility. We review recent progress towards clinical application of small interfering RNAs (siRNAs) for single-target therapy and microRNA (miRNA) activity modulators for multi-target therapy. siRNAs derive their potency from the fact that the underlying RNA interference (RNAi) mechanism is catalytic and reliant on post-transcriptional mRNA degradation. Therapeutic siRNAs can be designed against virtually any mRNA sequence in the transcriptome and specifically target a disease-causing mRNA variant. Two main classes of microRNA activity modulators exist to increase (miRNA mimics) or decrease (anti-miRNA inhibitors) the function of a specific microRNA. Since a single microRNA regulates the expression of multiple target genes, a miRNA activity modulator can have a more profound effect on global gene expression and protein output than siRNAs do. Both types of sncRNA-based drugs have been investigated in clinical trials and some siRNAs have already been granted FDA approval for the treatment of genetic, cardiometabolic, and infectious diseases. Here, we detail clinical results using siRNA and miRNA therapeutics and present an outlook for the potential of these sncRNAs in medicine.

Highlight report : Therapeutic miRNAs in mesenchymal stem cells and their derived extracellular vesicles in rheumatoid arthritis

Highlight report : Therapeutic miRNAs in mesenchymal stem cells and their derived extracellular vesicles in rheumatoid arthritis

Role of miRNAs in Neurodegeneration: From Disease Cause to Tools of Biomarker Discovery and Therapeutics.

Neurodegenerative diseases originate from neuronal loss in the central nervous system (CNS). These debilitating diseases progress with age and have become common due to an increase in longevity. The National Institute of Environmental Health Science’s 2021 annual report suggests around 6.2 million Americans are living with Alzheimer’s disease, and there is a possibility that there will be 1.2 million Parkinson’s disease patients in the USA by 2030. There is no clear-cut universal mechanism for identifying neurodegenerative diseases, and therefore, they pose a challenge for neurobiology scientists. Genetic and environmental factors modulate these diseases leading to familial or sporadic forms. Prior studies have shown that miRNA levels are altered during the course of the disease, thereby suggesting that these noncoding RNAs may be the contributing factor in neurodegeneration. In this review, we highlight the role of miRNAs in the pathogenesis of neurodegenerative diseases. Through this review, we aim to achieve four main objectives: First, we highlight how dysregulation of miRNA biogenesis led to these diseases. Second, we highlight the computational or bioinformatics tools required to identify the putative molecular targets of miRNAs, leading to biological molecular pathways or mechanisms involved in these diseases. Third, we focus on the dysregulation of miRNAs and their target genes leading to several neurodegenerative diseases. In the final section, we highlight the use of miRNAs as potential diagnostic biomarkers in the early asymptomatic preclinical diagnosis of these age-dependent debilitating diseases. Additionally, we discuss the challenges and advances in the development of miRNA therapeutics for brain targeting. We list some of the innovative strategies employed to deliver miRNA into target cells and the relevance of these viral and non-viral carrier systems in RNA therapy for neurodegenerative diseases. In summary, this review highlights the relevance of studying brain-enriched miRNAs, the mechanisms underlying their regulation of target gene expression, their dysregulation leading to progressive neurodegeneration, and their potential for biomarker marker and therapeutic intervention. This review thereby highlights ways for the effective diagnosis and prevention of these neurodegenerative disorders in the near future.

Nanoparticulate strategies for thedelivery of miRNA mimics and inhibitors in anticancer therapy and its potential utility in oral submucous fibrosis.

MicroRNAs (miRNAs) are naturally occurring noncoding RNAs with multiple functionalities. They are dysregulated in several conditions and can serve as disease biomarkers, therapeutic targets andtherapeutic agents. Translation of miRNA therapeutics to the clinic poses several challenges related to the safe and effective delivery of these agents to the site of action. Nanoparticulate carriers hold promise in this area by enhancing targeting efficiency and reducing off-target effects. This paper reviews recent advances in thedelivery strategies of miRNAs in anticancer therapy, with a focus on lipid-based, polymeric, inorganic platforms, cell membrane-derived vesicles and bacterial minicells. Additionally, this review explores the potentiality of miRNAs in the treatment of oral submucous fibrosis, a potentially premalignant condition of the oral cavity with no definitive treatment to date.

Turing miRNA into infinite coordination supermolecule: a general and enabling nanoengineering strategy for resurrecting nuclear acid therapeutics

BackgroundClinical translation of therapeutic nuclear acid, particularly those targeting tumor progression, has been hampered by the intrinsic weaknesses of nuclear acid therapeutic including poor systemic stability, rapid clearance, low membrane permeability and lack of targeting ability. Small nuclear acid engineered into carrier-free nanodrugs with structural stability and disease targeting may be viable to overcome pharmaceutical obstacles of nuclear acid.MethodsA general method through a mild and simple chemistry was established to convert therapeutic miRNA into an infinite Auric-sulfhydryl coordination supramolecular miRNA termed IacsRNA with near-spherical nanostructure, high colloid as well as anti-hydrolysis stability and low macrophage uptakes.ResultsIacsRNA presented the increased half-life period in circulation and accumulation at tumor sites in comparison to normal miRNA. Moreover, Iacs-miR-30c showed no toxicity of viscera and sanguis system in the 5-time injection dosage of the treatment. More importantly, Iacs-miR-30c potently suppressed the Wnt signaling pathway in vitro and in vivo, and effectively sensitized both potency of 5-Fu in PDX model of colon cancer and Anti-PD1 in B16F10 homograft model of melanoma.ConclusionCollectively, this work amply confirmed the design of IacsRNA as a general and viable strategy of nano-pharmaceutic to concert flimsy therapeutic miRNA into potential drugs. Considering from a broader perspective, the miRNA-initiated infinite coordination self-assembly strategy has distinct advantages in resurrecting nuclear acid therapeutics, probably bringing new inspiration to RNA-derived therapeutics of a great variety of human diseases including cancer.Graphical

Machine Learning Based Methods and Best Practices of microRNA-Target Prediction and Validation.

Within the last years, more and more noncoding RNAs (ncRNAs) became the focal point to understand cell regulatory mechanisms because one class of ncRNAs, microRNAs (miRNAs), plays an essential role in translation repression or degradation of specific mRNAs and is implicated in disease etiology. miRNAs can serve as oncomiRs (oncogenic miRNAs) and tumor suppressor miRNAs, thus, miRNA therapeutics in clinical trials have become a vital component with respect to cancer treatment. To circumvent side-effects and allow an accurate effect it is crucial to accurately predict miRNAs and their mRNA targets. Over the last two decades, different approaches for miRNA prediction as well as miRNA target prediction have been developed and improved based on sequence and structure features. Nowadays, the abundance of high-throughput sequencing data and databases of miRNAs and miRNA targets from different species allow the training, testing, and validation of predicted miRNAs and miRNA targets with machine learning methods. This book chapter focuses on the important requirements for miRNA target prediction tools using ML like common features used for miRNA-binding site prediction. Furthermore, best practices for the prediction and validation of miRNA-mRNA targets are presented and set in the context of possible applications for cancer diagnosis and therapeutics.

Therapeutic Potential of miRNAs for Type 2 Diabetes Mellitus: An Overview.

MicroRNA(miRNA)s have been identified as an emerging class for therapeutic interventions mainly due to their extracellularly stable presence in humans and animals and their potential for horizontal transmission and action. However, treating Type 2 diabetes mellitus using this technology has yet been in a nascent state. MiRNAs play a significant role in the pathogenesis of Type 2 diabetes mellitus establishing the potential for utilizing miRNA-based therapeutic interventions to treat the disease. Recently, the administration of miRNA mimics or antimiRs in-vivo has resulted in positive modulation of glucose and lipid metabolism. Further, several cell culture-based interventions have suggested beta cell regeneration potential in miRNAs. Nevertheless, few such miRNA-based therapeutic approaches have reached the clinical phase. Therefore, future research contributions would identify the possibility of miRNA therapeutics for tackling T2DM. This article briefly reported recent developments on miRNA-based therapeutics for treating Type 2 Diabetes mellitus, associated implications, gaps, and recommendations for future studies.

Epigenetic regulation of inflammation by microRNAs in post-infectious bronchiolitis obliterans.

ObjectivesPost‐infectious bronchiolitis obliterans (PiBO) is a rare, chronic disease initiated by severe infection and followed by perpetuating inflammation and obliteration of the small airways. MicroRNAs (miRNAs) have been proposed to play a central role as epigenetic regulators, which control resolution and prevent the uncontrolled progress of inflammation. The aim of this study was to define biomarkers on the level of post‐transcriptional gene regulation in order to characterise PiBO.MethodsA total of 39 patients with well‐defined PiBO and 31 controls from two centres, Barcelona, Spain, and Frankfurt, Germany, were analysed by next‐generation sequencing (NGS). The evaluation of the biological targets of the miRNAs was performed by pathway enrichment analysis and protein–protein interaction network analysis respectively.ResultsPatients with PiBO had significantly lower lung function values and increased airway inflammation in induced sputum as indicated by total cell counts, neutrophils, IL‐1β, IL‐6, IL‐8 and TGF‐β compared to controls.Next‐generation sequencing analysis revealed a total of 22 dysregulated miRNAs, which passed significance threshold for Padj ≤ 0.001 with 17 being upregulated and 5 being downregulated. Of these dysregulated miRNAs, miR‐335‐5p, miR‐186‐5p, miR‐30b‐5p and miR‐30c‐5p were further validated using qRT‐PCR. Interestingly, these miRNAs are functionally implicated in cytokine–cytokine receptor interaction, TGF‐β signalling and FoxO signalling pathway and significantly correlated with lung function values (FEV1).ConclusionOur results demonstrate an aberrant miRNA expression profile in PiBO, which impacts pathways responsible for the regulation of inflammation and fibrosis. The defined miRNAs are useful biomarkers and should be assessed as potential target in the field of miRNA therapeutics.

Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation.

Extracellular vesicles (EVs) are a population of particles naturally released by cells to transport biological messages, including nucleic acids. Thus, EVs represent an ideal vehicle to deliver therapeutic miRNAs. The current challenge is the development of efficient protocols to load EVs with exogenous miRNAs. Human plasma is an abundant source of EVs which can be manipulated for therapeutic applications. Despite numerous techniques are currently available to load EVs, all of them present issues which limit their clinical application. Among all, electroporation was shown to be superior to other protocols and to efficiently load plasma-derived EVs with miRNAs. However, also the electroporation procedure presents issues that can reduce the miRNA delivery. In this chapter, we describe a protocol to isolate EVs from human plasma, to load synthetic miRNA mimics using electroporation, to evaluate EV integrity and miRNA loading into EVs. Finally, the analysis of EV functionality allows to investigate the ability of engineered EVs to transfer the miRNAs to target cells and to exploit their biological effects.

Differential Expression of DDX3 and microRNAs in Response to Hormone and Cisplatin Against Cervical Cancer

Objectives: To discover micro ribonucleic acids (miRNAs) involved in the regulation of DDX3 expression using sexual hormones in combination with the well-known anticancer medication cisplatin. Methods: SiHa cells were treated with estradiol, dihydrotestosterone, and cisplatin and evaluated the expression of ER beta, Ki67, and DDX3 via quantitative reverse transcription–polymerase chain reaction. We generated a chimeric fusion construct five untranslated region (UTR)–hLUC–3UTR in the pEZX-MT06 miRNA vector under the control of the SV40 promoter. Reporter activity is measured with/without hormones, and their activity is compared with 5'- and 3'-UTR respectively. Various reporter deletion constructs were generated to identify the minimal UTR region in regulating the expression of DDX3. We identified the potential miRNA binding sites on the DDX3 UTR region, and their expression is monitored in cancer patients and cisplatin-treated SiHa cells. Results: Hormones increased the proliferation of SiHa cells and expression of DDX3. The 3'-UTR region 2135–4307bp contains miRNA sites that regulate DDX3 expression. miRNAs hsa-miR-671-5p, hsa-miR-361-5p, hsa-miR-140-5p, hsamiR-564, and hsa-miR-769-5p downregulated in patient samples but upregulated in cisplatin-treated cells. miRNA hsamiR-671-5p and hsa-miR-564 were associated with patient data and cisplatin-treated cancer cells. Conclusion: We discovered that sexual hormones enhanced DDX3 expression in SiHa cells. MiR-671-5p and miR-564 are two potential therapeutic miRNAs that can be used to treat DDX3-related malignancies. Keywords: Cancer patients, Cervical cancer, Cisplatin, DDX3, miRNA

CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/\u03b2-catenin axis

BackgroundEpstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown.MethodsCircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting.ResultsWe identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream β-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth.ConclusionsCircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/β-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

MicroRNA-Related Strategies to Improve Cardiac Function in Heart Failure

Heart failure (HF) describes a group of manifestations caused by the failure of heart function as a pump that supports blood flow through the body. MicroRNAs (miRNAs), as one type of non-coding RNA molecule, have crucial roles in the etiology of HF. Accordingly, miRNAs related to HF may represent potential novel therapeutic targets. In this review, we first discuss the different roles of miRNAs in the development and diseases of the heart. We then outline commonly used miRNA chemical modifications and delivery systems. Further, we summarize the opportunities and challenges for HF-related miRNA therapeutics targets, and discuss the first clinical trial of an antisense drug (CDR132L) in patients with HF. Finally, we outline current and future challenges and potential new directions for miRNA-based therapeutics for HF.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.