miRNA Inhibitors Research Articles

Multiple microRNAs regulate tacrolimus metabolism through CYP3A5

The CYP3A5 gene polymorphism accounts for the majority of inter-individual variability in tacrolimus pharmacokinetics. We found that the basal expression of CYP3A5 in donor grafts also played a significant role in tacrolimus metabolism under the same genetic conditions after pediatric liver transplantation. Thus, we hypothesized that some potential epigenetic factors could affect CYP3A5 expression and contributed to the variability. We used a high-throughput functional screening for miRNAs to identify miRNAs that had the most abundant expression in normal human liver and could regulate tacrolimus metabolism in HepaRG cells and HepLPCs. Four of these miRNAs (miR-29a-3p, miR-99a-5p, miR-532-5p, and miR-26-5p) were selected for testing. We found that these miRNAs inhibited tacrolimus metabolism that was dependent on CYP3A5. Putative miRNAs targeting key drug-metabolizing enzymes and transporters (DMETs) were selected using an in silico prediction algorithm. Luciferase reporter assays and functional studies showed that miR-26b-5p inhibited tacrolimus metabolism by directly regulating CYP3A5, while miR-29a-5p, miR-99a-5p, and miR-532-5p targeted HNF4α, NR1I3, and NR1I2, respectively, in turn regulating the downstream expression of CYP3A5; the corresponding target gene siRNAs markedly abolished the effects caused by miRNA inhibitors. Also, the expression of miR-29a-3p, miR-99a-5p, miR-532-5p, and miR-26b-5p in donor grafts were negatively correlated with tacrolimus C/D following pediatric liver transplantation. Taken together, our findings identify these miRNAs as novel regulators of tacrolimus metabolism.

The Role of HSA21 Encoded Mirna in Down Syndrome Pathophysiology:Opportunities in miRNA-Targeted Pharmacotherapy and Diagnosis of the Down Syndrome

Trisomy 21 is the most prevalent aneuploidy disorder among live-born children worldwide. Itresults from the presence of an extra copy of chromosome 21 which leads to a wide spectrum ofpathophysiological abnormalities and intellectual disabilities. Nevertheless human chromosome21 (HSA21) possess protein non-coding regions where HAS-21 derived-microRNA genes aretranscribed from. In turn, these HSA21-derived miRNAs curb protein translation of severalgenes which are essential to meet memory and cognitive abilities. From the genetics andmolecular biology standpoints, dissecting the mechanistic relationship between DS pathology/symptoms and five chromosome 21-encoded miRNAs including miR-99a, let-7c, miR-125b-2,miR-155 and miR-802 seems pivotal for unraveling novel therapeutic targets. Recently,several studies have successfully carried out small molecule inhibition of miRNAs function,maturation, and biogenesis. One might assume in the case of DS trisomy, the pharmacologicalinhibition of these five overexpressed miRNAs might open new avenues for amelioration of theDS symptoms and complications. In this review, we primarily elucidated the role of HSA21-encoded miRNAs in the DS pathology which in turn introduced and addressed importanttherapeutic targets. Moreover, we reviewed relevant pharmaceutical efforts that based theirgoals on inhibition of these pathological miRNAs at their different biogenesis steps. We havealso discussed the challenges that undermine and question the reliability of miRNAs as noneinvasivebiomarkers in prenatal diagnosis.

Interactions Between lncRNA TUG1 and miR-9-5p Modulate the Resistance of Breast Cancer Cells to Doxorubicin by Regulating eIF5A2.

PurposeBreast cancer (BC) is one of the leading causes of cancer-related deaths. Chemoresistance of BC remains a major unmet clinical obstacle. TUG1 (taurine-upregulated gene 1), a long noncoding RNA (lncRNA), and microRNAs (miRNA) are implicated in therapeutic resistance. However, the interactions between TUG1 and miRNAs that regulate doxorubicin (Dox) resistance in BC remain elusive.Materials and MethodsExpression of TUG1 and miR-9 was measured by real-time PCR. EIF5A2 (eukaryotic translation initiation factor 5A-2) was detected by Western blot. Transfection of siRNAs or miRNA inhibitors was applied to silence lncRNA TUG1, eIF5A2 or miR-9. Cell viability, proliferation, and apoptosis were determined by CCK-8 (cell counting kit-8), flow cytometry, and EdU (5-ethynyl-2ʹ-deoxyuridine) assays, respectively. The regulatory relationship between TUG1 and miR-9 was determined by a luciferase assay.ResultsLncRNA TUG1 was highly expressed in BC tissues and positively associated with Dox resistance in BC cell lines. SiRNA knockdown of TUG1 reversed Dox resistance in MCF-7/ADR cells. Mechanistically, TUG1 acted as a “sponge” for miR-9 and downregulated miR-9. Treatment with a miR-9 inhibitor blocked the effect of TUG1 siRNA, and knockdown of TUG1 inhibited the effects of miR-9. Furthermore, TUG1 inhibition of apoptosis induced by Dox involved miR-9 targeting of eIF5A2.ConclusionTUG1 modulates the susceptibility of BC cells to Dox by regulating the expression of eIF5A2 via interacting with miR-9. These results indicate that the lncRNA TUG1 may be a novel therapeutic target in breast cancer.

NRF2 in Cardiovascular Diseases: a Ray of Hope!

Heart failure is a worldwide pandemic influencing 26 million individuals worldwide and is expanding. Imbalanced redox homeostasis in cardiac cells alters the structure and function of the cells, which leads to contractile dysfunction, myocardial hypertrophy, and fibrosis in chronic heart failure. Various targets and agents acting on these such as siRNA, miRNA, interleukin-1, opioids, vasodilators, and SGLT2 inhibitors are being evaluated for heart failure, and nuclear factor erythroid 2-related factor 2 (NRF2) is one of them. NRF2 is a master transcription factor which is expressed in most of the tissues and exhibits a major role in amplification of the antioxidant pathways associated with the enzymes present in myocardium. Increased ROS generation and PI3K-Akt signaling can activate the receptor NRF2. Various in vitro and in vivo and few clinical studies suggested NRF2 may possess a potential for targeting oxidative stress-induced cardiovascular diseases including heart failures. All these studies collectively propose that upregulation of NRF2 will attenuate the increase in hemodynamic stress and provide beneficial role in cardiovascular diseases. The current review shall familiarize readers about the regulations and functions of NRF2. We have also discussed the current evidences suggesting beneficial role of NRF2 activators in heart failure. Graphical abstract.

Leonurine Attenuates Myocardial Fibrosis Through Upregulation of miR-29a-3p in Mice Post-myocardial Infarction.

Myocardial fibrosis (MF) is a pathological process that accelerates cardiac remodeling in myocardial infarction (MI), and miR-29 has become one of the foci of research into MF. As an alkaloid extracted from Herba leonuri, leonurine (LE) has been found to be an effective natural active ingredient for inhibiting fibrosis in many preclinical experiments. However, whether LE protects against MF after MI through modifying miR-29 remains unclear. The present study aimed to investigate the therapeutic effects of LE on MF, and to elucidate the underlying mechanisms involved. A mouse model of MI was established, followed by administration of LE for 4 weeks. We found that LE effectively improved cardiac function, and attenuated fibrosis and cardiac remodeling in mice post-MI. In vitro, LE simultaneously inhibited proliferation and migration of neonatal mouse cardiac fibroblasts (CFs) exposed to angiotensin II (Ang II), and the activation of collagen synthesis and myofibroblast generation was markedly suppressed by LE. Notably, we found that all mature miR-29 family members were downregulated in the myocardial tissues of mice post-MI, whereas LE significantly upregulated miR-29a-3p expression, and such upregulation was also detected in LE-treated CFs under Ang II stimulation. Knockdown of miR-29a-3p by a specific miRNA inhibitor upregulated the protein levels of TGF-β, collagen III, and collagen I in CFs, and completely reversed the antifibrotic effects of LE on CFs. Our study suggests that LE exerts cardioprotective effects against MF, possibly through the upregulation of miR-29a-3p.

Targeted delivery of small noncoding RNA for glioblastoma.

Aberrant expression of certain genes and microRNAs (miRNAs) has been shown to drive cancer development and progression, thus the modification of aberrant gene and miRNA expression presents an opportunity for therapeutic targeting. Ectopic modulation of a single dysregulated miRNA has the potential to revert therapeutically unfavorable gene expression in cancer cells by targeting multiple genes simultaneously. Although the use of noncoding RNA-based cancer therapy is a promising approach, the lack of a feasible delivery platform for small noncoding RNAs has hindered the development of this therapeutic modality. Recently, however, there has been an evolution in RNA nanotechnology, in which small noncoding RNA is loaded onto nanoparticles derived from the pRNA-3WJ viral RNA motif of the bacteriophage phi29. Preclinical studies have shown the capacity of this technology to specifically target tumor cells by conjugating these nanoparticles with ligands specific for cancer cells and resulting in the endocytic delivery of siRNA and miRNA inhibitors directly into the cell. Here we provide a systematic review of the various strategies, which have been utilized for miRNA delivery with a specific focus on the preclinical evaluation of promising RNA nanoparticles for glioblastoma (GBM) targeted therapy.

The role of noncoding RNAs during aortic valve stenosis

Abstract Background Aortic valve stenosis (AVS) is the most common valve disease worldwide. Thought to be a purely degenerative disease, it is now clear that shear stress/endothelial dysfunction, lipid deposition and inflammation lead to calcification and stenosis of the valve. There is evidence, that extracellular vesicles (EVs) are actively involved in calcification processes. Practically all cells, including endothelial cells, can generate EVs, which can be shed into the blood stream and into the interstitial space. EVs contain lipids, proteins and nucleic acids, including noncoding RNAs (ncRNAs). EVs can be taken up by acceptor cells and their cargo, especially the ncRNA content, can change the phenotype of these cells. NcRNAs have been shown to have protective and damaging properties in AVS, which can lead to disease progression. EVs are actively involved in atherosclerosis and vascular calcification, but their role during AVS formation remains largely unknown. Purpose We hypothesize, that EV-derived ncRNAs play a crucial role during calcification of the aortic valve through regulation of endothelial to mesenchymal transition (EndMT) and calcification of valvular interstitial cells. Methods and results In initial screening experiments, we investigated ncRNA (micro RNA, miRNA and long noncoding RNA, lncRNA) content in aortic valve tissue from explanted human aortic valves from patients undergoing surgical aortic valve replacement. There is a differential expression of miRNAs and lncRNAs in aortic valve tissue from patients with AVS and patients without AVS. We could also show a differential packaging of ncRNAs into EVs generated from patient aortic valve tissues. Furthermore, ncRNA expression in aortic valve tissue is altered in a “wire-injury” mouse model of AVS. We can demonstrate in vitro that EVs and their content can be transferred from valvular endothelial cells (VECs) to valvular interstitial cells (VICs) and vice versa. Additionally, we have established an isolation method of VECs and VICs from human samples. To identify ncRNAs involved in EndMT, we are investigating the effect of laminar and pulsatile flow on the expression of ncRNAs in vitro. We can demonstrate that different flow patterns lead to a vast change in ncRNA expression in primary VECs. Downstream effects of identified ncRNAs are currently under investigation in our in vitro calcification and EndMT models. Transfection of VICs with miRNA mimics and inhibitors and lentiviral transduction of our identified targets lead to an altered calcification potential of valve cells. Conclusion The analysis of the cell type specific expression of ncRNAs and the intercellular communication via EVs will greatly help our understanding of the pathomechanisms leading to valvular calcification. Pathway analysis will generate new targets that could be used to develop therapeutics to ameliorate disease progression. EV-based miRNA mimics and inhibitors could be used to treat valvular calcification. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, TRR259; Else-Kröner-Fresenius

Drugging the "undruggable" microRNAs.

As a naturally occurring class of gene regulators, microRNAs (miRNAs) have attracted much attention as promising targets for therapeutic development. However, RNAs including miRNAs have long been considered undruggable, and most efforts have been devoted to using synthetic oligonucleotides to regulate miRNAs. Encouragingly, recent findings have revealed that miRNAs can also be drugged with small molecules that directly target miRNAs. In this review paper, we give a summary of recently emerged small-molecule inhibitors (SMIs) and small-molecule degraders (SMDs) for miRNAs. SMIs are small molecules that directly bind to miRNAs to inhibit their biogenesis, and SMDs are bifunctional small molecules that upon binding to miRNAs induce miRNA degradation. Strategies for discovering SMIs and developing SMDs were summarized. Applications of SMIs and SMDs in miRNA inhibition and cancer therapy were also introduced. Overall, SMIs and SMDs introduced here have high potency and specificity in miRNA inhibition. We envision that these small molecules will pave the way for developing novel therapeutics toward miRNAs that were previously considered undruggable.

MicroRNAs in laser-induced choroidal neovascularization in mice and rats: their expression and potential therapeutic targets.

Choroidal neovascularization characterizes wet age-related macular degeneration. Choroidal neovascularization formation involves a primarily angiogenic process that is combined with both inflammation and proteolysis. A primary cause of choroidal neovascularization pathogenesis is alterations in pro- and anti-angiogenic factors derived from the retinal pigment epithelium, with vascular endothelium growth factor being mainly responsible for both clinical and experimental choroidal neovascularization. MicroRNAs (miRNAs) which are short, non-coding, endogenous RNA molecules have a major role in regulating various pathological processes, including inflammation and angiogenesis. A review of recent studies with the mouse laser-induced choroidal neovascularization model has shown alterations in miRNA expression in choroidal neovascularization tissues and could be potential therapeutic targets for wet age-related macular degeneration. Upregulation of miR-505 (days 1 and 3 post-laser), miR-155 (day 14) occurred in retina; miR-342-5p (days 3 and 7), miR-126-3p (day 14) in choroid; miR-23a, miR-24, miR-27a (day 7) in retina/choroid; miR-505 (days 1 and 3) in retinal pigment epithelium/choroid; downregulation of miR-155 (days 1 and 3), miR-29a, miR-29b, miR-29c (day 5), miR-93 (day 14), miR-126 (day 14) occurred in retinal pigment epithelium/choroid. Therapies using miRNA mimics or inhibitors were found to decrease choroidal neovascularization lesions. Choroidal neovascularization development was reduced by overexpression of miR-155, miR-188-5p, miR-(5,B,7), miR-126-3p, miR-342-5p, miR-93, miR-126, miR-195a-3p, miR-24, miR-21, miR-31, miR-150, and miR-184, or suppression of miR-505, miR-126-3p, miR-155, and miR-23/27. Further studies are warranted to determine miRNA expression in mouse laser-induced choroidal neovascularization models in order to validate and extend the reported findings. Important experimental variables need to be standardized; these include the strain and age of animals, gender, number and position of laser burns to the eye, laser parameters to induce choroidal neovascularization lesions including wavelength, power, spot size, and duration.

Identification of infectious spleen and kidney necrosis virus (ISKNV)-encoded microRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by complementary binding to target mRNAs. Virus-encoded miRNAs play important roles in virus life cycle and virus-host interactions. Viruses from the Megalocytivirus genus, family Iridoviridae, infect a wide range of fishes, bringing great challenges to aquaculture. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. In this study, using Illumina sequencing coupled with miRNA precursor prediction and stem-loop real-time PCR, 14 putative ISKNV-encoded miRNAs were preliminarily identified from ISKNV-infected mandarin fish MFF-1 cells. To initially study their functions, inhibitors of the 14 viral miRNAs were synthesized and transfected into MFF-1 cells, which were further infected with ISKNV. The results showed that these viral miRNAs could affect the virus titers in the supernatant of ISKNV-infected cells and the expression of major capsid protein (MCP). Moreover, we observed that inhibition of several ISKNV miRNAs had different effects on MCP expression and on titer of released virus, suggesting complex roles of viral miRNAs in ISKNV infection. The current study may provide a fundamental information for further identification and functional studies on miRNAs encoded by Megalocytivirus.

Treatment of human airway epithelial Calu-3cells with a peptide-nucleic acid (PNA) targeting the microRNA miR-101-3p is associated with increased expression of the cystic fibrosis Transmembrane Conductance Regulator () gene.

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3'-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p.

Suppression of lncRNA MALAT1 by betulinic acid inhibits hepatocellular carcinoma progression by targeting IAPs via miR-22-3p.

Betulinic acid (BA) is a natural product extracted from a broad range of medicinal and edible herbal plants. Previous studies showed that BA induces cell death in tumors derived from multiple tissues; however, the underlying mechanism remains obscure. The present study aimed to study the effects of BA on autophagy and apoptosis of hepatocellular carcinoma (HCC). Human HCC cell lines and orthotopic HCC implanted mice were employed to examine the BA‐induced tumor suppression; RT2 long noncoding RNA (lncRNA) PCR array and database analysis were used to explore the possible mechanisms; validation of pathways was performed using siRNA and miRNA inhibitors. The results indicated that BA regulated autophagy and induced apoptosis in HCC. The degradation of inhibitor of apoptosis proteins (IAPs), the conversion of LC3‐I to LC3‐II, and p62 accumulation were enhanced by BA, thereby suggesting that the downregulation of IAPs and autophagic cell death are induced by BA. The addition of autophagy and lysosomal inhibitors indicated that BA induced autophagy‐independent apoptosis via degradation of IAPs. Moreover, RT2 lncRNA PCR array and database analysis suggested that BA downregulated the levels of lncRNA MALAT1, which is considered to be an oncogene. Further investigations demonstrated that lncRNA MALAT1 functioned as a ceRNA (competing endogenous RNA) to contribute to BA‐mediated degradation of IAPs by sponging miR‐22‐3p. Therefore, BA could be developed as a potential anticancer agent for HCC.

Proteomic and deep sequencing analysis of extracellular vesicles isolated from adult male and female Schistosoma japonicum.

Schistosomes are the causative agent of schistosomiasis, which affects more than 200 million people worldwide. Unlike other trematode parasites, schistosomes (along with the Didymozoidae) have evolved separate sexes. Pairing of males and females is a prerequisite for female sexual development and subsequent egg production. However, the mechanisms underlying these processes remain poorly understood. Extracellular vesicles (EVs) have been shown to play important roles in many biological processes. In the present study, we characterized EVs isolated from adult male and female Schistosoma japonicum. Proteomic analyses of the isolated EVs revealed that some proteins are significantly enriched in male or female EVs. RNA-sequencing analysis of a small RNA population associated with EVs identified 18 miRNAs enriched in male and female S. japonicum EVs. Among these, miR-750 was specifically enriched in female EVs. Additionally, the inhibition of miR-750 by a miRNA inhibitor led to decreased egg production in female schistosomes cultured in vitro. Collectively, our results suggest that miR-750 within female EV cargo may be involved in regulating ovary development and egg production in S. japonicum females.

Targeting respiratory diseases using miRNA inhibitor based nanotherapeutics: Current status and future perspectives

MicroRNAs (miRNAs) play a fundamental role in the developmental and physiological processes that occur in both animals and plants. AntagomiRs are synthetic antagonists of miRNA, which prevent the target mRNA from suppression. Therapeutic approaches that modulate miRNAs have immense potential in the treatment of chronic respiratory disorders. However, the successful delivery of miRNAs/antagomiRs to the lungs remains a major challenge in clinical applications. A range of materials, namely, polymer nanoparticles, lipid nanocapsules and inorganic nanoparticles, has shown promising results for intracellular delivery of miRNA in chronic respiratory disorders. This review discusses the current understanding of miRNA biology, the biological roles of antagomiRs in chronic respiratory disease and the recent advances in the therapeutic utilization of antagomiRs as disease biomarkers. Furthermore our review provides a common platform to debate on the nature of antagomiRs and also addresses the viewpoint on the new generation of delivery systems that target antagomiRs in respiratory diseases.

Deoxycholic acid-stimulated macrophage-derived exosomes promote intestinal metaplasia and suppress proliferation in human gastric epithelial cells

The crosstalk between macrophages and gastric epithelial cells has emerged as a player in chronic inflammation during intestinal metaplasia. However, the role of bile acid on this modulation remains to be studied. We hypothesized that deoxycholic acid-induced macrophages secreted exosomes to mediate intercellular communication and promoted intestinal metaplasia in human gastric epithelial cells (GES-1 cells). Macrophage-derived exosomes (M-Exos) and deoxycholic acid-induced macrophage-derived exosomes (D-Exos) were isolated by ultracentrifugation. EdU staining and CCK-8 assay were utilized to evaluate the effects of exosomes on the proliferation of GES-1 cells. Intestinal metaplasia was assessed by the expression of caudal-related homeobox transcription factor 2 (CDX2) at both mRNA and protein level. MicroRNA sequencing revealed the microRNA (miRNA) expression profiles of M-Exos and D-Exos. The role of a specific miRNA and mRNA was analyzed by using miRNA mimics, miRNA inhibitors and siRNAs. D-Exos promoted the expression of CDX2 and suppressed the proliferation of GES-1 cells, compared to M-Exos. The miRNA profiles and quantitative real-time PCR examination showed D-Exos enriched a higher level of hsa-miR-30a-5p than M-Exos. Overexpressed has-miR-30a-5p increased CDX2 expression and inhibited the proliferation in GES-1 cells via targeted Forkhead Box D1 (FOXD1), a potential regulatory factor in the process of intestinal metaplasia. D-Exos may promote intestinal metaplasia and suppress proliferation of GES-1 cells via hsa-miR-30a-5p targeting FOXD1, which may be involved in the action mechanism of bile acid on gastric mucosa.

A three serum miRNA panel as diagnostic biomarkers of radiotherapy-related metastasis in non-small cell lung cancer.

Serum microRNAs (miRNAs) have been implicated as noninvasive biomarkers for lung cancer diagnosis. However, there are no sensitive and specific biomarkers for the detection of radiotherapy-related non-small cell lung cancer (NSCLC) metastasis. The present study aimed to investigate the role of three serum miRNAs, namely miRNA (miR)-130a, miR-25 and miR-191*, in diagnosing NSCLC, and their biological functions in radiation-mediated development of metastatic properties in A549 cells. To determine this, serum samples were collected from 84 patients with NSCLC and 42 age- and sex-matched healthy controls. Differential expression of serum miRNAs was analyzed by quantitative PCR. Significant associations between miRNA expression and overall survival of patients with NSCLC were identified using the Cox proportional regression model. A receiver operating characteristic curve was generated to evaluate diagnostic accuracy. The functions of miR-130a, miR-25 and miR-191* in lung cancer cells were studied by transfecting A549 cells with miRNA mimics and inhibitors. The results of the present study demonstrated that the expression levels of miR-130a, miR-25 and miR-191* in the serum of patients with NSCLC were increased compared with those in healthy controls, and these increases were associated with advanced age (≥60 years), radiotherapy, histological type (squamous carcinoma), low survival rate and low median survival time. Additionally, irradiation induced the upregulation of miR-130a, miR-25 and miR-191* expression in A549 cells in vitro and in a xenograft mouse model. Irradiation also promoted the invasiveness of A549 cells in vitro and metastasis in vivo. In conclusion, miR-130a, miR-25 and miR-191* may be potential biomarkers for the diagnosis of patients with NSCLC and may serve oncogenic roles in radiation-mediated metastasis of NSCLC.

MicroRNA-25 as a novel modulator of circadian Period2 gene oscillation

Circadian clock controls an organism’s biological rhythm and regulates its physiological processes in response to external time cues. Most living organisms have their own time-keeping mechanism that is maintained by transcriptional–translational autoregulatory feedback loops involving several core clock genes, such as Period. Recent studies have found the relevance between the modulation of circadian oscillation and posttranscriptional modifications by microRNAs (miRNAs). However, there are limited studies on candidate miRNAs that regulate circadian oscillation. Here, we characterize the functions of novel miRNA-25 regulating circadian Period2 (Per2) expression. Using several in silico algorithms, we identified novel miR-25-3p that, together with miR-24-3p, targets the Per2 gene. Luciferase reporter assays validated that miR-25-3p and miR-24-3p repressed Per2 expression and confirmed their predicted binding sites in the 3′-untranslated region (UTR) of Per2 mRNA. Real-time bioluminescence analyses using Per2::Luc mouse embryonic fibroblasts confirmed that PER2 protein oscillation patterns were responsive to miR-25-3p and miR-24-3. The overexpression of miR-25-3p or miR-24-3p resulted in the dampening and period shortening of the PER2::LUC oscillation, while inhibition of either miRNA increased the relative amplitude of the PER2::LUC oscillation. Notably, endogenous miR-25-3p expression in the suprachiasmatic nucleus (SCN) showed no circadian rhythmicity, but the expression levels differed in various brain regions and peripheral tissues. These results suggest that the posttranscriptional regulation of miR-25-3p and miR-24-3p may differ according to Per2 gene expression in different tissue regions. In summary, we found that novel miR-25-3p was involved in fine-tuning circadian rhythmicity by regulating Per2 oscillation at the posttranscriptional level and that it functioned synergistically with miR-24-3p to affect Per2 oscillation.

Progress in systemic co-delivery of microRNAs and chemotherapeutics for cancer treatment by using lipid-based nanoparticles.

MicroRNAs (miRNAs) hold the potential to boost therapeutic efficacy and/or reverse drug resistance associated with traditional cancer chemotherapy. Both miRNA mimics and inhibitors have been explored in cancer therapy. Systemic co-delivery of chemotherapeutics and miRNA therapeutics represents an attractive treatment approach, but safe and efficient delivery systems are greatly needed. The regulatory approval of Onpattro® paved the way for lipid-based nanoparticles to deliver RNA therapeutics in different settings, including in combination with chemotherapeutics to treat cancer. In this Special Report, we discuss the significance of systemic co-delivery of chemotherapeutics and miRNA therapeutics for cancer therapy and highlight the representative examples of this strategy using lipid-based nanoparticles. We also present outstanding roadblocks to clinical translation and provide the latest perspectives.

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

Background and AimsChanges in single microRNA (miRNA) expression have been associated with chemo‐resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy.Approach and ResultsHigh‐throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA‐sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR‐CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide–treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo‐resistant CCLP cells. MIR1249 modulated the chemotherapy‐induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt‐regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild‐type models showed only stable disease over treatment.ConclusionsMIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.

Next generation miRNA inhibition using short anti-seed PNAs encapsulated in PLGA nanoparticles

Selective inhibition of microRNAs (miRNAs) offers a new avenue for cancer therapeutics. While most of the current anti-miRNA (antimiR) reagents target full length miRNAs, here we investigate novel nanoparticle-delivered short PNA probes containing cationic domains targeting the seed region of the miRNA for effective antimiR therapy. For proof of concept, we tested PNAs targeting miRNA-155 and employed poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle formulation for delivery. A comprehensive evaluation of PLGA nanoparticles (NPs) containing short PNA probes showed significantly superior loading, release profile, and uniform size distribution, compared to conventional non-cationic PNA probes. Confocal microscopy and flow cytometry analyses showed efficient transfection efficiency and uniform distribution of PLGA NPs containing short PNA probes in the cytoplasm. Functional analysis also confirmed efficient miRNA-155 inhibition including an effect on its downstream target proteins. Further, reduced tumor growth was observed after systemic delivery of PLGA nanoparticles containing short PNA probes in vivo in a xenograft mouse model following inhibition of miR-155. There was no evidence of acute or chronic toxicity associated with systemic delivery of PLGA NPs containing short PNA probes in the mice. Overall, in this paper we present a novel antimiR strategy based on PLGA nanoparticle delivered short PNA probes for potential cancer therapy.