Antisense miRNAs Research Articles

MicroRNAs as novel endogenous targets for regulation and therapeutic treatments.

MicroRNAs (miRNAs) are small non-coding RNAs that have been identified as key endogenous biomolecules that are able to regulate gene expression at the post-transcriptional level. The abnormal expression or function of miRNAs has been demonstrated to be closely related to the occurrence or development of various human diseases, including cancers. Regulation of these abnormal miRNAs thus holds great promise for therapeutic treatments. In this review, we summarize exogenous molecules that are able to regulate endogenous miRNAs, including small molecule regulators of miRNAs and synthetic oligonucleotides. Strategies for screening small molecule regulators of miRNAs and recently reported small molecules are introduced and summarized. Synthetic oligonucleotides including antisense miRNA oligonucleotides and miRNA mimics, as well as delivery systems for these synthetic oligonucleotides to enter cells, that regulate endogenous miRNAs are also summarized. In addition, we discuss recent applications of these small molecules and synthetic oligonucleotides in therapeutic treatments. Overall, this review aims to provide a brief synopsis of recent achievements of using both small molecule regulators and synthetic oligonucleotides to regulate endogenous miRNAs and achieve therapeutic outcomes. We envision that these regulators of endogenous miRNAs will ultimately contribute to the development of new therapies in the future.

Assessing the Off-Target Effects of miRNA Inhibitors on Innate Immune Toll-Like Receptors.

MicroRNAs (miRNAs) are involved in most cellular processes and are deregulated in several diseases. Antisense miRNA oligonucleotides (AMOs) therefore present novel therapeutic opportunities. Currently, in vivo delivery of AMOs often relies on high doses of nucleic acids, with nonspecific uptake by most tissues. Critically, AMOs accumulate in phagocytic cells where they can interfere with immune functions, such as the activation of Toll-Like Receptors (TLRs). In this chapter, we describe a method to assess the possible off-target effects of AMOs on TLR7, 8, and 9 sensing.

Anti-apoptotic function of herpes simplex virus -2 latency-associated transcript RL1 sequence and screening of its encoded microRNAs.

The latency-associated transcript (LAT) gene of herpes simplex virus (HSV)-2 is the only detectable viral gene expressed during latent infection in neurons. LAT inhibits apoptosis and maintains latency by promoting the survival of infected neurons. However, whether LAT functions during HSV-2 infection via its encoded RNAs or via its encoded proteins remain unknown. Increasing evidence has indicated that LAT is likely to functionally promote the establishment of latent infection via LAT-encoded microRNAs (miRNAs). To explore whether the RL1 fragment of the five adjacent miRNAs has an effect on cell apoptosis, then provide supporting evidence to elucidate the potential role of these miRNAs and to aid screening of their cellular targets. A number of techniques, including MTT assay, flow cytometry and DNA ladder analysis, were used to verify the role of the RL1 fragment and the contribution of the individual miRNAs to the anti-apoptotic effect. Five miRNAs (miR-H3, miR-H4-3p, miR-H4-5p, miR-H24 and miR-H19) were detected by quantitative PCR in PC12 cells stably expressing RL1 after pEGFP-RL1 plasmid transfection invitro. The data indicated that expression of HSV-2 LAT RL1 seems to provide protection against apoptosis of PC12 cells induced by ActD. Antisense miRNAs specifically inhibiting these five miRNAs could efficiently reduce their expression. Transfection of antisense-miR-H3, antisense-miR-H4-5p and antisense-miR-H19 into PC12 cells stably expressing RL1 were able to partly reverse the anti-apoptotic effect of these miRNAs. These findings indicate that the apoptotic role of the RL1 fragment is likely to be related to overexpression of miR-H3, miR-H4-5p and miR-H19 in PC12 cells.

Inhibition of microRNA-155 sensitizes lung cancer cells to irradiation via suppression of HK2-modulated glucose metabolism.

MicroRNAs (miRNAs) are small non-coding regulatory RNAs, which are involved in the post-transcriptional regulation of gene expression. miRNA (miR)-155, which has previously been reported to be overexpressed in lung cancer, is correlated with poor patient prognosis. The present study aimed to investigate the effects of miR‑155 on the radiosensitivity of human non‑small cell lung cancer (NSCLC) cells. To explore the roles of miRNAs in the regulation of irradiation sensitivity of human lung cancer cells, the expressions of miR‑155 in response to irradiation, have been studied by RT‑qPCR, and the putative direct target of miR‑155 was identified by western blot and luciferase assays. The results of the present study revealed that the expression of miR‑155 was induced by irradiation, thus suggesting a positive correlation between miR‑155 and radiosensitivity. Furthermore, overexpression of miR‑155 rendered lung cancer cells resistant to irradiation. In addition, hexokinase2 (HK2) was identified as an indirect target of miR‑155; exogenous overexpression of miR‑155 upregulated the expression of HK2, whereas inhibition of miR‑155 by antisense miRNA suppressed HK2 expression. In addition, HK2‑modulated glucose metabolism was significantly upregulated by overexpression of miR‑155. Notably, inhibition of miR‑155 sensitized lung cancer cells to irradiation via suppression of glucose metabolism. In conclusion, the present study reported a novel function for miR‑155 in the regulation of NSCLC cell radiosensitivity, thus suggesting that miR‑155 may be considered a therapeutic target for the development of anticancer drugs.

ChemInform Abstract: Furan Oxidation Based Cross‐Linking: A New Approach for the Study and Targeting of Nucleic Acid and Protein Interactions

AbstractReview: 121 refs.

Expression of Tim4 in Glioma and its Regulatory Role in LN-18 Glioma Cells.

BackgroundTim4 is a transmembrane protein known as T cell immunoglobulin and mucin domain containing protein-4. We speculated that Tim4 might be associated with glioma. This study aimed to investigate the expression level of Tim4 in gliomas and the regulatory role of Tim4 on the growth and apoptosis of LN-18 glioma cells.Material/MethodsTumor tissues and adjacent normal tissues were collected from patients with glioma. The expression level of Tim4 mRNA and protein was determined by RT-PCR and Western blot analyses, respectively to evaluate their association with glioma. Tim4 was overexpressed or silenced by siRNA interference in cultured human glioma cells LN-18. The growth and apoptosis of LN-18 cells was detected by MTT assay and flow cytometry. The colony-forming ability of LN-18 cells was assessed by the colony formation assay. The collection of human tissues was approved by the Research Ethics Committee at the Harbin Medical University Cancer Hospital and performed in strict accordance with international standards. All patients were required to sign the informed consent.ResultsThe expression level of Tim4 mRNA and protein in tumor tissues was significantly higher compared with adjacent normal tissues. Antisense miRNA targeting Tim4 inhibited the growth of LN-18 cells, induced their apoptosis, and reduced their clonogenic capacity. In contrast, overexpression of Tim4 promoted the growth of LN-18 cells, inhibited their apoptosis, and enhanced their clonogenic potential.ConclusionsThe expression level of Tim-4 is closely associated with glioma. Decreased expression of Tim4 inhibited the growth and colony-forming ability of LN-18 cells and induced their apoptosis, whereas increased expression of Tim4 stimulated the growth and clonogenic potential of LN-18 cells and suppressed their apoptosis.

Hypoxia-upregulated microRNA-630 targets Dicer, leading to increased tumor progression.

MicroRNAs (miRNAs) are small RNA molecules that affect cellular processes by controlling gene expression. Recent studies have shown that hypoxia downregulates Drosha and Dicer, key enzymes in miRNA biogenesis, causing a decreased pool of miRNAs in cancer, and resulting in increased tumor growth and metastasis. Here, we demonstrate a previously unrecognized mechanism by which hypoxia downregulates Dicer. We found that miR-630, which is upregulated under hypoxic conditions, targets and downregulates Dicer expression. In an orthotopic mouse model of ovarian cancer, delivery of miR-630 using DOPC nanoliposomes resulted in increased tumor growth and metastasis and decreased Dicer expression. Treatment with the combination of anti-miR-630 and anti-vascular endothelial growth factor antibody in mice resulted in rescue of Dicer expression and significantly decreased tumor growth and metastasis. These results indicate that targeting miR-630 is a promising approach to overcome Dicer deregulation in cancer. As demonstrated in the study, use of DOPC nanoliposomes for anti-miR delivery serves as a better alternative approach to cell line based overexpression of sense or anti-sense miRNAs, while avoiding potential in vitro selection effects. Findings from this study provide a new understanding of miRNA biogenesis downregulation observed under hypoxia and suggest therapeutic avenues to target this dysregulation in cancer.

Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions.

Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.

Systematic study of cis-antisense miRNAs in animal species reveals miR-3661 to target PPP2CA in human cells.

MicroRNAs (miRNAs) suppress targeting gene expression through blocking translation or triggering mRNA degradation and, in general, act in trans, through a partially complementary interaction with the 3′ untranslated region (3′ UTR) or coding regions of a target gene. Although it has been reported previously that some miRNAs suppress their target genes on the opposite strand with a fully complementary sequence (i.e., natural antisense miRNAs that act in cis), there is no report to systematically study such cis-antisense miRNAs in different animal species. Here we report that cis-antisense miRNAs do exist in different animal species: 48 in Caenorhabditis elegans, 17 in Drosophila, 36 in Mus musculus, and 52 in Homo sapiens using a systematical bioinformatics approach. We show that most of these cis-antisense miRNAs can efficiently reduce the expression levels of their target genes in human cells. We further investigate hsa-miR-3661, one of the predicted cis-antisense miRNAs, in detail and demonstrate that this miRNA directly targets the coding sequence of PPP2CA located on the opposite DNA strand and inhibits the PPP2CA expression. Taken together, these results indicate that cis-antisense miRNAs are conservative and functional in animal species including humans.

Novel antisense therapeutics delivery systems: In vitro and in vivo studies of liposomes targeted with anti-CD20 antibody

Antisense gene therapy using molecules such as antisense oligodeoxynucleotides, siRNA or miRNA is a very promising strategy for the treatment of neoplastic diseases. It can be combined with other treatment strategies to enhance therapeutic effect. In acute leukemias, overexpression of the antiapoptotic gene BCL2 is observed in more than 70% of cases. Therefore, reduction of the Bcl-2 protein level could, in itself, prevent the development of cancer or could possibly help sensitize cancer cells to apoptosis inducers.The main objective of our work is to develop therapeutic liposome formulations characterized by high transfection efficiency, stability in the presence of serum, as well as specificity and toxicity for target (leukemic) cells. Each of our liposomal formulations consists of a core composed of antisense oligonucleotides complexed by either cationic lipid, DOTAP, or a synthetic polycation, polyethyleneimine, encapsulated within liposomes modified with polyethylenoglycol. In addition, the liposomal shells are enriched with covalently-bound antibodies recognizing a well characterized bio-marker, CD20, exposed on the surface of leukemia cells. The resulting immunoliposomes selectively and effectively reduced the expression of BCL2 in target cells. Model animal experiments carried out on mice-engrafted tumors expressing the specific marker showed high efficiency of the liposome formulations against specific tumor development. In conclusion, we show that lipid formulations based on a polyplex or lipoplex backbone additionally equipped with antibodies are promising non-viral vectors for specific oligonucleotide transfer into human tumor cells.

MiRNA therapeutics: a new class of drugs with potential therapeutic applications in the heart.

miRNAs are small non-coding RNAs (ncRNAs), which regulate gene expression. Here, the authors describe the contribution of miRNAs to cardiac biology and disease. They discuss various strategies for manipulating miRNA activity including antisense oligonucleotides (antimiRs, blockmiRs), mimics, miRNA sponges, Tough Decoys and miRNA mowers. They review developments in chemistries (e.g., locked nucleic acid) and modifications (sugar, 'ZEN', peptide nucleic acids) and miRNA delivery tools (viral vectors, liposomes, nanoparticles, pHLIP). They summarize potential miRNA therapeutic targets for heart disease based on preclinical studies. Finally, the authors review current progress of miRNA therapeutics in clinical development for HCV and cancer, and discuss challenges that will need to be overcome for similar therapies to enter the clinic for patients with cardiac disease.

The emerging role of dendritic cells in the host immune response against Helicobacter pylori.

The emerging role of dendritic cells in the host immune response against Helicobacter pylori.

Identification of microRNAs in six solanaceous plants and their potential link with phosphate and mycorrhizal signaling.

To date, only a limited number of solanaceous miRNAs have been deposited in the miRNA database. Here, genome-wide bioinformatic identification of miRNAs was performed in six solanaceous plants (potato, tomato, tobacco, eggplant, pepper, and petunia). A total of 2,239 miRNAs were identified following a range of criteria, of which 982 were from potato, 496 from tomato, 655 from tobacco, 46 from eggplant, 45 were from pepper, and 15 from petunia. The sizes of miRNA families and miRNA precursor length differ in all the species. Accordingly, 620 targets were predicted, which could be functionally classified as transcription factors, metabolic enzymes, RNA and protein processing proteins, and other proteins for plant growth and development. We also showed evidence for miRNA clusters and sense and antisense miRNAs. Additionally, five Pi starvation- and one arbuscular mycorrhiza (AM)-related cis-elements were found widely distributed in the putative promoter regions of the miRNA genes. Selected miRNAs were classified into three groups based on the presence or absence of P1BS and MYCS cis-elements, and their expression in response to Pi starvation and AM symbiosis was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These results show that conserved miRNAs exist in solanaceous species and they might play pivotal roles in plant growth, development, and stress responses.

Fibrosis: a novel approach for an old problem

Dupuytren’s disease is a fibroproliferative disease of the hand palm that results in permanent finger flexion and is the most common inherited condition to affect the body’s connective tissue with a global presence of 3-6%. It was originally known as the Viking’s disease and thought to be originated in the seventh or early eight century AD. In 1832 Baron Guillaume Dupuytren named and gave the first anatomo-clinical description of the disease. To date the causes underlying Dupuytren’s disease are unknown and surgery remains the standard of care. Multiple recurrences are a common feature of this disease, following surgical removal of the primary fibrotic formations. Our research focuses on understanding of the molecular mechanisms that are at the basis of the progression of fibrosis. In our recently published manuscript we described how modulation of the TGFβ signaling pathway results in “reduction” of the fibrotic content of the disease. Dupuytren’s resection specimens can now be maintained ex vivo providing a suitable model for preclinical screening of different classes of potential antifibrotic drugs; antisense oligonucleotides, chemical inhibitors, and miRNA- based therapies. Our findings indicate that decrease of collagen deposition, which is pathological characteristic of Dupuytren’s, can be achieved by blocking the main regulatory pathway of collagen expression and remodeling. Short term gene expression modulation could have prolonged antifibrotic effects, even upon withdrawal of the modulatory factor (e.g. pharmacological inhibitor). Dupuytren’s field of research is shifting direction from symptom-oriented studies towards molecular pathogenesis and gene expression “correction” attempts. Future studies shall focus on the molecular, epigenetic or immune modulation of key fibrotic stimuli in combination with current treatments with ultimate goal, to not only relief the primary symptoms, but mainly to prevent recurrence of the disease. Here, we discuss the applicability of ex vivo screening of potential pre-drugs on Dupuytren’s-derived tissue for the treatment of various fibrotic diseases.

Cząsteczki mikroRNA jako istotny składnik mechanizmów regulacji ekspresji genów związanych z nowotworami

This paper presents the relationship between changes in the expression of specific microRNAs and the formation and development of selected cancers. An overview of reports is provided on the usefulness of research on microRNA expression, which in the future may become valuable and desirable prognostic and prediction factors for diagnosticians and clinicians. miRNA will presumably become the cornerstone for the development of new therapeutic approaches using antisense miRNAs (antagomirs) or drugs aimed at miRNA offsetting in the case of deletion or damage to their genes. It would offer the potential possibility of the regulation of gene expression which is of great significance for the origin and development of cancers.

Global analysis of miRNA gene clusters and gene families reveals dynamic and coordinated expression.

To further understand the potential expression relationships of miRNAs in miRNA gene clusters and gene families, a global analysis was performed in 4 paired tumor (breast cancer) and adjacent normal tissue samples using deep sequencing datasets. The compositions of miRNA gene clusters and families are not random, and clustered and homologous miRNAs may have close relationships with overlapped miRNA species. Members in the miRNA group always had various expression levels, and even some showed larger expression divergence. Despite the dynamic expression as well as individual difference, these miRNAs always indicated consistent or similar deregulation patterns. The consistent deregulation expression may contribute to dynamic and coordinated interaction between different miRNAs in regulatory network. Further, we found that those clustered or homologous miRNAs that were also identified as sense and antisense miRNAs showed larger expression divergence. miRNA gene clusters and families indicated important biological roles, and the specific distribution and expression further enrich and ensure the flexible and robust regulatory network.

Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells

The transcripts encoded by the microRNA mir-142 gene are highly active in hematopoietic cells, but expressed at low levels in many other cell types. Treatment with the demethylating agent 5-Aza-2′-deoxycytidine increased both the 1,636 nucleotide primary transcript and mature miR-142-5p/3p in mesenchymal cells, indicating that mir-142 is epigenetically repressed by DNA methylation. The transcription start site was determined to be located 1,205 base pairs upstream of the precursor sequence within a highly conserved CpG island. In addition, a second CpG island overlapped with the precursor. A TATA-box, several promoter-proximal elements and enrichment of conserved transcription factor binding sites within the first 100 base pairs upstream of the transcription start site, suggests that this region represents the core/proximal mir-142 promoter. Moreover, both CpG islands were heavily methylated in mesenchymal cells, having low levels of miR-142-5p/3p, and unmethylated in hematopoietic cells where both miRNAs were abundantly expressed. We show that treatment with 5-Aza-2′-deoxycytidine significantly reduced the DNA methylation of the upstream CpG island, which led to increased expression, and that in vitro DNA methylation of the upstream region of the mir-142 precursor repressed its transcriptional activity. When overexpressed, miR-142-5p/3p reduced proliferation of cells with epigenetic silencing of endogenous mir-142. This finding is interesting as miR-142-5p/3p have been reported to be deregulated in tumors of mesenchymal origin. We provide the first experimental evidence that transcription of mir-142 is directly repressed by DNA methylation. In addition, we discovered that the antisense strand of mir-142 might act as a precursor for functional mature antisense miRNAs. Thus, our study expands the current knowledge about the regulation of mir-142 and function of miR-142-5p/3p, and adds novel insight into the rapidly increasing field of microRNA regulation.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment

MicroRNAs (miRNAs or miRs) are a family of small non‑coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) and tumor suppressor miRNAs; and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancer; ii) the oncosuppressor p27Kip1, a validated miR-221 target is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the downregulation of Slug and the upregulation of p27Kip1.

MicroRNA miR-378 regulates adipocytokine fate by targeting transcriptional factors in human visceral and subctaneous adipose tissue

Purpose: MicroRNAs (miRNA), single-stranded non-protein coding gene products, regulate gene expression through post-transcriptional inhibition, and known to be involved in essential biological processes including obesity and insulin resistance. miR-378 and miR-378*, which are encoded by PGC-1β gene and counterbalance the metabolic actions of PGC-1β. Mice genetically lacking miR-378 and miR-378* are resistant to high fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. We conducted to elucidate the role of miR378 expression in human adiposity and adipose tissue inflammation. Methods: Pair samples were obtained from subcutaneous (SAT) and visceral adipose tissue (VAT) during elective operation in 71 men and 42 women. Mature miRNA levels were determined by qRT-PCR and normalized to levels of U6 small nuclear ribonucleoprotein. Subcutaneous (SFA) and visceral fat area (VFA) was determined by abdominal multidetector CT scanning. Adipocyte sizing was determined by the osmium-fixed method on a Coulter counter. Results: Study 1 (clinical study): SFA were larger than VFA, and the mean size of adipocytes was greater in SAT than in VAT. In VAT, level of log(miR100) was positively correlated with level of adiponectin, TLR4 and HMGB1, while negatively correlated with CD68, NOX4 and with mean size of adipocyte. In SAT, level of log(miR100) was positively correlated with level of IL18, TLR4 and HMGB1, while negatively correlated with NOX4. Study 2 (in vitro study): expression levels of miR100 as well as PPARγ2 and PGC1α were increased in adipocyte-like 3T3L1 cells during differentiation. Levels of PPARγ2 and PGC1α were inhibited by antisense miRNA transfection. The expression levels of computer-predicted target genes, ACSL1, acyl-CoA synthetase long-chain family member 1 and AGPAT6, 1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase ζ), were partially inhibited by anti-sense miR-378 overexpression. Conclusion: Adipose tissue miR378 is derived mostly from adipocytes and may play a role in regulating expressions of adipocyte inflammatory cytokines and insulin sensitivity in human. Results suggested that mir-378 regulates adipocytokine expression by targeting transcriptional factors and controls adipose tissue inflammation in human.