mir-124-5p Regulates Phagocytosis of Human Macrophages by Targeting the Actin Cytoskeleton via the ARP2/3 Complex.

Phagocytosis is a cellular process crucial for recognition and removal of apoptotic cells and foreign particles, subsequently initiating appropriate immune responses. The process of phagocytosis is highly complex and involves major rearrangements of the cytoskeleton. Due to its complexity and importance for tissue homoeostasis and immune responses, it is tightly regulated. Over the last decade, microRNAs (miRNAs) have emerged as important regulators of biological pathways including the immune response by fine-tuning expression of gene regulatory networks. In order to identify miRNAs implicated in the regulation of phagocytosis, a systematic screening of all currently known, human miRNAs was performed using THP-1 macrophage-like cells and serum-opsonized latex beads. Of the total of 2,566 miRNAs analyzed, several led to significant changes in phagocytosis. Among these, we validated miR-124-5p as a novel regulator of phagocytosis. Transfection with miR-124-5p mimics reduced the number of phagocytic cells as well as the phagocytic activity of phorbol-12-myristate-13-acetate (PMA)-activated THP-1 cells and ex vivo differentiated primary human macrophages. In silico analysis suggested that miR-124-5p targets genes involved in regulation of the actin cytoskeleton. Transcriptional analyses revealed that expression of genes encoding for several subunits of the ARP2/3 complex, a crucial regulator of actin polymerization, is reduced upon transfection of cells with miR-124-5p. Further in silico analyses identified potential binding motifs for miR-124-5p in the mRNAs of these genes. Luciferase reporter assays using these binding motifs indicate that at least two of the genes (ARPC3 and ARPC4) are direct targets of miR-124-5p. Moreover, ARPC3 and ARPC4 protein levels were significantly reduced following miR-124-5p transfection. Collectively, the presented results suggest that miR-124-5p regulates phagocytosis in human macrophages by directly targeting expression of components of the ARP2/3 complex.

Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.

Induction of apoptosis in ovarian cancer cells by miR-493-3p directly targeting AKT2, STK38L, HMGA2, ETS1 and E2F5.

Apoptosis is a form of directed programmed cell death with a tightly regulated signalling cascade for the destruction of single cells. MicroRNAs (miRNAs) play an important role as fine tuners in the regulation of apoptotic processes. MiR-493-3p mimic transfection leads to the induction of apoptosis causing the breakdown of mitochondrial membrane potential and the activation of Caspases resulting in the fragmentation of DNA in several ovarian carcinoma cell lines. Ovarian cancer shows with its pronounced heterogeneity a very high death-to-incidence ratio. A target gene analysis for miR-493-3p was performed for the investigation of underlying molecular mechanisms involved in apoptosis signalling pathways. Elevated miR-493-3p levels downregulated the mRNA and protein expression levels of Serine/Threonine Kinase 38 Like (STK38L), High Mobility Group AT-Hook 2 (HMGA2) and AKT Serine/Threonine Kinase 2 (AKT2) by direct binding as demonstrated by luciferase reporter assays. Notably, the protein expression of RAF1 Proto-Oncogene, Serine/Threonine Kinase (RAF1) was almost completely downregulated by miR-493-3p. This interaction, however, was indirect and regulated by STK38L phosphorylation. In addition, RAF1 transcription was diminished as a result of reduced transcription of ETS proto-oncogene 1 (ETS1), another direct target of miR-493-3p. Taken together, our observations have uncovered the apoptosis inducing potential of miR-493-3p through its regulation of multiple target genes participating in the extrinsic and intrinsic apoptosis pathway.

MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression. The binding to target messenger RNAs (mRNAs) results in mRNA cleavage or inhibition of the translational machinery leading to decreased protein levels. Various signalling pathways, including apoptosis are modulated by miRNAs. Here, we investigated the role of miR-744-5p in apoptosis signalling in ovarian cancer cell lines. MiR-744-5p expression was reduced in the cancer cell lines independent of the host gene MAP2K4. Overexpression of miR-744-5p activated the intrinsic apoptotic pathway in SKOV3, OVCAR3 and Cisplatin resistant (A2780-cis) and non-resistant A2780 cells leading to cell death. Notably, miR-744-5p overexpression together with Carboplatin treatment led to at least additive pro-apoptotic effects. Investigation of the apoptotic signalling pathways mediated by miR-744-5p revealed that its elevated expression directly downregulated mRNA and protein expression of nuclear factor I X (NFIX) and heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC caused diminished miR-21 expression and AKT phosphorylation, while NFIX decreased Bcl2 levels, leading to the detected pro-apoptotic effects. Finally, Kaplan-Meier-Plots showed a prolonged median disease-free survival in ovarian serous cystadenocarcinoma patients with high miR-744 expression.

miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells.

Apoptosis is a genetically directed process of programmed cell death. A variety of microRNAs (miRNAs), endogenous single-stranded non-coding RNAs of about 22 nucleotides in length have been shown to be involved in the regulation of the intrinsic or extrinsic apoptotic pathways. There is increasing evidence that the aberrant expression of miRNAs plays a causal role in the development of diseases such as cancer. This makes miRNAs promising candidate molecules as therapeutic targets or agents. MicroRNA (miR)-217-5p has been implicated in carcinogenesis of various cancer entities, including colorectal cancer. Here, we analyzed the pro-apoptotic potential of miR-217-5p in a variety of colorecatal cancer cell lines showing that miR-217-5p mimic transfection led to the induction of apoptosis causing the breakdown of mitochondrial membrane potential, externalization of phosphatidylserine, activation of caspases and fragmentation of DNA. Furthermore, elevated miR-217-5p levels downregulated mRNA and protein expression of atypical protein kinase c iota type I (PRKCI), BAG family molecular chaperone regulator 3 (BAG3), integrin subunit alpha v (ITGAV) and mitogen-activated protein kinase 1 (MAPK1). A direct miR-217-5p mediated regulation to those targets was shown by repressed luciferase activity of reporter constructs containing the miR-217-5p binding sites in the 3' untranslated region. Taken together, our observations have uncovered the apoptosis-inducing potential of miR-217-5p through its regulation of multiple target genes involved in the ERK-MAPK signaling pathway by regulation of PRKCI, BAG3, ITGAV and MAPK1.

Investigation on tissue specific effects of pro-apoptotic micro RNAs revealed miR-147b as a potential biomarker in ovarian cancer prognosis.

The development and progression of cancer can be ascribed to imbalances in gene regulation leading to aberrant cellular behavior. The loss of micro RNAs (miRNAs) exhibiting tumor-suppressive function has been demonstrated to be often causative for uncontrolled cell proliferation, migration or tissue infiltration. The installation of de novo tumor suppressive function by using pro-apoptotic miRNAs might be a promising therapeutic approach. In addition, there is a great demand for novel biomarkers for the prognosis of cancer, which prompted us to transfer a high content miRNA screening initially performed to identify bioprocess relevant miRNAs in Chinese hamster ovary (CHO) cells to human cancer cell lines . Analysis of screened miRNAs exhibiting strongest pro-apoptotic effects discovered globally and cross-species active candidates. The recovery rate of apoptosis inducing miRNAs was highest in the human ovarian carcinoma cell line SKOV3. Focusing on ovarian cell lines miR-1912, miR-147b and miR-3073a showed significant apoptosis induction in cell lines with different genetic background (SKOV3p53null, OVCAR3p53R248Q, TOV21G, TOV112Dp53R175H, A2780, A2780-cisp53K351N) alone and additive effects in combination with carboplatin. While expression analysis revealed a low endogenous expression of miR-1912 and miR-147b in SKOV3, miRNA expression was highly upregulated upon apoptosis induction using chemotherapeutics. Ectopic introduction of these miRNAs lead to enhanced activation of caspase-dependent death signaling and an induction of the pro-apoptotic proteins Bak1 and Bax and a reduced expression of Bcl2 and Bcl-xL. Finally, analysis of The Cancer Genome Atlas data revealed the expression of hsa-miR-147b-5p to show a positive influence on the median survival of ovarian cancer patients.

miR-483 is a self-regulating microRNA and can activate its own expression via USF1 in HeLa cells.

MicroRNAs (miRNAs) are small non-coding RNAs that constitute a fundamental part of post-transcriptional gene regulation in mammalian cells. We have recently identified the intronic miR-483, which functions as an important regulator of protein synthesis during mild hypothermia in human and rodent cells. Since only very little is known about transcriptional regulation of intronic miRNAs and their host genes, we thoroughly investigated the regulation of miR-483 expression and its host gene IGF2 in HeLa cells. We demonstrate that miR-483 is regulated and expressed independently of its host gene IGF2 during mild hypothermia. Strikingly, we also discovered that miR-483 enhances its own transcription by up-regulation of the transcription factor USF1, which activates a promoter element upstream of the MIR483 gene. However, since the USF1 mRNA lacks binding sites for miR-483-5p and -3p, USF1 expression is likely enhanced in an indirect manner. Our results suggest that miR-483 may self-regulate its own expression independently of its host gene IGF2 in human HeLa cells. This points towards a novel feed-forward mechanism, in which selected intronic miRNAs may activate their own expression by transcriptional activation of upstream regulators.

Enhanced protein production by microRNA-30 family in CHO cells is mediated by the modulation of the ubiquitin pathway.

Functional genomics represent a valuable approach to improve culture performance of Chinese hamster ovary (CHO) cell lines for biopharmaceutical manufacturing. Recent advances in applied microRNA (miRNAs) research suggest that these small non-coding RNAs are critical for the regulation of cell phenotypes in CHO cells. However, the notion that individual miRNAs usually control the expression of hundreds of different genes makes miRNA target identification highly complex. We have recently reported that the entire miR-30 family enhances recombinant protein production in CHO cells. To better understand the pro-productive effects of this miRNA family, we set out to identify their downstream target genes in CHO cells. Computational target prediction combined with a comprehensive functional validation enabled the discovery of a set of twenty putative target genes for all productivity enhancing miR-30 family members. We demonstrate that all miR-30 isoforms contribute to the regulation of the ubiquitin pathway in CHO cells by directly targeting the ubiquitin E3 ligase S-phase kinase-associated protein 2 (Skp2). Finally, we provide several lines of evidence that miR-30-mediated modulation of the ubiquitin pathway may enhance recombinant protein expression in CHO cells. In summary, this study supports the importance of non-coding RNAs, especially of miRNAs, in the context of cell line engineering.