A novel microRNA mmu-miR-466h affects apoptosis regulation in mammalian cells

This study determined the changes in microRNA (miRs) expression in mammalian Chinese hamster ovary (CHO) cells undergoing apoptosis induced by exposing the cells to nutrient-depleted media. The apoptosis onset was confirmed by reduced cell viability and Caspase-3/7 activation. Microarray comparison of known mouse and rat miRs in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells subjected to depleted media. The mmu-miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation in depleted media was confirmed with qRT-PCR. Since miRs suppress mRNA translation, we hypothesized that up-regulated mmu-miR-466h inhibits anti-apoptotic genes and induces apoptosis. A combination of bioinformatics and experimental tools was used to predict and verify mmu-miR-466h anti-apoptotic targets. 8708 predicted targets were obtained from miRecords database and narrowed to 38 anti-apoptotic genes with DAVID NCBI annotation tool. Several genes were selected from this anti-apoptotic subset based on nucleotide pairing complimentarity between the mmu-miR-466h seed region and 3' UTR of the target mRNAs. The qRT-PCR analysis revealed reduced mRNA levels of bcl2l2, dad1, birc6, stat5a, and smo genes in CHO cells exposed to depleted media. The inhibition of the mmu-miR-466h increased the expression levels of those genes and resulted in increased cell viability and decreased Caspase-3/7 activation. The up-regulation of mmu-miR-466h in response to nutrients depletion causes the inhibition of several anti-apoptotic genes in unison. This suggests the pro-apoptotic role of mmu-miR-466h and its capability to modulate the apoptotic pathway in mammalian cells.     

Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells

MiRNAs have been shown to be involved in regulation of multiple cellular processes including apoptosis. Since a single miRNA can affect the expression of several genes, the utilization of miRNAs for apoptosis engineering in mammalian cells can be more efficient than the conventional approach of manipulating a single gene. Mmu-miR-466h-5p was previously shown to have a pro-apoptotic role in CHO cells by reducing the expression of several anti-apoptotic genes and its transient inhibition delayed both the activation of Caspase-3/7 and the loss of cell viability. The present study evaluates the effect of stable inhibition of mmu-miR-466h-5p in CHO cells on their ability to resist apoptosis onset and their production properties. The expression of mmu-miR-466h-5p in the engineered anti-miR-466h CHO cell line was significantly lower than in the negative control and the parental CHO cells. These engineered cells reached higher maximum viable cell density and extended viability compared with negative control and parental CHO cells in batch cell cultures which resulted in the 53.8% and 41.6% increase of integral viable cells. The extended viability of anti-miR-466h CHO cells was the result of delayed Caspase-3/7 activation by more than 35 h, and the increased levels of its anti-apoptotic gene targets (smo, stat5a, dad1, birc6, and bcl2l2) to between 2.1- and 12.5-fold compared with the negative control CHO in apoptotic conditions. The expression of secreted alkaline phosphatase (SEAP) increased 43% and the cell-specific productivity increased 11% in the stable pools of anti-miR-466h CHO compared with the stable pools of negative control CHO cells. The above results demonstrate the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression.     

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.     

MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3'-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3'-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.     

MicroRNA-146b-5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4

Alteration of tissue inhibitors of matrix metalloproteinases (TIMP)/matrix metalloproteinases (MMP) associated with collagen upregulation has an important role in sustained atrial fibrillation (AF). The expression of miR-146b-5p, whose the targeted gene is TIMPs, is upregulated in atrial cardiomyocytes during AF. This study was to determine whether miR-146b-5p could regulate the gene expression of TIMP4 and the contribution of miRNA to atrial fibrosis in AF. Collagen synthesis was observed after miR-146b-5p transfection in human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs)-fibroblast co-culture cellular model in vitro. Furthermore, a myocardial infarction (MI) mouse model was used to confirm the protective effect of miR-146b-5p downregulation on atrial fibrosis. The expression level of miR-146b-5p was upregulated, while the expression level of TIMP4 was downregulated in the fibrotic atrium of canine with AF. miR-146b-5p transfection in hiPSC-aCMs-fibroblast co-culture cellular model increased collagen synthesis by regulating TIMP4/MMP9 mediated extracellular matrix proteins synthesis. The inhibition of miR-146b-5p expression reduced the phenotypes of cardiac fibrosis in the MI mouse model. Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated (at both mRNA and protein levels) by miR-146b-5p inhibition in cardiomyocytes of MI heart. We concluded that collagen fibres were accumulated in extracellular space on miR-146b-5p overexpressed co-culture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR-146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR-146b-5p might become an effective therapeutic approach for preventing atrial fibrosis.     

Epigenetic control of a VDR-governed feed-forward loop that regulates p21(waf1/cip1) expression and function in non-malignant prostate cells

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.     

Phospho-ΔNp63α/microRNA network modulates epigenetic regulatory enzymes in squamous cell carcinomas

The tumor protein (TP) p63/microRNAs functional network may play a key role in supporting the response of squamous cell carcinomas (SCC) to chemotherapy. We show that the cisplatin exposure of SCC-11 cells led to upregulation of miR-297, miR-92b-3p, and miR-485-5p through a phosphorylated ΔNp63α-dependent mechanism that subsequently modulated the expression of the protein targets implicated in DNA methylation (DNMT3A), histone deacetylation (HDAC9), and demethylation (KDM4C). Further studies showed that mimics for miR-297, miR-92b-3p, or miR-485-5p, along with siRNA against and inhibitors of DNMT3A, HDAC9, and KDM4C modulated the expression of DAPK1, SMARCA2, and MDM2 genes assessed by the quantitative PCR, promoter luciferase reporter, and chromatin immunoprecipitation assays. Finally, the above-mentioned treatments affecting epigenetic enzymes also modulated the response of SCC cells to chemotherapeutic drugs, rendering the resistant SCC cells more sensitive to cisplatin exposure, thereby providing the groundwork for novel chemotherapeutic venues in treating patients with SCC.     

Extremely Low-Frequency Electromagnetic Fields Affect the miRNA-Mediated Regulation of Signaling Pathways in the GC-2 Cell Line

Extremely low-frequency electromagnetic fields (ELF-EMFs) can affect male reproductive function, but the underlying mechanism of this effect remains unknown. miRNA-mediated regulation has been implicated as an important epigenetic mechanism for regulatory pathways. Herein, we profiled miRNA expression in response to ELF-EMFs in vitro. Mouse spermatocyte-derived GC–2 cells were intermittently exposed to a 50 Hz ELF-EMF for 72 h (5 min on/10 min off) at magnetic field intensities of 1 mT, 2 mT and 3 mT. Cell viability was assessed using the CCK–8 assay. Apoptosis and the cell cycle were analyzed with flow cytometry. miRNA expression was profiled using Affymetrix Mouse Genechip miRNA 3.0 arrays. Our data showed that the growth, apoptosis or cell cycle arrest of GC–2 cells exposed to the 50 Hz ELF-EMF did not significantly change. However, we identified a total of 55 miRNAs whose expression significantly changed compared with the sham group, including 19 differentially expressed miRNAs (7 miRNAs were upregulated, and 12 were downregulated) in the 1 mT exposure group and 36 (9 miRNAs were upregulated, and 27 were downregulated) in the 3 mT exposure group. The changes in the expression of 15 selected miRNAs measured by real-time PCR were consistent with the microarray results. A network analysis was used to predict core miRNAs and target genes, including miR-30e-5p, miR-210-5p, miR-196b-5p, miR-504-3p, miR-669c-5p and miR-455-3p. We found that these miRNAs were differentially expressed in response to different magnetic field intensities of ELF-EMFs. GO term and KEGG pathway annotation based on the miRNA expression profiling results showed that miRNAs may regulate circadian rhythms, cytokine-cytokine receptor interactions and the p53 signaling pathway. These results suggested that miRNAs could serve as potential biomarkers, and the miRNA-mediated regulation of signaling pathways might play significant roles in the biological effects of ELF-EMFs.     

Enhanced peripheral blood miR-324-5p is associated with the risk of metabolic syndrome by suppressing ROCK1

The current study aims to evaluate whether peripheral blood miR-324-5p could be used to differentiate patients with metabolic disorders and healthy controls. Our data showed that miR-324-5p levels were elevated in the peripheral blood of patients with hyperglycemia or hyperlipidemia. In addition, the expression of miR-324-5p was enhanced in the peripheral blood and liver of db/db mice. Further study indicated that overexpression of miR-324-5p reduced the activation of the AKT/GSK pathway and increased lipid accumulation, while the inhibition of miR-324-5p activated the AKT/GSK pathway and decreased lipid accumulation. A dual luciferase assay revealed that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) was a target gene of miR-324-5p. In addition, silencing ROCK1 deteriorated lipid and glucose metabolism. More importantly, knockdown of ROCK1 reversed the miR-324-5p inhibitor-induced improvement of glucose and lipid metabolism. In summary, miR-324-5p plays a regulatory role in glucose and lipid metabolism by targeting ROCK1, which is involved in metabolic disorders. The use of miR-324-5p in diagnosing metabolic syndrome is worth investigating and may benefit patients.