Dynamic and differential regulation in the microRNA expression in the developing and mature cataractous rat lens

Recent evidence supports a role for microRNAs (miRNAs) in regulating gene expression, and alterations in gene expression are known to affect cells involved in the development of ageing disorders. Using developing rat lens epithelial cells (LECs), we profiled the expression of miRNAs by a microarray‐based approach. Few gene expression changes known to be involved in pathogenesis or cytoprotection were uniquely influenced by miRNA expression. Most miRNAs increased or decreased in abundance (let 7b, let 7c, miR29a, miR29c, miR126 and miR551b) in LECs/lenses during late embryonic and post‐natal development and in cataract. Among them, miR29a, miR29c and miR126 were dramatically decreased in cataractous LECs from Shumiya Cataract Rats (SCRs). Specifically, the cytoskeleton remodelling genes tropomyosin (Tm) 1α and 2β, which have been implicated in the initiation of pathophysiology, were targets of miR29c and were over‐stimulated as demonstrated by inhibitor experiments. In transfection experiments, increasing the level of miR29c caused a corresponding decrease in the expression of Tm1α and 2β, suggesting that miR29c may regulate the translation of Tm1α and 2β. 3′UTR luciferase activity of Tm1α, not 2β, was significantly decreased in miR29c‐transfected mouse LECs. These findings demonstrate changes in miRNAs expression, and target molecules have potential as diagnostic indicators of ageing and as a foundation of miR‐based therapeutics for age‐related diseases.     

Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways

In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT‐PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR‐29a, miR‐99a, and miR‐125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca²⁺‐dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR‐125a, additional miR‐125a was bound to Ago2 in the synaptosome, and expression of the miR‐125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA‐induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.     

MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

microRNAs in the blastocoel fluid as accessible indicators of chromosomal normality

MicroRNAs (miRNAs) derived from the pre-implantation blastocoel fluid (BF) have attracted interest as accessible biomarkers indicative of embryonic health in ongoing IVF cycles. Therefore, we investigated expression levels of some aneuploidy-associated miRNAs and implantation-related mRNAs as predictive markers for embryo chromosomal normality. In this study, the BF of 25 blastocysts that had been checked for aneuploidy (aneuploid=17 and euploid=8) was aspirated and the expression of 10 miRNAs (miR-20a, miR-30c, miR‐661, miR-372, miR-142, miR-191, miR‐345, miR‐339, miR‐141, and miR-27b) and four genes (ERBB4, SELL, ITGB3, ITGAV) were evaluated using real time-PCR. Results showed that the levels of miR-661 and miR-20a were significantly higher in the BF of the aneuploid embryos compared to the euploid group (p = 0.0017 and 0.004, respectively). A comparison of the mRNA levels between the aneuploid and euploid groups also demonstrated a significant difference in ITGAV (p = 0.013) and SELL (p = 0.0317) levels. In the euploid group, a negative correlation was found between ITGB3 and miR-30c (r = ?0.71, p = 0.08), and in the aneuploid group, a positive correlation was found between ERBB4 and miR-345 (r = 0.71, p = 0.02). It can be suggested that miR-20a, miR-661, and ITGAV levels of BF could be used as less-invasive biomarkers to evaluate embryonic health. Moreover, aneuploidy-related miRNA levels were associated with levels of genes involved in embryo implantation.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

CSPGs inhibit axon branching by impairing mitochondria‐dependent regulation of actin dynamics and axonal translation

Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin‐dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria‐dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl‐l ‐carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Serum miRNAs are potential biomarkers for the detection of disc degeneration,among which miR‐26a‐5p suppresses Smad1 to regulate disc homeostasis

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling.     

Activation of PI3K and R‐ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are extracellular inhibitors of axon extension and plasticity, and cause growth cones to exhibit dystrophic behaviors. Phosphoinositide 3‐kinase (PI3K) is a lipid kinase activated by axon growth promoting signals. In this study, we used embryonic chicken dorsal root ganglion neurons to determine if CSPGs impair signaling through PI3K. We report that CSPGs inhibit PI3K signaling in axons and growth cones, as evidenced by decreased levels of phosphorylated downstream kinases (Akt and S6). Direct activation of PI3K signaling, using a cell permeable phosphopeptide (PI3Kpep), countered the effects of CSPGs on growth cones and axon extension. Both overnight and acute treatment with PI3Kpep promoted axon extension on CSPG‐coated substrates. The R‐Ras GTPase is an upstream positive regulator of PI3K signaling. Expression of constitutively active R‐Ras promoted axon extension and growth cone elaboration on CSPGs and permissive substrata. In contrast, an N‐terminus‐deleted constitutively active R‐Ras, deficient in PI3K activation, promoted axon extension but not growth cone elaboration on CSPGs and permissive substrata. These data indicate that activation of R‐Ras‐PI3K signaling may be a viable approach for manipulating axon extension on CSPGs. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 918–933, 2014