miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.     

MiR-508-5p Inhibits the Progression of Glioma by Targeting Glycoprotein Non-metastatic Melanoma B

Glioma is a severe and highly lethal brain cancer, a malignancy largely stemming from growing in a relatively restrained area of the brain. Hence, the understanding of the molecular regulation of the growth of glioma is critical for improving its treatment. MicroRNA has become a hotspot in research on diseases, especially in the initiation and progression of different types of cancer. However, the molecular function and mechanisms of miR-508-5p in gliomagenesis are still unclear. The aim of this study was to investigate miR-508-5p expression in glioma and determine its effects on proliferation. miR-508-5p expression levels, both in glioma cell lines and in tissue, were significantly lower than in a normal human astrocyte cell line or adjacent tissues. Cell growth was analyzed using a MTT assay and over-expression of miR-508-5p was found to decrease glioma cell growth. Moreover, a bioinformatic analysis was performed, showing that glycoprotein non-metastatic melanoma B (GPNMB) was a direct target for miR-508-5p in glioma cells. Furthermore, in vivo treatment with miR-508-5p reduced GPNMB protein levels in the tumor. Additionally, overexpression of GPNMB without 3′-UTR partially reversed the cell growth arrest induced by miR-508-5p over-expression in glioma cells. In conclusion, these results indicate that increased expression of miR-508-5p might be related to glioma progression, indicating a potential role of miR-508-5p for clinical therapy.     

MiR-674-5p Suppresses the Proliferation and Migration of Glioma Cells by Targeting Cul4b

Neurochemical Research - Glioma multiforme (GBM) is the most common malignant primary brain tumors. Despite the considerable advances in GBM treatment, it is still one of the most lethal forms of...     

MiR-183-5p Alleviates Chronic Constriction Injury-Induced Neuropathic Pain Through Inhibition of TREK-1

MicroRNAs have been implicated in nerve injury and neuropathic pain. In the previous study we had shown that miR-96 can attenuate neuropathic pain through inhibition of Nav1.3. In this study, we investigated the role of miR-183, a same cluster member of microRNA with miR-96, in neuropathic pain and its potential mechanisms. We found that the expression level of miR-183-5p in dorsal root ganglion was decreased with the development of neuropathic pain induced by chronic constriction sciatic nerve injury (CCI). By contrast, the TREK-1, a K⁺ channel, was increased. Further investigation identified that intrathecal injection of miR-183-5p mimic efficiently ameliorated neuropathic pain and inhibited the expression of TREK-1, a predicted target gene of miR-183-5p. Luciferase assays confirmed the binding of miR-183-5p and TREK-1. In addition, over-expression of TREK-1 blocked the roles of miR-183-5p in neuropathic pain. Our findings suggested that miR-183-5P participated in the regulation of CCI-induced neuropathic pain through inhibiting the expression of TREK-1.     

MiR-210-3p Inhibits Proliferation and Migration of C6 Cells by Targeting Iscu

Neurochemical Research - Glioma is the most common primary brain tumor and the most malignant type of glioma is glioblastoma with the character of high mortality, high recurrence rate and poor...     

microRNA-17-5p Modulates Bacille Calmette-Guerin Growth in RAW264.7 Cells by Targeting ULK1

To explore the potential roles of miRNAs in controlling the survival of mycobacteria in macrophages, miR-17-5p in the regulation of Bacillus Calmette-Guérin(BCG)growth in the macrophage RAW264.7 cells was interrogated. Our results reveal that an infection of BCG shows a time-dependent up-regulation of miR-17-5p in RAW264.7 cells in early phase; importantly, excessive expression of miR-17-5p in these cells exhibits an increased propagation of intracellular BCG. Mechanistically, the Unc-51 like autophagy activating kinase 1 (ULK1), an initial molecular of autophagy are identified as novel target of miR-17-5p, the miR-17-5p is capable of targeting down-regulating the expression of ULK1 protein. In addition, an overexpression of miR-17-5p in RAW264.7 cells is correlated with repression of ULK1 and the autophagosome related proteins LC3I/II. These results imply that miR-17-5p may be able to arrest the maturation of mycobacterial phagosomes in part by targeting ULK1, subsequently reduces the ability of host cells to kill intracellular BCG.     

The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.     

MiR-122-5p Mitigates Inflammation,Reactive Oxygen Species and SH-SY5Y Apoptosis by Targeting CPEB1 After Spinal Cord Injury Via the PI3K/AKT Signaling Pathway

Neurochemical Research - Spinal cord injury (SCI) is a threatening disease that lead to severe motor and sensory deficits. Previous research has revealed that miRNAs are involved in the...     

Involvement of MAP3K8 and miR-17-5p in Poor Virologic Response to Interferon-Based Combination Therapy for Chronic Hepatitis C

Despite advances in chronic hepatitis C treatment, a proportion of patients respond poorly to treatment. This study aimed to explore hepatic mRNA and microRNA signatures involved in hepatitis C treatment resistance. Global hepatic mRNA and microRNA expression profiles were compared using microarray data between treatment responses. Quantitative real-time polymerase chain reaction validated the gene signatures from 130 patients who were infected with hepatitis C virus genotype 1b and treated with pegylated interferon-alpha and ribavirin combination therapy. The correlation between mRNA and microRNA was evaluated using in silico analysis and in vitro siRNA and microRNA inhibition/overexpression experiments. Multivariate regression analysis identified that the independent variables IL28B SNP rs8099917, hsa-miR-122-5p, hsa-miR-17-5p, and MAP3K8 were significantly associated with a poor virologic response. MAP3K8 and miR-17-5p expression were inversely correlated with treatment response. Furthermore, miR-17-5p repressed HCV production by targeting MAP3K8. Collectively, the data suggest that several molecules and the inverse correlation between mRNA and microRNA contributed to a host genetic refractory hepatitis C treatment response.     

The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K⁺ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.     

MiR-21-5p but not miR-1-3p expression is modulated by preconditioning in a rat model of myocardial infarction

Molecular Biology Reports - Isoflurane (Iso) preconditioning (PC) is known to be cardioprotective against ischemia/reperfusion (I/R) injury. It was previously shown that microRNA-21-5p (miR-21-5p)...     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

MiR-628-5p decreases the tumorigenicity of epithelial ovarian cancer cells by targeting at FGFR2

Micro RNAs (miRNAs) are small non-coding RNAs which are 19–24 nucleotides in length. MiRNAs play a vital role in the whole process of tumour development, but how they influence the tumourigenecity of epithelial ovarian cancer (EOC)cells is rarely researched. In our study, it was verified that miR-628-5p decreased the stem like cell percentage of EOC cells by inducing their apoptosis. The animal experiments showed that miR-628-5p decreased the tumourigenecity of EOC cells. Besides, we found miR-628-5p targeted at and down-regulated the expression of fibroblast growth factor receptor 2 (FGFR2). FGFR2 expressed higher in ovarian cancer tissues and was correlated with worse prognosis. Our findings indicated that miR-628-5pplays an important role in ovarian cancer stem cell driven tumorigenesis.     

MiR-195-5p inhibits proliferation and invasion of nerve cells in Hirschsprung disease by targeting GFRA4

Molecular and Cellular Biochemistry - Studies have reported that miR-195-5p plays a role in the Hirschsprung disease (HSCR). Our previous work found GDNF family receptor alpha 4 (GFRA4) is also...     

MiR-122-5p suppresses the proliferation,migration, and invasion of gastric cancer cells by targeting LYN

Gastric cancer (GC) is one of malignant tumors with high mortality and morbidity in the world.MicroRNA-122 (miR-122) acts as a tumor suppressor in a variety of ...     

MicroRNA-137-3p Protects PC12 Cells Against Oxidative Stress by Downregulation of Calpain-2 and nNOS

The imbalance between excess reactive oxygen species (ROS) generation and insufficient antioxidant defenses contribute to a range of neurodegenerative diseases. High ROS levels damage cellular macromolecules such as DNA, proteins and lipids, leading to neuron vulnerability and eventual death. However, the underlying molecular mechanism of the ROS regulation is not fully elucidated. Recently, an increasing number of studies suggest that microRNAs (miRNAs) emerge as the targets in regulating oxidative stress. We recently reported the neuroprotective effect of miR-137-3p for brachial plexus avulsion-induced motoneuron death. The present study is sought to investigate whether miR-137-3p also could protect PC12 cells against hydrogen peroxide (H₂O₂) induced neurotoxicity. By using cell viability assay, ROS assay, gene and protein expression assay, we found that PC-12 cells exposed to H₂O₂ exhibited decreased cell viability, increased expression levels of calpain-2 and neuronal nitric oxide synthase (nNOS), whereas a decreased miR-137-3p expression. Importantly, restoring the miR-137-3p levels in H₂O₂ exposure robustly inhibited the elevated nNOS, calpain-2 and ROS expression levels, which subsequently improved the cell viability. Furthermore, the suppressive effect of miR-137-3p on the elevated ROS level under oxidative stress was considerably blunted when we mutated the binding site of calpain-2 targted by miR-137-3p, suggesting the critical role of calpain-2 involving the neuroprotective effect of miR-137-3p. Collectively, these findings highlight the neuroprotective role of miR-137-3p through down-regulating calpain and NOS activity, suggesting its potential role for combating oxidative stress insults in the neurodegenerative diseases.

     

Interactions between Multiple Phosphorylation Sites in the Inactivation Particle of a K+ Channel : Insights into the Molecular Mechanism of Protein Kinase C Action

Protein kinase C inhibits inactivation gating of Kv3.4 K⁺ channels, and at least two NH₂-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403–1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine → alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ∼85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S → A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S → D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S → D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH₂-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating.