MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE32122 | GEO | 2012/05/16
SECONDARY ACCESSION(S): PRJNA147443
REPOSITORIES: GEO
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiae into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNAM-cM-^@M-^@microarray analysis using total RNAs harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis confirmed increased expression of miR-221, miR-181a* and miR-326, and decreased expression of miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Overexpression of miR-221 induced neurite outgrowth of PC12 cells in the absence of NGF treatment, and also enhanced neurite outgrowth caused by low-dose NGF. More importantly, knockdown of miR-221 by antagomir attenuated NGF-mediated neurite outgrowth. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are involved in apoptosis in PC12 cells. Our results indicate that miR-221 plays a critical role for neuronal differentiation as well as protection against apoptosis in PC12 cells. NGF induced miRNAs expression in rat pheochromocytoma PC12 cells was measured in cells treated with 100 ng/ml NGF for 0, 12, 24 and 48 hr.
2012-05-15 | E-GEOD-32122 | biostudies-arrayexpress
Project description:Multiple sclerosis (MS) begins as an inflammatory/demyelinating disease but axon injury and neurodegeneration underlie chronic progression. Extracellular matrix (ECM) heparan sulfate proteoglycans (HSPG) are catabolized in acute inflammatory MS lesions and HSPG catabolic products may contribute to failure of neuron repair. Neurite outgrowth and axon elongation of PC12 cells are inhibited when these cells are grown on heparanase-digested HSPG. This inhibition of differentiation is counteracted by nerve growth factor (NGF) but failure to completely reverse inhibition may be due to loss of other growth factors affected by HSPG breakdown. To understand effects of the digested ECM in this model, expression of genes that control differentiation in PC12 cells grown on digested and undigested HSPG will be analyzed. This will permit identification of new potential therapeutic targets for specifically counteracting inhibitory effects of ECM breakdown on neuron regrowth/regeneration in MS. We will compare gene expression patterns of PC12 cells grown on undigested and digested HSPG in the presence and in the absence of NGF over a two week time course to identify the alterations induced by the catabolized HSPG on expression of genes involved in their acquisition of a neuronal phenotype and to determine which genes are and are not targeted by NGF. We are studying ECM HSPG breakdown in human and experimental demyelinating diseases and have developed an in vitro model of ECM catabolism-induced inhibition of neuronal differentiation. Our data indicate that: 1) the numbers of PC12 cells that exhibit neurite outgrowth are decreased when the ECM contains digested, as opposed to intact HSPG; and 2) in the presence of NGF, neurite outgrowth inhibition is counteracted, but complete axonal growth/elongation is not achieved. We will use gene microarray analysis to identify the specific gene expression alterations that digested ECM HSPG induces in PC12 cells in this model of inhibition of neural differentiation. We hypothesize that the genes that are critical for PC12 cell differentiation will be altered by the pathologically altered ECM. Rat pheochromocytoma PC12 cell lines are widely used for analyzing neuronal differentiation. Microarray analyses have identified gene expression patterns associated with their differentiation and response to growth factors. We developed a model of catabolized HSPG inhibition of PC12 cell differentiation that mimics the ECM in MS lesions. HSPG coated on tissue culture dishes is digested with heparanases prior to plating of the PC12 cells. Cells grown on digested ECM without NGF show inhibition of neuronal differentiation (decreased numbers with neurite outgrowth) from days 7 to 14. NGF can partially, but not completely counteract the inhibition, suggesting that signaling pathways in addition to those affected by NGF may be affected by the ECM HSPG digestion. Therefore, we will compare gene expression patterns in samples of cells grown on digested and undigested HSPG ECM both in the presence and absence of NGF. The cells are plated on washed, undigested or digested HSPG-coated 100 mm Petri dishes and grown with or without NGF. Total RNA will be extracted from cells grown in the 4 conditions (w/without digestion; w/without NGF) on 100mm Petri dishes using the RNeasy extraction kit (Qiagen). We propose: 2 duplicate samples/culture condition (8 samples) per time point X 5 time points (days 1, 5, 7, 10, and 14 of culture) = 40 samples/experiment. The RNA will be sent to the Microarray Consortium center for QC check and microarray analysis. Previous analyses of PC12 cells have used the NIA Mouse 15K cDNA clone set scanned with the Agilent Oligo array platform (Vaudry, 2002; Grumolato, 2003). Therefore, this methodology will be useful for comparisons to published studies. (Number of experiments to be determined). Vaudry D, et al, 2002. J Neurochem 83:1272-1284 Grumolato L, et al, 2003. Endocrinology 144:2368-2379
2008-06-13 | E-GEOD-5015 | biostudies-arrayexpress
Project description:We analyzed the comprehensive gene expression changes by nerve growth factor (NGF) in PC12 cells using Agilent microarrays. The genes of 1920 NGF treated were up-regulated and 2670 genes were down-regulated significantly compared with the control group. The expression level of genes involved in cell structure and proteolysis were up-regulated. On the other hand, the genes of cell cycle and some metabolism were rather down-regulated. Therefore, it is suggested that it is necessary to stop the cell cycle and down-regulate metabolism to neurite outgrowth. Gene regulation associated with the differentiation program in PC12 cells still needs to be elucidated. We analyzed the genes expression with Agilent microarrays after 6 hours of NGF treatment by the one-color method, and found 2099 genes were up-regulated and 2781 genes were down-regulated.
2012-04-24 | E-GEOD-37564 | biostudies-arrayexpress
Project description:Background: Though exosomes, as the by-products of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), it remains unclear through which manner exosomes act. Aim: In order to figure out whether exosomes attenuate lesion size of SCI by the amelioration of neuronal injury triggered by secondary inflammatory storm and induction of inner motivation of neurite outgrowth, we designed and performed this experiment. Methods: We determined absolute contents of all exosomal miRNAs and investigated the potential mechanisms of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and vitro. Results: miR-199a-3p/145-5p, which were relatively top-ranking miRNAs in exosomes, promoted PC12 cells differentiation suppressed by lipopolysaccharide (LPS) in vitro through the modulation of NGF/TrkA pathway. We also demonstrated that Cblb was the direct target of miR-199a-3p and Cbl was the direct target of miR-145-5p. Cblb and Cbl genes knock down revealed that TrkA ubiquitylation level significantly decreased, subsequently, activating the NGF/TrkA downstream pathways Akt and Erk. In return, overexpression of Cblb and Cbl suggested that TrkA ubiquitylation level significantly increased, subsequently, inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and co-immunoprecipitation confirmed the direct interaction between TrkA and Cblb, TrkA and Cbl. In vivo experiment, exosomal miR-199a-3p/145-5p were found to up-regulate TrkA expression in the lesion site and promote locomotor function of SCI rats as well. Conclusion: In summary, our study suggested that hUC-MSCs derived exosomes may treat SCI by transferring miR-199a-3p/145-5p to neurons, and modulating TrkA ubiquitylation, and strengthening the NGF/TrkA signaling pathway in SCI rats.
2020-12-08 | GSE159814 | GEO
Project description:Multiple sclerosis (MS) begins as an inflammatory/demyelinating disease but axon injury and neurodegeneration underlie chronic progression. Extracellular matrix (ECM) heparan sulfate proteoglycans (HSPG) are catabolized in acute inflammatory MS lesions and HSPG catabolic products may contribute to failure of neuron repair. Neurite outgrowth and axon elongation of PC12 cells are inhibited when these cells are grown on heparanase-digested HSPG. This inhibition of differentiation is counteracted by nerve growth factor (NGF) but failure to completely reverse inhibition may be due to loss of other growth factors affected by HSPG breakdown. To understand effects of the digested ECM in this model, expression of genes that control differentiation in PC12 cells grown on digested and undigested HSPG will be analyzed. This will permit identification of new potential therapeutic targets for specifically counteracting inhibitory effects of ECM breakdown on neuron regrowth/regeneration in MS. We will compare gene expression patterns of PC12 cells grown on undigested and digested HSPG in the presence and in the absence of NGF over a two week time course to identify the alterations induced by the catabolized HSPG on expression of genes involved in their acquisition of a neuronal phenotype and to determine which genes are and are not targeted by NGF. We are studying ECM HSPG breakdown in human and experimental demyelinating diseases and have developed an in vitro model of ECM catabolism-induced inhibition of neuronal differentiation. Our data indicate that: 1) the numbers of PC12 cells that exhibit neurite outgrowth are decreased when the ECM contains digested, as opposed to intact HSPG; and 2) in the presence of NGF, neurite outgrowth inhibition is counteracted, but complete axonal growth/elongation is not achieved. We will use gene microarray analysis to identify the specific gene expression alterations that digested ECM HSPG induces in PC12 cells in this model of inhibition of neural differentiation. We hypothesize that the genes that are critical for PC12 cell differentiation will be altered by the pathologically altered ECM. Rat pheochromocytoma PC12 cell lines are widely used for analyzing neuronal differentiation. Microarray analyses have identified gene expression patterns associated with their differentiation and response to growth factors. We developed a model of catabolized HSPG inhibition of PC12 cell differentiation that mimics the ECM in MS lesions. HSPG coated on tissue culture dishes is digested with heparanases prior to plating of the PC12 cells. Cells grown on digested ECM without NGF show inhibition of neuronal differentiation (decreased numbers with neurite outgrowth) from days 7 to 14. NGF can partially, but not completely counteract the inhibition, suggesting that signaling pathways in addition to those affected by NGF may be affected by the ECM HSPG digestion. Therefore, we will compare gene expression patterns in samples of cells grown on digested and undigested HSPG ECM both in the presence and absence of NGF. The cells are plated on washed, undigested or digested HSPG-coated 100 mm Petri dishes and grown with or without NGF. Total RNA will be extracted from cells grown in the 4 conditions (w/without digestion; w/without NGF) on 100mm Petri dishes using the RNeasy extraction kit (Qiagen). We propose: 2 duplicate samples/culture condition (8 samples) per time point X 5 time points (days 1, 5, 7, 10, and 14 of culture) = 40 samples/experiment. The RNA will be sent to the Microarray Consortium center for QC check and microarray analysis. Previous analyses of PC12 cells have used the NIA Mouse 15K cDNA clone set scanned with the Agilent Oligo array platform (Vaudry, 2002; Grumolato, 2003). Therefore, this methodology will be useful for comparisons to published studies. (Number of experiments to be determined). Vaudry D, et al, 2002. J Neurochem 83:1272-1284 Grumolato L, et al, 2003. Endocrinology 144:2368-2379 Keywords: time-course
2006-06-07 | GSE5015 | GEO
Project description:We analyzed the comprehensive gene expression changes by nerve growth factor (NGF) in PC12 cells using Agilent microarrays. The genes of 1920 NGF treated were up-regulated and 2670 genes were down-regulated significantly compared with the control group. The expression level of genes involved in cell structure and proteolysis were up-regulated. On the other hand, the genes of cell cycle and some metabolism were rather down-regulated. Therefore, it is suggested that it is necessary to stop the cell cycle and down-regulate metabolism to neurite outgrowth.
2012-04-25 | GSE37564 | GEO
Project description:miRNA profiling of PC12 rat pheochromocytoma cells during NGF-induced differentiation and apoptosis of differentiated cells after 24h of NGF deprivation. miRNA expression in differentiating and deprivated cells was compared to untreated and terminally differentiated PC12 cells respectivelly.
2013-05-11 | GSE46827 | GEO
Project description:Ongoing neuronal activity during development and plasticity acts to refine synaptic connections and contributes to the induction of plasticity and ultimately long term memory storage. Activity-dependent post-transcriptional control of mRNAs occurs through transport to axonal and dendritic compartments, local translation, and mRNA stability. We have identified a mechanism that contributes to activity-dependent regulation of mRNA stability during synaptic plasticity. In this study we demonstrate rapid, post-transtriptional control over process-enriched mRNAs by neuronal activity. Systematic analysis of the 3'-UTRs of destablized transcripts, identifies enrichment in sequence motifs corresponding to miRNA binding sites. The miRNAs that were identified, miR-326-3p/miR-330-5p, miR-485-5p, miR-666-3p, and miR-761 are predicted to regulate networks of genes important in plasticity and development. We find that these miRNAs are developmentally regulated in the hippocampus, many increasing by postnatal day 14. We further show that miR-485-5p controls NGF-induced neurite outgrowth in PC12 cells, tau expression, and axonal development in hippocampal neurons. miRNAs can function at the synapse to rapidly control and affect short- and long-term changes at the synapse. These processes likely occur during refinement of synaptic connections and contribute to the induction of plasticity and learning and memory. 12 hippocampal cell culture samples analysed, 3 coverslips pooled per sample. Treatments are as follows: Block: an inhibitor cocktail containing 50 µM D-APV, 40 µM CNQX, and 100 nM TTX for 3 hrs Activity: 50 µM bicuculline (BiC)/500 µM 4-Aminopyridine ActD: 25 µM actinomycin D
2014-03-12 | E-GEOD-55777 | biostudies-arrayexpress
Project description:Ongoing neuronal activity during development and plasticity acts to refine synaptic connections and contributes to the induction of plasticity and ultimately long term memory storage. Activity-dependent post-transcriptional control of mRNAs occurs through transport to axonal and dendritic compartments, local translation, and mRNA stability. We have identified a mechanism that contributes to activity-dependent regulation of mRNA stability during synaptic plasticity. In this study we demonstrate rapid, post-transtriptional control over process-enriched mRNAs by neuronal activity. Systematic analysis of the 3'-UTRs of destablized transcripts, identifies enrichment in sequence motifs corresponding to miRNA binding sites. The miRNAs that were identified, miR-326-3p/miR-330-5p, miR-485-5p, miR-666-3p, and miR-761 are predicted to regulate networks of genes important in plasticity and development. We find that these miRNAs are developmentally regulated in the hippocampus, many increasing by postnatal day 14. We further show that miR-485-5p controls NGF-induced neurite outgrowth in PC12 cells, tau expression, and axonal development in hippocampal neurons. miRNAs can function at the synapse to rapidly control and affect short- and long-term changes at the synapse. These processes likely occur during refinement of synaptic connections and contribute to the induction of plasticity and learning and memory. 4 samples analysed, 3 coverslips pooled per sample. Mouse DRG neuron cell bodies and axons were separated in multicompartment cell cultures allowing electrical stimulation of axons, growing under a high-resistance partition between compartments, through platinum electrodes in the lid of the culture dish (Reference: http://www.ncbi.nlm.nih.gov/pubmed/9295372)
2014-03-12 | E-GEOD-55779 | biostudies-arrayexpress
Project description:Ongoing neuronal activity during development and plasticity acts to refine synaptic connections and contributes to the induction of plasticity and ultimately long term memory storage. Activity-dependent post-transcriptional control of mRNAs occurs through transport to axonal and dendritic compartments, local translation, and mRNA stability. We have identified a mechanism that contributes to activity-dependent regulation of mRNA stability during synaptic plasticity. In this study we demonstrate rapid, post-transtriptional control over process-enriched mRNAs by neuronal activity. Systematic analysis of the 3'-UTRs of destablized transcripts, identifies enrichment in sequence motifs corresponding to miRNA binding sites. The miRNAs that were identified, miR-326-3p/miR-330-5p, miR-485-5p, miR-666-3p, and miR-761 are predicted to regulate networks of genes important in plasticity and development. We find that these miRNAs are developmentally regulated in the hippocampus, many increasing by postnatal day 14. We further show that miR-485-5p controls NGF-induced neurite outgrowth in PC12 cells, tau expression, and axonal development in hippocampal neurons. miRNAs can function at the synapse to rapidly control and affect short- and long-term changes at the synapse. These processes likely occur during refinement of synaptic connections and contribute to the induction of plasticity and learning and memory. 12 hippocampal cell culture samples analysed, 3 coverslips pooled per sample. Treatments are as follows: Block: an inhibitor cocktail containing 50 µM D-APV, 40 µM CNQX, and 100 nM TTX for 3 hrs Activity: 50 µM bicuculline (BiC)/500 µM 4-Aminopyridine ActD: 25 µM actinomycin D
2014-03-12 | E-GEOD-55780 | biostudies-arrayexpress