Neural activity is significantly reduced in the spinal cords of SMA mice.

<p><b>A</b>. Example of a slice from the lumbar spine with only the ventral horn positioned over the electrodes of the MED probe. <b>B</b>. Two littermates from the Smn^−/−;SMN2 strain of mice at P4 showing the difference in size of the wild type and mutant pups. <b>C</b>. Average number of active electrodes recorded for each slice. One Way ANOVA, p<0.0001 <b>D</b>. Average spike frequency recorded by each active electrode. <i>p</i> = 0.46, One way ANOVA.</p

Excitatory and inhibitory input in motor neurons cultured from wild type and SMA mice.

<p><b>A</b>. Sample traces of the sEPSCs recorded on day 7 and day 17 in control and SMA cells. <b>B</b>. Summary data of the frequency of sEPSCs during the 2^nd and 3^rd week of culture in control and SMA cells. <b>C</b>. Summary data of the amplitude of sEPSCs during the 2^nd and 3^rd week culture in control and SMA cells. <b>D</b>. Sample traces of the sIPSCs at day7 and day17 in control and SMA mice. <b>E</b>. Summary data of the frequency of sIPSCs during the 2^nd and 3^rd week of culture in control and SMA cells. <b>F</b>. Summary data of the amplitude of sIPSCs during the 2^nd and 3^rd week culture in control and SMA mice. We did not find any significant difference in the frequency or amplitude of either excitatory or inhibitory PSPs between the control and SMA cultures (<i>p</i>>0.05, One-way ANOVA).</p

Sample traces show the reduced activity recorded from the ventral horns of SMA mice.

<p>Sample traces showing spike activity recorded from all active electrodes in a single slice from a control and an SMA mouse showing activity in both basal and serotonin-stimulated conditions. Slices from SMA mice had many fewer active electrodes than controls, and showed fewer neurons active after serotonin treatment.</p

In SMA slices the overall distribution of spike frequencies is not significantly changed by serotonin.

<p>Histograms of spike frequency comparing slices from control and SMA mice. The number of observations was normalized to the total number of observations in each slice and divided into. 0.5 Hz bins. The distributions were fitted with mixed Gaussian approximations using the Expectation-Maximization (EM) algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011696#pone.0011696-Alpaydin1" target="_blank">[26]</a>. Serotonin significantly shift the frequency distribution in control slices (p<0.0001), but the shift is not significant in the SMA slices (p = 0.1433; Kruskal-Wallis).</p

Pharmacological agents cause little change in activity of control or SMA slices.

<p>Acetylcholine (100 µM), bicuculline (10 µM) or GABA (20 µM) were bath applied to the slice and recording was resumed after 2 minutes. For wash, slices were perfused with aCSF for 10 minutes before recording was resumed. Averages are calculated for 12 slices from 3 control mice and 11 slices from 3 SMA mice.</p><p>*p<0.05 (One-way ANOVA). Active electrodes are those electrodes recording spiking activity of at least 1 Hz over a 50 second period. Number of spikes per minute totals the number of spikes recorded on all electrodes recording neural activity from a slice.</p

Motor neuron activity in both control and SMA slices is stimulated by serotonin.

<p>50 µM of serotonin was added to the bath and neuronal activity was recorded after 2 minutes. For the wash condition, slices were perfused with aCSF for 10 minutes before recording resumed. <b>A</b>. Average number of active electrodes/slice (active electrodes are those that recorded more than 50 spikes in 50 seconds). One way ANOVA, p<0.0001 for both control and SMA. <b>B</b>. Average frequency of spikes recorded on active electrodes. One way ANOVA, p<0.0001 for control, p = 0.137 for SMA.</p

Image_2_MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.tif

BackgroundGastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development.MethodsThe expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining.ResultsMiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly.ConclusionsIn conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.</p

Table_1_MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.docx

BackgroundGastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development.MethodsThe expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining.ResultsMiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly.ConclusionsIn conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.</p

Sequences of primers used for qRT-PCR.

<p>Sequences of primers used for qRT-PCR.</p

Expression of total SMN protein in undifferentiated as well as MN-differentiated control and SMA cells.

<p>Representative immunoblots of both differentiated and undifferentiated mESCs revealed a significantly reduced protein expression of SMN in A2 SMA cell lines compared to Hb9 control cells. SMN levels were normalized to Gapdh. C =  control, S =  SMA. Asterisks indicate significant differences compared to controls (*p<0.05, Student's <i>t</i> test).</p