Regulation of autophagy by two products of one gene: TRPM3 and miR-204.
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ABSTRACT:
SUBMITTER: Cost NG
PROVIDER: S-EPMC4905337 | biostudies-other | 2015 Oct-Dec
REPOSITORIES: biostudies-other
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Project description:Previously, we established a microRNA (miRNA) expression signature in pancreatic ductal adenocarcinoma (PDAC) tissues using RNA sequencing and found significantly reduced expression of miR-204-5p. Here, we aimed to investigate the functional significance of miR-204-5p and to identify miR-204-5p target genes involved in PDAC pathogenesis. Cancer cell migration and invasion were significantly inhibited by ectopic expression of miR-204-5p in PDAC cells. Comprehensive gene expression analyses and in silico database searches revealed 25 putative targets regulated by miR-204-5p in PDAC cells. Among these target genes, high expression levels of RACGAP1, DHRS9, AP1S3, FOXC1, PRP11, RHBDL2 and MUC4 were significant predictors of a poor prognosis of patients with PDAC. In this study, we focused on RACGAP1 (Rac guanosine triphosphatase-activating protein 1) because its expression was most significantly predictive of PDAC pathogenesis (overall survival rate: p = 0.0000548; disease-free survival rate: p = 0.0014). Overexpression of RACGAP1 was detected in PDAC clinical specimens, and its expression enhanced the migration and invasion of PDAC cells. Moreover, downstream genes affected by RACGAP1 (e.g., MMP28, CEP55, CDK1, ANLN and S100A14) are involved in PDAC pathogenesis. Our strategy to identify antitumor miRNAs and their target genes will help elucidate the molecular pathogenesis of PDAC.
| S-EPMC6468488 | biostudies-literature
Project description:Hepatitis B virus (HBV) is a major human pathogen. In this study, we found that miR-204 and miR-1236 were down-regulated in HBV-producing cells, and each could suppress HBV replication. Using a bioinformatic approach and a reporter assay, we identified miR-1236, which can reduce HBV replication and protein production by directly targeting at HBV specific mRNA. In contrast, miR-204 was identified by a microarray approach, and had no effect on HBV RNA and protein production. Surprisingly, miR-204 could inhibit HBV pregenomic RNA encapsidation and capsid assembly. We further demonstrated that HBV suppressed miR-204 expression via activating a host transcription factor STAT3. We established a positive feed-forward loop between HBV, miR-204 and STAT3. Interestingly, miR-204 has been considered as a tumor suppressor in some literature. Since the risk for hepatocellular carcinoma (HCC) is significantly increased in chronic HBV patients, it is possible that chronic suppression of miR-204 by HBV contributes to HCC incidence. Both miR-204 and miR-1236 might be useful for developing new therapeutics against HBV.
| S-EPMC5062086 | biostudies-other
Project description:PurposePax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action.MethodsMutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells.ResultsPAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site.ConclusionsThese studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.
| S-EPMC3945805 | biostudies-literature
Project description:Background & aimsThere is high need of novel diagnostic and prognostic tools for tumors of the digestive system, such as gastric cancer and cholangiocarcinoma. We recently found that miR-204 was deeply downregulated in gastric cancer tissues. Here we investigated whether this was common to other tumors of the digestive system and whether this elicited a miR-204-dependent gene target signature, diagnostically and therapeutically relevant. Finally, we assessed the contribution of the identified target genes to the cell cycle progression and clonogenicity of gastric cancer and cholangiocarcinoma cell lines.MethodsWe employed quantitative PCR and Affymetrix profiling for gene expression studies. In silico analysis aided us to identifying a miR-204 target signature in publicly available databases (TGCA). We employed transient transfection experiments, clonogenic assays and cell cycle profiling to evaluate the biological consequences of miR-204 perturbation.ResultsWe identified a novel miR-204 gene target signature perturbed in gastric cancer and in cholangiocarcinoma specimens. We validated its prognostic relevance and mechanistically addressed its biological relevance in GC and CC cell lines.ConclusionsWe suggest that restoring the physiological levels of miR-204 in some gastrointestinal cancers might be exploited therapeutically.
| S-EPMC5444686 | biostudies-literature
Project description:During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.
| S-EPMC3597499 | biostudies-literature
Project description:Chicken atrophic ovaries have decreased volume and are indicative of ovarian failure, presence of a tumor, or interrupted ovarian blood supply. Ovarian tumor is accompanied by an increase in follicular atresia, granulosa cell (GC) apoptosis, and autophagy. In a previous study, we found using high throughput sequencing that miR-204 is highly expressed in chicken atrophic ovaries. Thus, in the present study, we further investigated its function in GC apoptosis and autophagy. We found that overexpression of miR-204 reduced mRNA and protein levels of proliferation-related genes and increased apoptosis-related genes. Cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and flow cytometry assays revealed that miR-204 inhibited GC proliferation and promoted apoptosis. Furthermore, we confirmed with reporter gene assays that Forkhead box K2 (FOXK2) was directly targeted by miR-204. FOXK2, as a downstream regulator of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathways, promoted GC proliferation and inhibited apoptosis. Subsequently, we observed that miR-204 was involved in GC autophagy by targeting Transient Receptor Potential Melastatin 3 (TRPM3). The luciferase activities of the two binding sites of TRPM3 were decreased in response to treatment with a miR-204 mimic, and the autophagic flux was increased after miR-204 inhibition. However, overexpression of miR-204 had opposite results in autophagosomes and autolysosomes. miR-204 inhibits GC autophagy by suppressing the protein expression of TRPM3/AMP-activated protein kinase (AMPK)/ULK signaling pathway components. Inhibition of miR-204 enhanced autophagy by accumulating and degrading the protein levels of LC3-II (Microtubule Associated Protein Light Chain 3B) and p62 (Protein of 62 kDa), respectively, whereas miR-204 overexpression was associated with contrary results. Immunofluorescence staining showed that there was a significant reduction in the fluorescent intensity of LC3B, whereas p62 protein was increased after TRPM3 silencing. Collectively, our results indicate that miR-204 is highly expressed in chicken atrophic ovaries, which promotes GC apoptosis via repressing FOXK2 through the PI3K/AKT/mTOR pathway and inhibits autophagy by impeding the TRPM3/AMPK/ULK pathway.
| S-EPMC7676916 | biostudies-literature
Project description:In this short report, we pinpoint some technical and conceptual flaws that we found in the article entitled "miR-204-5p and miR-211-5p contribute to BRAF inhibitor resistance in melanoma" (Díaz-Martínez et al., Cancer Research 2018). We also discuss how, in our opinion, these flaws led Díaz-Martínez and colleagues to incorrect conclusions about the biological role that miR-204 and miR-211 play in melanoma and about the terms of their involvement in the phenomenon of resistance to BRAF inhibitors.
| S-EPMC6142896 | biostudies-other
Project description:MicroRNAs (miRNAs or miRs) are small molecules known to be involved in post-transcriptional gene expression. Many of them have been shown to influence risk for various diseases. Recent studies suggest that lower expression of miR-204, a gene coding for miRNA-204, is correlated with shorter survival in patients with acute myeloid leukaemia (AML). This observation prompted us to analyse the effect of two polymorphisms of the miR-204 gene, one in the upstream flanking region (rs718447 A?>?G) and the other inside the gene itself (rs112062096 A?>?G), both also in intron 3 of the TRPM3 gene.The study was conducted on DNA samples isolated from AML patients (n?=?95) and healthy individuals (n?=?148), who were genotyped using the Light SNiP assays.The miR-204 rs718447 GG homozygosity was found to constitute a risk factor associated with susceptibility to AML (73/95 vs 92/148, AML patients vs healthy controls, OR?=?2.020, p?=?0.017). Additionally, this genotype was more frequent in patients with subtypes M0-M1 in the French-American-British (FAB) classification as compared to patients with subtypes M2-M7 (23/25 vs 39/57, p?=?0.026). We also found that presence of allele A was linked to longer survival of AML patients.Our results show that polymorphism in miR-204 flanking region may constitute a risk and prognostic factor in AML.
| S-EPMC5791219 | biostudies-literature
Project description:Exercise-induced autophagy is associated with physiological left ventricular hypertrophy (LVH), and a growing body of evidence suggests that microRNAs (miRNAs) can regulate autophagy-related genes. However, the precise role of miRNAs in exercise induced autophagy in physiological LVH has not been fully defined. In this study, we investigated the microRNA-autophagy axis in physiological LVH and deciphered the underlying mechanism using a rat swimming exercise model. Rats were assigned to sedentary control (CON) and swimming exercise (EX) groups; those in the latter group completed a 10-week swimming exercise without any load. For in vitro studies, H9C2 cardiomyocyte cell line was stimulated with IGF-1 for hypertrophy. We found a significant increase in autophagy activity in the hearts of rats with exercise-induced physiological hypertrophy, and miRNAs showed a high score in the pathway enriched in autophagy. Moreover, the expression levels of miR-26b-5p, miR-204-5p, and miR-497-3p showed an obvious increase in rat hearts. Adenovirus-mediated overexpression of miR-26b-5p, miR-204-5p, and miR-497-3p markedly attenuated IGF-1-induced hypertrophy in H9C2 cells by suppressing autophagy. Furthermore, miR-26b-5p, miR-204-5p, and miR-497-3p attenuated autophagy in H9C2 cells through targeting ULK1, LC3B, and Beclin 1, respectively. Taken together, our results demonstrate that swimming exercise induced physiological LVH, at least in part, by modulating the microRNA-autophagy axis, and that miR-26b-5p, miR-204-5p, and miR-497-3p may help distinguish physiological and pathological LVH.
| S-EPMC7042403 | biostudies-literature
Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA). HEK-293 cells were transfected with either miR-204 or a control, and gene expression was analyzed using the Agilent Human Whole Genome 4x44K array. A dye-swap was performed.
2012-03-30 | E-GEOD-28400 | biostudies-arrayexpress