Proteinase-activated Receptor 2 Promotes Cancer Cell Migration through RNA Methylation-mediated Repression of miR-125b

Proteinase activated-receptor 2 (PAR₂) participates in cancer metastasis promoted by serine proteinases. The current study aimed to test the molecular mechanism by which PAR₂ promotes cancer cell migration. In different cancer cells, activation of PAR₂ by activating peptide (PAR₂-AP) dramatically increased cell migration, whereas knock down of PAR₂ inhibited cellular motility. The PAR₂ activation also repressed miR-125b expression while miR-125b mimic successfully blocked PAR₂-induced cell migration. Moreover, Grb associated-binding protein 2 (Gab2) was identified as a novel target gene of miR-125b and it mediated PAR₂-induced cell migration. The correlation of PAR₂ with miR-125b and Gab2 was further supported by the findings obtained from human colorectal carcinoma specimens. Remarkably, knock down of NOP2/Sun domain family, member 2 (NSun2), a RNA methyltransferase, blocked the reduction in miR-125b induced by PAR₂. Furthermore, PAR₂ activation increased the level of N⁶-methyladenosine (m⁶A)-containing pre-miR-125b in NSun2-dependent manner. Taken together, our results demonstrated that miR-125b mediates PAR₂-induced cancer cell migration by targeting Gab2 and that NSun2-dependent RNA methylation contributes to the down-regulation of miR-125b by PAR₂ signaling. These findings suggest a novel epigenetic mechanism by which microenvironment regulates cancer cell migration by altering miRNA expression.     

Nidogen‐1 is a common target of microRNAs MiR‐192/215 in the pathogenesis of Hirschsprung's disease

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1.

     

Growth factor TGF-β induces intestinal epithelial cell (IEC-6) differentiation: miR-146b as a regulatory component in the negative feedback loop

TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.