Anti-RND3 polyclonal antibody

PURPOSE. To evaluate the role of miR-200b expression in the proliferation of human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor-beta 1 (TGF-beta 1). METHODS. Human Tenon's capsule fibroblasts were treated with various doses of TGF-beta 1 for 24 hours. Cell proliferation was quantified by the cell counting kit-8 (cck-8) assay, cell cycle analysis, 5-ethynyl-2-deoxyuridine (EdU) assay, and analysis of cyclin E, cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. MicroRNA-200b (miR-200b) was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by the luciferase assay and Western blot analysis. The effect of miR-200b on the proliferation of HTFs was analyzed using both miR200b-mimic and inhibitor-transfected HTFs and confirmed in p27/kip1 and RND3 (the target of miR-200b) knockdown cells. RESULTS. The proliferation of the TGF-beta 1-treated HTFs increased significantly in a dose-and time-dependent manner. Treatment with 5 ng/mL TGF-beta 1 caused an upregulation of miR-200b. The luciferase assay identified p27/kip1 and RND3 as target genes for miRNA-200b, which was confirmed by the expression of p27/kip1 and RND3 and their downstream products (cyclinE and cyclinD1) in the TGF-beta 1-treated cells. Transforming growth factor-beta 1 and miR-200b mimics enhanced the proliferation of HTFs; suppressed the expression of p27/kip1 and RND3; and subsequently stimulated the expression of cyclinE, cyclinD1, and PCNA. The miR-200b inhibitor attenuated the effects of TGF-beta 1 on HTFs. Furthermore, knockdown of p27/kip1 and RND3 resulted in an increase in cell proliferation and expression of the proliferation-related genes. CONCLUSIONS. MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.

Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation through the Rho kinase-dependent signaling pathway. We report a role of Rnd3 in the pathogenesis of hydrocephalus disorder. Mice with Rnd3 genetic deletion developed severe obstructive hydrocephalus with enlargement of the lateral and third ventricles, but not of the fourth ventricles. The cerebral aqueducts in Rnd3-null mice were partially or completely blocked by the overgrowth of ependymal epithelia. We examined the molecular mechanism contributing to this Rnd3-deficiency-mediated hydrocephalus and found that Rnd3 is a regulator of Notch signaling. Rnd3 deficiency, through either gene deletion or siRNA knockdown, resulted in a decrease in Notch intracellular domain (NICD) protein degradation. However, there was no correlated change in mRNA change, which in turn led to an increase in NICD protein levels. Immunoprecipitation analysis demonstrated that Rnd3 and NICD physically interacted, and that down-regulation of Rnd3 attenuated NICD protein ubiquitination. This eventually enhanced Notch signaling activity and promoted aberrant growth of aqueduct ependymal cells, resulting in aqueduct stenosis and the development of congenital hydrocephalus. Inhibition of Notch activity rescued the hydrocephalus disorder in the mutant animals.