ABSTRACT
CD44 is a marker of cancer stem cell (CSC) in many types of tumors. Alternative splicing of its 20 exons generates various CD44 isoforms that have different tissue specific expression and functions, including the CD44 standard isoform (CD44s) encoded by the constant exons and the CD44 variant isoforms (CD44v) with variant exon insertions. Switching between the CD44v and CD44s isoforms plays pivotal roles in tumor progression. Here we reported a novel mechanism of CD44 alternative splicing induced by TGF-ß1 and its connection to enhanced epithelial-to-mesenchymal transition (EMT) and stemness in human prostate cancer cells. TGF-ß1 treatment increased the expression of CD44s and N-cadherin while decreased the expression of CD44v and E-cadherin in DU-145 prostate cancer cells. Other EMT markers and cancer stem cell markers were also upregulated after TGF-ß1 treatment. RNAi knockdown of CD44 reversed the phenotype, which could be rescued by overexpressing CD44s but not CD44v, indicating the alternatively spliced isoform CD44s mediated the activity of TGF-ß1 treatment. Mechanistically, TGF-ß1 treatment induced the phosphorylation, poly-ubiquitination, and degradation of PCBP1, a well-characterized RNA binding protein known to regulate CD44 splicing. RNAi knockdown of PCBP1 was able to mimic TGF-ß1 treatment to increase the expression of CD44s, as well as the EMT and cancer stem cell markers. In vitro and in vivo experiments were performed to show that CD44s promoted prostate cancer cell migration, invasion, and tumor initiation. Taken together, we defined a mechanism by which TGF-ß1 induces CD44 alternative splicing and promotes prostate cancer progression.
Subject(s)
DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition/drug effects , Hyaluronan Receptors/metabolism , RNA-Binding Proteins/metabolism , Transforming Growth Factor beta1/pharmacology , Alternative Splicing , Animals , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Humans , Hyaluronan Receptors/antagonists & inhibitors , Hyaluronan Receptors/genetics , Male , Mice , Mice, Nude , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase Inhibitors/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , RNA, Small Interfering/therapeutic use , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transplantation, HeterologousABSTRACT
Ubiquitin-like with PHD and ring finger domains 2 (Uhrf2) is distributed in many brain regions, including the cortex and hippocampus. Decreased Uhrf2 expression is involved in neurodegenerative disease. A recent study showed Uhrf2 deletion impaired spatial memory; however, the mechanism remains elusive. In our study, we determined that Uhrf2+/- and Uhrf2-/- mice had significant learning and memory deficiencies in contextual fear conditioning (CFC) and the novel place recognition test but not in the novel object recognition test. Interestingly, there were no changes in the Uhrf2 protein levels in the hippocampus of C57BL6 mice after CFC training, which suggests Uhrf2 in adult mice may not be related to the formation of CFC long-term memory. Based on Nissl staining, Uhrf2 deletion caused neuropathological changes specifically in the crest of the dentate gyrus (DG), such as cell swelling, a vague outline and confused boundary; however, no changes were identified in the medial prefrontal cortex (mPFC). Transmission electron microscope assay further indicated a series of abnormal ultrastructure changes in neurons and glia in the DG crest. These results suggested that Uhrf2 deletion selectively blocked the development of the DG crest and impaired hippocampus-dependent learning and memory. Our study will facilitate a better understanding of the role of Uhrf2 protein in the central nervous system.
Subject(s)
Dentate Gyrus/pathology , Memory Disorders/genetics , Memory Disorders/pathology , Ubiquitin-Protein Ligases/deficiency , Animals , Conditioning, Classical/physiology , Dentate Gyrus/ultrastructure , Disease Models, Animal , Exploratory Behavior/physiology , Fear/psychology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron , Recognition, Psychology/physiology , Spatial Learning/physiology , Ubiquitin-Protein Ligases/geneticsABSTRACT
OBJECTIVE: To study the expression of long nonîcoding RNA (lncRNA) H19 in human prostate cancer tissue and its effect on the glycometabolism and growth of human prostate cancer cells. METHODS: Realîtime quantitative RTîPCR (qRTîPCR) was employed to detect the expression of lncRNA H19 in human prostate tissues from 20 patients with prostate cancer (10 cases of highîGleason score prostate cancer ï¼»HGPCï¼½ and 10 cases of lowîGleason score prostate cancer ï¼»LGPCï¼½) and another 5 with benign prostatic hyperplasia (BPH). After transfection of H19 siRNA into the DU145 and PCî3 prostate cancer cells, the growth of the cells and the H19 expression in the cells were determined by MTT and qRTîPCR respectively, and the changes in the glycometabolism of the prostate cancer cells were analyzed by measuring the contents of glucose and lactate in the culture medium. Nonîtransfected and transfected negative vectors were used as blank and negative controls respectively. RESULTS: The relative expression of H19 was significantly increased in both the HGPC and LGPC tissues (0.725±0.385 and 2.086±0.542) as compared with that in the BPH tissue (0.210±0.068) (P< 0.01), even higher in the HGPC than in the LGPC tissue (P< 0.01). After transfection of H19 siRNA, the expressions of H19 were remarkably decreased in the DU145 and PCî3 prostate cancer cells in comparison with those in the blank control and negative control groups (P< 0.01), and so were the proliferation of and the glucose and lactate levels in the DU145 and PCî3 cells (P< 0.01).