RESUMO
Selenoprotein P (Sepp1), a glycoprotein rich in selenium, is thought to function in selenium transport throughout the body. The sepp1 gene locus potentially produces three alternative transcripts that differ only in their 5' untranslated regions (5'UTRs) and not in their protein coding regions, as indicated by transcript information in genomic databases. Here we investigated the distribution, relative expression, and biological significance of these transcript variants. We confirmed the expression of Sepp1 transcript variants using PCR and sequencing. Using 5'-RACE, we identified multiple 5'-termini upstream from three different splice donor sites, and a single splice acceptor site for exon 2. We found regional and temporal changes in variant expression in select adult and neonate murine tissue and brain regions. Distribution of variants in heart and kidney varied with stage of development. Notably, the Sepp1b variant was localized specifically to the hippocampus in brain. Targeted silencing of individual variants using RNAi demonstrated the biological importance for all transcript variants in cell viability. Additionally, we determined that the Sepp1b variant is a specific target for the miR-7 microRNA by means of its unique 5'UTR structure. Our results emphasize the importance of non-coding transcript variations as a regulatory means for Sepp1 expression in different tissues and stages of development. The presence of a variant localized in the hippocampus and regulated by a microRNA may have implications for the known deficits in synaptic function caused by genetic deletion of Sepp1.
Assuntos
Processamento Alternativo/genética , RNA não Traduzido/genética , Selenoproteína P/genética , Selenoproteína P/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Transporte de Íons , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Isoformas de Proteínas , Interferência de RNA , Sítios de Splice de RNA , RNA Interferente Pequeno , Selênio/metabolismo , Análise de Sequência de RNA , Regiões não Traduzidas/genéticaRESUMO
The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.