[MicroRNA-34a regulates cell cycle by targeting CD44 in human bladder carcinoma cells].
Yu Gan, Kai Xu, Shian Xu, Xiaolan Zhang, Qianhua Huang, Bin Lang
Abstract
Yu Gan, Kai Xu, Shian Xu, Xiaolan Zhang, Qianhua Huang, Bin Lang
Abstract
OBJECTIVE: To investigate the role of microRNA-34a (miR-34a) in regulating the cell cycles of bladder cancer cell line J82 and explore the underlying mechanism. METHODS: J82 cells were transfected with a miR-34a mimic or an inhibitor to induce miR-34a overexpression or silencing. The RNA level of miR-34a in the transfected cells was detected by real-time PCR, and CD44 expressions at the mRNA and protein levels were detected by real-time PCR and Western blotting. Luciferase reporter assay was used to detect the activation of 3'UTR of CD44, and flow cytometry was performed to analyze the cell cycle changes. RESULTS: The expression level of miR-34a was significantly increased and CD44 expression significantly lowered in cells transfected with miR-34a mimic; miR-34a inhibitor transfection caused reverse effects on miR-34a and CD44 expressions. MiR-34a mimics downregulated while miR-34a inhibitor enhanced the activation of 3'UTR of CD44 with corresponding changes in the expressions of some cell cycle-related proteins. MiR-34a mimics and miR-34a inhibitor induced opposite changes in J82 cell cycle, which were partly reversed by CD44. CONCLUSION: MiRNA-34a regulates cell cycles by targeting CD44 in human bladder carcinoma cell line J82.
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OBJECTIVE: To investigate the role of microRNA-34a (miR-34a) in regulating the cell cycles of bladder cancer cell line J82 and explore the underlying mechanism. METHODS: J82 cells were transfected with a miR-34a mimic or an inhibitor to induce miR-34a overexpression or silencing. The RNA level of miR-34a in the transfected cells was detected by real-time PCR, and CD44 expressions at the mRNA and protein levels were detected by real-time PCR and Western blotting. Luciferase reporter assay was used to detect the activation of 3'UTR of CD44, and flow cytometry was performed to analyze the cell cycle changes. RESULTS: The expression level of miR-34a was significantly increased and CD44 expression significantly lowered in cells transfected with miR-34a mimic; miR-34a inhibitor transfection caused reverse effects on miR-34a and CD44 expressions. MiR-34a mimics downregulated while miR-34a inhibitor enhanced the activation of 3'UTR of CD44 with corresponding changes in the expressions of some cell cycle-related proteins. MiR-34a mimics and miR-34a inhibitor induced opposite changes in J82 cell cycle, which were partly reversed by CD44. CONCLUSION: MiRNA-34a regulates cell cycles by targeting CD44 in human bladder carcinoma cell line J82.
Key concepts: CD44, Transfection, microRNA, Flow cytometry, Cell cycle, Gene silencing, Cell culture, Cancer research