ABSTRACT
MicroRNAs (miRNAs or miRs) are a class of short, non-coding RNAs that participate in various oncological processes. This study aims to explore the roles of microRNA-34a (miR-34a) in invasive urothelial bladder carcinoma. miR-34a was transfected into bladder cancer cell lines 253J and J82. The miR-34a expression levels in tissues and cells were detected by using qRT-PCR. The Notch1 expression was detected by qRT-PCR and Western blotting. Cell migratory and invasive abilities were measured by Transwell chamber assay. Bioinformatics and luciferase assay were performed to predict and analyze the binding sites between miRNA-34a and Notch1. It was found that there was aberrant expression of miR-34a in bladder cancer tissues. Moreover, we revealed that ectopic expression of miR-34a suppressed cell migration and invasion, while forced expression of Notch1 increased cell migratory and invasive abilities. Finally, we observed that miR-34a transfection significantly down-regulated luciferase activity and reduced the mRNA and protein levels of Notch1. Our study concluded that microRNA-34a antagonizes Notch1 and inhibits cell migration and invasion of bladder cancer cells, which indicates the tumor-suppressive function of microRNA-34a in bladder cancer.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Cell Movement , Genetics , Down-Regulation , Genetics , Gene Targeting , MicroRNAs , Genetics , Neoplasm Invasiveness , Receptor, Notch1 , Physiology , Transfection , Tumor Cells, Cultured , Urinary Bladder Neoplasms , PathologyABSTRACT
MicroRNAs (miRNAs or miRs) are a class of short, non-coding RNAs that participate in various oncological processes. This study aims to explore the roles of microRNA-34a (miR-34a) in invasive urothelial bladder carcinoma. miR-34a was transfected into bladder cancer cell lines 253J and J82. The miR-34a expression levels in tissues and cells were detected by using qRT-PCR. The Notch1 expression was detected by qRT-PCR and Western blotting. Cell migratory and invasive abilities were measured by Transwell chamber assay. Bioinformatics and luciferase assay were performed to predict and analyze the binding sites between miRNA-34a and Notch1. It was found that there was aberrant expression of miR-34a in bladder cancer tissues. Moreover, we revealed that ectopic expression of miR-34a suppressed cell migration and invasion, while forced expression of Notch1 increased cell migratory and invasive abilities. Finally, we observed that miR-34a transfection significantly down-regulated luciferase activity and reduced the mRNA and protein levels of Notch1. Our study concluded that microRNA-34a antagonizes Notch1 and inhibits cell migration and invasion of bladder cancer cells, which indicates the tumor-suppressive function of microRNA-34a in bladder cancer.
ABSTRACT
ObjectiveTo investigate the effects of over expression of miR-34a on cellular proliferation and migration in bladder cancer cell line J82 by targeting Notchl.MethodsmiR-34a was predicted as a putative gene which can target Notchl through bioinformatics analysis,qRT-PCR and Western blot were performed to measure the expression levels of Notchl and miR-34a in invasive transitional cell carcinoma of bladder (TCCB) tissues and J82 cells transfected with miR-34a.Luciferase assay was employed to determine if miR-34a could target Notchl through binding to the 3'-untranslated region (3'UTR) of Notchl mRNA.J82 cells were transfected with pcDNA3.0-miR-34a or pcDNA3.0 control plasmid.MTS colorimetry was used to evaluate the effect of miR-34a on cell proliferation.The effect of miR-34a on cell migration was assessed by transwell migration assay.ResultsThe expression level of miR-34 in invasive TCCB tissues was lower than in adjacent bladder tissues (0.016(0.018) vs 0.042 (0.059),N =16; P =0.0006).On the contrary,the average levels of Notchl mRNA and protein were higher in tumors than in adjacent bladder tissues (2.765(2.156) vs 2.312(1.365),N =16; P =0.0025 and 0.857 ±0.197 vs 0.648 ±0.171 ;P ＜0.0001 ).After the transfection of miR-34a,the expressive level of miR-34a in J82 was highly induced ( (2.408 ±0.789) × 10-4 vs(0.153 ±0.029) × 10-4; P =0.0026).However,the expressive levels of Notchl mRNA and protein were obviously decreased (3.001 ± 0.106 vs 4.998 ± 1.053 ; P =0.0308 and 0.747 ± 0.050 vs 0.988 ± 0.102 ; P =0.0215 ).The results of luciferase assay showed that firefly activity was highly dimished (0.422 ± 0.028 vs 2.392 ± 0.148 ; P ＜ 0.0001 ).Cellular proliferation was inhibited after the transfection of miR-34a in J82 (P ＜ 0.0001 ).Moreover,number of migration cells of J82 was significantly reduced after the ectopic expression of miR-34a ( 179.3 ± 21.02 vs 269.7 ± 23.71 ; P =0.0078 ).ConclusionsmiR-34a inhibits the cellular proliferation and migration of bladder cancer cell line J82 via binding to the 3UTR of Notchl mRNA.