Abstract:

Objective To identify and verify the target gene of miRNA-449 and explore the molecular mechanism of regulating the proliferation activity of neuroblastoma cell SH-SY5Y by miRNA-449. Methods Expressions of miRNA-449 and NOTCH1 protein in neuroblastoma tissues and adjacent tissues were detected and their correlation was analyzed. The binding site between miRNA-449 and 3′UTR region of NOTCH1 gene was predicted with bioinformatic software and verified with luciferase reporter gene assay. The mimic, inhibitor, and scrambled sequence of miRNA-449 were chemically synthesized and transfected to SH-SY5Y cells via liposome. Expressions of miRNA-449 and NOTCH1 protein were detected by real-time PCR and Western blotting 48 h after transfection, respectively. The proliferation activity of neuroblastoma cells was measured by CCK-8 method 24, 48, and 72 h after transfection. Results Compared with adjacent tissues, the miRNA-449 expression in neuroblastoma tissues was significantly higher and the NOTCH1 protein expression was significantly lower. The differences were statistically significant (P=0.002; P=0.021). There was a binding site 5′-CACTGCC-3′ in 3′UTR region of NOTCH1 gene. Gene expression was negatively regulated via the binding site. Transfection of SH-SY5Y cells with miRNA-449 mimic significantly inhibited the NOTCH1 protein expression (P=0.006), whereas transfection with miRNA-449 inhibitor significantly increased the NOTCH1 protein expression (P=0.018).miRNA-449 mimic significantly inhibited the proliferation activity of SH-SY5Y cells and miRNA-449 inhibitor significantly enhanced the proliferation activity at time points of 48 and 72 h. Conclusion miRNA-449 can inhibit the proliferation activity of SH-SY5Y cells via inhibiting the expression of its target gene NOTCH1.

Key words: miRNA-449, SH-SY5Y cell, luciferase, NOTCH1, cell viability