Objective      To analyze the effect of miR-34a on NOTCH1 gene expression and SW480 cell proliferation.       Methods      NOTCH1 was predicted to be the specific target gene of miR-34a by bioinformatics. The dual luciferase vector containing 3′-UTR of NOTCH1 gene was constructed

and the 3′-UTR was reg

arded as the binding site of miR-34a. The effect of miR-34a interaction with the 3′-UTR of NOTCH1 on luciferase activity was detected with a dual luciferase assay system. The expression level of NOTCH1 protein affected by miR-34a was detected by Western blotting. The proliferation of SW480 cells transfected with miR-34a was measured by MTT assay and flow cytometry.       Results      The dual luciferase recombinant vector containing the 3′-UTR of NOTCH1 gene was successfully constructed and verified by enzyme digestion and gene sequencing. The luciferase activity significantly reduced to 53.4% in the SW480 cells co-transfected with the recombinant vectors and miR-34a mimics as compared with the SW480 cells transfected with empty vectors (P=0.003 8)

while the luciferase activity significantly was enhanced to 145% in the HEK293 cells co-transfected with the recombinant vectors and miR-34a inhibitors as compared with the HEK293 cells transfected with empty vectors (P=0.002 1). The results of the luciferase assay revealed that miR-34a could negatively regulate the luciferase activity by interacting with the 3′-UTR of NOTCH1. Western blotting results demonstrated that the NOTCH1 protein level in the SW480 cells transfected with miR-34a mimics decreased by 53.6% as compared with the untreated SW480 cells (P0.05)

while the NOTCH1 protein level in the HEK 293 cells transfected with miR-34a inhibitors increased by 78.9% as compared with the untreated HEK293 cells (P=0.03). NOTCH1 protein expression was negatively regulated by miR-34a. The growth of SW480 cells transfected with miR-34a was much slower than that of the untreated SW480 cells

and the cells were arrested at G

0

-G

1

phase

suggesting miR-34a overexpression could inhibit the proliferation of SW480 cells.       Conclusion      miR-34a negatively targetedly regulates the expression of NOTCH1 and inhibits the proliferation of SW480 cells.