Abstract: Objective To construct a colorectal cancer cell line stably expressing mir-101 and identify the target gene of mir-101.
Methods Quantitative real-time PCR was used to detect mir-101 expression in colorectal cancer cell lines. The recombined
lentiviral vector GV209-mir101 or the empty lentiviral vector GV209 was transfected into human colorectal cancer cells SW620.
The recombinant psiCHECK-2-Rac1 vector containing RAC1 3’UTR was constructed, and site-directed mutagenesis of RAC1
3’UTR was induced to construct the psiCHECK-2-Rac1-Mut vector. In HEK293A and SW480 cells co-transfected with mir-101
inhibitors or negative control (NC) and these recombined vectors, luciferase activities was examined with a dual-luciferase
reporter assay. Results SW620 cells transfected with GV209-mir101 lentivirus exhibited higher mir-101 expression level than
cells transfected with GV209 lentivirus. Mir-101 inhibitors significantly increased the luciferase activities of RAC1 3’UTR.
Overexpression of mir-101 increased the expression of RAC1 while inhibition of mir-101 suppressed RAC1 expression.
Conclusion We have successfully constructed a SW620 cell line stably overexpressing mir-101. mir-101 can suppress RAC1
gene expression by targeting the specific sequence of RAC1 3’UTR.