Construction of colorectal cancer cell line stably expressing mir-101 and identification of the target gene of mir-101
LIU Yan
LU Yanxia
ZHOU Min
ZHANG Chao
LI Xuenong
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.
Keywords：Has-mir-101lentiviruscolorectal cancer cell linesdual-luciferase reporter assayRAC1
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 928-933 )