Construction of colorectal cancer cell line stably expressing mir-101 and identification of the target gene of mir-101
10.3969/j.issn.1673-4254.2014.07.03
- VernacularTitle:稳定过表达mir-101结直肠癌细胞株的建立及其靶基因的鉴定
- Author:
Yan LIU
1
;
Yanxia LU
;
Min ZHOU
;
Chao ZHANG
;
Xuenong LI
Author Information
1. 南方医科大学病理学系/广东省分子肿瘤病理学重点实验室
- Keywords:
Has-mir-101;
lentivirus;
colorectal cancer cell lines;
dual-luciferase reporter assay;
RAC1
- From:
Journal of Southern Medical University
2014;(7):928-933
- CountryChina
- Language:Chinese
-
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.
