Effect of small interfering RNA targeting Rac1 gene on colony formation of SW480 cells in vitro.
- Author:
Qing-zhen NAN
1
;
Lei GAO
;
Bing XIAO
;
Zhen-shu ZHANG
;
Bo JIANG
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; pathology; Down-Regulation; Humans; Molecular Sequence Data; RNA Interference; RNA, Small Interfering; genetics; Transfection; rac1 GTP-Binding Protein; genetics
- From: Journal of Southern Medical University 2010;30(6):1339-1342
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a vector expressing small interfering RNA (siRNA) against Rac1 gene and observe its effect on soft agar colony formation of SW480 cells in vitro.
METHODSOligos of 64 base pairs for hairpin RNA targeting Rac1 were chemically synthesized and annealed. The siRNA constructs for Rac1, produced by inserting the annealed oligos into the downstream of H1 promoter of linearized pSUPER, were confirmed by restriction digestion and DNA sequencing. The constructed Rac1-siRNA was transfected into SW480 cells and Western blotting was performed to assess the expression and interference efficiency of siRNAs against Rac1.The soft agar colony formation assay was used to study the effect of Rac1 gene silencing on SW480 cells.
RESULTSRestriction digestion and DNA sequencing showed that the siRNA targeting Rac1 gene was successfully constructed. The siRNA could effectively down-regulate the expression of Rac1 in SW480 cells. Soft agar colony formation assay showed that the colony number and diameter of SW480 cells was reduced after siRNA transfection.
CONCLUSIONA vector expressing hairpin RNA against Rac1 gene are successfully produced, which significantly reduces the colony numbers and size of SW480 cells in vitro, suggesting that Rac1 plays an important role in the growth of colorectal cancer in vitro.
