Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation
Authors Qi Y, Zhou F, Zhang L, Liu L, Xu H, Guo H
Received 15 February 2015
Accepted for publication 10 March 2015
Published 14 May 2015 Volume 2015:9 Pages 2647—2652
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 3
Editor who approved publication: Prof. Dr. Wei Duan
Yanmei Qi,1 Fengqiang Zhou,2 Lu Zhang,2 Lei Liu,2 Hong Xu,2 Huiguang Guo2
1Department of Gastroenterology, 2Department of General Surgery, Binzhou People’s Hospital, Binzhou, Shandong, People’s Republic of China
Background: Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells.
Methods: In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Results: The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P<0.01). Western blot analysis results showed that Ep-CAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (P<0.01). MTT assay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (P<0.05).
Conclusion: Silencing of Ep-CAM can significantly inhibit the proliferation of colorectal cancer cells.
Keywords: epidermal growth factor, Ep-CAM, colorectal cancer, vector construction
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