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Published on: July 3, 2013
Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation
Yanmei Qi1, Fengqiang Zhou2, Lu Zhang2
1Department of Gastroenterology, 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.
Insights
Silencing the Ep-CAM gene significantly inhibits colorectal cancer cell proliferation. This study constructed an interference plasmid to target Ep-CAM, demonstrating its suppressive effect on cancer cell growth.
Area of Science:
- Oncology
- Molecular Biology
- Gene Expression Regulation
Background:
- Abnormal protein expression and function are linked to colorectal cancer (CRC) development and progression.
- Gene expression alterations play a critical role in CRC pathogenesis.
Purpose of the Study:
- To create an interference plasmid targeting the Ep-CAM gene.
- To evaluate the impact of Ep-CAM gene silencing on colorectal cancer cell proliferation.
Main Methods:
- Utilized HT-29 and HCT-116 colorectal cancer cell lines.
- Constructed miRNA interference plasmids targeting Ep-CAM and transfected them using Lipofectamine™ 2000.
- Assessed transfection efficiency via RT-PCR, Ep-CAM protein levels by Western blot, and cell proliferation using MTT assays.
Main Results:
- Ep-CAM mRNA and protein expression were significantly reduced in the interference group compared to control and empty plasmid groups (P<0.01).
- Cell proliferation was significantly inhibited in the Ep-CAM interference group (P<0.05).
Conclusions:
- Ep-CAM gene silencing effectively suppresses colorectal cancer cell proliferation.
- Targeting Ep-CAM presents a potential therapeutic strategy for colorectal cancer.
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