Combining siRNAs at Two Different Sites in the EGFR to Suppress its Expression, Induce Apoptosis, and Enhance 5-Fluorouracil Sensitivity of Colon Cancer Cells

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• 作者：Xiangbai Wu ; Yongzhi Deng ; Guobin Wang ; Kaixiong Tao
• 刊名：Journal of Surgical Research
• 出版年：2007
• 出版时间：March 2007
• 年：2007
• 卷：138
• 期：1
• 页码：56-63
• 全文大小：1181 K

文摘

Background

The epidermal growth factor receptor (EGFR) has played an important role in the growth and apoptosis of colon cancer. The RNA interference (RNAi) technique can suppress gene expression, but the effects of double combining sites RNAi targeting EGFR have not been well understood.

Methods

pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors were transfected to the LoVo cells. Five groups were selected for the study: Group 1, the control cells; group 2, the negative control plasmid vector HK; group 3, pU6-EGFR-shRNA-1; group 4, pU6-EGFR-shRNA-2; group 5, pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed by Real Time quantitative PCR and Western blot. Apoptosis was determined via flow cytometry. IC₅₀ and the inhibition ratio of 5-fluorouracil (5-FU) were carried out by CCK-8.

Results

In groups 3, 4, and 5, the mRNA expression was decreased by (80.22 ± 3.42) % , (81.30 ± 2.83) % , and (90.58 ± 2.76) % , respectively, and the protein expression was decreased by (74.11 ± 4.02) % , (73.39 ± 2.30) % , and (90.39 ± 3.34) % , respectively. Meanwhile, the cell apoptosis increased by (10.43 ± 0.49) % , (10.13 ± 0.39) % , and (14.17 ± 0.53) % , respectively. The IC₅₀ of 5-FU and cell inhibition ratio analysis demonstrated that there were significant differences between the following three: group 5, groups 3 and 4, and groups 1 and 2.

Conclusions

Both pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 are capable of suppressing EGFR expression of the LoVo cell and can promote apoptosis and increase the cell toxicity of 5-FU. The double combining sites RNAi technique is significantly better than a single site.