Combining siRNAs at two different sites in the EGFR to suppress its expression, induce apoptosis, and enhance 5-fluorouracil sensitivity of colon cancer cells.
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. IC(50) 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 IC(50) 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.
Wu
Xiangbai
X
Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Deng
Yongzhi
Y
Wang
Guobin
G
Tao
Kaixiong
K
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy*; D003131:Combined Modality Therapy / Q000379:methods; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D066246:ErbB Receptors; D005472:Fluorouracil