[Double sites short hairpin RNAs targeting epidermal growth factor receptor to promote colon cancer cells apoptosis and enhance 5-fluorouracil chemotherapy effect].
OBJECTIVE
To investigate the differences about RNA interference (RNAi) technique which focuses on single or multiple sites to suppress colon cancer LoVo cell line's epidermal growth factor receptor (EGFR) mRNA and protein expression, induce cell apoptosis and enhance 5-fluorouracil (5-FU) sensitivity.


METHODS
The human colon cancer LoVo cells were transfected by liposome with pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors which were established by p Genesil-1 plasmid and EGFR short hairpin RNA (shRNA) synthesized in vitro, then were selected for 4 weeks by using G418. Five groups were selected for the study: Group 1: the normal cultured LoVo cells; Group 2: the negative control plasmid HK; Group 3: pU6-EGFR-shRNA-1 plasmid vector; Group 4: pU6-EGFR-shRNA-2 plasmid vector; Group 5: pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed using Real Time PCR and Western blot, the cell apoptosis was determined via flow cytometry, and the suppressive rate and IC(50) to LoVo cells by 5-FU of different concentrations and time points were carried out by using Cell Counting Kit-8 (CCK-8).


RESULTS
Expression plasmids encoding shRNA were successfully established and transfected into the LoVo cells. In group 3, 4 and 5, the mRNA expression was decreased by (80.2 +/- 3.4)%, (81.3 +/- 2.8)% and (90.6 +/- 2.8)%, respectively, and protein expression was decreased by (74.1 +/- 4.0)%, (73.4 +/- 2.3)% and (90.4 +/- 3.3)%, respectively; meanwhile, cell apoptosis increased by (10.4 +/- 0.5)%, (10.1 +/- 0.4)% and (14.2 +/- 0.5)%, respectively. The IC(50) of 5-FU and cell suppressive rate analysis demonstrated that there were significant differences among group 5, groups 3 and 4, and groups 1 and 2, but there were no significant difference between group 1 and group 2, as well as group 3 and group 4.


CONCLUSIONS
Both pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were capable of suppressing EGFR expression of LoVo cells, and therefore promoting apoptosis and increasing the cell toxicity of 5-FU. The targeting double combined sites RNAi technique was significantly better than single site interference. The new therapeutic modalities in the treatment of human colon cancer are suggested by this study.
Wu
Xiang-bai
XB
Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Tao
Kai-xiong
KX
Wang
Guo-bin
GB
Tian
Yuan
Y
Zhang
Jing-hui
JH
Chen
Dao-da
DD
D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D003131:Combined Modality Therapy; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D005472:Fluorouracil / Q000494:pharmacology*; D015316:Genetic Therapy; D006801:Humans; D012313:RNA / Q000235:genetics; D034622:RNA Interference; D014162:Transfection
D012313:RNA; D066246:ErbB Receptors; D005472:Fluorouracil