EXPERIMENTAL STUDY
Effect of blocking Ras signaling pathway with K-Ras siRNA on apoptosis in esophageal squamous carcinoma cells

https://doi.org/10.1016/S0254-6272(13)60179-XGet rights and content
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Abstract

Objective

To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells.

Methods

EC9706 cells were treated in the following six groups: blank group (no transfection), negative control group (transfection no-carrier), transfection group (transfected with pSilencer-siK-ras), taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and western blotting were used to analyze the expression of MAPK1 (mitogen-activated protein kinases 1) and cyclin D1 in response to siRNA (small interfering RNA) transfection and taxol treatment.

Results

K-Ras (K-Ras gene) siRNA transfection of EC9706 esophageal squamous carcinoma cells decreased the expression of K-Ras, MAPK1 and cyclin D1 at the mRNA and protein level. Reverse transcription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group (P<0.05). Western blotting showed that 72 h after EC9706 cell transfection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the expression levels in the transfection group were significantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as compared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe.

Conclusion

Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-mediated inhibition of Ras signaling, with the most obvious increase observed in the transfection plus taxol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeutic agent, taxol. Ras knockdown also down-regulated the expression of the downstream genes, MAPK1 and cyclin D1, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.

Key words

RNA, small interfering
Genes, ras
Ras signal pathway
Esophageal neoplasms
Taxol
Apoptosis
Chemotherapy sensitivity

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Supported by the Natural Science Research Program of the Education Department of Henan Province (No. 2011B320012)