[Retrovirus-mediated RNA inhibition of EphA2 gene expression in colon adenocarcinoma HCT-8 cells].
OBJECTIVE
To explore novel cancer gene therapy by retrovirus-mediated RNAi technique to suppress the endogenous EphA2 oncogene expression in colon adenocarcinoma HCT-8 cells.


METHODS
Sequence information of EphA2 mRNA was selected and two complementary oligonucleotides with hairpin loop were designed. Retrovirus-mediated RNAi expression vector (pSIREN-EphA2) was then constructed and transfected into the HCT-8 cells. Inhibition of EphA2 protein expression was quantitatively determined by Western blot and immunohistochemistry assay (SP method).


RESULTS
The construction of pSIREN-EphA2 vector was successful and confirmed by restriction enzyme analysis and DNA sequencing. The post-transfection level of EphA2 protein expressions was greatly reduced in HCT-8 cells transfected with pSIREN-EphA2, as compared with those of untransfected cells and the vector control (P < 0.001).


CONCLUSIONS
EphA2 protein expression in HCT-8 cell line can be suppressed using recombinant retrovirus-mediated RNAi technique. This approach may provide a novel gene therapy against colonic adenocarcinoma.
Chen
Ren-yin
RY
Department of Pathology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China.
Li
Shan-shan
SS
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D036104:Receptor, EphA2 / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D036104:Receptor, EphA2