Retrovirus-mediated RNA inhibition of EphA2 gene expression in colon adenocarcinoma HCT-8 cells.
- Author:
Ren-yin CHEN
1
;
Shan-shan LI
Author Information
- Publication Type:Journal Article
- MeSH: Adenocarcinoma; metabolism; pathology; Cell Line, Tumor; Colonic Neoplasms; metabolism; pathology; Down-Regulation; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; RNA Interference; RNA, Messenger; biosynthesis; genetics; Receptor, EphA2; biosynthesis; genetics; Recombinant Proteins; biosynthesis; genetics; Retroviridae; genetics; Reverse Transcriptase Polymerase Chain Reaction; Transfection
- From: Chinese Journal of Pathology 2006;35(2):101-105
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore novel cancer gene therapy by retrovirus-mediated RNAi technique to suppress the endogenous EphA2 oncogene expression in colon adenocarcinoma HCT-8 cells.
METHODSSequence 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).
RESULTSThe 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).
CONCLUSIONSEphA2 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.
