Inhibited Proliferation Of Human Breast Carcinoma Cells By RNA Interference-based Knockdown Of ErbB2/HER2

RNA interference (RNAi) represents a phenomenon of double-stranded RNA (dsRNA)-mediated post-transcriptional gene silencing (PTGS). During this process, exogenous cellular dsRNA is cleaved by an ribonuclease, Dicer, to generate 19-23 bp small RNA fragments, referred to as small interfering RNA (siRNA). By joining an effector complex termed RISC (RNA-induced silencing complex), siRNA binds to the cellular RNA with homologous sequences, and contributes to the degradation of the corresponding RNA. At present, RNAi has been widely applied in the research of gene fuction and gene therapy as an efficient tool for specific gene silencing.The c-ErbB receptor tyrosine kinase family is composed of four known members, epidermal growth factor receptor (EGFR/c-ErbB-1), c-ErbB-2,c-ErbB-3, and c-ErbB-4. c-ErbB-2 is a 185-kDa transmembrane phosphoglycoprotein and is the most commonly studied member of this family in breast cancer. There are at least 9 ligands with selectivity for one or more c-ErbB receptors. EGF, TGFα, AR, HB-EGF, BTC (beta-cellaulin), EPR (epiregulin), HRG (heregulin), NRG-2 (neuregulin-2) and NRG-3 (neuregulin-3). It is postulated that coexpression of c-ErbB receptors and ligands leads to receptor activation, stimulation of tumor cell proliferation, and apoptosis resistance, thus providing a survival advantage of cells. In addition, both clinical and experimental evidence suggests a link of c-ErbB-2, not just to tumor growth but also to breast cancer progression, including acquisition of a metastatic phenotype and some forms of drug resistance. However, c-ErbB-2 cannot be considered separately since it functions as part of a multimolecular complex with other receptors, their ligands, and many other components of the cell membrane and intracellular machinery.In principle, RNAi can be achieved by chemically synthesized RNA duplexes, or hairpin RNAs of 19-23 base pair stem-loop structures, which are expressed from an H1 or U6 promoter of an eukaryotic expression vector, e.g. a pSUPER vector harboring a H1 promoter. Here we constructed the expression vectors of 2 siRNA, i.e. sihe1 and sihe2, which targeted to the 548–566 nt and 1811–1829 nt of the transcript of the oncogene erbB2/HER2/neu, respectively. The constructs were introduced into the human breast carcinoma SKBr3 cells which overexpress HER2, followed by selection with G418. The resulting cell clones were subject to a further examination. As expected, both reverse transcription PCR (RT-PCR) and flow cytometry (FCM) revealed a potent inhibition of the expression of HER2. The transfected cells exhibited a much lower rate of growth and proliferation in comparison with the untransfected cells and cells mock transfected with a pSUPER vector. The present study provides in vitro validation for a novel strategy to treat HER2-positive tumors by RNA interference-based knockdown of the corresponding oncogene.