Experimental Research On Breast Cancer Therapy Using SiRNA Targeting HER-2

HER-2 (also known as neu, c-erbB-2) is a member of the epidermal growth factor receptor (EGFR) family. Overexpression of HER-2 is found in 20-40% of human breast cancers, and correlates with breast cancer occurrence, metastasis and prognosis. HER-2 overexpression is a poor prognostic factor and may be associated with increased resistance to cancer chemotherapy, radiotherapy, endocrine therapy and co-adjuvant therapy. In recent years, HER-2 gene and p185^HER-2 have already become the ideal targets for the treatment of breast cancer. Herceptin, a humanized monoclonal antibody directed against the extracellular domain of HER-2, is the first approved biotherapy drug targeting breast cancer. Addition Herceptin to chemotherapy produces significant survival benefits in HER-2 positive metastatic breast cancer patients. However, Herceptin recognizes p185^HER-2 protein, it could not inhibit HER-2 gene expression directly. Gene therapy based on antisense technologies could directly and effectively inhibit the expression of HER-2 gene. At present, RNA interference (RNAi) against special mRNA supplies a new method for gene therapy. The purpose of this study is to find appropriate small interfering RNAs (siRNAs) targeting HER-2 for breast cancer gene therapy.First, six sequences of human HER-2 mRNA were obtained from GenBank. Among these sequences, 4624bp mRNA sequence (serial number: NM₀04448.2) has higher alignment score with other five sequences and contains complete coding DNA sequences (CDS). So this mRNA sequence was used as the target to design siRNA against HER-2. Begin with the AUG start codon and scan the length of the gene for AA dinucleotide sequences. Record the AA and its 3' adjacent 19 nucleotides as potential siRNA target sites. Finally, five siRNAs with 40-50% G/C content and no similarity to other mRNA were selected from the CDS of this mRNA. Then five plasmids expressing hairpin siRNA were constructed and used to transfect MCF-7 breast carcinoma cells. After selection, five stable siRNA-producing cell lines were obtained. The levels of HER-2 mRNA and protein in the cells were quantitated by RT-PCR and flow cytometry, respectively. The results showed that all the five kinds of siRNA could effectively decrease HER-2 mRNA level in these cells. Of them, siRNA 3P could almost completely eliminate HER-2 mRNA in the cells. As to HER-2 protein expression, siRNA 3P and 2P exhibited obvious inhibition effects.