Study Of HER2 Gene Silencing By RNA Interference

The HER2 gene is a member of the epidermal growth factor (EGF)-receptor (EGFR) family and encodes a 185 kDa protein with tyrosine kinase activity. The amplification or enhanced transcription of the HER2 gene is associated with the oncogenesis or increased severity of several types of cancers. The HER2 protein can dimerize with other members of the EGFR family, and it is thought that overexpression of the HER2 gene product provides mitogenic signals to tumor cells, resulting in increased growth potential and rendering them resistant to apoptotic factors. Overexpression of HER2 occurs primarily through amplification of the wild-type HER2 gene and is associated with poor disease-free survival and may be associated with resistance to certain types of chemotherapy.HER2 has become an important therapeutic target in breast cancer for several reasons. (1) HER2 levels correlate strongly with the pathogenesis and prognosis of breast cancer. (2) The level of HER2 in human cancer cells with gene amplification is much higher than that in normal adult tissues, potentially reducing the toxicity of HER2 -targeting drugs. (3) HER2 is present in a very high proportion of tumor cells, and tumors with high expression often show uniform, intense immunohistochemical staining, suggesting that anti- HER2 therapy would target most cancer cells in a given patient. (4) HER2 overexpression is found in both the primary tumor and metastatic sites, indicating that anti- HER2 therapy may be effective in all disease sites.HER2-targeting strategies include:(1) Strategies target HER2 protein, include exogenous blocking antibodies, heterdimerization disrupting antibodies, endogenous blocking antibodies induced by HER2 specific vaccines, tyrosine kinase inhibitors, single chain antibodies, etc. Trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2, is currently used for clinical therapy. Trastuzumab combined with chemotherapy increases response rates, time to progression, and survival. However, the majority of cancers that initially respond to trastuzumab begin to progress again within 1 year. (2) Strategies target HER2 gene, such as transcription repressors, antisense oligonucleotides, ribozymes, antisense RNA, etc. Each strategy has its advantages and disadvantages.RNA interference (RNAi) is a form of post-transcriptional gene silencing(PTGS) in which double-stranded RNA(ds RNA) induces degradation of the homologous endogenous transcripts, mimicking the effect of the reduction, or loss, of gene activity. Small interfering RNAs (siRNAs) of approximately 21-23bp mediate this sequence-specific mRNA degradation. RNAi blocks the expression of endogenous gene in a sequence-specific and high-effective manner. Its effects can be delivered between cells, even are hereditary. These characteristics make it a high-selective and powerful strategy for gene therapy and gene functional study.In our study, we constructed 3 siRNAs against separate regions of the HER2 mRNA, whose sequences are 5'-TCTCTGCGGTGGTTGGCATTC -3' (named H1), 5'-GGGAAACCTGGAACTCACCTAC -3' (named H2), 5'-AAGGGGCTGGCTCCGATGTATTT -3' (named H3), separately; and one nonsense control sequence 5'-GACTTCATAAGGCGCATGC -3' (named CON). They were directed into plasmids- pGenesil-1 vector. DNA sequencing of the plasmids verified the successful construction of 3 HER2-specific and one control siRNA vectors. Then, they were transfected into HER2- positive tumor cells, SK-BR-3 and SK-OV-3, with METAFECTENE. Positive clones of infected cells were selected for 21 days using G418 (250μg /ml). The ability of siRNAs to downregulate HER2 expression was detected by RT-PCR and Western blot. CCK- 8(Cell counting kit- 8) colorimetry was used for growth assays and taxol (paclitaxel) resistance detection. Cell cycle analysis was done with flow cytometry.After stable transfection with HER2-specific siRNAs, SK-BR-3 cells became wrinkled, rounded, some sheded from the growing surface under microscopy. The positive clones grew too slow to form a layer on the growing surface, which indicated significant growth suppression of HER2-specific siRNAs to SK-BR-3 cells. Further detections to these clones were not done because of the difficulty to acquire enough cells.Stable transfection with HER2-specific siRNAs in SK-OV-3 cells resulted in slower growth while similar morphology to parental cells. Detections with RT-PCR and Western Blot showed sequence-specific decrease in HER2 mRNA and protein levels. Survival fractions of HER2 gene silenced cells were statistically decreased compared with HER2 gene highly expressed cells (P =0.000). Cell cycle analysis showed increased numbers of HER2 gene silenced cells in non-proliferative and apoptotic stages (P all lower than 0.01), while decreased number in synthetic stage compared with those of HER2 gene highly expressed cells (P ≤0.001). The cell cycles of HER2 gene silenced cells mainly arrested at G0/G1. siRNAs treatment also resulted in markedly increased cellular taxol resistance in SK-OV-3 cells, consistent with a powerful RNA silencing effect.Knockdown of HER2 expression by siRNAs was also associated with decreased expression of apoptosis inhibitor survivin and the pro-angiogenic vascular endothelial growth factor(VEGF), suggesting that HER2 stimulates tumor growth at least in part by regulating apoptosis and angiogenesis. siRNA-mediated gene silencing of HER2 and decreasing the expressions of survivin and VEGF may be a useful therapeutic strategy for HER2-overexpressing breast or ovarian cancer.Conclusions: 1. Transfection with HER2-specific siRNA plasmids can suppress the growth of SK-BR-3 cells.2. Transfection with HER2-specific siRNA plasmids resulted in sequence-specific decrease in HER2 mRNA and protein levels stably and permanently in SK-OV-3 cells.3. HER2 gene silencing by HER2-specific siRNAs transfection resulted in antiproliferative and apoptotic responses in SK-OV-3 cells, caused cell cycle arrest at G0/G1.4. HER2 gene silencing also resulted in markedly increased cellular taxol resistance in SK-OV-3 cells.5. Knockdown of HER2 expression by siRNAs was also associated with decreased expression of apoptosis inhibitor survivin and the pro-angiogenic vascular endothelial growth factor(VEGF), suggesting that HER2 stimulates tumor growth at least in part by regulating apoptosis and angiogenesis.