The Molecular Mechanism And RNAi Research About HER2 Positive Metastatic Gastric Cancer

At present, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing. 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 a eukaryotic expression vector, e.g. a pSUPER vector harboring a H1 promoter. HER2 (neu/ErbB2) is widely overexpressed on tumours of epithelial origin, and is intimately related to metastasis. Therefore, HER2 has been addressed as an important therapeutic target. Here we constructed the expression vectors of 2 siRNA, sihe1 and sihe2, which targeted to the 548–566 nt and 1811–1829 nt of the transcript of the oncogene erbB2/HER2, respectively. The constructs were introduced into the human gastric carcinoma cells which overexpress HER2, followed by selection with G418. 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 level of growth and invasion in comparison with the untransfected cells and cells transfected with a mock vector. In the further study, we had observed that CXCR4 was downregulated. and we observed that expression of CXCR4 not only changed in protein level but also changed significantly in mRNA level. Real-time quantitative RT-PCR showed that CXCR4 mRNA decreased in a time-dependent manner in SGC-7901 and MKN-45 cells transiently transfected with HER2 siRNA construct. We observed that HER2 promotes CXCR4 expression in gastric cancer cells via post-transcriptional regulation, which is different from the mechanisms established in other tumour cells. We also demonstrate that HER2 knockdown by siRNA in gastric cancer cell lines activates a stress response involving the upregulation of CD44 through a crosstalk between MAPK and PI3K pathways. CD44 binds HER2 directly through a cytoplasmic disulfide linkage and assists HER2 in downregulating miR-139, a microRNA which inhibits CXCR4 expression through binding to the 3'UTR of its mRNA. HER2 and CD44-mediated miR-139 suppression is achieved by histone H3 deacetylation in the promoter region resulting from upregulation of the metastasis-associated protein MTA1 and recruitment of the nucleosome remodeling and deacetylase (NuRD) complex. The histone deacetylase (HDAC) inhibitor 4-phenylbutyric acid (PBA) treatment or MTA1 knockdown relieves the supression of miR-139. Furthermore, a significant correlation between lymph node metastasis and high HER2, CD44, CXCR4 but undetectable miR-139 expressions is observed in human gastric cancer cases. Combined knockdown of HER2 and CD44 abrogates the mobility and invasion of cultured gastric cancer cells and suppressed tumour growth in BALB/c nude mice.Taken together, these results provide novel insight into the epigenetic mechanism of action of the HER2/CD44 in regulation of CXCR4 expression in gastric cancer cells.