The Study Of The Effects Of HER-2on Invasion And Metastasis Of Gastric Cancer Cells And Its Mechanisms

Gastric carcinoma (GC) is a malignant neoplasm harming the health of people around the world and accounting for7.8%of all cancers, with the second highest morbidity and mortality in the global. China is also a country with high incidence of gastric cancer. Because of lacking typical clinical manifestations in early time, most patients have been in the advanced stage and the tumor has metastasized when diagnosed, resulting in a poor prognosis. Invasion and metastasis are the major causes for the death of patients with GC. Therefore, further investigations on the mechanism of invasion and metastasis are very important for the development of new treatment strategies.HER-2oncogene, also known as neu, ErbB-2, c-erbB-2or P185, is a transmembrane glycoprotein with tyrosine protein kinase activity, and is a member of the epidermal growth factor receptors (EGFRs). Generally, HER-2is extensively expressed during fetal period, and it is only expressed a few normal tissues in adulthood. Under normal circumstances, HER-2expression is very low and regulates the biological functions such as cell growth, proliferation and differentiationof cells. When HER-2is subject to certain cancer-causing factors, it is activated with the structure or expression out of control, then induceing cell malignant transformation, accelerating tumor cell motility and leading to tumor invasion and metastasis. Most studies have found that HER-2amplification and over-expression play a key role in initiation, progression, invasion, lymph node metastasis and liver metastasis of gastric carcinoma.The human Fascin1gene belongs to the fascin family, encoding a55KD cytoskeletal protein which is able to bind with F-actin proteins. Fascin1localizes in the core actin bundles of microspikes, filopodia and actin-based protrusions underneath the plasma membrane and that has been implicated in cell motility, cancer cell invasion and metastasis. Fascinl is predominantly expressed in neuronal tissue and is absent from normal epithelial cells. However, high level of Fascin1expression was reported in many epithelial different types of carcinomas, including breast, pancreatic and ovarian carcinoma. Despite Fascinl expression is not high (25%) in gastric cancer, but its expression is closely associated with invasion, lymph node metastasis, TNM stage and recurrence of the tumor.Twist is a highly conserved transcriptional factor of basic helix-loop-helix (bHLH), encoding a26KD protein. It is highly expressed in the placenta and the tissues and cells derived from mesodermal, involving in regulating organ development, tumorigenesis, cellular proliferation and differentiation process. Recently, Twist is considered as an oncogene protein, affecting the apoptosis of tumor cells, and playing an important role in epithelial-mesenchymal transition (EMT). In recent years, researches have found that twist is closely associated with degree of differentiation, depth of invasion, lymph node metastasis, TNM stage and distant metastasis of gastric carcinoma.ObjectiveOur research attempts to study the effect of HER-2gene on invasion and metastasis of gastric cancer cells, to investigate the relationship between HER-2gene and transfer molecules such as Fascinl and Twist in the gastric cancer cells, to provide a theoretical basis and experimental evidence for HER-2as a target of gastric cancer gene therapy.Methods1. Culturing gastric cancer cell lines SGC7901, BGC823and MKN45.2. Using the liposome-mediated transfection techniques transfect HER-2(CA), HER-2(KD), HER-2(WT) plasmid vector and blank plasmid vector (PCDNA3) into MKN45cell and SGC7901cell. The NC group was served as negative control and the blank transfection group as the blank control.3. Using the liposome-mediated transfection techniques transfect interference plasmid Neu-shRNA, the Control-shRNA into BGC823cell, The NC group was the negative control and the blank transfection group was the blank control.4. The HER-2and Fascinl mRNA levels of MKN45, BGC823and SGC7901cell lines were investigated by Real-time PCR, and the correlation between HER-2and Fascin1was analyzed.5. The HER-2, Fascin1, Twist and E-cadherin protein levels of MKN45, BGC823and SGC7901cell lines were investigated by Western blotting and the correlations of HER-2, Fascin1, Twist and E-cadherin were analyzed.6. The invasive ability of MKN45cells transfected with HER-2plasmid and of BGC823cell transfected with interference plasmid Neu-shRNA were detected by transwell chamber assay, transwell migration assay and in vitro wound healing assay.Results1. Compared with BGC823and Control-shRNA-BGC823cells, HER-2and Twist expression were decreased (P<0.05), the E-cadherin expression was increased (P<0.05) and the Fascinl expression was unchanged (P>0.05) in the Neu-shRNA-BGC823group.2. Compared with MKN45and PCDNA3-MKN45cells, the HER-2and Twist expression were increased (P<0.05), the E-cadherin expression was decreased (P<0.05) and the Fascin1expression was unchanged (P>0.05) in the groups of HER2-MKN45cell transfected with plasmid (HER-2CA, HER-2KD, HER-2WT). While in another cell line of SGC7901reached the same conclusion.3. Compared with BGC823and Control-shRNA-BGC823cells, the cell migration, invasion and metastasis ability of Neu-shRNA-BGC823cell was decreased obviously (P<0.05).4. Compared with MKN45cell and PCDNA3-MKN45cells,the cell migration, invasion and metastasis ability of HER2CA-MKN45and HER2WT-MKN45cells were increased obviously (P<0.05).Conclusions1. HER-2promoted the invasion and migration of gastric cancer cells.2. HER-2could down-regulate the E-cadherin expression by raising Twist to promote the invasion and migration of gastric cancer cells.3. There seems no relationship between HER-2gene and Fascinl in gastric cancer cell.