The Study Of Effects Of SHP-1 On Breast Caner Cell By RNA Interference

Background and objectiveIt shows that breast cancer accounts for 7%～10% of all human malignant tumors. According to statistics, millions women suffer from breast cancer, and about half of million breast cancer patients died of it in the world every year. In America, the new cases of breast cancer were more than 190 thousand in 2009, which was the top of the female cancer, another 40 thousand died from it. The incidence of breast cancer is increasing gradually, which increased about 40% in Shanghai, Beijing, Shenzheng and other cities during 10 years. So how to prevent and treat of breast cancer has become the current focus of cancer research.With the development of the modern molecular biology, researchers have realized the phosphorylation levels of tyrosine residues in most signal proteins were significant up-regulated in eukaryotic cells, and also tumor cells. More current researches focus on protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Some scientists considered that the imbalance of intracellular phosphorylation happened in the development of breast caner.Tyrosine kinases and tyrosine phosphatases are play key roles in regulating the intracellular phosphorylation. The epidermal growth factor receptor tyrosine kinases are the most tyrosine kinases of them, which include EGFR(HER1/ErbB-1),HER2 (ErbB-2/neu),HER3 (ErbB-3),HER4 (ErbB-4). When the specific ligands are combinded with ErbB, tyrosine resides of ErbB in intracellular are phosphorylated, which play important roles in the cell proliferation, differentiation and signal transduction. There are 20%～30% breast cancer patients with high expression of HER2. Some researches showed that HER2 gene amplification was the independent prognostic indicator of breast cancer and could guide treatment and predict the efficacy. Breast cancers with overexpression of HER2 are prone to resistance, early metastasis and recurrence and poor prognosis. Currently, the trastuzumab is the most important molecular targeted anti-cancer drugs for breast cancers with overexpression of HER2. The application of trastuzumab has achieved encouraging efficacy, but in clinical applications most patients emerge acquired drug-resistance in one year. It is necessary for us to study how to reverse the resistance of trastuzumab and other molecular drugs. Studies have shown that the resistance may cause from increased the levels of intracellular phosphorylation because of interacting activation of HER family themselves or with other adaptor molecules. The activating of HER3 in breast cancers may be an important factor in drug resistance. HER3 lacks kinase activity, but it has the role of transphosphorylation. When NRG ligand stimulated HER2 receptor, HER2 and HER3 would form signaling complex, which can activate HER3 cytoplasmic tyrosine phosphorylation. Then the phosphorylated protein activated the PI3K/Akt signaling pathway to inhibit apoptosis of tumor cells. The cytoplasmic tyrosine phosphorylation of HER3 could not be blocked by trastuzumab, so the appearance of HER3 presents a considerable challenge to molecular targeted therapy.When emergence of drug resistance in the pattern of HER2 as the therapy target, we assume that we could regulate the levels of intracellular phosphorylation balance by dephosphorylating the tyrosine phosphatase. More than 40 PTPs family members have been purified, which include SHP-1 gene, an important candidate tumor suppressor gene found in recent years. Yip and his colleagues had demonstrated the role of SHP-1 in the development of breast cancer, also found its dephosphorylation by specifically bind to the phosphorylation tyrosine residues. In our previous work, 30 specimens were detected including early stage specimens, HER2 positive specimens and metastatic breast cancer specimens respectively. We found that the expression of SHP-1 was significantly lower in HER-2 positive and metastatic breast cancer specimens than these in early stage specimens. When MDA-MB-231 cells were transfected with SHP-1 gene, the proliferation of tumor cells were inhibited. Combined with our previous work, we concluded that dephosphorylation of SHP-1 may reverse the excessive activation of the signaling pathway. The expression of HER-2 up-regulation and SHP-1 down-regulation damage balance of phosphorylation and dephosphorylation, then excessive activation of phosphorylated signal ultimately enhance the degree of malignance and invasion of the cells. The important negative regulatory role of SHP-1 mainly affected the dephosphorylation of growth factor receptors or non-receptors protein tyrosine kinase. Matrix metalloproteases (MMPs) are a multigene family of zinc-dependent endopeptidases that share similar structure and which have the capacity to degrade virtually every component of the extracellular matrix (ECM). Emerging evidences show that the members of MMPs family can serve not only as potential markers for diagnosis and prognosis, early detection, and risk assessment, but also as indicators of tumor recurrence, metastasis, and response to primary and adjuvant therapy for breast cancer. MMP9 is an important member of MMPs, whose level in tumor tissue as well as serum, plasma, and urine are significantly elevated in patients with breast cancer. The relationship between MMP9 and SHP-1 would be investigated in this study. Methods1. Cells were transiently transfected with siRNA of the SHP-1 mRNA using lipofectamine 2000 according to the manufacturer's instructions. Then cells were analyzed for the presence of SHP-1 using Real-time PCR and western blot analysis;2. Forty-eight hours after transient transfection SHP-1 gene, total RNA were collected and then reverse transcriptase into cDNA, prepared reaction mixture under the instructions, and then the expression of MMP-9 was detected by Real-time PCR. Meanwhile cells were adjusted to a certain concentration and prepared reaction mixture needed for analyze the cell invasion ability using transwell chamber invasion assay. After 12 hours, the cells that migrated through the membrane were counted in random elds.3. Prepare the reaction solution used the cell cycle kit, after collecting the cells, then detected the cell cycle.4. After transient interference, when the cells growth to logarithmic phase, cultured for 24 hours after cells were adjusted to a certain concentration. NC, Mock and siRNA three group cells were tested for cell proliferation by MTT assay respectively.Statistical methods All data were treated with SPSS 13.0, P<0.05 was represented statistically significant.Results1. The expression levels of mRNA and protein of SHP-1 in the siRNA group were both significantly lower than those in NC and Mock group by Real-time PCR and western blot. The results showed that we have successfully transient transfected SHP-1 gene. 2. The expression levels of mRNA of MMP9 gene in the siRNA group were significantly higher than those in NC and Mock group by Real-time PCR. The perforated cells were much more than the NC and Mock group using the transwell chamber invasion assay.3. The G1 phase cells accounted for 55.68% in NC group compared with 42.93% in siRNA group by cell assay.4. The proliferation rate of siRNA group was significantly higher than that of NC and Mock group by MTT assay.Conclusions1. The perforated cells were much more than the NC and Mock group by the transwell chamber invasion assay. The results showed that the ability of invasion was enhanced by silencing the expression of SHP-1 gene.2. The expression levels of mRNA of MMP9 gene in the siRNA group were significantly higher than those of NC and Mock group by Real-time PCR. The results may show that there may have potential association with SHP-1 protein and the activating MMP9 protein pathway.3. The expression of SHP-1 can slow the process of the cell cycle by cell cycle assay.4. The proliferation rate of siRNA group was significantly higher than that of NC and Mock group by MTT assay. These showed that expression of SHP-1 decreased the proliferation of the cells.