The Changes Of Proliferation And Phosphatase Activity In Breast Cancer Cells Transfected With SHP-1

Background and objectiveBreast cancer is one of the common malignant diseases that threaten human health seriously, and it accounts for 7%～10% of all of human malignant turmors. The incidence of breast cancer is increasing gradually, which has already ranked the first in female malignant turmors. How to prevent the occurrence and progression of breast cancer is a very important question today.With the development of the modern molecular biology, scientists recognized that the phosphorylation level of tyrosine residues in most signal proteins is significant in regulate eukaryotic cells signal transduction. The dynamic equilibrium of the phosphorylation of protein tyrosine kinases (PTKs) and the dephosphorylation of protein tyrosine phosphatases (PTPs) play considerable roles in cells proliferation, differentiation and cell cycle control and so on. In more in-depth studies, it can be objected that the cellular phosphotyrosine imbalance due to deviation from the dynamic phosphotyrosine equilibrium is a common feature in human breast cancer. Decreased PTP activity and increased PTK activity have been regarded as an important diagnostic parameter in breast cancer. Epidermal growth factor receptor (EGFR) is an essential component of PTKs family including four members:Her-1 (EGFR), Her-2, Her-3 and Her-4. When EGFR is combinds with its specific ligand, its tyrosine residues in intracellular are phosphorylated and the cellular down-stream signal path are stimulated.SHP-1 Gene is considered as one of the important tumor suppressor genes found in recent years, since mainly expressed in hematopoietic cells, it was called Hemato-poietic cells Phosphatase (HCP, SHPTP-1, SHP-1, PTP1C, PTPN6). It is an SH2 domain-containing cytosolic PTP and a key regulator that controls the intracellular phosphotyrosine level in cellular. SHP-1 protein expression was dramatically decreased in most lymphocytic-related cancers, and was decreased in some non-lymphocytic cancers (eg. breast cancer, ovarian cancer, prostate cancer and pancreatic cancer). SHP-1 plays a signifcant role during the development of breast cancer, which mainly involves to phorphatase growth factor receptor or non-receptor protein Tyrosine Kinase's phorphate groups to play negative regulation. As a tyrosine phosphatase, the function of SHP-1 protein in signal transduction as well as its substrate molecules and expression regulation has not been completely clear so far. To explore all of these problems could help researchers to illustrate the function of SHP-1 in regulating cellular signal transduction. We constructed four Vectors contained SHP-1 gene in our study, which respectively named as pEGFP-C3-SHP-1, pEGFP-C3-SHP-1 C/S,pReceiver-B03-SHP-1 and pReceiver-B03-SHP-1 C/S. Then, we objected the difference beween MDA-MB-231 cells not be transfected and MDA-MB-231 cells tranfected of their individual vectors above.Methods:1. To construct four Vectors contained SHP-1 gene, which respectively named as pEGFP-C3-SHP-1, pEGFP-C3-SHP-1 C/S, pReceiver-B03-SHP-1 and pReceiver-B03-SHP-1 C/S, using biology technique. We obtained SHP-1 gene by digesting pcDNA3.1(+)-SHP-1 with restriction enzyme HindⅢand EcoRI, or KpnI and XhoI, than linked SHP-1 gene respectively with linear plasmid pEGFP-C3 and pReceiver-B03 using T4 ligase. pEGFP-C3-SHP-1 C/S and pReceiver-B03-SHP-I C/S were constructed by using the method of point mutation PCR. These four vectors were transformed into competent BL21 E coli., and store at 4℃.2. To transfected human breast cancer cell line MDA-MB-231 with the vectors pEGFP-C3-SHP-1, pEGFP-C3-SHP-1 C/S and pEGFP-C3, respectively named the stable cell line of expression the insert gene as MDA-MB-231-SHP-1, MDA-MB-231-SHP-1 C/S and MDA-MB-231-pEGFP-C3. Then, we observed the biological characteristics of these stable cell lines. First of all, we chosed the experimental methods of RT-PCR to identificated the expression of SHP-1 mRNA in the stable cell lines we constructed previously and in MDA-MB-231 cells and Western Blot to identificated the expression of SHP-1 protein. Through MTT and p-nitrophenyl phosphate (p-NPP) reveal the difference of proliferation and phosphatase activity in MDA-MB-231-SHP-1, MDA-MB-231-SHP-1 C/S, MDA-MB-231-pEGFP-C3 and MDA-MB-231 cells.3. To extract pReceiver-B03, pReceiver-B03-SHP-1 and pReceiver-B03-SHP-1 C/S plasmids from E coli. BL21, and induce expression of GST fusion protein with IPTG in E coli.BL21.Results:1. We have successfully constructed four vectors, and named them as pEGFP-C3-SHP-1, pEGFP-C3-SHP-1 C/S, pReceiver-B03-SHP-1 and pReceiver-B03-SHP-1 C/S.2. We transfected human breast cancer cell line MDA-MB-231 with the vectors pEGFP-C3-SHP-1, pEGFP-C3-SHP-1 C/S and pEGFP-C3, and established the cell lines expressed the insert gene stablely. 3. Compared with MDA-MB-231-SHP-1 C/S, MDA-MB-231-pEGFP-C3 and MDA-MB-231 cells, the proliferation rate of MDA-MB-231-SHP-1 cells is slower and the phosphatase activity is increased.4. In BL21 E coli. transformed with pReceiver-B03-SHP-1 or pReceiver-B03-SHP-1 C/S plasmids, we observed IPTG could induce the expression of GST fusion protein. However, it could not be found in BL21 E coli. transformed with pReceiver-B03 plasmids.Conclusions:1. The cell lines we established in our study named as MDA-MB-231-SHP-1, MDA-MB-231-SHP-1 C/S, MDA-MB-231-pEGFP-C3 and MDA-MB-231 cells could individually express their insert gene.2. Inserted SHP-1 gene in MDA-MB-231 cells could increase the phophatase activity and decrease the proliferation of the cells.3. IPTG could induce the expression of GST fusion protein In BL21 E coli. transformed with pReceiver-B03-SHP-1 or pReceiver-B03-SHP-1 C/S plasmids.