Preliminary Study Of Effect Of ZIP10 Over-expression On Invasion Of Breast Cancer Cells

BackgroundBreast cancer is the most frequently diagnosed cancer among women, the attack rate of it is the first place of all women cancer in developed countries in Europe and American, and only in American, breast cancer patients accounts for about 30 percent of all diagnosed cancer women. Although china belongs to lower incidence of breast cancer countries in the world, with the change of lifestyle and environment, and the incidence of breast cancer among women in our country shows a rapid upward trend. In recent years, along with the increase of discovery rate, the improve of surgery methods and the application of comprehensive therapy, the cure rate of breast cancer has been advanced. But the recrudescence and transfer after surgery are still difficult problems. If breast cancer patients receive early diagnosis and treatment, about 90 percent of them can survive, therefore it is important to find a novel non-surgical targeting in intervention for preventing and treating breast cancer.Zinc transporter is a kind of transmembrane protein involved in cellular zinc transport, which belong to two gene families:ZnT and ZIP. ZnT transporters reduce intracellular zinc by promoting zinc efflux from cells or into intracellular vesicles, while ZIP transporters work in the opposite effect, ZIP transporters increase intracellular zinc by promoting intracellular zinc uptake and, perhaps, vesicular zinc release into the cytoplasm. Zinc is an essential element for organism, and is a cofactor for many enzymes, it is involved in diverse cellular processes, from catalysis to gene expression to cell proliferation. Zinc redistribution both within and outside cells is to maintain the stability of zinc levels in vivo, and zinc transporters plays an important role in the maintenance of this balance.As well known, zinc content often changes in tumor patients, zinc levels of serum and tissue are significantly lower in malignant tumor such as liver, gallbladder and prostate cancer. In patients with breast cancer, serum zinc level decrease but cancer tissue zinc level increase significantly, and some zinc transporters have the same motif with matrix metalloproteinases, a family of zinc-dependent endopeptidases involved in breast cancer initiation, invasion and metastasis, which suggests zinc or zinc transporters may plays a role in the development and progression of breast cancer. It has been found that the expression of zinc transporter ZIP 10 was positively correlated with malignancy and metastasis of breast cancer; the expression of ZIP 10 mRNA was higher in the invasive and metastatic breast cancer cell line MDA-MB-231 than in less metastatic breast cancer cell line such as MCF-7, besides, the depletion of zinc transporter ZIP 10 by RNA interference inhibited the migratory activity of breast cancer cells.ObjectiveTo construct the pEGFP-N1-ZIP10expression vector, and transfect transiently expression vector to breast cell lines MCF-7 and MDA-MB-231, then explore effects of over-expression of ZIP 10 on invasion of breast cancer cells.MethodsWe construct the expression vector of ZIP 10 pEGFP-N1-ZIP10, then identify the recombinant vector by enzyme digestion and gene blast, transfect transiently expression vector pEGFP-N1-ZIP10 and control vector to the breast cancer cell lines MCF-7 (ER+) and MDA-MB-231 (ER-). After 48 hours, we use FACS and fluorescence microscope to detect the expression of GFP in transfected cells in order to measure the transfection rate of breast cancer cells, ZIP 10 mRNA and protein levels were determined by RT-PCR and Western-blot respectively, and other zinc transporters ZnTl, ZIP1 and ZIP6 were detected by RT-PCR, MTT was used to analyze the effect on the proliferation of MCF-7 and MDA-MB-231 cells, breast cancer cell invasion in vitro was detected with Transwell Matrigel invasion assay.ResultsIdentification of pEGFP-N1-ZIP10 by enzyme digestion and PCR showed that the length, location of insertion and direction of target gene inserted into the recombinant were correct. The results of gene transfection revealed that the amount of positive cells expressing GFP exceed 60% after transfection about 48h respectively; RT-PCR results demonstrated that pEGFP-N1-ZIP10 could up-regulate the expression of ZIP 10 mRNAin MCF-7 and MDA-MB-231 cells respectively; Western-blot results showed that pEGFP-N1-ZIP10 could up-regulate the expression of ZIP 10 protein in MCF-7 and MDA-MB-231 cells respectively; RT-PCR detected that pEGFP-N1-ZIP10 could inhibit the expression of ZIP1 mRNA, but there were no significant effects on the expression of ZnTl and ZIP6; MTT results displayed that pEGFP-N1-ZIP10 could not significantly affect the growth of MCF-7 and MDA-MB-231 cells; Transwell Matrigel invasion assay showed pEGFP-N1-ZIP10 could increased invasiveness of beast cancer cells MCF-7 and MDA-MB-231.ConclusionGene vector pEGFP-N1-ZIP10 can up-regulate the expression of ZIP 10 mRNA effectively, and inhibit the expression of ZIP 1 from the same zinc transporters, can not significantly affect the proliferation of cells, can significantly improve the invasion of breast cancer cells MCF-7 and MDA-MB-231. This further confirmed that there is a close connection between the expression of ZIP 10 gene and the invasiveness of breast cancer, it is feasible for the utilization of ZIP 10 gene as a target of breast cancer's diagnosis and therapy.