Effect Of Exogenous Zinc On Cells Growth And Certain Genes Expression

Zinc is an essential trace metal element in human body, participating in many important physiological activities such as cell proliferation, differentiation, nucleic acids and protein metabolism. In human body, zinc deficiency can lead to some diseases such as decreased immunity, male reproductive dysfunction, while zinc excess can also cause iron-deficiency anemia and cardiovascular disease. So, maintaining body zinc homeostasis is extremely important for human health. More and more evidences suggested that zinc play important roles in at all levels of cell signal transduction. Early in1986, Grummt and his colleagues proposed that zinc is a second messenger, participating in the intracellular signal transduction. But until now, expect for some assumptions, people still have not yet fully understood how the zinc exercises the regulatory function in cell signal pathways. People only get the knowledge of zinc mostly from the abnormal status of cells or organisms (apoptosis, gene irregular expression et al) and until now, there are few studies to reveal zinc functions in normal status of cells or living organisms.Therefore, we firstly studied the different concentrations of zinc ion on the growth of different cell lines (HCT116, A549, MDA-MB-231,293T, HUVEC, HepG2). CCK-8(WST-8) Growth Assay showed that under the concentration of125 μM, ZnSO4doesn’t have detectable effect on the cell growth, while above the concentration of300μM, ZnSO4will completely inhibit cell growth. However, the tolerance to ZnSO4of different cell lines is slightly different. We then tested the mRNA transcription patterns of some key proteins such as transporter proteins, zinc-related transcription factors and zinc binding proteins after the gradient concentration of ZnSO4(0μM、100μM、200μM ZnSO4) stimulated cells using RP-PCR. The results showed that after six hours stimulation with ZnSO4, the mRNA of ZnT family protein or ZIP zinc transporter family protein obviously increased as the increased concentration of ZnSO4, but the relative increased extents are different from one cell line to another. At the same time, we used laser confocal fluorescence microscopy to observe the changes of zinc ions concentration and distribution after gradient ZnSO4stimulation in living cells. The results showed that zinc ions concentration increased significantly with heterogeneous distribution in the cytoplasm of different cells.Then we selected A549cell line to study the time-dependent change of MT1, MTF1, ZnTl and ZIP1mRNA transcription after stimulation with100μM ZnSO4. The results showed that the mRNA reached to its maximum level after the6-8hours stimulation with ZnSO4. We next tested the changes of mRNA level of MT1, MTF1, ZnTl and ZIP1after6hours stimulation with different concentration of ZnSO4. The results showed that there was significant increased in MT1mRNA level, while there was little change in MTF1mRNA level. We finally studied the expression of zinc-related proteins at different time points after stimulated A549cells with100μM ZnSO4by Western Blot. The results showed that ZnTl protein expression level reached to the highest point at about10hours and keep relatively stable till24hours. After48hours, the protein level returned to its ground status. There were no significant expression change of MTF1、ZIP1、ZIP9and ZnT6proteinOur preliminary study showed that different cell line have different tolerant level of ZnSO4concentration and above certain high level of concentration of ZnSO4, cell growth can be totally inhibited; Zinc ions are significantly increased and mainly distributed in the cytoplasm after stimulated by exogenous zinc. Although, ZnT and ZIP family proteins responded differently in different cell line after exogenous zinc stimulation, the overall trend of protein levels is increased. MT1as a major zinc binding protein is more sensitive to exogenous zinc stimulation, while MTF1as a major zinc-related transcription factor protein is not so sensitive. This trend is accordance with the MTF1protein function as a transcription factor protein which shuttles from cytoplasm to nuclear and activate target gene transcription after binding to zinc.