ROS-dependent HMGA2 Upregulation Mediates Cd-induced Proliferation In MRC-5 Cells

Cadmium(Cd) is a heavy Metal widely found in a number of environmental matrices, affecting human health through occupational and environmental exposure. Cadmium is mainly applied to the cadmium battery, ceramic glaze, camera and carbon paper and synthetic fiber dyeing and printing auxiliaries. With the application of cadmium in production and life, people are paying more and more attention to the human health effects of cadmium exposure. A large number of epidemiological studies have confirmed that people who exposed to cadmiumhave been correlated with the development of lung cancer. But there are no conclusive studies demonstrated that cadmium relates directly to lung cancer.Objective: In this study, our aim is to access the cadmium-induced proliferation of MRC-5 and to clarify if the mechanisms related to high mobility group protein HMGA2, and the role of the reactive oxygen species(ROS) in this process.Method: In this study, human embryonic lung fibroblast cells(MRC-5) were used as in vitro model. 3-(4, 5 – Dimethylthiazol- 2- yl)- 2, 5- diphenyltetrazolium bromide(MTT) was used to detect the proliferated effects of different concentrations of cadmium chloride on MRC-5 cells. Cell viability was detected with reagents 3-(4, 5 – Dimethylthiazol- 2- yl)- 2, 5- diphenyltetrazolium bromide(MTT) when treated MRC-5 cells with different concentration of cadmium chloride. We examined the intracellular reactive oxygen species(ROS) with 2 ’, 7 ’- dihydro dichlorofluorescein(DCFH). Western blot was used to detect the intracellular protein level of high mobility group A2(HMGA2) and cell cycle related protein CyclinD1. NAC pre-treatment was performed to detect the involvement of ROS in Cd-induced expression of HMGA2 and cell proliferation. The expression level of HMGA2 gene was detected by qPCR method, and cell cycle changes were measured by flow cytometry. HMGA2 gene was interfered by siRNA to determine the role of HMGA2 in cadmium-induced expression of Cyclin D1, and cell cycle change. SPSS 17.0 statistical software was used for statistical analysis.Results: Treated with cadmium chloride(2μM)for 48 hours, the cell viability of MRC-5 cells increased significantly(P<0.05), this suggests that low concentration of cadmium chloride could promote proliferation of MRC-5 cells. The exposure to cadmium chloride caused the content of intracellular ROS increased significantly(P<0.05). The expression of HMGA2 protein was increasing with the exposure time of cadmium chloride(P<0.05). After 2-hour pretreatment by ROS scavenger NAC, the ROS level, cell viability of MRC-5 cells, and the expression of HMGA2 protein were all significantly decreased compared to treated with cadmium chloride only(P<0.01). Cell cycle was detected by flow cytometry, the number of cells in S phase of cadmium chloride group was higher than control group, the G0/G1 phase was shortened compared to the control(P<0.05). The CyclinD1 protein expression of cadmium chloride group(2μM) was higher than control(P<0.05). When HMGA2 gene was interfered by siRNA, there is no significant change of the expression of CyclinD1 and cell cycle between Cd-treatment group and siRNA control group.Conclusion: Low dose of cadmium chloride could promote MRC-5 cell proliferation, the proliferation associated with oxidative stress, and intracellular ROS induced by cadmium chloride caused expression of HMGA2 mRNA and protein levels, and the expression of HMGA2 protein increased also promoted the expression of cell cycle protein CyclinD1, thus changing the cell cycle distribution of cells. The fact that S phase increasing and G0/G1 phase shortening eventually lead to accelerating of cell proliferation, therefore, this research preliminarily indicates that oxidative stress induced by cadmium gives rise to increasing expression of HMGA2, and further effect on CyclinD1, and distribution of cell cycle, which is the main molecular mechanism of the proliferation of MRC-5 cells, therefore, this research preliminarily indicates that oxidative stress induced by cadmium gives rise to increasing expression of HMGA2, and further effect on CyclinD1, and distribution of cell cycle, which is the main molecular mechanism of the proliferation of MRC-5 cells.