Gadd45β和Gadd45γ在亚砷酸钠所致的MIHA细胞周期改变中的作用

目的 探索Gadd45β和Gadd45γ在亚砷酸钠所致的MIHA细胞周期改变中的作用，为砷中毒的人群防治提供依据。方法 收集贵州省兴仁县雨樟镇交乐病区砷中毒人群以及格沙屯正常人群分别作为砷中毒组和对照组，实时荧光定量PCR检测各组人群Gadd45β和Gadd45γ基因mRNA表达水平。同时，分别以不同剂量亚砷酸钠、不同时间处理MIHA细胞，流式细胞术测定细胞周期改变情况，实时荧光定量PCR和免疫印迹法分别检测Gadd45β和Gadd45γ基因mRNA表达水平和蛋白表达情况。在上述实验基础上，针对Gadd45β和Gadd45γ基因分别设计的siRNA作用于染砷的MIHA细胞，反向验证Gadd45β和Gadd45γ基因在砷致细胞周期改变过程中的影响。统计学分析采用独立样本t检验比较两组间差异，采用单因素方差比较多组间差异。结果 人群实验研究发现，与对照组相比，砷中毒患者Gadd45β和Gadd45γ基因mRNA表达水平升高（t = 2.576，P = 0.011；t = 2.312，P = 0.022）；MIHA细胞中，随亚砷酸钠染毒剂量、染毒时间增加，细胞G2/M期比例明显升高（F剂量 = 340.136，P＜0.001；F时间 = 49.194，P＜0.001）；在相对较低亚砷酸钠浓度（低于20 μmol/L）、一定时间内（低于48 h）Gadd45β和Gadd45γ基因mRNA和蛋白表达水平随染砷剂量、染砷时间升高而升高，超过该浓度范围和作用时间后，出现表达下降的现象（Gadd45β:F剂量-蛋白 = 37.568，P＜0.001；F剂量- mRNA = 9.771，P＜0.001；F时间-蛋白 = 61.144，P＜0.001；F时间- mRNA = 46.366，P = 0.001；Gadd45γ:F剂量-蛋白 = 12.989，P = 0.001；F剂量- mRNA = 23.613，P＜0.001；F时间-蛋白 = 27.425，P＜0.001；F时间- mRNA = 37.969，P＜0.001）。转染siRNA分别下调Gadd45β和Gadd45γ的表达后，细胞周期都出现G2/M期比例下调（t = 3.053，P = 0.038；t = 14.47，P＜0.001）。结论 砷致Gadd45β和Gadd45γ基因表达水平升高在其诱导MIHA细胞出现G2/M期阻滞过程中发挥重要作用。

Role of Gadd45β and Gadd45γ in sodium arsenite-induced alterations of cell cycle in MIHA cell

Objective To explore the role of Gadd45β and Gadd45γ in MIHA cell cycle changes caused by sodium arsenite, to provide a basis for the prevention and control of arsenic poisoning population. Methods Arsenic poisoning population in Jiaoluo Ward, Yuzhang Town, Xingren County, Guizhou Province and normal population in Geshatun were collected as arsenic poisoning group and control group, respectively. The mRNA expression levels of Gadd45β and Gadd45γ genes were detected with qRT-PCR. At the same time, MIHA cells were treated with different doses of sodium arsenite or at different times, and the cell cycle changes were determined by flow cytometry, respectively, and the expression levels of mRNA and protein of the Gadd45β and Gadd45γ genes were measured by qRT-PCR and Western-blot. Based on the above experiments, siRNA designed for the Gadd45β and Gadd45γ genes, respectively, acts on arsenic-stained MIHA cells for reverse validation of the effects of the Gadd45β and Gadd45γ genes during arsenic-induced cell cycle changes. Statistical analyses used independent sample t-test to compare differences between two groups and one-way variance was used to compare differences between multiple groups. Results Compared with the control group, higher mRNA expression levels of Gadd45β and Gadd45γ genes were found in arsenic poisoning patients (t=2.576, P=0.011; t=2.312, P=0.022). In the MIHA cells, with the infection dose of NaAsO2 and the infection time increased, the proportion of cell G2/M phase increased significantly (Fdose=340.136, P<0.001; Ftime=49.194, P<0.001). The mRNA and protein expression levels of Gadd45β and creased with increasing arsenic dose and time of arsenic staining at a relatively low sodium arsenite concentration (below 20 μmol/L), for a certain time (below 48h), while the expression of Gadd45β and Gadd45γ decreased beyond this concentration range and the time of action (Gadd45β: Fdose-protein=37.568, P<0.001; Fdose-mRNA=9.771, P<0.001; Ftime-protein=61.144, P<0.001; Ftime-mRNA=46.366, P=0.001; Gadd45γ: Fdose-protein=12.989, P=0.001; Fdose-mRNA=23.613, P<0.001; Ftime-protein=27.425, P<0.001; Ftime-mRNA=37.969, P<0.001). Both cell cycles showed down-regulation of G2/M phase ratio after down-regulation of Gadd45β and Gadd45γ expression by transfected siRNA, respectively (t=3.053, P=0.038; t=14.47, P<0.001). Conclusion Elevated expression levels of Gadd45β and Gadd45γ genes induced by arsenic play an important role in the induction of G2/M phase arrest in MIHA cells.