Modulation Of MiR-21 In Cadmium Induced HEK293 Cell Oxidative Stress Injury And Apoptosis

Objective（1）To explore the effects of CdCl₂ with different concentrations and different action time on the oxidative stress injury,apoptosis and the content of miR-21 in HEK293 cells（human embryonic kidney cells）induced by cadmium.（2）To explore the modulation of miR-21 on oxidative stress injury with 15μmol/L CdCl₂ for 1h,miR-21 mimic and inhibitor transfection and had transfected with 15μmol/L CdCl₂ for 1h.（3）To explore the modulation of miR-21 on apoptosis with 15μmol/L CdCl₂ for 1h,miR-21 mimic and inhibitor transfection and had transfected with 15μmol/L CdCl₂ for 1h.Methods HEK293 cells were cultured with different treatments.Cell number and cell morphology was observed using an inverted microscope.MTT was used to test cell growth.The levels of apoptosis and the contents of ROS were measured both by flow cytometry.TBA method was used to detect MDA contents.GSH-PX activities were determined by colorimetric method.WST-1 method was used to measure the activities of SOD.Real-time quantitative reverse transcription polymerase chain reaction assay miR-21 content.Results（1）Cell adhesion decreased,rounded and pseudopodia disappeared followed different concentrations of CdCl₂ exposure at different times.With the increasing of concentrations of CdCl₂ as well as action time extending,the growth of cells was inhibited.And this inhibition will reached maximum after treatment by 120μmol/L CdCl₂ for 12 h.Comparing with the group of 0μmol/L CdCl₂,the rates of apoptosis increased（P<0.05）.The apoptosis increased with action time extending at 30μmol/L and 60μmol/L CdCl₂（Ptrend=0.001,Ptrend=0.009）.With the increasing of concentrations of CdCl₂,apoptosis increased at action time of 1,3,6 hours,respectively（Ptrend=0.003 or Ptrend=0.001）.Comparing with the group of 0μmol/L CdCl₂,the contents of MDA and ROS increased with increasing concentrations of CdCl₂（Ptrend<0.001 or Ptrend=0.001）.The contents of MDA and ROS increased gradually with action time extending（Ptrend<0.05）at the concentrations of CdCl₂ of 30,60 and 120μmol/L,respectively.Comparing with the group of 0μmol/L CdCl₂,the activities of SOD and GSH-PX decreased（P<0.05）in groups of 30,60 and 120μmol/L,and the activities of SOD and GSH-PX decreased following with the increasing of CdCl₂ concentrations（Ptrend<0.001）.The activities of SOD and GSH-PX decreased with action time extending at 30,60 and 120μmol/L CdCl₂（Ptrend<0.01）,respectively.At the same time,significantly statistical difference on the expression of miR-21 was showed in different concentration of CdCl₂（P<0.001 or P=0.006）,and the expression of miR-21 upregulated with increasing concentrations of CdCl₂（Ptrend=0.002 or Ptrend=0.004）.At the same concentration of CdCl₂,the different action time of CdCl₂ showed different expression level of miR-21（P<0.001 or P=0.042）.With action time extending of CdCl₂,the expression of miR-21 upregulated significantly at 30μmol/L and 120μmol/L CdCl₂（Ptrend=0.030,Ptrend=0.011）.And the expression of miR-21 will reached maximum after treatment by 120μmol/L CdCl₂ for 12 h.（2）The contents of MDA and ROS increased（P<0.05）by miR-21 mimic transfection compared with control and miR-21 mimic Negative Control,the activities of SOD and GSH-PX decreased（P<0.05）compared with control,and the activities of SOD decreased（P<0.05）compared with miR-21 mimic Negative Control.The contents of MDA and ROS decreased（P<0.05）by miR-21 inhibitor transfection compared with control,ROS decreased（P<0.05）compared with miR-21 inhibitor Negative Control,the activities of SOD and GSH-PX increased（P<0.05）compared with control and miR-21 inhibitor Negative Control.The contents of MDA and ROS increased and the activities of SOD and GSH-PX decreased（P<0.05）with both miR-21 mimic transfected and 15μmol/L CdCl₂ for 1h compared with groups of control and 15μmol/l CdCl₂.（3）Apoptosis（P<0.001）and expression of miR-21（P<0.05）increased with miR-21 mimic transfected compared with control,but apoptosis（P<0.001）decreased with miR-21 inhibitor transfected.Apoptosis（P<0.05）and expression of miR-21（P<0.05）increased with both miR-21 mimic transfected and 15μmol/l CdCl₂ for 1h,but apoptosis（P<0.05）and expression of miR-21（P<0.05）decreased with both miR-21 mimic transfected and 15μmol/l CdCl₂ for 1h compared with groups of control and 15μmol/L CdCl₂.The correlation existed between apoptosis and miR-21,and correlation coefficients was 0.732.Conclusion（1）Cadmium can induce oxidative stress injury and apoptosis in HEK293 cells.（2）miR-21 attenuates oxidative stress injury of HEK293 induced by cadmium.（3）miR-21 attenuates apoptosis of HEK293 induced by cadmium.