MiR-155 Regulates Apoptosis Of Podocyte Induced By Mitophagy And Its Clinical Significance In Patients With Lupus Nephritis

Objective:To further explore the regulatory mechanism of mi R-155 on mitophagy mediated by FOXO1/PINK1 in podocytes and its effect on ROS and apoptosis-related proteins,and to analyze the effect of mi R-155 on renal function of lupus nephritis（LN）patients and its clinical significance.Methods:（1）The venous blood and urine of patients with LN were collected,and the transcription level of mi R-155 in patients with this disease was studied by quantitative PCR.（2）ELISA and colorimetry were used to detect renal function impairment indexes and podocyte-specific protein content in urine of patients with LN.（3）A transiently transfected podocyte model that overexpressed and suppressed mi R-155 was established（After pretreated with H₂O₂）,and mitohagy activators and inhibitors were used to intervene.Quantitative PCR and Wsetern blot were used to detect the effects of mi R-155 on the transcription and protein expression levels of podocyte mitophagy proteins（FOXO1,PINK1,Parkin,LC3,Beclin1,p62）and apoptosis-related proteins（Caspase-3,Bax,Bcl-2）.（4）The effects of mi R-155 on podocyte mitophagy were examined by JC-1 probe detection of mitochondrial membrane potential,Immunofluorescence observation of LC3Ⅱexpression,and ELISA kit detection of intracellular ROS content changes.（5）Flow cytometry and CCK8 kit were used to detect the effects of mi R-155 on podocyte status,cell activity and apoptosis.Results:（1）Compared with the normal population,the expression level of mi R-155 in urine and blood of LN patients was increased significantly（p<0.05）.（2）Compared with normal people,the levels of urinary acetylglucosidase,microalbumin,transferrin,and urinary creatinine,as well as the concentration of podocyte-specific proteins Nephrin,podocalyxin,and podocin in the urine of patients with LN were increased significantly（p<0.05）,and mi R-155 was positively correlated with renal function impairment index and podocyte-specific protein concentration.（3）Compared with the control group,the expression of FOXO1,PINK1,Parkin,LC3Ⅱ,Beclin1,and Bcl-2 in the mi R-155 inhibition group were increased significantly,cell activity and mitochondrial membrane potential were increased significantly（p<0.05）,and the expression of p62,Caspase-3 and Bax were decreased significantly,the content of ROS and apoptotic rate were decreased significantly（p<0.05）,while the expression of FOXO1,PINK1,Parkin,LC3Ⅱ,Beclin1,and Bcl-2 in the mi R-155overexpression group were decreased significantly,cell activity and mitochondrial membrane potential were decreased significantly（p<0.05）,and the expression of p62,Caspase-3 and Bax were increased significantly,the content of ROS and apoptotic rate were increased significantly（p<0.05）.（4）Overexpressed mi R-155 could significantly inhibit the expression levels of PINK1,Parkin,LC3,and Beclin1 that induced by mitochondrial autophagy inducers,increase the expression levels of p62,Caspase-3,Bax,the ROS content and apoptotic rate（p<0.05）,decreased Bcl-2 expression and podocyte activity（p<0.05）.However,suppressed mi R-155 could significantly increase the expression levels of PINK1,Parkin,LC3,and Beclin1 that down-regulated by mitochondrial autophagy inhibitors,reduce the expression levels of p62,Caspase-3,Bax,and podocyte ROS content and apoptosis number（p<0.05）,increase Bcl-2 expression and podocyte activity（p<0.05）.It is indicated that mi R-155 causes podocyte injury and apoptosis by regulating mitophagy.Conclusion:（1）In this study,it was found that the expression level of mi R-155 in the urine of lupus nephritis patients was positively correlated with the levels of acetylglucosidase,microalbumin,transferrin and urine creatinine in the indicators of renal injury,as well as the concentrations of Nephrin,podocalyxin and podocin in podocyte-specific proteins.（2）In this study,it was found that the up-regulated expression of mi R-155 could inhibit the PINK1/Parkin mitochondrial autophagy pathway by targeting FOXO1,thereby increasing oxidative stress injury and apoptosis in podocytes.（3）This study indicated that mi R-155could be used as a potential biomarker for the diagnosis and treatment of lupus nephritis.