Regulatory Effect Of MiR-96 In Malathion-induced Human Kidney Cells Toxicity

microRNAs（miRNAs）are the main players in the cell’s response to exogenous compounds and toxins.However,their role in organophosphorus-induced cytotoxicity remains unclear.In this study,we investigated the toxic effects of miR-96-5p on human kidney cells（HK-2 cells）and mouse kidney tissue of malathion,providing evidence for the study on the non-cholinergic toxicological mechanism of organophosphorus.First,malathion reduced HK-2 cell viability and miR-96-5p expression in a dose-and time-dependent manner.In addition,transfection of miR-96-5p mimics can decrease malathion-induced apoptosis of HK-2 cells,while transfection of miR-96-5p inhibitors can increase HK-2 cell apoptosis.Luciferase assay showed that miR-96-5p can directly bind to the 3’UTR region of DDIT3,which is a well-known marker of endoplasmic reticulum stress.Further analysis of the expression of apoptosis-related proteins indicated that miR-96-5p may reduce malathion-induced HK-2 cells apoptosis through the DDIT3/BCL-2/Caspase-3signaling pathway.In vitro assay,the mice were orally administered with malathion at sublethal doses of 1/3LD₅₀,1/10LD₅₀,1/25LD₅₀for 18 days respectively,and then the hematoxylin-eosin staining was used to observe the pathology of the kidney tissues.Fluorescence quantitative PCR and immunocytochemistry methods were also to determine the mRNA and protein expression of DDIT3 gene in mice kidney tissue after the malathion treatment.The sublethal dose of malathion can increase the mice blood urea nitrogen,serum creatine,and uric acid.HE staining showed that the1/3LD₅₀and 1/10LD₅₀dose treatment groups had significant damage to the glomerulus,renal capsule cavity,and renal vein blood vessels in mice.Fluorescence quantitative PCR and IHC results showed that malathion can significantly increase the mRNA level and protein expression of DDIT3 in the mice kidney tissue.In conclusion,the results of this study indicate that miR-96-5p could target DDIT3 and relieve the HK-2 cells apoptosis which induced by malathion.Malathion can significantly increase the mRNA level and protein expression of DDIT3,a marker gene of endoplasmic reticulum stress in mice kidney tissue.Our research results suggest that the low dosage malathion can induce renal cell apoptosis by increase the cellular endoplasmic reticulum stress in vivo and sublethal doses of malathion can cause renal tissue damage in vitro.