MiR-122 Regulates Arsenic-induced Liver Damage By Targeting PKM2/autophagy Axis In Chickens

The liver is the largest detoxification organ in poultry and the main target organs for studying arsenic poisoning.Previous studies have found that arsenic poisoning can cause autophagy in chicken liver cells,but the mechanism is still unclear.Studies have reported that miRNAs can participate in the autophagy process induced by NaAsO2 by regulating its downstream signaling pathways.MiR-122 is a liver-specific microRNA that accounts for about 72%of total liver microRNA and is involved in the regulation of liver growth,development,and the development and progression of related diseases.Our previous study indicated that miR-122 was highly expressed in NaAsO2-treated cells and tissues.Whether miR-122 is involved in NaAsO2-induced autophagy and its molecular mechanism has not been reported.In this study,the arsenic-induced broiler model was used to determine the damage of arsenic to the liver by pathological observation and anti-oxidation level detection(GSH,CAT,SOD and MDA);ultrastructural observation,q-PCR,Western Blot were used to detect autophagosome formation in the liver of arsenic poisoning and mRNA and protein levels of autophagy-related genes(Beclinl,LC3,TORC1,ATG5 and p62).To construct a miR-122 overexpression and knockdown model of primary chicken hepatocytes to study the biological function of miR-122 in sodium arsenite-induced hepatotoxicity;the target gene of miR-122 and its regulatory role in sodium arsenite-induced hepatotoxicity were identified and verified in hepatocytes.The results show:1.After 90 days of feeding broiler chickens with arsenic(30 mg/kg)diet,liver tissue damage(HE)was found in broilers,hepatocytes showed obvious necrosis and apoptosis(transmission electron microscopy),and oxidative stress(biochemical experiment)and other pathological changes.Hepatocytes were observed to have autophagy in the ultrastructure of liver tissue of the experimental group.The q-PCR and Western Blot results also showed that the autophagy-related genes in this group were significantly up-regulated compared with the control group.2.Culture chicken primary hepatocytes,determine the optimal conditions of NaAsO2 by MTT test and liver function biochemical experiment(ALT,AST)under different NaAsO2 concentration/time.Evaluation of oxidative stress autophagy model by anti-oxidation index(biochemical experiment)and autophagy-related protein expression level(q-PCR+Western Blot).3.Based on the establishment of chicken primary hepatocyte autophagy model,the miR-122 overexpression and knockdown model was successfully constructed.The overexpression efficiency was 22 times and the knockdown efficiency was 0.015 times.The miR-122 target gene was predicted using bioinformatics prediction software TargetScan and MiRDB,and PKM2 was identified as a target gene of miR-122 by constructing a dual luciferase reporter gene.An increase in miR-122 levels and a decrease in PKM2 levels were detected in both the experimental groups of Models 1 and 2 by q-PCR and Western Blot methods.4.Overexpression of miR-122 increased the level of autophagy in the arsenic poisoning cell model,and miR-122 knockdown showed the opposite result,which was reverted by si-PKM2 via the PI3K/AKT/mTOR autophagy inhibitory pathway.In summary,the high expression of miR-122 induced by NaAsO2 can increase the sensitivity of hepatocytes to NaAsO2 by negative regulation of PKM2,and promote autophagy of oxidative stress cells in hepatocytes.This study laid the foundation for exploring the mechanism of microRNA regulation of arsenic poisoning and provided a theoretical basis for the prevention and treatment of arsenic poisoning in poultry.