Mechanisms Of MiR-155 And Its Target Gene Rheb In Cadmium-induced Autophagy In Rat Liver

The heavy metal Cadmium is a widely distributed pollutant in the environment.Cadmium enters the body through the food chain and respiratory tract and accumulates in many organs in the body.Liver is one of the important target organs for cadmium accumulation.MiR-155 is associated with alcoholic,drug-induced,and inflammatory liver disease.Studies have shown that miR-155 can regulate autophagy by targeting Rheb and Rictor under hypoxia conditions.Previous studies in our laboratory have shown that cadmium can increase the level of autophagy by down-regulating mTOR signaling pathway in BRL 3 A cells,but the specific mechanism remains to be further studied.This experiment use SD rats and BRL 3A cell as the research object,by dealing with cadmium,use of cell and molecular biological methods,explore the miR-155 and its target genes Rheb role in cadmium induced-autophagy mechanism,in order to further explore the cadmium to the pathogenesis of liver injury and provides the theory basis for prevention and treatment measures.1.Effects of cadmium-induced autophagy on miR-155 and its target gene Rheb in rat liverEighteen male SD rats were randomly divided into two groups:control group and cadmium group.Rats in the cadmium group were free to drink cadmium water(50 mg/L)for 90 days.After the experiment,liver,urine,whole blood and serum were collected for subsequent experiments.Determination of liver coefficient;alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(ALP)were determined by blood biochemical analyzer.The histopathological changes of liver were observed by HE staining.The ultrastructure was observed by transmission electron microscope.The activities of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),reduced glutathione(GSH)and glutathione peroxidase(GSH-Px)in liver tissues were determined by colorimetry.LC3 expression was observed by immunohistochemistry.The expression levels of miR-155 and Rheb in liver were detected by qRT-PCR.The expression levels of autophagy related proteins and Rheb/mTOR pathway related proteins in liver were detected by Western blot.The results showed as follows:(1)Compared with the control group,the liver coefficient of rats in cadmium group was significantly decreased(P<0.01),and the contents of AST and ALP in serum were significantly increased(P<0.05).(2)The results of HE staining showed that the limits of liver lobules were not clear in the cadmium exposed group.The results of transmission electron microscopy showed that the chromatin in the liver of the cadmium group was contracted,the nucleus were pyknosis and concentrated,the mitochondrial cristae were blurred and disintegrated,and the production of autophagosomes was increased.(3)The activities of SOD in liver were significantly decreased(P<0.05),and the activities of MDA,CAT,GSH and GSH-Px were significantly increased(P<0.01).(4)Immunohistochemical results showed that LC3 expression was increased in liver of rats in the cadmium group.(5)The expression level of miR-155 in liver of rats in cadmium group was significantly higher than that in control group(P<0.01),and the expression level of Rheb gene was significantly decreased(P<0.05).(6)The expression levels of autophagy related proteins Atg5,Beclin1 and LC3 in liver of cadmium-treated group were significantly increased(P<0.01),and the expression levels of Rheb,p-mTOR/mTOR were significantly lower than those in control group(P<0.05 or P<0.01).According to the above results,the conclusions can be drawn:cadmium exposure can cause damage in rat liver;Cadmium can up-regulate the expression level of miR-155 in rat liver,down-regulate the expression level of Rheb/mTOR pathway protein,and increase the level of autophagy in rat liver.2.The mechanism of miR-155 and its target gene Rheb in cadmium-induced autophagy of rat hepatocytesBRL 3A cells were divided into control group and cadmium group(5 μM CdAC2);Or NC-mimics group and miR-155 mimics group(50 nM);Or NC-inhibitor group,miR-155 inhibitor(200 nM)group,cadmium group,cadmium+inhibitor group;Or NC-siRheb group,siRheb(100 nM)group.CCK-8 assay was used to detect cell viability in each group.The change of LC3 polymeric point was observed by immunofluorescence.The ultrastructure of cells was observed by transmission electron microscopy.The expression levels of miR-155 and Rheb genes were detected by qRT-PCR.The expression levels of autophagy-related proteins and Rheb/mTOR pathway proteins were detected by Western blot.Dual luciferase reporter gene assay was used to detect the interaction between miR-155 and Rheb.The results showed as follows:(1)Compared with the control group,the expression level of miR-155 was significantly increased(P<0.01),the expression level of Rheb gene was significantly decreased(P<0.05),the protein expression level was significantly decreased(P<0.01),the p-mTOR/mTOR ratio was significantly decreased(P<0.05),and the expression levels of autophagy related proteins Atg5,Beclin1 and LC3 were significantly increased(P<0.01)after treated with 5 μM CD for 6 h.(2)compared with NC-mimics group,50 nM miR-155 mimics transfection cells after 24 h,cell viability decreased significantly(P<0.05),the expression of miR-155 levels significantly increased(P<0.01),Rheb transcription level decreased significantly(P<0.05),the amount of protein expression significantly lower(P<0.01),p-mTOR/mTOR ratio significantly reduced(P<0.05),the amount of autophagy related protein expression were significantly increased(P<0.01),LC3 accumulation point increase,the number of autophagosome increased;(3)Compared with NC-inhibitor group,the cell viability was not significantly changed after transfection with 200 nM miR-155 inhibitor for 24 h,the expression level of Rheb protein was significantly increased(P<0.01),and the p-mTOR/mTOR ratio was significantly increased(P<0.01).(4)Compared with cadmium group,cell viability,expression level of Rheb,p-mTOR/mTOR ratio,expression level of autophagy related protein,LC3 polymeric point and autophagosome production were significantly decreased in cadmium+inhibitor group(P<0.05).(5)The fluorescence ratio of cells transfected with miR-155 mimics in the wild group was significantly lower than that in the NC group(P<0.01).(6)Compared with NC-siRheb group,the expression of Rheb and p-mTOR/mTOR protein were significantly decreased(P<0.01)and the expression of autophagy related protein was significantly increased(P<0.05 or P<0.01)after transfection of 100 nm siRheb group for 24 h.According to the above results,the following conclusions can be drawn:cadmium can up-regulate the expression level of miR-155 in BRL 3A cells,down-regulate the expression level of Rheb/mTOR pathway protein,and increase the level of autophagy;miR-155 has a targeted negative regulation effect on the Rheb gene.Conclusion:Cadmium can cause toxic damage to rat liver,increase the expression level of miR-155 in rat liver,down-regulate the Rheb/mTOR pathway and induce the increase of autophagy.Rheb is a direct target of miR-155,and miR-155 negatively regulates the expression level of Rheb.