| Background and objectiveDrug-induced liver injury(DILI)is one of the main causes leading to drug withdrawals or failure of drug development.Therefore,drug-induced liver injury has become one of the most concerned issues in the drug research and development.Currently,Alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH),total bilirubin(TBIL)and alkaline phosphatase(ALP),is the principal reference standard biomarker to diagnose DILI.But these indicators reflect liver injury that has been occurred,and in the early stages of DILI only 40% of patients show abnormity.In recent years,the more popular biomarkers(miRNAs),especially miRNA-122,which are specifically expressed in liver tissue,have higher specificity and sensitivity than traditional biomarkers,and have been studied and confirmed in clinical,preclinical and in vitro studies.Although a number of studies have confirmed that miRNA-122 can be used as a biomarker of DILI,its role and mechanism in DILI is still unclear.This study has been established on the basis of the establishment of DILI model of SD rats.In the present study,acetaminophen(APAP)-induced hepatotoxicity was established in HepG2 cells.Through overexpression or inhibition of miRNA-122,we investigated the role and mechanism of mi RNA-122 in DILI from tissue,cellular and molecular levels.It will provide a theoretical basis for further establishment of miRNA-122 as a biomarker of DILI.Methods and Results Part ?: Establishment of acetaminophen induced hepatocyte injury model.To determine the concentration of IC50 and IC75,the cell viability of HepG2 treated by APAP for 48 h was measured by Cell Counting Kit-8(CCK-8)assay.And we investigate the effects of 7.5 mM and 20 mM of acetaminophen on the biochemical activity of HepG2 cells by detecting AST and LDH.Compared with the control group,in 20 mM APAP group,AST level was increased by 1.6-fold and 4.3-fold at the 24 h and 48 h after APAP treatment,and LDH level was increased by 2.0-fold at 6 h.From 12 h to 48 h,the LDH level was increased continuously.The changes of AST and LDH level in the cell supernatant showed dose and time dependent,indicating that acetaminophen induced hepatocyte injury model has established successfully.Part ?: Based on APAP-treated HepG2 cell model,we investigate the effects of miRNA-122 on APAP hepatotoxicity in HepG2 cells.HepG2 cells were treated with 7.5 mM and 20 m M APAP.After 3 h,6 h,12 h,24 h and 48 h of APAP treatment,total RNA were extracted from HepG2 cells.Expression of miRNA-122 was measured by RT-qPCR.HepG2 cells were transfected with miRNA-122 mimic and the cell viability was detected by CCK-8 assay.After transfection with miRNA-122 mimic/inhibitor for 24 h,HepG2 cells were treated with APAP for another 24 h.The effect of mi RNA-122 silencing or overexpression on APAP-induced HepG2 cell injury was examined by CCK-8 assay.The results showed that,compared with the NC group,in the 20 mM APAP group,the level of miRNA-122 was significantly increased by 2.82-fold and 2.97-fold at 3 h and 24 h after treatment and reached its peak at 24 h.At 48 h after treatment,the level of miRNA-122 returned to normal levels(0.71-fold).In the 7.5 mM APAP group,the level of miRNA-122 was significantly increased by 2.10-fold at 12 h after treatment when compared with control group.At 24 h after treatment,the level of miRNA-122 returned to normal levels(1.40-fold).The effects of overexpression of mi RNA-122 on HepG2 cells were detected by CCK-8.The results showed that,the cell viability of 200 nM mi RNA-122 mimic group was 84.4%,which is significantly decreased compared with the NC group.The cell viability of 100 nM and 200 nM miRNA-122 mimic + APAP group was 10.3% and 14.3%,which is significantly decreased compared with the NC group.Compared with the LNA NC + APAP group,the cell viability of 20 nM LNA-antimiR-122 + APAP group was 14.5%,which is a significant difference.The above results showed that the expression of miRNA-122 in HepG2 cells after APAP treatment is dose-and time-dependent.In HepG2 cells,overexpression of miR-122 dramatically decreased cell viability.In APAP-treated HepG2 cell model,overexpression of miR-122 aggravated APAP-induced cell damage,while inhibition of miR-122 significantly alleviated APAP-induced cytotoxicity detected by CCK-8 assay.Part ?: Based on male SD rats DILI model,the effect of miRNA-122 on acetaminophen-induced liver injury in rats was investigated.Male SD rats were randomly divided into three groups,each group of four.Three group animals were treated by LNA-antimiR-122(group 3),PBS(group 2),PBS(group 1)respectively via tail vein injection for 3 days,on the third day,group 3,group 2,group 1 were treated with 1250 mg/kg APAP,1250 mg/kg APAP and 0.5% CMC-Na,respectively.After 12 h of APAP exposure,the animals were euthanized.The assessment of liver injury in rat was performed according to the changes in ALT,AST,or histopathology.In LNA-antimiR-122 + APAP group,the serum level of AST was lower than that in NC + APAP group,decreased by 1.7-fold,although no statistical significance was noted for ALT(decreased by 1.1-fold).In histopathology examination,inflammatory cell infiltration and centrilobular necrosis in the liver tissue was observed in NC + APAP group and significantly reduced in LNA-antimi R-122 + APAP group,as compared to the NC group.The above results show that miRNA-122 down-regulation can alleviate APAP-induced liver injury.Part ?: The potential mechanism on miRNA-122 in APAP induced liver injury.Based on miRNA target analysis using the websites miRanda,TargetScan and miRBase,and compared with results in the literature,we found that Bcl-w was a target gene of miRNA-122.HepG2 cells were treated with 20 mM APAP or culture,after 3 h,6 h,12 h and 24 h of APAP treatment,total RNA were extracted from HepG2 cells.HepG2 cells were transfected with 50 nM,100 nM and 200 nM miRNA-122 mimic or mimic NC for 48 h.Total RNA were extracted from HepG2 cells.Expression of Bcl-w was measured by RT-q PCR.The results showed that,compared with the NC Bcl-w expression was decreased by 2.0-fold and 2.3-fold respectively after 12 h and 24 h APAP treatmnet.Compared with the mimic NC,100 nM and 200 nM miRNA-122 mimic significantly decreased Bcl-w expression by 2.4-fold and 2.9-fold.Based on the results above,we can conclude that miRNA-122 was involved in APAP-induced liver injury by inhibiting the anti-apoptotic gene Bcl-w.Conclusions1.It was confirmed that the change of intracellular miRNA-122 was closely related to APAP-induced injury of HepG2 cells.2.APAP-induced liver injury is mediated by the overexpression of miRNA-122,and mi RNA-122 participates in APAP-induced liver injury by inhibiting the anti-apoptotic gene Bcl-w. |
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