MicroRNA-200a Induces Hepatocyte By Targeting ZEB2 In Alcoholic Liver Disease

Background: Liver disease is still one of the most frequent causes of death in China,especially when people's living standards are rising and abuse of alcohol in life,the incidence of alcoholic liver disease(ALD)is also increased rapidly.In China about 6.18% of drinking people have chance to be infected by ALD.Increasing evidence suggests that the fine-tuning role of micro RNA(mi RNA)in lipid metabolism,apoptosis,inflammation are affected by alcohol in ALD.Mi RNAs are highly conserved small noncoding RNAs,about 20-22 nucleotides long,although they can't encode protein,they can reduced or increased target gene expression by binding specifically to the 3' or 5' untranslated(UTR)region of m RNA.The roles of mi RNAs in initiation and progression of hepatic fibrosis and various cancers have been well documented.However,the biological significance of mi RNA in alcoholic liver disease is still unclear.It is one of the important way to cure ALD that the role of mi R-200 a is investigated during ALD.E-box zinc finger binding protein(ZEB)1 and 2 inhibit expression of E-cadherin and induce epithelial-mesenchymal transition(EMT)during the process of epithelial cells transformed into interstitial cells,the abnormal activation of EMT is an important factor in tumor development.ZEB2 play an important role in the process of apoptosis,but its role in ALD is not clear.However,recent study suggest that there are closed relationship between mi R-200 a and ZEB2.Our group intend to take ethanol-feed mice as subject to study the pro-apoptosis regulation role of mi R-200 a and its mechanism.Methods: 8-week-old male C57BL/6J mice were feed by common diet about 1 weak to adapt to the environment.For all experiments,mice were divided into CD-fed group and Et OH-fed group,and Et Oh-fed mice were administered to alcohol gavage.The period of entire molding is 16 days.All of the groups were sacrificed 9h after the last injection,blood and liver tissue were prepare for further experiments.The expression of mi R-200 a and ZEB2 in primary hepatocytes was detected by immunohistochemistry,RT-q PCR and Western Blot.RT-q PCR ?Western Blot and TUNEL taining were used to test the influence of mi R-200 a on hepatocyte apoptosis after tail vein injection of lentivirus.Normal mice hepatocyte AML-12 was stimulated with different concentrations of ethanol,and the expression of mi R-200 a and ZEB2 was detected by RT-q PCR and Western Blot.Annexin V-FITC,TUNEL staining and Western Blot were used todetect the change of hepatocyte-apoptosis rate after mi R-200 a inhibitor and mimics infection.We found that ZEB2 is a possible binding site of mi R-200 a by computer simulation and luciferase reporter assay,and then test the expression change of ZEB2 by RT-q PCR and Western Blot after infected by mi R-200 a inhibitor and mimics.Finally,mi R-200 a and ZEB2 were co-transfected to determine whether the pro-apoptosis role of mi R-200 a is realized by binding ZEB2.Result: We found that the apoptosis rate of hepatocyte was increased significantly in Et OH-fed mice versus CD-fed mice,and the slice of mi R-200 a induce the increased expression of ZEB2 in Et Oh-fed mice.The mi R-200 a expression was increased gradiently but the ZEB2 expression was decreased gradiently after different ethanol stimulation in AML-12.We also find that the hepatocyte apoptosis rate was decreased in cells infected by inhibitor however increased in cells infected by mimics versus control group.Luciferase reporter assay found that ZEB2 may have mi R-200 a binding sites.The ZEB2 expression was increased accompany with the silence of mi R-200 a,which suggest the negative regulation of mi R-200 a on ZEB2.Conclusion: In alcoholic liver disease model,the mi R-200 a expression was increased gradiently and ZEB2 was decreased gradiently.And we find that the elevated expression of mi R-200 a can induce hepatocyte apoptosis,however,the depressed expression of ZEB2 can prevent hepatocyte apoptosis.Our study also suggests that mi R-200 a may regulate hepatocyte apoptosis and thus affect alcoholic liver progression is by targeting ZEB2.