Effects And Mechanisms Of HMGA2 Hypermethylation In CCl₄ Induced Acute Liver Injury

Acute liver injury(ALI)is featured of abrupt hepatic dysfunction and inflammatory response.Liver injury is the basis of acute liver failure,severe or persistent liver injury can ultimately lead to liver failure.Acute liver injury may be caused by many factors including virus infection,improper use of drugs,excessive intake of ethanol and so on.Show a categorical medical history within 72 hours,including abnormal indicators of liver function,jaundice and other clinical manifestations.Activaion of Kupffer cells(KCs)and the release of various inflammatory cytokines play a central role in the development of ALI.KCs could develop into distinct subpopulations upon the surrounding stimulation.A large number of studies have shown that inflammatory response is closely related to the occurrence,development and prognosis of hepatocellular carcinoma.It has been demonstrated that HMGA2 was highly expressed in hepatocellular carcinoma and contributed to hepatocellular carcinoma cells proliferation and metastasis,and the inhibition of HMGA2 could promote apoptosis of hepatocellular carcinoma cells.Moreover,it has been reportd that HMG AT-hook transcription factor was widely linked to inflammation,and inflammation itself is linked to acute liver injury.Therefore,we would investigate the role of HMGA2 in activation of macrophage during ALI.Data showed that the expression of HMGA2 in primary Kupffer cells and LPS-induced RAW264.7 cells were changed significantly,the expression of HMGA2 was obviously increased at 24 h,but there was a dramatically decreased at 48 h.Moreover,loss-and gain-of-function studies suggested that HMGA2 could promote the expression of pro-inflammatory cytokines such as TNF-?,IL-6 and IL-1?.These results suggested that HMGA2 may play an crucial role in macrophages during ALI.NF-?B is a transcription factor that plays a critical role in inducing and resolving inflammatory response,in order to verify whether HMGA2 regulate the expression of LPS-induced inflammatory cytokines through activating NF-?B pathway.gain-and loss-of-function studies showed that HMGA2 could promote or inhibit the expression of NF-?B signaling pathway related transcription factors.Thus,HMGA2 might be involved in the regulation of inflammatory cytokines through NF-?B signaling pathway.In addition,in order to verify whether the downregulation of HMGA2 involved epigenetic modification at 48 h.Further results showed that the expression of HMGA2 could be restored by treating with 5-azadC in macrophages after LPS stimulation 48 h.To further gain insight into which DNMTs mainly influence HMGA2 methylation,DNMT1-siRNA,DNMT3a-siRNA and DNMT3b-siRNA were respectively used to knockdown DNMT1,DNMT3 a and DNMT3 b in LPS-treated RAW264.7 cells respectively,then the expression of HMGA2 could be significantly reversed at 48 h.CHIP results further proved that DNMT1,DNMT3 a participated in the regulation of HMGA2 expression.In conclusion,HMGA2 may regulate the release of inflammatory cytokines by the NF-?B signaling pathway,and the down-regulation of HMGA2 at 48 h might involve with methylation.