The Role And Mechanism Of SIX1 In Nonalcoholic Fatty Liver Disease

Backgrounds:Non-alcoholic fatty liver disease(NAFLD)is the most common liver disease worldwide and is becoming a major global health threat which is recognized as the most common risk factor for HCC.NAFLD encompasses a disease spectrum ranging from simple steatosis(NAFL)to nonalcoholic steatohepatitis(NASH),which has the manifestation of lipid accumulation,inflammation,and varying degrees of fibrosis.Aberrant lipid metabolism and accumulation of extracellular matrix proteins are the hallmark of the disease.A variety of metabolic conditions such as obesity,insulin resistance,and diabetes have been linked to NAFLD.But the underlying mechanisms are largely unknown.Sine oculis homeobox homolog 1(SIX1)is a transcription factor that is conserved from Drosophila to humans and is reported to be involved in a wide range of biological processes.SIX1 is a key regulator of organogenesis but remains at low or undetectable levels in most normal adult tissues.SIX1 has also been reported to be highly expressed in HCC and is related to the clinicopathological characteristics of HCC patients.Interference with SIX1 in HCC cells can reduce the stemness of HCC cells and inhibit the metastatic ability of HCC cells.In addition,the previous study of our group also found that the expression level of SIX1 was increased in HCC,and the overexpression of SIX1 promoted the malignant phenotype of HCC by regulating c-MYC and cyclin-D1.These studies demonstrate the key role of SIX1 in the development of HCC.However,the hepatic function of the SIX1 and the extent of its activation in non-tumor disease in liver remains unknown.A recent study reported that SIX1 can increase the transcription of glycolytic genes has attracted attention to its ability to modulate energy metabolism.These results prompted us to explore the potential effect of SIX1 in NAFLD progression.Aims:1.To evaluate the expression of SIX1 in NAFLD patients and mouse models.2.To explore the role of SIX1 in NAFLD.3.To find out the potential target proteins or signal pathways that SIX1 may participate.Methods:1.NAFLD patients’ tissues and mouse model tissues were used to test the expression level of SIX1.2.Alb-Cre mice were administered the adeno-associated virus AAV9 vector for SIX1liver-specific overexpression.Then the SIX1-overexpressing mice and control mice were fed with high-fat high-cholesterol(HFHC)diet and methionine-choline deficient(MCD)diet to induce NAFLD mouse model.The hematoxylin-eosin(H&E)staining and oil red O staining were used to evaluate the steatosis of mouse liver.The serum level of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were used as the indicator for the liver injury.The sirus red staining and m RNA level of transforming growth factor-β1(TGFB1)and smooth muscle alpha 2 actin(α-SMA)were used to detect the fibrosis extent.3.The mouse hepatocyte AML12 and human hepatoma Hep G2 were used to identify the role of SIX1 in vitro.SIX1 lentiviral were used to obtain SIX1-overexpression cell lines and palmitic acid(PA)was used to establish the NAFLD model in vitro.Western blot and immunofluorescence were used to test the expression level of SIX1.The NAFLD parameters were examined by Oil red O staining and ALT/AST levels.4.High throughput CUT&Tag analysis was performed to screen the direct target genes of SIX1.Dual-luciferase report and western blot were used to confirm the direct function of SIX1 upon the target genes.PCR were conducted to test the expression of DNL and fibrogeneic genes with or without the presence of specific inhibitors.5.Transwell chamber coculture was used to observe the effect of SIX1 in Hep G2 cells on the acitivation of HSC cell line LX-2.Results:1.Increased SIX1 expression was detected in patients and mouse models with NAFLD.SIX1 was positively correlated with ALT,AST and triglyceride TG levels of NAFLD patients.2.Liver-specific overexpression of SIX1 in mice using adeno-associated virus provoked more severe inflammation,triglyceride accumulation and fibrosis in HFHC and MCD mouse model.SIX1 overexression also promoted lipid accumulation and cell injury in cultured hepatocytes.3.Based on the CUT & Tag results,we demonstrated that SIX1 can directly activate the expression of Liver X receptor(LXR)α and β,thus inducing the de novo lipogenesis.4.Our results also illustrated a critical role of SIX1 in regulating HSC activation by increasing the levels of TGFB1 in liver cells and TGF-β receptors in hepatic stellate cells.Conclusion:Our findings suggest a detrimental function of SIX1 in the progression of NAFLD.The direct regulation of LXRα/β and TGF-β signaling by SIX1 provides a new regulatory mechanism in hepatic steatosis and fibrosis.