The Role Of Aspartate ?-Hydroxylase On The Development And Progression Of Liver Fibrosis

Background and ObjectivesHepatocellular carcinoma?HCC?is the most common form of primary liver cancer and is the fifth most common cancer worldwide,with more than half of the affected population found in China.HCC is usually associated with a poor patient prognosis and is the second leading cause of cancer-related deaths.A predominant proportion of HCC cases develop on the basis of severe liver fibrosis and cirrhosis caused by chronic liver inflammation.Chronic viral infection,alcoholic liver disease and hepatic steatosis are major risk factors for cirrhosis and subsequent HCC development.Therefore,since the risk of HCC depends on the pathologic background of the liver,where liver fibrosis is one of the most common culprits,it is of substantial importance to investigate the underlying molecular mechanism of liver fibrosis and HCC as a whole.Aspartate?-hydroxylase?ASPH?is an?-ketoglutarate-dependent dioxygenase that has been initially found elevated in a human HCC cell line?FOCUS?.It catalyzes post-translational hydroxylation of?-carbons of aspartate and asparagine residues in certain epidermal growth factor-like domains present in various functional proteins such as receptors and receptor ligands.Currently,studies on ASPH showed that this enzyme is highly expressed in various malignancies such as glioma,small cell lung cancer,colon cancer,pancreatic cancer,hepatocellular carcinoma and cholangiocarcinoma,playing an important role in the malignant transformation of tumor cells.Our previous studies have also found that increased expression of ASPH is associated with poorer clinical outcome and more aggressive tumor progression in patients with HCC.Meanwhile,some studies have found that ASPH regulates cellular senescence through glycogen synthesis kinase 3?to promote the development and progression of liver cancer.However,present studies mainly investigated the role of ASPH on HCC at clinical and cellular levels and on other malignancies,and the impact of ASPH on liver fibrosis and HCC animal models has not been thoroughly studied.Our previous experiments have found that the expression of ASPH in HCC tissues was significantly higher compared to paracancerous tissues.Similarly,ASPH is highly expressed in liver fibrosis tissues compared with normal liver tissues.Moreover,in carbon tetrachloride?CCl₄?-induced liver fibrosis mouse model and diethylnitrosamine?DEN?-induced HCC mouse model,similar results were observed:The ASPH expression in mouse with liver fibrosis and HCC was significantly higher compared to the control group.Therefore,studying the role of ASPH in liver fibrosis and HCC and its underlying molecular mechanism would contribute to a more comprehensive understanding of the biological function of this molecule,and provide a theoretical foundation for the clinical transformation of ASPH as a molecular target in the treatment of liver diseases.Methods1.Investigation of the association between ASPH and liver fibrosis and HCC in clinical sample:To study the relationship between ASPH and hepatic fibrosis and hepatocellular carcinoma:The role of elevated ASPH expression in the development and progression has been demonstrated in our previous studies.In this study,quantitative PCR was used to detect ASPH expression in liver fibrosis tissues in comparison to normal adjacent liver tissues.The expression of ASPH was also observed in immunohistochemistry and confirmed with western blot,and the associations between ASPH and liver fibrosis and liver cancer,respectively were studied.Results of immunohistochemistry were rated with the Ishak fibrosis scoring system to investigate the association between ASPH expression and the degree of liver fibrosis.Hepatic stellate cells?HSCs?have been demonstrated to be one of the major effector cells in the pathogenesis of liver fibrosis.Therefore,we examined the molecular indicators for hepatic stellate cell activation in tissues with liver fibrosis,including Collagen??,TIMP-1,MMP-2,?-SMA and TGF-?1.Meanwhile,we confirmed the specific expression of ASPH in HSCs with a double staining of?-SMA and ASPH in immunofluorescence test.2.In vivo studies:Accoding to the previously described methods,two types of liver fibrosis mouse model,which is induced either by intraperitoneal injection of carbon tetrachloride?CCl₄?or bile duct ligation?BDL?,and a were used in this study.Meanwhile,the HCC mouse model was induced by serial diethylnitrosamines?DEN?and carbon tetrachloride?CCl₄?administration.Furthermore,the exon 23 of His-2region?the gene responsible for enzymatic activity?in the ASPH genome was knocked down by using CRISPR-Cas9 genome editing technology.Serum liver function test and histopathological examination in the liver,as well as genetic sequencing analysis were performed in ASPH knockout(ASPH^-/-)and wild type mice in both disease models.Changes in mRNA expression were detected by microarray studies and relevant molecules and pathways were analyzed.Meanwhile,mice were injected with MO-I-1100,a small molecule inhibitor of ASPH,during the modeling process to verify whether ASPH can act as a potential therapeutic target to prevent liver fibrosis and HCC.3.In vitro studies:The effect of ASPH on HSC activation and subsequent liver fibrosis was investigated by overexpression and knockdown of ASPH in HSC cell line Lx2.The effects of ASPH expression changes on the proliferation,apoptosis and migration of HSCs were analyzed.Meanwhile,the expression of downstream markers such as collagen??,TIMP-1,MMP-2,?-SMA and TGF-?1 were also detected.4.Statistical analysis:All data were presented as mean±standard deviation.Statistical analyses were performed with Graphpad Prism 5.0 software for and mapping.One-way ANOVA was used to analyze the differences among the groups.P value of<0.05 was defined as statistically significant.Results1.We collected 34 liver fibrosis tissue samples and 36 normal liver tissue samples in this study.Compared with normal tissues,the expression of ASPH in liver fibrosis tissues was significantly higher?P<0.01?.Further,Ishak fibrosis scoring indicated that ASPH was positively correlated with the degree of liver fibrosis?P<0.01?.2.In mice with liver fibrosis or HCC,the ASPH expression in the liver of wild-type mice was significantly higher than those in the control and sham-operated groups.Meanwhile,the degree of liver cirrhosis in ASPH^-/-mice was less severe than that in wild type animals,with decreased deposition of collagen in the portal area and lower fibrosis index.The expressions of Collagen??,TIMP-1,MMP-2,?-SMA and TGF-?1 were also significantly lower than those in wild-type mice.Similarly,the diameter and number of induced HCC tumor nodules in ASPH^-/-mice were significantly lower than those observed in wild-type mice.3.Microarray test identified a total of 86 and 259 gene up-and down-regulations when comparing ASPH^-/-mice with the wild type,of which Cyp4a14 gene had the largest fold change in expression.We subsequently selected the Cyp4a14 gene in the retinol metabolic pathway and found that the Cyp4a14 pathway was significantly down-regulated in ASPH^-/-mice.The proliferation and ability to migration in Lx2cells with knockout of ASPH through lentiviral-siRNA or administration of small molecule inhibitor MO-I-1100 were significantly inhibited when compared with control group.Meanwhile,the expressions of molecular markers related to liver fibrosis were also significantly down-regulated in Lx2 cells with ASPH knockout or MO-I-1100 administration.ConclusionASPH expression is elevated in patients with liver fibrosis and in mice,and the severity of liver fibrosis and liver cancer in ASPH knockout mice is reduced.Inhibition of ASPH can lead to alleviation of HSC activation.Cyp4a14 may serve as a downstream molecule of ASPH and participate in the activation of HSC directly or through the retinol metabolic pathway.This study indicates that ASPH can promote HSC activation through Cyp4a14/retinol metabolic pathway to promote the occurrence and development of liver fibrosis.