Roles And Mechanism Of MORC2 In Hepatocytes Steatosis

Objective:Nonalcoholic fatty liver disease(NAFLD) is characterized by excessive accumulation of triglyceride in hepatocytes. Liver diseases that are caused by distinct factors such as alcohol or drugs are not considered as NAFLD. Due to the fact that the incidence of NAFLD is increasing and that the disease may lead to chronic liver failure and hepatocellular carcinoma, NAFLD has become an important public health issue. Up to now, the pathogenesis of NAFLD is not completely understood. One of the theories is the “two hits” hypothesis. The first hit is that hepatocytes are more sensitive to damage factors when steatosis and insulin resistance. The second hit is that lipotoxic injury caused by steatosis resulted in damage on the integrity of cell membrane, intracellular organelle, and DNA, which finally leads to death of hepatocytes.Disorder of lipid metabolism in hepatocyte plays a key role in the pathogenesis of NAFLD. Microrchidia family CW-type zinc finger 2(MORC2, also named as ZCWCC1) is composed of 970 amino acid residues. As a transcriptional factor, MORC2 contains nuclear localization signal, which makes mature MORC2 mainly locate in nucleus. Moreover, zinc finger domain of MORC2 is crucial for its binding with DNA fragments and regulation of gene transcription. Furthermore, MORC2 contains conservative structure GHKL-ATPase domain, which is required for heterochromatin condensation and gene silencing. MORC2 may also interact with histone deacetylase 4(HDAC4) and regulate transcription negatively through epigenetic mechanisms. For example, Carbonic anhydrase 9(CAIX) is the first identified gene which is directly down-regulated by MORC2.Although MORC2 is mainly located in nucleus, cytoplasmic MORC2 can also perform non-transcriptional functions. During preadipocyte differentiation, the abundance of cytoplasmic MORC2 is elevated. MORC2 is reported to interact with ATP-citrate lyase(ACLY), an enzyme that catalyzes the formation of acetyl-CoA and plays a central role in lipogenesis. Acetyl-Co A is a central knot of energy metabolism network for sugar and lipid. Since preadipocyte differentiation and hepatocyte steatosis exhibit similar process such as transcriptional activation of lipogenic genes and accumulation of triglyceride, we propose that MORC2 may play a role in the pathogenesis of NAFLD.To test our hypothesis, we examined the expression of MORC2 in the process of hepatocyte steatosis and further manipulated MORC2 expression to confirm its role in lipid homeostasis. Our study may help clarify the mechanism of "first hit" and provide a potential target for the treatment of NAFLD.Methods:1. Hepatocyte steatosis was modeled using L02 cells and oleic acid. To identify the model, cells were stained with ORO reagent, cellular triglycerides content was also measured after the steatosis induction.2. Immunofluorescence and western blotting were used to observe nuclear and cytoplasmic levels of MORC2 protein in the process of steatosis.3. Detection of MORC2 protein synthesis and degradation before and after L02 hepatocytes steatosis induction.3.1 qRT-PCR was performed to detect mRNA level of morc2 after the steatosis induction.3.2 Ubi Pred website and UbiProber software were used to predict possible ubiquitin binding sites in MORC2 amino acid sequence.3.3 Western blotting was used to detect the alteration of MORC2 protein in L02 hepatocytes treated by NEM or MG-132, which enhances or inhibits the ubiquitinproteasome pathway respectively.4. Eco RV enzyme digestion and plasmid sequencing were used to confirm the plasmid we constructed. Western blotting was performed to identify the overexpression of MORC2 in lentivirus-transfected L02 hepatocytes.5. ORO staining and triglyceride content measurement were used to evaluate the alteration of steatosis among L02-WT, L02-ctrl and L02-MORC2 cells after steatosis induction.6. c DNA microarray analysis was used to identify differentially expressed genes or pathways after MORC2 overexpression in L02 hepatocytes.7. Western blotting was used to detect protein level of p53 after MORC2 was overexpressed in L02 hepatocytes.8. Western blotting was used to identify the overexpression efficiency of p53.9. ORO staining and triglyceride content measurement were used to observe the effect of p53 overexpression on MORC2-overexpression-altered steatosis.10. StepOne and SPSS 18.0 software were used to analyze the data.Results:1. Establishment and identification of L02 hepatocytes steatosis model.ORO staining and triglyceride content measurement showed that triglyceride barely accumulated in control cells. However, the level of steatosis elevated in a time-dependent manner in L02 hepatocytes treated with oleic acid.2. Immunofluorescence and western blotting revealed that nuclear MORC2 was reduced during the process of steatosis.MORC2 was mainly located in nuclei. Nuclear and cytoplasmic protein levels of MORC2 were stable in the first 12 h of steatosis induction. After that, nuclear content of MORC2 was significantly reduced, while the protein level of MORC2 in cytoplasm was still unaffected.3. The reduction of MORC2 in the process of L02 hepatocyte steatosis was induced through ubiquitin-proteasome pathway.3.1 Compared with the control group, the level of morc2 m RNA was steady during the process of steatosis induction.3.2 Ubi Pred website and UbiProber software prediction showed that 9 lysine residues of MORC2 could be modified by ubiquitin, with a possibility over 80% for all sites.3.3 The nuclear level of MORC2, but not cytoplasmic level, was decreased by ubiquitination protector NEM.3.4 The reduction of MORC2 expression during steatosis was aborted by proteasome inhibitor MG-132.4. Establishment and identification of MORC2 overexpressed L02 hepatocytes.4.1 Eco RV enzyme digestion and sequencing results showed that the plasmid sequence was correct, thus morc2 gene was successfully inserted into the vector.4.2 Western blotting indicated that content of MORC2 was significantly increased in nuclei, cytoplasm and whole cell lysis of L02-MORC2 cells.5. MORC2 overexpression alleviated steatosis of L02 hepatocytes.ORO staining and triglyceride measurement showed that triglyceride accumulation was alleviated in L02-MORC2 cells after steatosis induction.6. Screening for lipid metabolism related genes or pathways affected by MORC2.Microarray data revealed significant alteration of gene expression profile after MORC2 overexpression. GO and pathway analysis results indicated that MORC2 was mainly associated with negative regulation of various biological processes, including the lipometabolism related p53 pathway.7. The protein level of p53 was down-regulated in L02-MORC2 cells.Western blotting result showed that level of p53 protein was remarkably reduced in MORC2 overexpressed L02 hepatocytes.8. Confirmation of p53 overexpression in L02 hepatocytes.The protein level of p53 was significantly increased after transfection of p CMV6-XL5-p53 plasmid. The protein level of p53 was slightly decreased in L02-MORC2 cells, as compared to L02-WT and L02-ctrl cells.9. Overexpression of p53 partially blocked the effect of MORC2 overexpression on steatosis in L02 hepatocytes.ORO staining and triglyceride measurement revealed that alleviation of steatosis in L02-MORC2 cells was partially cancelled by pCMV6-XL5-p53 plasmid transfection.Conclusions:1. Nuclear MORC2 is remarkably reduced during the process of L02 hepatocytes steatosis. Further investigation suggests that MORC2 protein may be degradated through ubiquitin-proteasome pathway.2. Overexpression of MORC2 alleviates steatosis and reduces lipid accumulation of L02 hepatocytes, indicating that MORC2 expression may inhibit steatosis of hepatocytes.3. Protein level of p53 is decreased after MORC2 overexpression. Ameliorative effect of MORC2-overexpression on steatosis is partially cancelled by p53 overexpression, indicating that the protective effect of MORC2 on hepatocyte steatosis may be associated with p53.4. MORC2 protein, which is down-regulated by ubiquitin-proteasome pathway during the process of steatosis, inhibits the development of NAFLD. The cellular function of MORC2 in hepatocyte steatosis may be associated with inhibiton of p53.