Expression Of NDRG2in Liver Injury-Repair And Preliminary Study On Its Function

Human NDRG2, initially identified in our laboratory, was involved in the regulationof cell proliferation and differentiation. However, to data, the detailed biology function ofNDRG2was not fully explored.Accumulated data suggest that NDRG2is closely involved in liver histogenesis andorganogenesis as NDRG2mRNA and protein levels are generally lower in the early stagesand markedly higher during the later stages of histogenesis in mouse and human fetallivers of different gestational ages.As a tumor suppressor gene, NDRG2also plays a critical role in HCC. Comparedwith adjacent normal tissues, the expression levels of NDRG2mRNA in correspondingcancer tissues reduced significantly. Low expression of NDRG2might be related to themutation and methylation of the NDRG2promoter. The NDRG2gene-transfected HepG2 cells were shown a bad condition, structure damage, and a great number of died cells.Flow cytometry analysis showed G1phase arrest and apoptosis peak appeared. The typicalmanifestation of cell apoptosis could be observed under transmission electron microscope.Furthermore, high NDRG2expression levels correlate positively with tumordifferentiation and negatively with clinical parameters relevant to tumor metastasis (TNMstage, differentiation grade, portal vein thrombi, infiltrative growth pattern, nodal/distantmetastasis, recurrent tumor and shorter patient survival rates). Ectopically expressedNDRG2suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, andexperimental tumor metastasis in vivo, whereas small interfering RNA-mediated NDRG2knockdown resulted in increased invasion and migration of a weakly invasive cell line,PLC/PRF/5. In addition, NDRG2could antagonize transforming growth factor-β1mediated tumor cell invasion by specifically down-regulating the expression of matrixmetalloproteinase2and laminin332pathway components, with concomitant suppressionof Rho GTPase activity.Additionally, NDRG2was found been down-regulated in hepatocytes followingfibrogenesis induced by DMN administration. Meanwhile, our unpublished datademonstrated that in HeLa cells, enhanced expression of NDRG2could inhibitPDGFRA-a key factor for hepatic stellate cells (HSCs) activation and proliferation.Taken together, these data demonstrated that NDRG2participated in the regulation ofliver organogenesis, HCC tumorigenesis and metastasis. Liver regeneration (LR) is theprocess of hepatocyte proliferation and differentiation, liver re-organogenesis. Meanwhile,liver fibrosis is accompanied with hepatocyte proliferation, differentiation, apoptosis andHSCs activation. Therefore, we infer that as a critical regulator of cell proliferation,differentiation and organogenesis, NDRG2may involved in the regulation of LR and liverfibrosis, both are the process of liver injury.【Objectives】1. To find the distribution and expression pattern of NDRG2in rat regenerative livertissue and rat hepatocyte cell line;2. To explore the effect and regulation mechanism of NDRG2on hepatocyte cell cycle and apoptosis;3. To find the distribution and expressionpattern of NDRG2in HSCs; To explore the role and regulation mechanism of NDRG2inthe HSCs activation process;4. To demonstrate the therapeutic effect of NDRG2onfibrotic rat liver.【Methods】1. Establish rat partial hepatectomy model, using Real-time PCR, Western Blot andindirect immunofluorescence analysis to find the distribution and expression pattern ofNDRG2in rat regenerative liver tissue as well as the relationship between NDRG2andC-Myc;2. Enhance the level of NDRG2in rat hepatocyte cell line and analyze the changeof cell cycle and apoptosis by flow cytometry method (FCM);3. Analyze key regulatorsof cell cycle and apoptosis by Real-time PCR and Western Blot to explore the regulationmechanism of NDRG2on LR;4. Using Real-time PCR, Western Blot andimmunofluorescence analysis to find the distribution and expression pattern of NDRG2inHSCs. Enhance the level of NDRG2in HSCs to demonstrate the relationship betweenNDRG2and HSCs activation;5. Analyze the TGF-β1/Smad pathway and MMP2/TIMP2system by Real-time PCR and Western Blot to explore the regulation mechanism ofNDRG2in the HSCs activation process;6. Establish rat liver fibrosis model bydimethylnitrosamine (DMN) injection, explore the therapeutic effect and mechanism ofNDRG2on fibrotic rat liver by HE staining, Sirius Red staining, TUNEL analysis, IHC,Real-time PCR and Western Blot.【Results】1. NDRG2expression is down-regulated during hepatocyte proliferation andenhanced during hepatocyte differentiationThe mRNA and protein levels of NDRG2were strongly reduced when LR reached apeak of activity. In addition, we found that rat NDRG2expression and C-Myc expression were inversely correlated during this process. A low level of NDRG2was observed as theC-Myc expression increased during regeneration.2. Ectopic expression of NDRG2results in G1/S phase arrest and an increase inapoptosisA dramatic cell cycle arrest was found in normal rat liver-derived BRL cells48hoursafter being infected by adenoviral vectors expressing rat NDRG2. Meanwhile, theapoptotic rates were increased from9.4%in control group to64.7%in adenoviral vectorsexpressing rat NDRG2group. These phenomena could also be observed in BRL3A andL-02cells.3. NDRG2induces G1/S arrest and apoptosis via the modulation of Cyclin E andp53-mediated Bax inductionNDRG2overexpression may mediate the antiproliferative effect by inducing p53andp21regulated Bax/Bcl-2increase and Cyclin E-Cdk2inhibition.4. NDRG2shows an inverse relationship with HSCs activation and inhibits HSCsactivationNDRG2mRNA and protein levels were reduced during HSCs activation. In addition,NDRG2overexpression inhibited HSCs activation.5. NDRG2inhibits the TGF-β1/Smad signaling pathway and alters the relative levelsof MMP2to TIMP2Enhanced NDRG2expression reduced Smad3expression and phosphorylation,which inhibited HSCs activation by blocking the TGF-β1signal. Moreover, NDRG2contributed to an increase in the ratio of matrix metalloproteinase2(MMP2) to tissueinhibitor of matrix metalloproteinase2(TIMP2), which may facilitate the degradation ofthe extracellular matrix (ECM).6. NDRG2attenuates DMN-induced liver fibrosis, increases hepatocyte proliferationand improves liver functionIn DMN-induced fibrotic rat livers, adenovirus-mediated NDRG2overexpressionresulted in decreased ECM deposition and improved liver function compared with controls.The regulatory role of NDRG2in TGF-β1/Smad signaling and in the regulation of MMP2 and TIMP2expression contributes towards its therapeutic effects in the fibrotic liver.NDRG2facilitated the regression of liver fibrosis by enhancing the proliferation ofhepatocytes without inducing apoptosis.【Conclusions】In conclusion, our findings point to physiological roles for NDRG2in LR and liverfibrosis. Meanwhile, the modulation of NDRG2is a promising strategy for the treatmentof LR and liver fibrosis.