| Hepatic fibrosis, characterized by accumulation of excessiveextracellular matrix (ECM), is a wound-healing response to all chronic liverdiseases including viral infection, non-alcoholic and alcoholic steatohepatitis,immune injury, and others. This process is driven by a heterogeneouspopulation of hepatic myofibroblasts, which mainly derive from hepaticstellate cells (HSCs).During liver injury of any etiology, quiescent HSCs, which are vitaminA–rich cells residing between hepatocytes and sinusoidal endothelial cells inthe subendothelial space of Disse, lost vitamin A droplets and convert tomyofibroblast-like cells. This orchestrated response to liver Injury, named as“activation”, is the most important event of hepatic fibrosis. These activatedcells are capable to elevate deposition of ECM components, increaseproliferation and enhance migration. Although many potential anti-fibrotictargets have been fixed，there are no effective treatment of hepatic fibrosis upto now. Regression of liver fibrosis is associated with resorption of fibrousscar and the disappearance of collagen-producing myofibroblasts. It isbelieved that HSCs are the major collagen-producing cells of all fibrogenicmyofibroblasts. Clearance of activated HSCs is a key mechanism forachieving resolution of fibrosis and is linked with regression of fibrotictissue. Suppression of activated HSCs proliferation, or induction of apoptosisis considered as a useful strategy for antifibrotic therapy.Astrocyte elevated gene-1(AEG-1), also called metadherin (MTDH)and lysine-rich CEACAM1coisolated (LYRIC), was initially identified as aHIV-1and tumor necrosis factor-α-inducible gene in primary human fetalastrocytes. Over the past decade, AEG-1emerged as a positive regulator oftumorigenesis and a valuable prognostic marker of a diverse array of cancers, such as breast cancer, malignant glioma, neuroblastoma and liver cancer.AEG-1expression was utilized in stratification of hepatocelluar carcinoma(HCC) and high level of AEG-1is poportional to poor prognosis. Recently,studies increasingly focus on functions of AEG-1beyond cancer, such asdevelopment, inflammation and neurodegeneration.AEG-1is reported to be a downstream target of Ha-ras and participatesin diverse signaling pathways and interacts with phosphatidylinositol3-linase (PI3K)/Akt, nuclear factor-κB and extracellular signal-regulatedkinase (ERK), which are molecules also involved in liver fibrogenesis.Forced overexpression of AEG-1is demonstrated to increase proliferation,inhibit apoptosis and promote invasion and migration in different types ofcells. It is also noteworthy that up-regulation of AEG-1is associated withenhanced angiogenesis and inflammation, which are key factors of hepaticfibrosis formation. However, the relationship between AEG-1and hepaticfibrogenesis is not known.This study was performed to evaluate the function of AEG-1in hepaticfibrogenesis. Lentiviral delivery of shRNA was used to obtain stablesilencing of AEG-1in the rat HSC-T6cell line, and meanwhile, the effects ofAEG-1on cell proliferation, apoptosis, migration andphenotype wereexamined, as well as the possibly associated molecular mechanisms. Theexperiments contain four parts, shown as follows：Part1: The expression of AEG-1in fibrotic rats and HSCs.Objective: To explore the expression of AEG-1in fibrotic rats inducedby common bile duct ligated (BDL) or dimethylnitrosamine (DMN) andactivated HSCs stimulated by transforming growth factor-β (TGF-β) orlipopolysaccharide (LPS).Methods: hepatic fibrosis was induced by BDL and intraperitonealinjections of DMN．For the bile duct ligation (BDL) model of liver fibrosis,rats were laparotomized after anesthetized. The common bile duct wasdouble ligated and sectioned before the abdomen closed. The sham operationwas performed similarly without BDL. Rats were sacrificed2weeks after operation. For the DMN model of liver fibrosis, rats were injectedintraperitoneally with DMN, which was diluted1:100in0.15M NaCl, orwith vehicle (NaCl) at a dose of1μL/100g of body weight. The injectionswere given on the first three consecutive days of each week over a period4weeks. Livers were fixed in4%paraformaldehyde, embedded in paraffin andsectioned. Sections were used for hematoxylin and eosin (H&E), Sirius redstaining and immunohistochemistry following standard procedures.Furthermore，the expressions of AEG-1in above-mentioned groups wereexamined by RT-real time PCR and western blot. Primary rat HSCs wereisolated using pronase/collagenase perfusion digestion followed by densitygradient centrifugation. The quiescent HSCs immediately after plating weretested by fluorescence microscope to evaluate the purity of the cultures andα-SMA monoclonal antibody was used to identify the activated primaryHSCs by immunocytochemistry. To investigate the expression of AEG-1inactivated HSCs, we incubated cells with TGF-β and LPS of variousconcentrations (TGF-β:0ng/ml,5ng/ml,10ng/ml; LPS:0μg/ml,0.5μg/ml,1.5μg/ml) and timespans (0h,24h,48h), followed by western blot.Results:①To study the potential role of AEG-1in hepatic fibrosis, weused two well-established models, BDL and DMN, to induce liver fibrosis.As examined by H&E and Sirius red staining, the livers of BDL rats showedsever distortion of architecture, proliferating bile ductules and collagendeposition. Compared to the characters above, the liver of DMN ratsexhibited more hemorrhagic necrosis, sinusoidal congestion andinflammatory cells. These data confirmed that both fibrotic models weresuccessfully established.②AEG-1expression in fibrotic liver wassignificant increased, while little was detected in liver treated with sham orsaline detected by immunohistochemistry. AEG-1was localizedpredominantly in the cytoplasmic region.③Up-regulation of AEG-1infibrotic liver was also confirmed by RT-real time PCR analysis and westernblot (P<0.05).④The expression of AEG-1in HSC-T6stimulated by TGF-βwas up-regulated in a dose-and time-dependent manner.⑤The expression of AEG-1in HSC-T6stimulated by LPS was up-regulated in a dose-andtime-dependent manner.⑥Primary rat HSCs were isolated successfully bysequential digestion of the liver with pronase and eollagenase, followed bysingle step density gradient centrifugation with Nycodenz. Cell viability andcell purity were greater than90％, with a yield ranging from1.2×107to2.0×107HSCs/rat. The primary HSC immediately after plating is round andin rich of lipid droplet under the inverted microscope, while HSCs show blueunder the ultraviolet light (L=328nm). After14days culture，activated HSCslose retinoid and become fusiform myofibroblast-like cell.⑦To indentify theprimary HSCs identification α-SMA staining was performed at day l(quiescent, α-SMA negative cells)and day14(activated，α-SMA positivecells) by immunocytochemistry．⑧The expressions of AEG-1in primary ratHSCs stimulated by TGF-β (10ng/ml) or LPS (1.5μg/ml) for48h wereup-regulated.Conclusions: AEG-1expression in fibrotic liver was significantincreased. TGF-β, as well as LPS, induced AEG-1expression of HSCs in adose-and time-dependent manner.Part2: Knockdown of AEG-1inhibits activation of HSC-T6cellsObjective: To investigate the influences of down-regulation of AEG-1on HSC-T6activation.Methods: Three shRNA oligonucleotide duplexes targeting rat AEG-1sequence were synthesized and cloned into a lentivirus-based vector carryingthe green fluorescent protein (GFP) gene by GeneChem. Cells wereharvested after infection. Protein and mRNA were detected to determineAEG-1knockdown efficiency and screen for the most efficient shRNAwhich was then used for subsequent experiments. cell count kit-8was usedin cell proliferation assay. The cell cycle was analyzed by flow cytometer.The expression of collagen Ⅰ and α-SMA were examined by RT-real timePCR and western blot after knockdown of AEG-1.Results:①AEG-1expression in HSC-T6cells infected with sh-AEG-1lentivirus was significantly decreased at both mRNA and protein levels as compared to control cells. The protein interference efficiencies of threeshRNAs were75.22%,60.21%and64.36%respectively. The mRNAinterference efficiencies of three shRNAs were85.52%,69.96%and71.10%respectively.②Knockdown of AEG-1markedly suppressed cell viability ofHSC-T6cells examined by CCK-8.③Cell cycle analysis showed thatLv-shAEG-1infection led to an increase of cells population in G0/G1phase,but a corresponding decrease in G2/M phase, indicating that knockdown ofAEG-1induced cell cycle arrest in the G0/G1phase.④Gene abolishment ofAEG-1by shRNA interference significantly reduced the expression ofcollagen Ⅰ in both protein and mRNA levels (P<0.05).⑤Gene abolishmentof AEG-1by shRNA interference significantly reduced the expression ofα-SMAin both protein and mRNAlevels (P<0.05).Conclusion: Knockdown of AEG-1inhibits the activation of HSC-T6cellsPart3: Knockdown of AEG-1induces apoptosis and inhibits migrationof HSC-T6Objectives: To investigate the influences of knockdown of AEG-1oncell apoptosis and migration of HSC-T6.Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Theextent of apoptosis was quantified and visualized by Annexin V-PE/7AADstaining. The TUNEL technique using the In Situ Cell Death Detection kitPOD was performed to measure nuclear DNA fragmentation. Morphologicalexamination of HSC-T6was performed by scanning electron microscopy. Toverify the role of AEG-1on HSC-T6migration, wound-healing assay andtranswell insert chambers assay were performed.Results:①Knockdown of AEG-1by Lv-shAEG-1infection increasedTUNEL-positive cells distribution at10.43%, as compared to5.01%and5.43%in uninfected and LV-shCon infected cells, respectively.②A flowcytometric analysis also demonstrated that as compared to the LV-shConinfected cells, knockdown of AEG-1in the cells infected with Lv-shAEG-1significantly induced cell apoptosis (7.97±2.52%vs27.12±5.72%, P<0.05). ③Analysis of mRNA showed enhanced Caspase-3expression inLv-shAEG-1infected cells compared with LV-shCon cells.④Knockdown ofAEG-1reduced HSCs cell volume, shortened microvilli on cell surface andvanished dendritic pseudopodia.⑤In the wound-healing assay, the distancemoved by a wounded cell significantly decreased in Lv-shAEG-1group at24h (P<0.05) and even pronounced at48h (P<0.05) after treatment.⑥Inthe transwell insert chambers assay, the number of Lv-shAEG-1infectedcells (18.20±3.44) which penetrated the chamber membrane wassignificantly less than the uninfected cells (58.60±4.82; P<0.05) andLV-shCon infected cells (46.60±4.85; P <0.05).Conclusion: Knockdown of AEG-1induces apoptosis and reduces cellmigration capacity of HSC-T6Part4: The mechanism of intracellular signal transduction thatcontribute to the impacts of AEG-1on HSC-T6biologicalbehavioursObjectives: To explore the regulation of AEG-1knockdown onPI3K/Akt, ERK and P38MAPK signalling pathways in HSC-T6．Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Thephosphorylated and totle protein expressions of PI3K/Akt, ERK and P38MAPK were examined by Western blot.Results:①Knockdown of AEG-1in HSC-T6cells inhibitedphosphorylation of PI3-K and Akt (P<0.05), while total expression of PI3-Kand Akt were unchanged.②Knockdown of AEG-1in HSC-T6cellsinhibited phosphorylation of ERK and P38MAPK (P<0.05), while totalexpression of ERK and P38MAPK were unchanged.Conclusion: Knockdown of AEG-1changed HSC-T6biologicalbehaviours probably via down-regulation of phosphorylations of PI3-K, Akt,ERK and P38MAPK signaling pathways... |
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