| Objective： To explore the effect and molecular mechanism ofArginine-specificmono-ADP-ribosytransferases-1(ART1) onepithelial–mesenchymal transition of murine CT26cells.Methods: In this experiment, GFP-ART1group and GFP-shART1group as experimental group, GFP-Vector group and un-transfectiongroup as control group. Cell morphology in each group was observed byhematoxylin-eosin staining(HE). Expression of EMT inducers Snail1,mesenchymal marker protein N-cadherin, Vimentin and epithelial markerprotein E-cadherin were tested by Western blot. Abilities of Migration,invasion and adhesion were determined by Wound healing assay, Transwellinvasion and Adhesion assay respectively. BALB/C mouse model of livermetastasis were successfully built and liver metastasis was observed ineach group. Expression of β-catenin in each group was examed byImmunofluorescence assay and western blot. Adenovirus Ad-si-β-cateninwas transfected successfully into Lv-GFP-ART1group. Expression ofSnail1，N-cadherin, Vimentin and E-cadherin were tested by western blot after silencing β-catenin in Lv-GFP-ART1group. Expression of RhoA,ROCK1, Phospho-AKT（ser-473）, Phospho-GSK-3β（ser-9）in each groupwere determined by Western blot. Un-transfected CT26cells were treatedwith Y27632and Y294002respectivly and expression of ROCK1,Phospho-AKT（ser-473）were detected by western blot between treated anduntreated group. Actin polymerization was observed by confocalfluorescence microscopy. Expression of F-actin was measured by Flowcytometry(FCM) assay. Activity and expression of MMP-9and MMP-2were determined by western blot and Zymography assay respectively.Rusults:（1） Effect of ART1on CT26cells EMT and metastatic potential invivo and vitro. HE staining showed that compared with the control group,the number of spindle-like non-polarized mesenchymanl-like cellssignificantly increased, while apical–basal polarized epithelioid cellsdramaticlly decreased in GFP-ART1group. In contrast, silencing ART1inCT26cells induced a significant increase of epithelioid cells and a obviousdecrease of mesenchymanl-like cells. Western blot confirmed thatcompared with the control group, expression of EMT inducers Snail1,mesenchymal marker protein N-cadherin, Vimentin were increased andexpression of epithelial maker E-cadherin in GFP-ART1group wasdecreased in vivo and vitro, and GFP-shART1group showed the oppositephenomenon(p<0.05). Compared with the control group, GFP-ART1grouppromoted CT26cells abilities of adhesion, invasion and migration andGFP-shART1group inhibited CT26cells abilities of adhesion, invasionand migration in vitro(p<0.05). The mouse model of liver metastasis wassuccessfully built through under the splenic capsule with CT26cellsinjection. Compared with GFP-vector group and non-transfection group, Liver weight was much heavier in GFP-ART1group and was lighter inGFP-shART1group(p<0.05). The numer of metastasis nodules showed asignificant increase in GFP-ART1group and an observably decrease inGFP-shART1group, compared with GFP-vector group andnon-transfection group in vivo(p<0.05).（2）Research on possible pathway of EMT related protein by ART1regulation. Immunofluorescence confirmed that expression of β-cateninexisted in cytoplasm and the nucleus of CT26cells, and compared withnon-transfection group and GFP-vector group, mean fluorescence intensitywere increased in GFP-ART1group and decreased in GFP-shART1group(p<0.05). Western blot assay confirmed that compared with thecontrol group, expression of β-catenin in GFP-ART1group was increasedand was decreased in GFP-shART1group in vivo andvitro(p<0.05).Adenovirus Ad-si-β-catenin was sucessfully transfected intoGFP-ART1group. expression of Snail1, N-cadherin, Vimentin expressionwere significantly decreased and expression of E-cadherin was a significantincrease in GFP-ART1group after silencing β-catenin(p<0.05). Comparedwith the control group, expression of Phospho-AKT(ser-473) andPhospho-GSK-3β （ser-9） were increased in GFP-ART1group anddecreased in GFP-shART1group in vivo and in vitro(p<0.05). GFP-ART1group was treated with Y294002, showed that compared with untreatedgroup, expression of β-catenin was decreased in treated group. Expression of RhoA and ROCK1were increased in GFP-ART1group and decreased inGFP-shART1group in vivo and in vitro(p<0.05). Un-transfection groupwas treated with Y27632, showed that expression of ART1was nostatistically significant difference between treated group and untreatedgroup(p>0.05). Untransfected CT26cells were treated with Y294002andY27632, showed that ROCK1increased the phosphorylation of AKT, butthe latter failed to regulate ROCK1. In confocal laser scanning microscopy,compared with the other two control groups, GFP-ART1group displayedordered actin network, more actin fibers. However, GFP-shART1groupindicated disordered actin network, few actin fibers. Expression of F-actinwas determined by FCM. Compared with control group, Expression ofF-actin was increased in GFP-ART1group and decreased in GFP-shRNAgroup. Compared with control group, expression and activities of MMP-2and MMP-9were increased in GFP-ART1group and decreased inGFP-shART1group(p<0.05).Conclusion： We showed that ART1regulated CT26cells EMT at thevery least through activation of RhoA/ROCK1/AKT pathway, which leadto cytoskeleton reorganization, a decrease of epithelial maker E-cadherin,an increase of EMT inducers snail1, and mesenchymal marker includingVimentin, N-cadherin and MMP-2, MMP-9. Base on these molecularmechanism, ART1promoted CT26cells invasion and metastasis through EMT in vivo and vitro. And it is suggested that ART1plays an importantrole in colon cancer progression. |
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