PTPRO Overexpression In Human Hepatoma Cells Regulate The Epithelial-mesenchymal Transition

Objectives:To observe the effect of overexpression PTPRO in human hepatoma cells on the epithelial–mesenchymal transition(EMT),as well as the expression changes of important phenotypic markers such as E-Cadherin,N-Cadherin,Vimentin and ?-catenin in EMT.Methods:(1)HepG2,the research object,cells were treated with EGF(human epidermal growth factor)at concentration of 100ng/m L for 36 hours,observe the cell mesenchymal changes by the Reversed Fluorescence microscope to study whether EMT was induced by EGF in vitro.(2)To construct The p EGFP-PTPRO and p EGFP-C2 carriers,gene sequencing and RT-PCR confirmed the successful construction of the carriers.(3)Hep G2 cells were transfected with p EGFP-PTPRO and p EGFP-C2 empty carriers respectively.Reversed Fluorescence microscope and Western-bolt tested the efficiency of transfection.(4)The experiment was divided into three groups:overexpression group(transfected p EGFP-PTPRO group),empty carrier group(transfected p EGFP-C2 group),blank group(transfection reagent group).all groups were treated with EGF at concentration of 100ng/m L for 36 hours,observe the cell mesenchymal changes differences among the threegroops by the microscope after successful transfection.(5)Wound healing test to observe the migration ability of each group cells.(6)To test the expression of PTPRO,E-Cadherin,N-Cadherin,Vimentin and ?-catenin in cells of each group by Western-bolt.(7)The expression and distribution of ?-catenin in the cells were further observed by laser scanning confocal microscope.Results:(1)Epithelial-mesenchymal transition was obviously observed by Reversed Fluorescence microscopein in the Hep G2 cells which were treated with EGF at concentration of 100ng/m L for 36 hours.(2)Gene sequencing and RT-PCR confirmed the successful construction of the p EGFP-PTPRO and p EGFP-C2 carriers.(3)Hep G2 cells were transfected with p EGFP-PTPRO and p EGFP-C2 empty carriers respectively.After the plasmid was transfected into Hep G2 cells observe through the Reversed Fluorescence microscope,we can found that successfully transfected cells will distribute green fluorescence,the transfection efficiency more than 70%.Western-blot detection display that PTPRO protein expression was significantly increased(p<0.05)in the overexpression group cells of p EGFP-PTPRO with empty carrier group p EGFP-C2 had no obvious change(p>0.05).(4)All groups were were treated with EGF at concentration of 100ng/m L for 36 hours after successfully transfected,Under microscope,no obvious mesenchymal changes were observed in PTPRO overexpression group,but there were obviousmesenchymal changes in empty carrier group and blank control group.(5)Wound healing test display that cultivate the cells which transfected p EGFP-PTPRO carrier the cell scratch no significant healing.Conversely,the cells which transfected the corresponding empty carrier and the Hep G2 cells scratch significant healing after cultivate 48 hours.(6)Western blot showed that the expression of E-cadherin protein in the overexpression group(p EGFP-PTPRO group)was higher than that in the empty carrier group(transfected with p EGFP-C2 group)and blank control group(p<0.05).But The expression of Vimentin protein and N-cadherin protein was lower than the other two groups(p<0.05).In addition,The expression of E-cadherin protein,Vimentin protein and N-cadherin protein were not significantly different between empty carrier group and blank group(p>0.05).But the expression level of ?-catenin protein had no significant difference among the three groups(p>0.05).(7)Confocal microscopy revealed that ?-catenin was mainly distributed in the cytoplasm in the blank and empty carrier groups,while ?-catenin in the p EGFP-PTPRO group was partially transferred to the nucleus.Conclusion: PTPRO gene expression can inhibit liver cancer cells EMT phenomenon.