N-Mycdownstream-Regulatedgene2(NDRG2) Overexpression In Human Hep Atoma Cellsre Gulatethe Epithelial-Mesenchymaltransition(EMT)

Objectives :To observe the effect of NDRG2 overexpression in human hepatoma cell on the epithelial–mesenchymal transition(EMT). To explore that whether NDRG2 overexpression in Hep G2 cell through inhibiting Stat3 / Twist signaling pathway axis to suppresse the EMT.Methods:(1)Hep G2, the research object, Cell was treated with EGF at concentration of( 100ng/m L) for 24 hours,observing the cell morphology by the Reversed Fluorescence microscope to determine whether induce the Epithelial-mesenchymal transition.(2)To construct The PEGFP-NDRG2 and PEGFP-C2 carriers;(3)Make PEGFP-NDRG2 and PEGFP-C2 plasmid immediatedly transfected to Hep G2 respectively.Reversed Fluorescence microscope,RT-PCR and Western-bolt tested the efficiency of transfection.(4)Divided the test by four groups: PEGFP-NDRG2, PEGFP-C2,Transfection reagent and Blank.After the cells were successfully transfected PEGFP-NDRG2 and PEGFP-C2 respectively treated with EGF at the concentration of 100ng/ml for 36 hours,To test the expression of P-Stat3 in four groups by Western-Blot.(5)To test the Stat3,Twist,E-Cadherin m RNA in cells which was transfected PEGFP-NDRG2 and PEGFP-C2 respectively and treated with EGF for 36 hours by RT-PCR.Results:(1)At first the Hep G2 cells were treated with EGF at concentration of 100ng/m L for 24 hours, then observed the cells through the Reversed Fluorescence microscope. We found that the cells were induced the Epithelial-mesenchymal transition obviously.(2) We constructed the eukaryotic expression vector cells of PEGFP-C2 containing high NDRG2 expression and corresponding to the empty vector PEGFP-C2 and transfected them into Hep G2 cells.The sequenceing showed that the eukaryotic overexpression vectorcells of PEGFP-C2 which successfully constructed expression of NDRG2.After the plasmid was transfected into Hep G2 cells observed through the Reversed Fluorescence microscope,we could find that successfully transfected cells would distribute green fluorescence, the transfection efficiency more than 70%. RT-PCR and Western-blot detection displayed that NDRG2 protein expression was significantly increased(P<0.05) in the eukaryotic expression vector cells of PEGFP-NDRG2.The expression of Hep G2 cells,m RNA of empty vector PEGFP-C2 and protein had no obvious change(P>0.05).(3) The influence of transfection PEGFP-NDRG2 vector on Stat3 / Twist / E-Cadherin signaling pathway.RT-PCR detection display that the cells which transfected PEGFP-NDRG2 vector its Stat3 m RNA had no obvious change compared to the other groups,but the level of Twist m RNA was significantly reduced.Incontrast,the level of E-Cadherin was significantly increased.Western-boltdetection display that the cells which transfected PEGFP-NDRG2 vector its p-Stat3 protein expression was significantly reduced compared to other groups(P<0.05).Conclusion:(1)NDRG2 gene overexpression can inhibit liver cancer cells EMT phenomenon;(2) It may play a role by inhibiting Stat3 / Twist signaling pathway axis.