The Study On ERK2 Expression In Esophageal Cancer 109 Cell Line By Short Hairpin RNA Interference

Objective: Short hairpin RNA (shRNA) interference vector targeted ERK2 was constructed using pGeneclip U1 hairpin cloning systems,to observe ERK2 gene silencing effect and mechanisms in human esophageal cancer cell line(ECa109).Methods:The expression of phosphoralation ERK2 (p-ERK2), incentived by serum and U0126 (10μM-50μM) in ECa109 , were detected by western-blot technique. Two complement- tary oligo DNA strands targetedERK2 gene were designed and synthesized according to the principles of siRNA. After annealing, oligo DNAs were inserted into pGeneClipTM vector by T4DNA lingase. The recombinant plasmid shRNA-ERK2was transfected into ECa109with lipofectamine 2000The transfection efficiency was observed by fluorescence microscope and the proliferation ability of ECa109 cell were analyzed by MTT at 24h,48h,72h,96h. The expression of ERK2 and survivin were detected by western-blot at 72h and 96h. The cell apoptosis and the cell cycle was analyzed by flow cytometry(FCM)after transfection. Results: The expression of phosphorylated ERK2 rised gradually along with the time of serum stimulation, while it can be decreased the expression of phosphorylated ERK2 gradually with the increase of concentration of U0126.Based on enzyme digestion and DNA sequencing analysis, shRNA-ERK2were constructed successfully. The transfection efficiency of shRNA-ERK2 at 72 hours is higher than that at 48h and 96h. The proliferation rate of Eca109 cells after transfection with shRNA-ERK2, control vector shRNA-scrambled , or untransfected cells control for 4 days, were examined by MTT assay. The cells, transfected with shRNA-ERK2, were obviously retarded the proliferation at 96 hours, the inhibition rate is 10.45%, which is highest.There was no significant difference was no significant difference between control groups. there was a significant decrease of ERK2 and survivin expression at 72 hours and 96 hours compared to control (P<0.05), respectively. Moreover, there was also a statistical difference between 72 hours and 96 hours (P<0.05). The apoptosis rate of Eca109 cells transfected with shRNA-ERK2 was 9.7%. The cells proportion of in S phase in shRNA-ERK2 group was decreased compared to control cells.Moreover, the proportion of cells in G1 phase in shRNA-ERK2 group was increasedcompared to control cells.Conclusion: ERK2 targeted small interfering RNA and its expression vector were constructed and transfected into ECa109 successfully. The induction of cell apoptosis and inhibition of cell cycle may be affect the cell growth.ERK2 play an important role in the ECa109 cell growth.