Establishment of EC-1 cell line stably expressing p-Stat3 protein

Aim: To establish a EC-1 cell line stably expressing p-Stat3 protein. Methods: EC-1 cells were transfected with eukaryotic expression vector pXJ40-Stat3 by Lipofectamine 2000, screened by G418, and then stimulated by IL-6 for 20 min to obtain the cell line stably expressing p-Stat3 (pXJ40-Stat3+IL-6 group). At the same time EC-1 cells transfected with pXJ40-Stat3 but not stimulated by IL-6(pXJ40-Stat3 group), EC-1 cells stimulated by IL-6 alone, EC-1 cells transfected with blank plasmid and blank EC-1 cells were used as control. The expressions of Stat3 mRNA and p-Stat3 protein in each group were detected by RT-PCR and Western blot. Results: The expression of Stat3 mRNA and p-Stat3 had significant difference among the five groups(FStat3 mRNA=5.971,P=0.010;Fp-Stat3=7.334,P=0.005). The expression of Stat3 mRNA in pXJ40-Stat3+IL-6 group was the highest in the five groups and it was (0.72±0.01), followed by that in pXJ40-Stat3 group(0.65±0.01),and no significant differences were found in the other three groups. The expression of p-Stat3 in pXJ40-Stat3+IL-6 group was also the highest in the five groups and it was (0.72±0.03), followed by that in IL-6 alone group(0.65±0.01), and no significant differences were found in the other three groups. Conclusion: The EC cell line stably expressing p-Stat3 has been successfully constructed and high expression of p-Stat3 could be obtained after IL-6 treatment.