Research On Target Relationship Between NF-?b And CD133 By Dual Luciferase Report Gene Assay

Purpose: Our team found that NF-?B and CD133 are both high expression in cholesteatoma tissue. Flow cytometry was used to indicated that,compared with Rhek-ev(empty vector)cell line,the ratio of CD133-positive cells in Rhek-P65(Excessive expression of NF-?B) cell lines was increased.It is a positive correlation between the expression level of NF-?B and the expression level of CD133,and it is also suggesting that NF-?B can regulate the expression of CD133,and CD133 maybe a potential target gene of NF-?B,there is no enough evidence to prove it now.In this study,we construct a luciferase reporter plasmid(promoter-PGL3-basic CD133) of CD133 gene promoter, Applying Dual-Luciferase Reporter Gene System to verify the regulation between NF-?B and CD133,further to provide the theoretical basis for the possible mechanism of NF-?B and CD133 in cholesteatoma occurrence and development.Methods:Using Biological information website to predict the promoter sequence of CD133 gene which may be combined with NF-?B. After PCR amplifying, the CD133 gene promoter sequence was inserted into the luciferase reporter gene vector PGL3-basic to construct a luciferase reporter plasmid(promoter-PGL3-basic CD133) of CD133 gene promoter.Next CD133 promoter luciferase reporter plasmid?pc DNA-NF-?Bplasmid?PGL3-control plasmid?pc DNA3.0 plasmid and PRL-TK plasmid were cotransfected to 293 ft cell bygrouping. Dual-Luciferase Reporter Assay System was used to determine the luciferase activity after 48h-72 h to assay the luciferase activity and observe the changes of fluorescence value.Results:Restriction enzyme analysis showed that the sequence of this gene fragment was identical with CD133 promoter gene sequence reported in genbank.The CD133 promoter-p GL3-Basic eukaryotic expression vector was confirmed by sequencing. The activity of luciferase reporter gene was 46.62±4.62in 293 ft cells which transfected CD133 promoter-p GL3 eukaryotic expression vector and pc DNA-NF-?B plasmid,and the activity of luciferase reporter gene is 44.74±7.85 in 293 ft cells which transfected CD133 promoter-p GL3 eukaryotic expression vector?pc DNA3.0 plasmid. The p value is more than 0.05 between two groups.Conclusion : CD133 promoter-pGL3-Basic eukaryotic expression vector was successfully constructed. NF-?B may not regulate the activity of CD133 gene's promoter.