Construction Of The MIRNA RNAI Vector And The Eukaryotic Expression Vector Of Ago2, And Preliminary Study On The Role Of AGO2 In Claudin-11 Expression

Objective:In previous study,we found that Argonaute 2(Ago2) may be involved in the regulation of claudin-11 expression.Then to further investigate the cellular and molecular mechanism by which Ago2 regulate the expression of claudin-11,we constructed the miRNA RNAi vector and the eukaryotic expression vector of Ago2 and investigated the biological role of Ago2 in claudin-11 expression.Methods:To consturct the eukaryotic expression vector of Ago2,the coding region of Ago2 amplified by RT-PCR from mouse testis RNA was cloned into the pEGFP-C1 vector to form the recombinant pEGFP-C1-Ago2 plasmid.Then the recombinant plasmid was transfected into 15P-1 sertoli cells.Additionaly,BLOCK-iT? HiPerform? Lentiviral Pol II miR RNAi Expression System with EmGFP was adopted to construct lentiviral expression vector targeting for mouse Ago2, subsequently, Ago2 RNAi lentiviruses were transfected into 15P-1 sertoli cells. The expression of Ago2 and claudin-11 in 15P-1 sertoli cells were analyzed by RT-PCR and western blotting,respectively.Results:Ago2 coding region was successfully cloned into the eukaryotic vector pEGFP-C1.The recombinant vector was transfected into 15P-1 sertoli cells,results of RT-PCR and Western blotting showed that the expression of Ago2 was significantly up-regulated in 15P-1 sertoli cells. Ago2 RNAi lentiviruses were transfected into 15P-1,and a stable 15P-1 sertoli cell line bearing Ago2-miRNA RNAi lentivirus was successfully constructed by Blasticidin screening. Furthermore, Ago2 silenced induced a significant decrease in claudin-11 expression,which indicated that Ago2 is a positive regulator of claudin-11.Additionally,15P-1 sertoli cell with pEGFP-C1-Ago2 transfected exhibited high level of claudin-11 strengthen the evidence for claudin-11 is regulated by Ago2.Conclusion1. Ago2 RNAi lentivirus is successfully prepared,which could effectively inhibit Ago2 expression in 15P-1 cells.Also,We have constructed a recombinant vector expressing Ago2 and eGFP fusion protein, which is in favour of the further study on the gene function of Ago2.2. Ago2 is a positive regulator of claudin-11 expression.