Construction Of β-catenin-targeting RNAi Lentivirus And Investgation Of The Effect Wnt/β-catenin Signaling On The Proliferation Of Mice Pancreatic Beta Cell

Objective: The aim of this study was to construct an expressive RNAi lentivirus specific toβ-catenin in order to observe the post-transcriptional knockdown effect ofβ-catenin in mice pancreatic beta cell line(Min6 cell) ; and then we used this technical tool to additionally investigate the regulation of Wnt/β-catenin signaling on the proliferation aspect of Min6 cellMethods: In the first part, We designed and synthesized four pairs of complementary double strand DNA oligos, which could express short hairpin RNA targeting theβ-catenin mRNA, and linked them into the lentiviral plasmid vector pGCL-GFP; next we constructed anotherβ-catenin over-expression eukaryotic plasmid, which was cotransfected together with the recombined lentiviral plasmid into 293T cell in order to screen and verify the knockdown effect of the RNA interference by Western blot; and then we packaged the most efficient lentiviral plasmid vector assisted with the other two plasmids (pHelper 1.0 and Helper 2.0) in 293T cell line, and detected the viral titer by dilution method. In the second part, we infected the RNAi lentivirus to Min6 cell, and observed the inhibition ofβ-catenin mRNA and protein level in terms of Real time PCR (RT-PCR) and Western blot, meanwhile further detected the efficiency of RNAi lentivirus endogenously. Finally, we utilized the efficient RNAi lentivirus to attenuateβ-catenin level in Min6 cell in order to investigate the regulation effect by turning down Wnt/β-catenin signaling on the proliferation of Min6 cell through MTT method.Results: 1.We successfully constructed RNAi lentiviral plasmid vectors targeting different sits ofβ-catenin mRNA, of which the base sequences and insertion sites were confirmed to be completely correct by PCR and sequencing assay. We also successfully constructed aβ-catenin over-expression eukaryotic vector. After cotransfecting RNAi lenteiviral plasmid andβ-catenin over-expression eukaryotic vector into 293T cell, Western blot detection indicated a remarkable reduction ofβ-catenin protein level in the cell. Packaging the most efficient RNAi lentiviral plasmid with assistant packaging plasmids in 293T cell line, we obtained the RNAi lentiviral supernatant of high titer. 2. We detected a highly efficient inhibition to the expression ofβ-catenin gene through RT-PCR and Western blot after infecting the specific RNAi lentivirus to Min6 cell. 3. Down regulating the Wnt/β-catenin signaling byβ-catenin-targeting RNAi lentivirus could obviously suppress the proliferation of Min6 cell.Conclusions: Theβ-catenin-targeting RNAi lentivirus is efficient to inhibit the expression ofβ-catenin gene in pancreatic beta cell, which can be used as an important technical tool to study the Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling plays an important role in the proliferation of mice pancreatic beta cell.