Construction of β-catenin-targeting RNAi lentivirus and effect of Wnt/β-catenin signaling on proliferation of mice pancreatic beta-cell

Abstract

Objective To construct an expressive RNA interference RNAi lentivirus specific to β-catenin, and to investigate the regulation of Wnt/β-catenin signaling on the proliferation aspect of mice pancreatic β cell line (Min6 cell). Methods Homologous recombination and cloning techniques were used to create the lentivirual plasmid which contains the RNAi cassette targeting the β-catenin gene. Then, the lentivirual plasmid was transfected with the other two packaging plasmids into 293T cells to product and amplify lentivirus. Dilution assay was used to titer the lentivirual stock.The β-catenin gene silencing effect induced by the RNAi lentivirus in Min6 cell was detected by real time-PCR and Western blot analysis. Finally, we transducted the RNAi lentivirus to inhibit the expression of β-catenin protein and to explore the effect of Wnt/β-catenin pathway on proliferation of mice pancreatic β cell, with MTT cell proliferation assay. Results The RNAi lentivirus specific to β-catenin was produced with a titer of about 5.0×108 TU/ml.The RNAi lentivirus could be infect Min6 cells efficiently in 3 days, and the inhibition effect was detected in 5 days after infection. The results of MTT assay suggested that inhibiting the β-catenin with the RNAi lentivirus could suppress the proliferation of Min6 cells obviously. Conclusion RNAi lentivirus is an important tool to inhibit the expression of target gene efficiently. The Wnt/β-catenin pathway plays an important role in the regulation of proliferation of mice pancreatic β cells. Key words: RNAi; Wnt signaling; β-catenin; Beta cell of pancreatic islet; Proliferation