The Stabilization Of Novel Mutant H36-beta-catenin And Correlated Effects On Proliferation Of Hepatoma Cells

Background: The protein ?-catenin encoded by CTNNB1 gene have always played a crucial role in the canonical Wnt signaling, although which evolves conservatively, it affects diversely in functions. Moreover, its deregulation has been implicated in human diseases, most notably in cancer, but also in the association with the size, metastasis and clinic stage of liver cancer. DSG?XS, the ubiquitination destruction motif in ?-catenin, is the core domain binding the E3 ubiquitin ligase ?-Tr CP, that can trigger the proteosomal degradation of ?-catenin. Now generally believed that the S45?D32?T41?G34?S33 and S37 are the most frequently mutated nonserine/threonine residues in DSG?XS structure, which could alter the stabilization of ?-catenin and so further abnormally regulate cellular proliferation, invasion, differentiation and so on. Prof. Yosuke Hirotsu, Yamanashi Prefectural Central Hospital, Japan, through the targeteddeep sequencing analysis found for the first time that H36 of DSG?XS structure in ?-catenin is mutated frequently(42%) in hepatic tumor. Therefore, Several relevant work on novel mutant H36 was conducted.Aim: To reveal the stabilization and nuclear translocation of novel mutant H36-?-Catenin, and further explore the correlate effects of proliferation in hepatoma cells.Methods: Firstly, amplified CTNNB1 gene by PCR, then constructed plasmids including WT(negative control), G34R(positive control), H36P(novel mutant) gene and clone culture, meanwhile, the vector p-CMV5 was set as blank control. All the plasmids were equally transfected into Hep G2 and Huh7 hepatoma cells, respectively. The expression of WT-?-catenin, G34R-?-catenin and H36P-?-catenin was examined by Western Blotting. The degradation of related ?-catenin in Hep G2 and Huh7 was detected after treated with cycloheximide(CHX). CCK8 and Ed U assay was performedfor detecting the ability of proliferation. All the experimental data and images were deal with SPSS, Photo Shop and Graphad Prism software.Results: The results showed that the expression of novel mutant H36P-?-catenin was no difference compared to control. However, its proteosomal degradation was suppressed, which mean that the thestability of H36P-?-catenin was increased. In addition, the proliferation ability of Hep G2 and Huh7 transfected with H36 P was significantly improved compared with G34 R.Conclusions: The expression of novel mutant H36P-?-catenin performed no difference compare to WT-?-catenin and G34R-?-catenin, but the stabilization and nuclear translocation was certainly improved, as well as the proliferation ability of Hep G2 and Huh7 hepatoma cells.