Effect Of β-catenin Silenced By Recombinant Adenovirus-mediated SiRNA On Proliferation Of Human Amniotic Mesenchymal Stem Cell

OBJECTIVETo construct recombinant adenovirus containing Green fluorescent protein and P-catenin silenced specifically by RNAi, to inhibit the expression of P-catenin in the post-transcriptional, to investigate the effects of β-catenin siRNA on proliferation of amniotic mesenchymal stem cell (AMSC).METHODSIn the first part, we designed and synthesized three interference fragment, P-catenin siRNA was transfected instantaneously into AMSC by liposome, selected the most efficient interference fragment by Western blot. Then encoded single-stranded DNA matching targeting genes CTNNB1short hairpin RNA (shRNA) and acquired targeting genes double-stranded DNA. Then cutted targeting genes by two endonucleases and directional linked to the pAd track-cmv vector. Afterward, the correct recombinant was linearized by Pmel, following co-transformation with the backbone vector pAdEasy-1in E.coli BJ5183. The homologous recombinant was linearized by Pad, and then transfected into HEK293cell line to pack the adenovirus. And detected the viral titer by plaque assay. In the second part, the adenovirus infected AMSC. The expression of β-catenin and GSK-3P were assayed by Western blot. The change of cell proliferation was determined by CKK-8.RESULTS1. Transfection of β-catenin siRNA to AMSC could effectively inhibit the expression of P-catenin, we selected the best target gene sequence successfully by Western blot, designed and synthesized targeting genes double-stranded DNA. And we constructed recombinant adenovirus vector pAd-si-β-catenin successfully. We packaged and amplified recombinant adenovirus successfully, and we obtained the adenovirus supernatant of high titer.2. After AMSC infected by the adenovirus, we could observed the expression of GFP in infection group, and by Western blot we could detected that the expression of P-catenin was inhibited effectively, and the expression of β-catenin in infection group was less than control group obviously.3. After AMSC infected by the adenovirus, we could detected cell growth curve. And compared with control group, the cell proliferation of infection group was inhibited effectively.CONCLUSIONThe β-catenin-targeting RNAi recombination adenovirus is efficient to inhibit the expression of P-catenin gene in AMSC and to decrease the expression of β-catenin in AMSC. And P-catenin silenced could inhibit effectively cell proliferation in AMSC.