万江 1张海燕 1刘义红 1李新涛 1涂胜英 1吴丽敏1
作者信息
- 1. 武汉科技大学附属孝感医院儿科 湖北孝感,432000
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摘要
Abstract
Objective To construct lentivirus-mediated shRNA targeting β-catenin to infect DOAY cells, to observe the expression of β-catenin and the biological behavior of cells. Methods 3 kinds of shRNA targeting β-catenin were designed and inserted into the pCH-CMV-MCS-EF1-copGFP, the recombinant pCHD-shRNA was obtained. The pCHD-shRNA was transfected into HEK293T cells, and the lentivirus-shRNA was obtained. DOAY cells were infected by lentivirus-shRNA and divided into 5 groups:Lentivirus-shRNA 1group (LV-shRNA 1), Lentivirus-shRNA 2 group (LV-shRNA 2), Lentivirus-shRNA 3 group (LV-shRNA 3) , Lentivirus-NC group ( LV-NC ) and blank group ( Blank ) . After 72 h, the expression of green fluorescent protein (GFP) was observed, the expression ofβ-catenin was detected by reverse transcription PCR and Western Blot, theβ-catenin gene stable suppression cells were screened out by puromycin. Effects of silencing β-catenin expression on cell biological behavior were observed. There were 3 groups: LV-shRNA 1, LV-NC and Blank. The proliferation and apoptosis of cells were detected by MTT assay and flow cytometry, the invasion and migration of the cells were detected by Transwell and cell scratch test. ResuIts The lentivirus-mediated shRNA targeting β-catenin was constructed successfully. 72 h after injection, the expression of GFP was not found in the blank group, the infection efficiency of LV-shRNA 1, LV-shRNA 2, LV-shRNA 3 and LV-NC was more than 89％(P>0.05).The expression of β-catenin mRNA (0.24±0.09) and protein (0.15±0.07) in LV-shRNA 1 group was significantly lower than that in the other 4 groups, and the difference was statistically significant (P<0.05). Compared with LV-NC and blank group, the OD ( 570 nm) values of LV-shRNA 1 group at 24, 36, 48, 72 h were significantly decreased, the total apoptosis rate (31.28％) was significantly increased, the numbers of crossed cells (8.41±21.27) and migration distance (497.33±45.28) μm were significantly reduced, the difference was statistically significant (P<0.05).ConcIusion Lentivirus-mediated shRNA targeting β-catenin was successfully constructed. LV-shRNA 1 could significantly down-regulate the expression of β-catenin, significantly inhibit the proliferation of DOAY cells, reduce their invasion and migration ability, promote their apoptosis.关键词
β-连环蛋白/慢病毒/髓母细胞瘤/生物学行为Key words
β-catenin/Lentivirus/Medulloblastoma/Biological behavior
