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中国循证儿科杂志

中国循证儿科杂志 ›› 2017, Vol. 12 ›› Issue (6): 463-467.

• 论著 • 上一篇    下一篇

慢病毒介导shRNA抑制β-连环蛋白对儿童髓母细胞瘤细胞株生物学行为的影响

万江，张海燕，刘义红，李新涛，涂胜英，吴丽敏   

  1. 武汉科技大学附属孝感医院儿科 湖北孝感，432000
  • 收稿日期:2017-11-15 修回日期:2017-12-22 出版日期:2017-12-25 发布日期:2017-12-25
  • 通讯作者: 张海燕，E-mail: ZHY-zq@163.com

Effect of lentivirus-mediated shRNA targeting β-catenin on biological behavior in children medulloblastoma

WAN Jiang, ZHANG Hai-yan, LIU Yi-hong, LI Xin-tao, TU Sheng-ying, WU Li-min   

  1. Department of Pediatrics, Xiaogan Hospital affiliated to Wuhan University of Science and Technology, Xiaogan 432000, China
  • Received:2017-11-15 Revised:2017-12-22 Online:2017-12-25 Published:2017-12-25
  • Contact: ZHANG Hai-yan, E-mail: ZHY-zq@163.com

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摘要： 摘要 目的：构建并观察靶向β-连环蛋白（β-catenin）的shRNA慢病毒对儿童髓母细胞瘤（MB） 细胞株生物学行为的影响。方法：①设计靶向β-catenin的shRNA 1、2、3和阴性对照（NC），分别插入pCH-CMV-MCS-EF1-copGFP质粒中，通过慢病毒包装后获得相应的重组慢病毒。②培养儿童MB细胞株DOAY并分为Lentivirus（LV）-shRNA 1、2、3、 NC组和空白对照组（Blank），前4组感染相应的重组慢病毒，Blank组未感染病毒，感染72 h后在荧光显微镜下观察绿色荧光蛋白（GFP）表达，通过RT-PCR和Western Blot检测各组细胞中β-catenin的表达情况，选取对β-catenin抑制效果最佳的LV-shRNA，用嘌呤霉素分别筛选稳定感染该LV-shRNA和LV-NC的DOAY细胞系。③设3组，LV-shRNA组和LV-NC组为筛选得到的稳定感染的细胞系，Blank组为未感染病毒的细胞，采用MTT法检测细胞增殖能力，流式细胞术检测细胞凋亡情况，Transwell实验检测细胞侵袭能力，细胞划痕实验检测细胞迁移能力。结果：①LV-shRNA 1、2、3组β-catenin的mRNA和蛋白表达均低于LV-NC组和Blank组，LV-shRNA 1组低于LV-shRNA 2、3 组，差异均有统计学意义（P＜0.05），以4 μg·mL-1嘌呤霉素筛选得到稳定表达LV-shRNA 1和LV-NC的DOAY细胞株。②与LV-NC和Blank组比较，LV-shRNA 1组MTT实验第12、24、36、48、72 h时的OD值较低，细胞总凋亡率较高，穿膜细胞数目较少，细胞迁移距离较短，差异均有统计学意义（P＜0.05）。结论：慢病毒介导shRNA抑制β-catenin后，能显著抑制DOAY细胞的增殖能力，降低其侵袭和迁移能力，促进其凋亡。

Abstract: AbstractObjective: 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 of β-catenin was detected by reverse transcription PCR and Western Blot, the β-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. Results: 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).Conclusion: 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.