Construction of lentiviral vector harboring short hairpin RNA of RNA binding protein MSI1 and its effects on biological behaviors of human glioma cells

作者：付锴; 宫睿; 王伟

来源：中华实验外科杂志, 2016, 33(9).

DOI：10.3760/cma.j.issn.1001-9030.2016.09.005

• MSI1
• RNA interference
• Lentiviral vector
• Glioma

Objective To construct a lentiviral vector carrying short hairpin RNA (shRNA) of human MSI1 gene,and to study the relationship between the expression of MSI1 and the biological behaviors of human glioma U-87MG cells.Methods A lentiviral vector-mediated MSI1 shRNA (pGCL-GFP-MSI1) was constructed and transfected into the packaging cells 293T.The lentiviral vector was used to transfect into the human glioma U-87MG cells,and blank control group,negative group and pGCL-GFP-MSI1 group wer set up.The infection efficiency was evaluated.Fluorescence microscopy was used to detect the transfection efficiency after 72 h.The expression of MSI1 and cancer stem cells marker (HES1)in U-87MG cells was detected by Western blotting.Methyl thiazol tetrazolium (MTT) assay was used to observe the proliferation behaviors of U-87MG cells 1 to 7 days after transfection.Colony formation assay was used to observe the colony formation of U-87MG cells.Transwell assay was used to observe the invasive behaviors.Flow cytometry was used to observe the apoptosis.Results Lentiviral vector-mediated MSI1 shRNA was correctly constructed.Immunofluorescence assays demonstrated that the transfection efficiency was above 80％.Western blotting analyses demonstrated that pGCL-GFP-MSI1 could significantly inhibit the expression of MSI1 (31.64％) and HES1 (37.25％) in U-87MG cells (P ＜ 0.01).MTT results showed that pGCL-GFP-MSI1 could significantly inhibited the proliferation of U-87MG cells.Colony formation assays showed that pGCL-GFP-MSI1 could decrease the colony formation number in U-87MG cells (27.24 ± 2.52,P ＜ 0.01).Transwell assays showed that pGCL-GFP-MSI1 could reduce the invasive cell number to (18.24 ±3.52,P ＜0.01).FCM results showed that the apoptosis was increased significantly in U-87MG cells (22.81％,P ＜ 0.01).Conclusion pGCL-GFP-MSI1 could significantly inhibit the expression of MSI1 in human glioma U-87MG cells.It could suppress the growth and invasion,and induce apoptosis of human glioma U-87MG cells by regulating the cancer stem cells factors in its signal path.

• 单位
  武汉大学