Effect of interfering the β-catenin by lentiviral vector-mediated RNAi on the biological behavior of mouse bone marrow mesenchymal stem cells.
10.7534/j.issn.1009-2137.2013.06.034
- Author:
Cheng-Juan FU
1
;
Zhen-Yu LI
1
;
De-Peng LI
1
;
Zhi-Ling YAN
1
;
Wei CHEN
1
;
Chong CHEN
1
;
Qing-Yun WU
1
;
Xiu-Ying PAN
1
;
Kai-Lin XU
2
,
3
Author Information
1. Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
2. Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China. E-mail: Lizhenyumd@
3. com.
- Publication Type:Journal Article
- MeSH:
Animals;
Apoptosis;
Bone Marrow Cells;
cytology;
Cell Line, Tumor;
Cell Movement;
Genetic Vectors;
Lentivirus;
genetics;
Mesenchymal Stromal Cells;
cytology;
Mice;
RNA Interference;
RNA, Small Interfering;
Wnt Signaling Pathway;
beta Catenin;
metabolism
- From:
Journal of Experimental Hematology
2013;21(6):1546-1551
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to investigate the effect of Wnt/β-catenin signaling pathway on the biologic behavior of mouse bone marrow mesenchymal stem cells (mBM-MSC) by constructing a RNAi lentiviral vector specific to β-catenin. Three pairs of shRNA coding sequences directed against different sites of β-catenin mRNA were designed and were linked into lentiviral vector plasmid PLB for constructing the PLB-β-catenin/shRNA1, PLB-β-catenin/shRNA2 and PLB-β-catenin/ shRNA3. Those plasmids and lentiviral packaging plasmids were co-transfected into the packaging cells 293FT, then the virus particles were collected and the viral titer was assayed after concentration, and these viral particles were infected to MSC. The flow cytometry was used to sort GFP (+) cells, Western blot and RT-PCR were used to verify the inhibitory effect of those cells on expression of β-catenin gene in cells, CCK-8 method was used to detect the cell proliferation level, Annexin-V/7-AAD was used to determine the cell apoptosis after interference, the cell scratch and transwell tests were used to detect the migration capability of MSC. The results showed that the efficient inhibition of β-catenin mRNA and protein expression, and the suppression of MSC proliferation were observed in group of PLB-β-catenin/shRNA2 (interference group), while there was no significant changes of MSC proliferation between negative group (PLB group) and the normal group (control group). The flow cytometric detection indicated that after induced by serum starvation for 72 h, the apoptosis of MSC increased in interference group, but there was no difference between PLB and control groups (P > 0.05). The cell scratch and transwell tests demonstrated that the migration capability of MSC in interference group decreased significantly, while the migration capability of MSC in control group was not changed obviously. It is concluded that the constructed specific RNAi lentivirus can effectively inhibit the expression of β-catenin gene, decrease expression level of β-catenin mRNA and protein. The Wnt/β-catenin signaling pathway plays an important role in biological behavior of BM-MSC.
