Pinch-1 was up-regulated in leukemia BMSC and its possible effect.
Pinch-1, a widely expressed focal adhesion protein, has been demonstrated to be up-regulated in multiple solid tumor-associated stromal cells, particularly at invasive edges. It was supposed that Pinch-1 was intimately associated with development and progression of tumors. The expression of Pinch-1 in hematopoietic microenvironment in patients with leukemia remains unclear. This study focused on the expression of Pinch-1 in bone marrow stromal cells (BMSCs) from leukemia patients and its possible effect. BMSC was isolated and cultured from bone marrow in leukemia patients and normal healthy donors. RT-PCR and Western blot analysis were performed to determine Pinch-1 mRNA and protein level in BMSC, respectively. Lentiviral vector containing Pinch-1 siRNA was constructed, and the recombinant lentivirus particle was packaged in 293 cells. Effectiveness of Pinch-1 siRNA was determined by Western blot. The proliferation, apoptosis and motility of leukemia BMSC subjected to Pinch-1 knockdown using siRNA were tested by flow cytometry, TUNEL assay and Transwell system, respectively. Pinch-1 mRNA and protein were significantly up-regulated in ALL and AML BMSC compared to normal BMSC (p<0.01). Although there was no difference in Pinch-1 mRNA between ALL and AML BMSC, cellular levels of Pinch-1 protein in ALL BMSC were significantly higher than that in AML BMSC (p<0.01). Overexpressed Pinch-1 was significantly reduced in leukemia BMSC transfected with Pinch-1 siRNA evidenced by Western blot. Flow cytometry analysis showed that the percentage of cells in S+G2 phases in leukemia BMSC transfected with Pinch-1 siRNA was significantly lower than control (p<0.01). The percentage of apoptotic cells in leukemia BMSC transfected with Pinch-1 siRNA was 19.8±1.0%, significantly higher than controls (p<0.01). The number of leukemia BMSC transfected with Pinch-1 siRNA that migrated to the lower chamber after culturing for 24 h was 8.4±1.1 per field, significantly lower than controls (p<0.01). Pinch-1 mRNA and protein in leukemia BMSC were up-regulated drastically compared with BMSC from healthy donors. Leukemia BMSC displayed hypoproliferation, decreased migration and increased apoptosis after transfecting Pinch-1 siRNA.
Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China.
Dongfeng
D
Zeng
Lei
L
Hao
Wei
W
Xu
Zhihong
Z
Li
Weiyan
W
Li
Jieping
J
Li
Xi
X
Zhang
Xinghua
X
Chen
Peiyan
P
Kong
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D060588:LIM Domain Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D016086:Lentivirus; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000473:pathology*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D060888:Real-Time Polymerase Chain Reaction; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D060588:LIM Domain Proteins; C088088:LIMS1 protein, human; D008565:Membrane Proteins