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Effect of KLF4 on self-renewal and proliferation potential of tumor stem cells
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Abstract:
Objective: To explore the effect of Krüppel-like factors 4 (KLF4) on self-renewal and proliferation potential of tumor stem cells （T3A-A3）. Methods: A lentiviral vector carrying shRNA targeting KLF4 (pLVTHM-shKLF4) was constructed. Tumor stem cells (T3A-A3 cells) were isolated from a human hepatocarcinoma and were identified in our previous study. The expression of KLF4 mRNA and protein in T3A-A3 cells was analyzed by RT-PCR and Western blotting analysis after pLVTHM-shKLF4 infection. Self-renewal ability of T3A-A3 cells was evaluated by tumor sphere formation assay after pLVTHM-shKLF4 infection; clonogenic assay was used to determine the clonogenic ability of T3A-A3 cells; and cell cycle phase distribution was analyzed by flow cytometry. Influence of KLF4 knockdown on the growth of T3A-A3-transplanted tumors was examined in xenograft model of nude mice. Results: T3A-A3 expressed higher level of KLF4 than human hepatocarcinoma cell line Bel-7402 and HepG2. pLVTHM-shKLF4 infection significantly decreased the expression of KLF4 mRNA and protein in T3A-A3 cells. The formed tumor spheres of T3A-A3 cells were significantly smaller in pLVTHM-shKLF4 infection group compared with that in the pLVTHM-shNC control group (\[104.33±16.28\] μm vs \[186.67±28.15\] μm, P<0.01). pLVTHM-shKLF4 infection significantly inhibited the number of T3A-A3 cell-colonies compared with control group (83.5±7.78 vs 125±9.19, P<0.01). Flow cytometry analysis showed that pLVTHM-shKLF4 infection significantly increased G1 population when compared with the control vector (\[39.65±403\]% vs \[29.35±1.00\]%, P<0.01). Furthermore, the growth of T3A-A3-transplanted tumors in pLVTHM-shKLF4 infection group was significantly slower than that in the control group (33 days after cell inoculation, \[46.14±1294\] vs \[228.12±94.86\] mm3, P<0.01). Conclusion: KLF4 knockdown can inhibit the self-renewal of tumor stem cells (T3A-A3 cells), and inhibit the proliferation potential of T3A-A3 both in vitro and in vivo.
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Project Supported:
Project supported by the Major National Basic Research Development Program (973 program) of China（No. 2009CB521804）
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