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Archive > Volume 25 Issue 3 > 2018,25(3):229-235. DOI:10.3872/j.issn.1007-385X.2018.03.003 Prev Next
Basic Research
Knock-down of cytokine induced apoptosis inhibitor 1 gene sensitized leukemia K562 cells to imatinib
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Abstract:
Objective: To investigate the biological effects and the related mechanisms of cytokine induced apoptosis inhibitor 1 (CIAPIN1) on the sensitivity of K562 chronic myeloid leukemia cells to imatinib. Methods: Specific short hairpin RNA (shRNA) inter- ference vectors targeting CIAPIN1 (CIAPIN1-shRNA) were constructed. Interference efficiency of interference group (K562 cells trans- fected with CIAPIN1-shRNA) and control group (K562 cells transfected with scramble-shRNA) was evaluated using Real-time PCR, Western blotting and immunofluorescence staining. The interference group and control group were treated by 2 μmol/L imatinib. Cell viability was detected using MTT assay. Colony formation ability was detected using cell colony forming experiment. Cell cycle and apoptosis was detected using Flow cytometry and Western blotting. Results: CIAPIN1 expression was decreased effectively by specific shRNA targeting CIAPIN1. The CIAPIN1 mRNA content in CIAPIN1-shRNA group accounted (29.74±4.03)% of scramble-shRNA group, while the CIAPIN1 protein content in CIAPIN1-shRNA group accounted (21.57±2.18)% of scramble-shRNA group. CIAPIN1 knock-down significantly enhanced the inhibitory activity of imatinib on proliferation and colony forming ability of K562 cells. The col-ony number and radius of the CIAPIN1-shRNA+imatinib group was (15.60±1.03) and (2.63±0.55) μm, which were all less than those of the scramble-shRNA+imatinib group. The knock down also increased the activity of imatinib to block the cell cycle at G1 phase and to promot apoptosis of cells. The cell ratio at G1 phase of the CIAPIN1-shRNA+imatinib group was obviously increased while the ratio at S phase was obviously decreased compared with those of scramble-shRNA+imatinib group. Hoechst33258 staining and flow cytome- try showed that the proportion of apoptotic K562 cells in the CIAPIN1-shRNA+imatinib group increased. The results of Western blot- ting showed that CIAPIN1 knock-down not only up-regulated the expressions of apoptosis related proteins (p21, Bid and Bim), but also repressed expressions of cell cycle related proteins (Cyclin D1, Bcl-xl, Bcl-2 and Mcl-1), which had synergistic effects with imatinib. Conclusion: CIAPIN1 knock-down significantly sensitized K562 cells to imatinib treatment, and the mechanism might be related with cell cycle arrest and expression of apoptosis-associated proteins.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81541093), the Natural Science Foundation of Shandong Province (No. ZR2015HL075), the Shandong Provincial Foundation for Scientific Research Planning Project of College and University (No. J17KA252), the Shan- dong Provincial Foundation for Medical and Healthy Science Technology Development Program (No. 2015WS0056), and the Foundation of Weifang City for Scientific and Technological Development Program (No. 2014WS048)
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