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Archive > Volume 24 Issue 10 > 2017,24(10):1063-1069. DOI:10.3872/j.issn.1007-385X.2017.10.004 Prev Next
Basic Research
Inhibitory effect of target silencing Pim-3 gene on melanoma B16 cell line
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Abstract:
Objective: To observe the inhibitory effect of target silencing pim-3 on mouse melanoma B16 cell line. Methods: Pim-3-shRNA vector and pim-3 over-expressing vector (pim-3-MIGR1) were respectively transfect-ed into B16 melanoma cells by liposome, and the changes in mRNA and protein expression of pim-3 was measured by qPCR and Western blotting assay, respectively; the apoptosis of B16 cells was detected by AnnexinV/PI staining and TUNEL staining; the expressions of apoptosis-related factors, such as p-Bad, Bcl-2, Bcl-xl, Bax, caspase-3,were evaluated by qPCR and Western blotting; MTT assay was applied to confirm the proliferation of B16 cells;Flow cytometry was used to detect cell cycle; RTCAxCELLigence system was performed to confirm cell migration;and Transwell assay was applied to examine cell invasion. Results: pim-3-shRNAsignificantly reduced while pim-3-MIGR1 significantly enhanced pim-3 expression in B16 cells at both mRNA and protein levels (all P<0.05). Pim-3-shRNA also effectively induced the apoptosis of B16 cells; however, co-transfection with pim-3-MIGR1 obviously reversed the apoptosis induced by Pim-3 silencing (P<0.05). Further studies found pim-3-shRNAsignificantly down-regulated the expressions of apoptosis-related molecules (p-Bad, Bcl-2 and Bcl-xl), but remarkably up-regulated Bax expression, and finally resulted in increased caspase-3 activity (all P<0.05). The proliferation and migration of B16 cells were significantly inhibited by pim-3 silencing (all P<0.05); however, there was no obvious change in cell cy-cle. Conclusion: Pim-3-specific gene silencing effectively promoted the apoptosis and inhibited both the prolifera-tion and migration of B16 cells, suggesting that pim-3 may serve as a potential effective gene therapy target for mel-anoma.
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Project Supported:
Project supported by the National Natural Science Founda-tion of China （No.81472646, No.81273220, No.91442114）
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