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Archive > Volume 24 Issue 12 > 2017,24(12):1375-1380. DOI:10.3872/j.issn.1007-385X.2017.12.006 Prev Next
Basic Research
Livin gene silence by RNA interference enhances the chemotherapeutic sensitivity of drug resistant MG-63 osteosarcoma cells to doxorubicin
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Abstract:
Objective: To investigate the effect of Livin gene (an inhibitor of apoptosis protein) on reversing the drug-resistance of osteosarcoma. Methods: Drug-resistant MG-63 cell strain was established in vitro by continuous exposure to doxorubicin at gradually increased concentrations. The resistance index of drug-resistant MG-63 cells was examined by MTT method; Livin protein expressions in MG-63 cells and durg-resistant MG-63 cells were determined by Western blotting．Livin shRNA eukaryotic expression vector (pSilencer3.1-H1 neo-Livin si) was constructed and then transfected into drug-resistant MG-63 cells by using Lipofectmine 2000. Expression change of Livin mRNA and protein in drug-resistant MG-63 cells before and after the transfection was respectively measured by Real-time PCR and Western blotting. The distribution of cell cycle and apoptosis were determined by flow cytometry.The analysis of chemotherapeutic sensitivity of drug-resistant MG-63 cell to doxorubicin was performed by MTT. Results: The recombinant eukaryotic expression vector Silencer3.1-H1 neo-Livin si was successfully constructed.Resistance index to doxorubicin of drug-resistant MG-63 cells (MG-63/R) was 81.32±5.33. Livin shRNA could significantly inhibit the mRNA and protein expression of Livin in MG-63/R cells compared with untransfect-ed group and non-specific transfected group（down-regulated by 72% and 69% at mRNA and protein level respectively,all P<0.05）. The flow cytometry analysis showed there was significantly higher apoptosis rate in Livin shRNA transfected group than that of untransfected group and non- specific transfected group ([22.4±3.2]% vs [4.2±1.1]%, [4.7±0.6]%，P<0.05). After adding doxorubicin, the proliferation of three groups of cells was all inhibited at different levels in time-dependent manner; However, the cell survival rate in Livin shRNA transfected group was significantly lower than that in other two groups (P<0.05). Conclusion：Livin shRNA could efficiently promote the ,apoptosis of drug-resistant MG-63 cells, and thus increase its sensitivity to chemotherapy drugs.
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Project Supported:
Project supported by the National Natural Science Foundation of China（No.81660443）, and the Health and Family Planning Commision of Jiangxi Province（No. 20155278）
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