Silencing Livin gene expression by RNA interference enhanced the chemotherapeutic sensitivity of drug-resistant osteosarcoma cells to doxorubicin

Abstract

Background

Osteosarcoma is one of the most common malignant tumors in children and adolescents. It occurs in the metaphysis of long bones and is a type of aggressive malignant tumor. Although there are treatment methods such as surgery and chemotherapy, the mortality and disability of osteosarcoma patients are still high. With the emergence of more and more chemotherapy resistance, it is necessary to find new therapies to improve the chemotherapy sensitivity of osteosarcoma.

Methods

Drug-resistant MG-63 and U2OS cell strain was established in vitro by continuous exposure of human osteosarcoma cells to doxorubicin at gradually increasing concentrations，then determined for resistance index to doxorubicin by MTT method，for transcriptions of Livin mRNA by real-time polymerase chain reaction（RT⁃PCR），and for expressions of Livin proteins by Western blot．The technology of gene recombination was used to construct the eukaryotic expression vector pSilencer3.1-H1 neo-Livin. Then the pSilencer3.1-H1 neo-Livin was transfected into drug-resistant MG-63 cell by using Lipofectmine 2000. Expressions of Livin mRNA and protein in the transfected cells were respectively measured by RT-PCR and Western blot. The distribution of cell cycle phase 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 pSilencer3.1-H1 neo-Livin was successfully constructed. The result of inverted microscope revealed that the drug-resistant MG-63 cell were irregularity and morphological diversity. Compared with those in osteosarcoma cells，the transcription levels of Livin mRNA and protein in drug-resistant osteosarcoma cell increased（P＜0.05）．The flow cytometry analysis showed there was higher percentage of apoptosis in transfected drug-resistant MG-63 cell. Compared with control groups，the expression of Livin mRNA and protein were both significantly decreased in the transfected drug-resistant osteosarcomacell（P＜0.05）. We also observed that suppression of Livin expression in osteosarcoma cells increased their chemosensitivity to doxorubicin.

Conclusion

This study showed that Livin shRNA inhibited the proliferation level and increased the sensitivity of drug-resistant osteosarcoma cell to doxorubicin, suggested that Livin is involved in drug resistance of human osteosarcoma and may serve as a promising therapeutic target for osteosarcoma.