Despite great progress in chemotherapy and surgery, clinical treatment remains confronted with high recurrence rates and stagnant five-year survival rates(7, 8). Livin, also known as KIAP, ML-IAP and Birc7 protein, plays an important role in the occurrence and progression of different types of cancer cells(9), and its expression is extremely low or absent in most terminally differentiated tissues of normal adults(10–12). The expression level of Livin is closely related to tumorigenesis and associated with the high invasiveness of tumors. Thus, it may serve as a new target for inducing tumor cell apoptosis. Moreover, the level of Livin expression is related to the tumor case, clinical stage, metastasis and prognosis(13–15). Livin is also highly associated with chemotherapy resistance, radiation resistance, relapse rate and low survival(16). Studies have shown that silencing Livin expression can reduce ERK1/2 and AKT expression levels, thereby increasing breast cancer sensitivity to trastuzumab(17). Silencing Livin can also increase the chemotherapy sensitivity of lung cancer and liver cancer cells to cisplatin(18, 19), glioma cells to temozolomide(20) and bladder cancer cells to mitomycin(21). In our previous study, we proved that Livin was closely related to the 5-year overall survival rate in OS patients. We made a conclusion that compared with other OS cell lines, the expression levels of Livin in MG-63 cells were markedly increased(5). We also demonstrated that high levels of Livin were significantly associated with poorly differentiated OS and poor prognosis. The chemoresistance of OS is one of the important reasons for OS progression, as well as a major insurmountable difficulty in clinical treatment of OS. Thus, our study is the first to demonstrate the role of Livin in the treatment of drug-resistant OS cells. In the present study, we silenced Livin expression by transfecting with shRNA targeting Livin after established MG-63/R cells. Both CCK-8 assay and live/dead cell staining results revealed the proliferation of MG-63/R cells in the Livin shRNA group was significantly inhibited compared to the control and NC groups, suggesting that silencing Livin expression could reverse chemotherapy resistance in MG-63/R cells.
Malignant cells, when being conferred upon unlimited proliferation, metastatic spread, apoptosis evasion and resistance against therapy, which not only allow their proliferation and survival but also generate adverse effects on the patients diagnosis and prognosis(22). One of the most critical factors for tumor progression is migration via the extracellular matrix (ECM). In the current study, the wound healing assay and the Transwell invasion assay were conducted to determine the effects of Livin-transfected shRNA on the migration and invasion ability of MG-63/R cells. The results indicated that silencing Livin could significantly reduce the metastatic and invasive ability of MG-63/R cells. MMPs family has been documented as a family of endoproteases which can degrade the ECM. We detected MMP-2 and MMP-9 due to their high expression reporting in various cancers investigations(23). Western blot and PCR results also showed that knockdown Livin repressed protein and mRNA expression of MMP-2 and MMP-9. These findings revealed that Livin knockdown significantly repressed cell proliferation and migration through diminution MMP-2 and MMP-9 enzymatic activity in MG-63/R cells.
The current biological basis of tumor treatment is to eliminate tumor cells by apoptosis(24). It is generally believed that the tumor recurrence and drug resistance are caused by the imbalance between cell proliferation and apoptosis. Moreover, the chemoresistance of tumors is closely associated with apoptotic pathways, including mutations of proapoptotic proteins, overexpression of antiapoptotic proteins and the loss of caspase(25–27). In this study, we assessed cell apoptosis using flow cytometry analysis, and the results showed that cells in the Livin shRNA group presented obvious apoptosis, indicating that silencing Livin can induce apoptotic cell death and inhibit the proliferation of MG-63/R cells. In addition, these findings provide insights on the mechanisms by which silencing Livin could improve the chemosensitivity of MG-63/R cells. As predicted, our results suggested that Livin knockdown significantly accelerated the rate of apoptotic cells compared with control and negative transfected cell. Incidentally, management of the apoptotic pathways, including stimulation of proapoptotic markers and deactivation of antiapoptotic pathways to eliminate tumor cells, demonstrates significant roles. Both proteins from the Bcl-2 family and proteases from caspase family have been discovered to be involved in the regulatory process of apoptosis, among which Bcl-2 has been reported to possess an inhibitory effect on apoptosis, while caspase-3, on the contrary, yields a promotive effect(28). From the western blot and qRT-PCR results, it was also associated with the reduced expression of Bcl-2 and the elevated expression of Bax/Bcl-2 ratio, caspase-3 and cleaved caspase-3, suggesting silencing Livin in MG-63/R cells could promote resistant cells apoptosis, attenuating MG-63/R cells growth through apoptosis augmentation. Together with our previous findings, our data suggest that silencing Livin could promote drug resistant cells apoptosis and inhibit infiltration and metastasis in OS. It is possible that interfering with Livin-transfected shRNA may improve chemoresistance to doxorubicin in MG-63/R cells and provide a potential synergistic therapy for patients with osteosarcoma.
To further investigate the impacts of Livin, we intend to study the specific molecular mechanisms underlying the ability of Livin to reverse the resistance of osteosarcoma, such as the specific targets, the regulated miRNA and the related signal pathways affected by Livin. Moreover, we will explore the possibility of using the Livin gene as a new targeted tumor therapy and applying it in the targeted development of new antitumor drugs.