[Sensitivity of doxorubicin-resistant osteosarcoma cells to doxorubicin regulated by long non-coding RNA NR_036444]

Objective: To investigate the mechanism of long non-coding RNA (LncRNA) NR_036444 mediated sensitivity of multidrug-resistant osteosarcoma to doxorubicin. Methods: LncRNA-mRNA combined microarray was used to screen the differential expressions of lncRNA and mRNA in doxorubicin-resistant MG63/DXR osteosarcoma cells and their paired doxorubicin-sensitive MG63 cells; qRT-PCR was used to check the consistency of microarray. LncRNA NR_036444 was over-expressed in MG63/DXR cells by lentrvirus. CCK-8 array was used to evaluate cell proliferation and the sensitivity of cells to doxorubicin; Flow cytometry was used to evaluate cell cycle and apoptosis. The expressions of lncRNA NR_036444 in 60 cases of tumor tissues resected from osteosarcoma patients were detected by qRT-PCR and correlation analyses administrator were conducted. Results: Compared with those in MG63 cells, 1, 761 lncRNAs were significantly up-regulated and 1, 704 lncRNAs were dramatically down-regulated in MG63/DXR cells (P<0.05). 15 lncRNAs were selected randomly and their expressions were confirmed by qRT-PCR. The data showed that the differences in expression of 15 lncRNAs were consistent with microarray result. lncRNA NR_036444 with 22 folds down-regulation was identified as the most significant differential lncRNA in MG63/DXR cells. Half maximal inhibitory concentrations (IC(50)) in the blank group, negative control group and lncRNA NR_036444 overexpressed group were (12.73±0.50) μg/ml、(12.12±0.31) μg/ml and (5.77±0.25) μg/ml, respectively. Compared with that in negative control group, IC(50) in overexpressed group decreased by 49.6% (P<0.001). The percentages of G(1) phase cells in the overexpressed group, negative control group and blank group were(90.12±0.08)%, (33.36±0.85)% and (39.38±1.02)%. Compared with the negative control group and blank control group, the overexpression group had significantly more cells arrested in G(1) phase (both P<0.001). In the overexpressed group, negative control group and blank group, the percentages of late-apoptotic cells were (11.40±1.08)%, (4.23±1.12)% and (0.28±0.12)%, respectively. The percentages of early-apoptotic cells were (86.40±1.80)%, (4.35±2.03)% and (0.25±0.02) %, respectively. The percentages of early- and late-apoptotic cells were significantly increased in the overexpressed group when compared with those in control groups (P<0.01). Meanwhile, the expression of lncRNA NR_036444 in the tissues of chemoresistance group and chemosensitivity group was 19.67±1.53 and 77.67±2.52, respectively, with a statistically significant difference (P<0.01). The postoperative overall survival time of osteosarcoma patients with low expression of lncRNA NR_036444 was (24.6±2.4) months, significantly shorter than (48.2±1.8) months of patients with high expression of lncRNA NR_036444 (P<0.001). Conclusions: LncRNA NR_036444 may play a vital role in regulating osteosarcoma doxorubicin resistance and it may be a useful biomarker to assess the chemosensitivity and predict the prognosis of osteosarcoma patients in the future.