K P Zhu1, C L Zhang2. 1. Department of Orthopaedic Surgery, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China. 2. Department of Orthopaedic Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai 200072, China.
Abstract
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.
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 osteosarcomapatients 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 osteosarcomapatients 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 osteosarcomadoxorubicin resistance and it may be a useful biomarker to assess the chemosensitivity and predict the prognosis of osteosarcomapatients in the future.