长链非编码RNA PANDAR对骨肉瘤细胞阿霉素耐药的机制研究

Study on the mechanism of long non-coding RNA PANDAR against adriamycin resistance in osteosarcoma cells

目的观察长链非编码RNA(lncRNA)PANDAR对骨肉瘤细胞增殖、凋亡及对阿霉素化疗耐药的影响,并探讨其机制。方法采用实时荧光定量PCR(qPCR)检测骨肉瘤细胞系和人正常成骨细胞及阿霉素耐药骨肉瘤细胞中lncRNA PANDAR和B淋巴细胞瘤-2(Bcl-2)基因相对表达水平。抑制lncRNA PANDAR表达后,采用qPCR和蛋白质印迹法检测Bcl-2的相对表达水平;同时过表达Bcl-2,CCK-8检测各组细胞增殖情况,Annexin V-FITC PI染色流式细胞术检测各组细胞凋亡率。在耐药与非耐药的骨肉瘤细胞培养基中加入阿霉素检测各组细胞生存率。结果与人正常成骨细胞相比,骨肉瘤细胞中lncRNA PANDAR过表达(P<0.05)。抑制lncRNA PANDAR表达后,骨肉瘤细胞中Bcl-2表达下调(P<0.05)。抑制lncRNA PANDAR表达后,细胞增殖能力明显减弱(P<0.05),凋亡增加(P<0.05),而过表达Bcl-2后,凋亡减少(P<0.05)。在耐药与非耐药的骨肉瘤细胞培养基中加入阿霉素,抑制lncRNA PANDAR后,肿瘤耐药能力减弱,加入Bcl-2后部分恢复,si-lncRNA PANDAR对肿瘤耐药的抑制作用在非耐药细胞中效果更为明显(P<0.05)。结论lncRNA PANDAR可通过调控Bcl-2的表达而调节骨肉瘤细胞的增殖、凋亡和化疗耐药,参与骨肉瘤的发生、发展。

Objective To research the role and mechanism of long non-coding RNA(lncRNA)PANDAR in proliferation,apoptosis and adriamycin chemotherapy-resistant of osteosarcoma cells.Methods The expression of B lymphocyte tumor-2(Bcl-2)gene and lncRNA PANDAR normal osteoblast,osteosarcoma and osteosarcoma/adriamycin chemotherapy-resistant cells were detected by real-time fluorescent quantitative PCR(qPCR).The relative expression level of Bcl-2 in osteosarcoma cells after down-regulated lncRNA PANDAR were examined by qPCR and Western blot.After inihibited lncRNA PANDAR and overexpressed Bcl-2,the changes in abilities of proliferation of each group was examined by CCK-8.The apoptotic rates of each group were examined by Annexin V-FITC PI after transfection.Cell viabilities were detected after transfection and culture with resistant and non-resistant osteosarcoma cells of each group.Results Compared with normal osteoblasts,lncRNA PANDAR was overexpressed in osteosarcoma cells(P<0.05).After inhibiting the expression of lncRNA PANDAR,the expression of Bcl-2 in osteosarcoma cells was down-regulated(P<0.05).After inhibiting the expression of lncRNA PANDAR,the cell proliferation ability was significantly weakened(P<0.05)and apoptosis was increased(P<0.05),while after overexpression of Bcl-2,apoptosis was reduced(P<0.05).Adding adriamycin to drug-resistant and non-drug-resistant osteosarcoma cell culture media inhibited lncRNA PANDAR,the tumor resistance ability was weakened,and the tumor resistance was partially recovered after adding Bcl-2.The inhibitory effect of si-lncRNA PANDAR on tumor resistance was more obvious in non-resistant cells(P<0.05).Conclusion lncRNA PANDAR can regulate the proliferation,apoptosis and adriamycin chemotherapy-resistant of osteosarcoma cells by regulating the expression of Bcl-2,and adjust the occurrence and development of osteosarcoma.