[Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells impacts chemotherapeutics-induced apoptosis]

To investigate the effect of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells on apoptosis induced by chemotherapeutics. Methods: A total of 30 osteosarcoma tissues of sensitive and resistant to chemotherapeutics were divided into a chemotherapy-sensitive group and a chemotherapy-resistant group. The mRNA expression levels of miR-30a and high mobility group protein A2 (HMGA2) in the chemotherapy-sensitive group and the chemotherapy-resistant group, and the mRNA expression levels of miR-30a in osteosarcoma U2-OS cells treated by cisplatin, doxorubicin and methotrexate at different concentrations were detected by real-time PCR. The expression levels of autophagy related protein Beclin 1, microtubule associated protein 1 light chain 3B (LC3B) and autophagy factor P62 were detected by Western blotting. The osteosarcoma U2-OS cells were transfected with miR-30a mimics and miR-30a inhibitors to construct a miR-30a high expression group, a miR-30a low expression group and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after treatment of cisplatin and doxorubicin in these 3 groups were detected by Western blotting; the level of autophagy was detected by monodansylcada (MDC) staining; the level of ROS was detected by dihydroethidium (DHE); the level of cell surviving rate was detected by cell counting kit-8 (CCK-8); the level of apoptosis was detected by annexin APC/PI double staining; the level of mitochondria oxidative damage was detected by mitochondrial membrane potential assay kit with JC-1 (JC-1 method). The interaction between miR-30a and HMGA2 was detected by dual luciferase reporter assay. The osteosarcoma U2-OS cells were transfected with HMGA2 mimics and HMGA2-shRNA to construct a high HMGA2 group, a low HMGA2 group, and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after the treatment of cisplatin were detected by Western blotting. Results: The level of miR-30a in the chemotherapy-resistant tissues was significantly lower than that in the chemotherapy-sensitive tissues (P0.05), and the expression of HMGA2 was opposite comparing to that of miR-30a (P0.05). After the treatment by low concentration (5 μmol/L) of chemotherapeutics, the level of miR-30a was down-regulated in osteosarcoma U2-OS cells, accompanied with up-regulation of Beclin 1 and LC3B (P0.01) and down-regulation of P62 (P0.01). Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly decreased (P0.05), and the expression level of P62 was significantly increased (P0.05) in the miR-30a high expression group, which was opposite in the miR-30a low expression group. In the miR-30a high expression group treated by chemotherapeutics, the level of autophagy and the cell survival rate were lower than those in group with low expression of miR-30a, while the levels of ROS, the mitochondrial oxidative damage and the apoptosis were higher than those in group with low expression of miR-30a (all P0.05). The targeting interaction between HMGA2 and miR-30a were verified by dual luciferase reporter assay. Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly increased (P0.05), and the expression level of P62 was significantly decreased (P0.05) in the HMGA2 high expression group, which was opposite in the HMGA2 low expression group. Conclusion: Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells is likely to enhance the therapeutic effect of chemotherapeutics.目的：探讨微小RNA(microRNA，miR)-30a/高迁移率族蛋白A2(high mobility group protein A2，HMGA2)介导的骨肉瘤细胞自噬对化学药物治疗(以下简称化疗)药物诱导的细胞凋亡的影响。方法：随机选取30例经化疗药物治疗后表现为化疗敏感和化疗抵抗的骨肉瘤患者组织，分为化疗敏感组(n=15)和化疗抵抗组(n=15)，采用real-time PCR检测两组中miR-30a和HMGA2的mRNA表达水平，以及骨肉瘤细胞U2-OS经不同浓度的化疗药物(顺铂、阿霉素、氨甲蝶呤)处理后miR-30a的mRNA表达水平；采用蛋白质印迹法检测细胞内自噬相关因子Beclin 1，自噬微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B，LC3B)、自噬抑制因子P62的表达情况。在骨肉瘤细胞U2-OS中转染miR-30a模拟物和抑制剂，构建miR-30a高表达组、低表达组和对照组，采用蛋白质印迹法检测经过顺铂和阿霉素处理后上述3组细胞内Beclin 1，LC3B和P62的表达情况；采用单丹磺酰尸胺(monodansylcada，MDC)染色法检测细胞内自噬水平，ROS荧光探针-二氢乙啶(dihydroethidium，DHE)检测细胞内ROS水平，细胞计数试剂盒8(cell counting kit-8，CCK-8)检测细胞存活率，流式细胞术检测细胞凋亡程度，线粒体膜电位荧光探针JC-1检测细胞线粒体氧化损伤程度；采用双荧光素酶法检测miR-30a与HMGA2的相互作用，同时通过转染HMGA2模拟物和HMGA2-shRNA干扰质粒载体，构建HMGA2高表达组、低表达组和对照组，采用蛋白质印迹法检测经过顺铂和阿霉素处理后上述3组细胞内Beclin 1，LC3B和P62的表达情况。结果：化疗抵抗组中miR-30a的mRNA水平显著低于化疗敏感组(P0.05)，HMGA2的表达与miR-30a相反(均P0.05)。在相对低浓度(5 μml/L)的化疗药物刺激下，骨肉瘤细胞U2-OS内的miR-30a mRNA表达下调，Beclin 1和LC3B均显著上调(均P0.01)，P62显著下调(P0.01)。在miR-30a高表达组中，与对照组相比，Beclin 1和LC3B的表达水平与miR-30a明显下降(P0.05)，P62的表达水平明显升高(P0.05)，在miR-30a低表达组中则相反；在经过化疗药物处理后的miR-30a高表达组中，细胞自噬水平更低，细胞存活率更低，ROS水平更高，线粒体氧化损伤程度更高，细胞凋亡水平更高(均P0.05)，miR-30a低表达组则相反(均P0.05)。双荧光素酶报告基因检测验证了miR-30a与HMGA2的靶向互补配对关系；与对照组相比，HMGA2高表达组中Beclin 1和LC3B的表达水平与HMGA2明显升高(P0.05)，P62的表达水平明显下降(P0.05)，在HMGA2低表达组中则相反。结论：积极发挥miR-30a/HMGA2抑制骨肉瘤细胞自噬的功能，能够破坏细胞应对ROS介导的自噬与凋亡的平衡，增强化疗药物对细胞的杀伤作用。.