miRNA-34a调控ether-à-go-go 1基因抑制骨肉瘤细胞增殖的体外研究

MiRNA-34a inhibits human osteosarcoma proliferation by regulating the expression of ether-à-go-go 1 gene： a study in vitro

目的检测miRNA-34a（miR-34a）对于骨肉瘤细胞增殖的影响及可能的调控机制。方法选择人骨肉瘤细胞株MG-63、Saos-2和人成骨细胞株hFOB 1.19以及穿刺活组织检查并经病理确诊的10例骨肉瘤和10例正常骨组织标本。采用实时定量聚合酶链反应（qPCR）检测miR-34a在各细胞株和组织中的表达。构建pcDNA/miR-34a真核表达载体并转染骨肉瘤MG-63、Saos-2细胞，采用CCK-8法、克隆形成实验和裸鼠成瘤实验分别检测转染pcDNA/miR-34a前后细胞增殖、生长和裸鼠肿瘤体积的变化。采用Western blot法检测转染pcDNA/miR-34a和miR-34a抑制剂miR-34a-2＇-O-甲基反义核糖核苷酸（miR-34a-2＇-O-Me）前后骨肉瘤细胞株中ether-à-go-go 1（Eag1）蛋白的表达。结果与成骨hFOB 1.19细胞和正常骨组织相比，miR-34a在骨肉瘤细胞株和组织中表达下调。CCK-8法检测结果示，在MG-63细胞中，空白对照组、阴性对照组和miR-34a转染组72 h细胞存活率分别为（40.05±4.82）%、（36.88±4.66）%和（26.24±6.22）%，96 h细胞存活率分别为（83.55±5.95）%、（80.13±4.48）%和（30.21±7.26）%，转染pcDNA/miR-34a后72、96 h分别与空白对照组及阴性对照组比较，差异均有统计学意义（均P〈0.001）。Saos-2细胞中，空白对照组、阴性对照组和miR-34a转染组72 h细胞存活率分别为（46.45±8.15）%、（43.33±6.89）%和（26.81±3.17）%，96 h细胞存活率分别为（84.79±4.10）%、（80.14±3.11）%和（31.77±5.17）%，转染pcDNA/miR-34a后72、96 h分别与空白对照组及阴性对照组比较，差异均有统计学意义（均P〈0.001）。克隆形成实验结果示，转染pcDNA/miR-34a后，骨肉瘤MG-63和Saos-2细胞克隆形成数分别为（24.40±2.71）、（30.40±4.94）个，均低于空白对照组[（83.40±3.29）、（85.00±3.32）个]和阴性对照组[（80.00±3.06）、（80.60±3.29）个]（均P〈0.001）。裸鼠骨肉瘤生长结果显示，从转染pcDNA/miR-34a后第21天起，miR-34a转染组裸鼠肿瘤体积小于空白对照组和阴性对照组，两者差异均有统计学意义（均P〈0.001）。miR-34a表达上调可抑制骨肉瘤MG-63和Saos-2细胞中Eag1的表达，而miR-34a抑制剂可诱导骨肉瘤细胞株中Eag1的表达（均P〈0.001）。结论miR-34a作为抑癌基因可能通过调控Eag1的表达抑制骨肉瘤细胞增殖和生长，有望成为骨肉瘤治疗的新靶点。

ObjectiveTo detect the influence of miRNA-34a （miR-34a） on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation.MethodsThe osteoblastic cell line MG-63 and Saos-2, human osteoblastic cell line hFOB 1.19, 10 osteosarcoma tissues and 10 normal bone tissues were selected. The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction （qPCR）. Next, a eukaryotic expression vector named pcDNA/miR-34a was constructed. Then, osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8, colony formation and xenograft model of nude mice. Finally, Western blot analysis was used to detect the expression of ether-à-go-go 1 （Eag1） gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2＇-O-Methyl antisense oligoribonucleotide （miR-34a-2＇-O-Me）.ResultsCompared with normal bone tissues and osteoblastic cell line, miR-34a was down-regulated in osteosarcoma cell lines and tissues. Compared with the blank group and the control group, the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h： blank group （40.05±4.82）%, control group （36.88±4.66）%, miRNA-34a group （26.24±6.22）%; MG-63 96 h： blank group （83.55±5.95）%, control group （80.13±4.48）%, miRNA-34a group （30.21±7.26）%; Saos-2 72 h： blank group （46.45±8.15）%, control group （43.33±6.89）%, miRNA-34a group （26.81±3.17）%; Saos-2 96 h： blank group （84.79±4.10）%, control group （80.14±3.11）%, miRNA-34a group （31.77±5.17）%]. The similar results were obtained from colony formation assay （MG-63： blank group 83.40±3.29, control group 80.00±3.06, miR-34a group 24.40±2.71; Saos-2： blank group 85.00±3.32, control group 80.60±3.29, miR-34a group 30.40±4.94）. The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment （all P 〈 0.001）. Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1（P 〈 0.001）.ConclusionMiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1. Moreover, it may be a novel target for osteosarcoma therapy.