miR-219a-5p通过负调控HMGA2抑制骨肉瘤U2OS细胞增殖、侵袭和迁移

MiR-219a-5p inhibits the proliferation,invasion and migration of osteosarcoma U2OS cells by negatively regulating HMGA2

目的研究miR-219a-5p通过调控高迁移率蛋白A2(HMGA2)对骨肉瘤U2OS细胞增殖、侵袭和迁移的影响。方法采用实时定量PCR检测骨肉瘤U2OS细胞和正常成骨细胞hFOB1.19的miR-219a-5p mRNA表达水平。采用脂质体转染法将miR-219a-5p模拟物miR-219a-5p mimic(miR-219a-5p mimic组)以及阴性对照mimic NC(mimic NC组)转染U2OS细胞,通过实时定量PCR检测转染后细胞miR-219a-5p和HMGA2 mRNA的表达水平,蛋白质印迹法检测HMGA2蛋白水平,采用CCK-8法和克隆形成实验检测细胞增殖能力、划痕实验检测细胞迁移能力、Transwell小室侵袭实验检测细胞侵袭能力,利用双荧光素酶报告基因实验验证miR-219a-5p与HMGA2间的作用关系。结果实时定量PCR结果显示,骨肉瘤U2OS细胞中的miR-219a-5p表达水平(0.11±0.01)显著低于正常成骨细胞(1.00±0.06),差异具有统计学意义(t=26.83,P<0.001)。CCK-8法结果显示,mimic NC组和miR-219a-5p mimic组培养24 h后细胞吸光度值分别为0.52±0.02、0.42±0.02,48 h后分别为0.85±0.03、0.60±0.03,72 h后分别为1.12±0.02、0.72±0.02,miR-219a-5p mimic组细胞增殖活性显著低于mimic NC组,差异均具有统计学意义(t=6.97,P<0.001;t=16.65,P<0.001;t=26.78,P<0.001)。克隆形成实验结果显示,miR-219a-5p mimic组细胞克隆数为(157.00±15.39)个,明显低于mimic NC组的(294.00±15.51)个,差异具有统计学意义(t=9.70,P<0.001)。划痕实验结果显示,培养24 h后,miR-219a-5p mimic组细胞划痕面积百分比为(40.53±2.92)%,显著高于mimic NC组的(21.71±3.11)%,差异具有统计学意义(t=7.26,P=0.002)。Transwell小室侵袭实验结果显示,miR-219a-5p mimic组细胞的穿膜数量为(128.67±18.67)个,显著少于mimic NC组的(317.67±14.33)个,差异具有统计学意义(t=15.65,P<0.001)。双荧光素酶报告基因实验结果显示,在含有MUT-HMGA2细胞中,与mimic NC组(4.40±0.28)相比,转染miR-219a-5p mimic(4.30±0.26)对荧光素酶活性无明显影响,差异无统计学意义(t=0.85,P=0.690)。在含有WT-HMGA2细胞中,相比于NC mimic组(4.50±0.25),miR-219a-5p mimic组(2.88±0.16)荧光素酶活性显著降低,差异具有统计学意义(t=19.15,P<0.001)。且过表达miR-219a-5p后,骨肉瘤U2OS细胞中的HMGA2 mRNA和蛋白表达(0.77±0.01;0.37±0.01)相比于mimic NC组(1.00±0.02;1.00±0.01)均下调,差异均具有统计学意义(t=16.38,P<0.001;t=42.02,P<0.001)。结论在骨肉瘤细胞中,miR-219a-5p可通过下调HMGA2的表达抑制骨肉瘤细胞的增殖、迁移和侵袭。

Objective To investigate the effects of miR-219a-5p on proliferation,invasion and migration of osteosarcoma U2OS cells by regulating high mobility group A2(HMGA2).Methods Real-time quantitative PCR was used to detect miR-219a-5p mRNA expression levels in osteosarcoma U2OS cells and normal osteoblasts hFOB1.19.The U2OS cells were transfected with miR-219a-5p mimic(miR-219a-5p mimic group)and negative control mimic(mimic NC group)by liposome transfection.The expression levels of miR-219a-5p and HMGA2 mRNA in transfected cells were detected by real-time quantitative PCR.The level of HMGA2 protein was detected by Western blotting,cell proliferation ability was detected by CCK-8 assay and clonogenesis assay,cell migration ability was detected by scratching assay,cell invasion ability was detected by Transwell chamber assay,and the relationship between miR-219a-5p and HMGA2 was verified by double luciferase reporter gene assay.Results Real-time quantitative PCR showed that the expression level of miR-219a-5p in osteosarcoma U2OS cells(0.11±0.01)was significantly lower than that in normal osteoblasts(1.00±0.06),with a statistically significant difference(t=26.83,P<0.001).The results of CCK-8 showed that the cell absorbance values of the mimic NC group and miR-219a-5p mimic group were 0.52±0.02 and 0.42±0.02 after 24 h,0.85±0.03 and 0.60±0.03 after 48 h,and 1.12±0.02 and 0.72±0.02 after 72 h respectively.The proliferation activity of the miR-219a-5p mimic group was significantly lower than that of the mimic NC group,with statistically significant differences(t=6.97,P<0.001;t=16.65,P<0.001;t=26.78,P<0.001).The results of clonogenesis assay showed that the number of clones in the miR-219a-5p mimic group was 157.00±15.39,which was significantly lower than that in the mimic NC group(294.00±15.51),with a statistically significant difference(t=9.70,P<0.001).The results of scratch experiment showed that the percentage of scratch area in the miR-219a-5p mimic group was(40.53±2.92)%after 24 h culture,which was significantly higher than that in the mimic NC group[(21.71±3.11)%],with a statistically significant difference(t=7.26,P=0.002).The results of Transwell chamber assay showed that the number of cells penetrating the membrane in the miR-219a-5p mimic group was 128.67±18.67,which was significantly lower than that in the mimic NC group(317.67±14.33),with a statistically significant difference(t=15.65,P<0.001).The results of double luciferase reporter gene assay showed that in MUT-HMGA2 cells,transfection with miR-219a-5p mimic(4.30±0.26)had no significant effect on luciferase activity compared with the mimic NC group(4.40±0.28),with a statistically significant difference(t=0.85,P=0.690).In WT-HMGA2 cells,compared with the mimic NC group(4.50±0.25),the lucifase activity of the miR-219a-5p mimic group(2.88±0.16)was significantly decreased,with a statistically significant difference(t=19.15,P<0.001).After miR-219a-5p was overexpressed,HMGA2 mRNA and protein expressions in osteosarcoma U2OS cells(0.77±0.01;0.37±0.01)were downregulated compared with the mimic NC group(1.00±0.02;1.00±0.01),with statistically significant differences(t=16.38,P<0.001;t=42.02,P<0.001).Conclusion In osteosarcoma cells,miR-219a-5p can inhibit the proliferation,migration and invasion of osteosarcoma cells by down regulating the expression of HMGA2.