miR-99a抑制物对骨肉瘤细胞生物行为学的影响

Effect of MiR-99a Inhibitor on the Biological Behavior of Human Osteosarcoma Cells

目的:探索miR-99a在骨肉瘤中的表达情况及其对骨肉瘤细胞生物学行为特性的影响。方法:收集7例骨肉瘤组织及瘤旁骨组织,RT-PCR检测并比较miR-99a表达情况;RT-PCR技术检测miR-99a在骨肉瘤细胞系143B、MG-63和U2OS中的相对表达量,以人成骨细胞系hFOB1.19作为对照比较;RT-PCR检测hepG2、lovo、A549和MCF-7中的miR-99a相对表达量,以143B作对照比较。培养人骨肉瘤细胞系143B,瞬时转染miR-99ainhibitor后,CCK-8检测细胞增殖,流式细胞仪检测细胞凋亡,划痕试验检测细胞迁移,Transwell小室法检测细胞侵袭,Western Blot检测RECK、MMP-2、MMP-9蛋白的表达情况。结果:miR-99a在7例骨肉瘤组织中的相对表达量(0.81±0.06)要明显高于瘤旁骨组织(0.48±0.06)(P<0.01)。3组骨肉瘤细胞系miR-99a表达量均高于人成骨细胞hFOB1.19,差异有统计学意义(P<0.01);骨肉瘤细胞系中miR-99a相对表达明显高于四种非骨肉瘤细胞系(P<0.05)。miR-99ainhibitor转染组细胞增殖速率显著减慢,低于inhibitor-NC组和空白组(P<0.05);转染组早期凋亡和晚期凋亡均未明显增加(P>0.05);转染组细胞迁移能力明显受到抑制;转染组143B细胞穿过小室底膜的数目明显低于inhibitor-NC组和空白组(P<0.05);转染组RECK蛋白表达上调,MMP-2和MMP-9蛋白表达下调。结论:miR-99a在骨肉瘤组织和细胞系中呈现高表达,下调miR-99a能抑制骨肉瘤细胞系143B增殖、侵袭和转移能力,其机制可能是通过调控RECK/MMP-2蛋白表达而实现的。

Objective:To explore the expression of miR-99 ain osteosarcoma and effect of miRNA-99 a inhibitor on the biological behavior of osteosarcoma cell line and its mechanism.Methods:Seven cases of osteosarcoma tissue and tumor adjacent tissue were collected and RT-PCR was used to detected the miR-99 aexpression.Relative expression of miR-99 ain osteosarcoma cell lines 143 B,MG-63 and U2 OS were detected by RT-PCR in osteosarcoma cell lines 143 B,MG-63 and U2 OS,as compared with the hFOB 1.19.Relative expression of miR-99 ain HepG2,A549,MCF-7 were detected by RT-PCR and compared with U2 OS.After transient transfection of miR-99 ainhibitor in 143 Bcells,cell proliferation was measured by CCK-8 and flow cytometry,scratch assay was used to detect the cell migration,transwell assay was used to detect cell invasion.The protein expression of RECK,MMP-2,and MMP-9 were evaluated by Western Blotting.Results:The rela-tive expression of miR-99 ain 7 osteosarcoma tissues(0.81±0.06)was significantly higher than that in adjacent bone tissue(0.48±0.06)(P<0.01).The relative expression of miR-99 ain 3 osteosarcoma cell lines were higher than that of human osteoblast cell line hFOB1.19,and the difference was statistically significant(P<0.01).Relative expression of miR-99 ain osteosarcoma cell lines was significantly higher than those of four non osteosarcoma tumor cell lines(P<0.05).Furthmore,cells proliferation rate in miR-99 ainhibitor transfection group is significantly decelerated compared with the control group.However,The early apoptosis and late apoptosis were not significantly changed in these two groups.Downregulation of miR-99 ain miR-99 ainhibitor transfection group suppressed the cell invasion and migration compared with the control group.RECK protein expression was up-regulated,while MMP-2 and MMP-9 were down regulated in miR-99 ainhibitor transfection group as compared with the control group.Conclusion:MiR-99 ais highly expressed in osteosarcoma tissues and cell lines.Downregulation of miR-99 acan inhibit the proliferation,invasion and migration of osteosarcoma cell line 143 B,and its mechanism may be through regulating the expression of MMP-2/RECK signal pathway.