miR-639在骨肉瘤组织中的表达及其模拟物转染对人骨肉瘤细胞U20S增殖、迁移、侵袭能力的影响

Expression of miR-639 in osteosarcoma and its effects on proliferation,migration,and invasion of U20S cells

目的观察微小RNA-639(miR-639)在骨肉瘤组织中的表达及miR-639模拟物(miR-639 mimics)转染对人骨肉瘤细胞U20S增殖、迁移、侵袭能力的影响。方法通过GEO数据库和TCGA数据库分析骨肉瘤组织及癌旁组织miR-639表达情况,另分析miR-639高表达与骨肉瘤临床病理参数的关系。取U20S细胞,将miR-639 mimics和模拟物阳性对照(miR-NC)转染入U20S细胞中(分别计为观察组和对照组),转染后继续培养24 h,后收集细胞进行后续实验。采用CCK-8实验检测两组细胞集落形成数,Ed U实验检测细胞Ed U染色数,克隆形成实验检测细胞迁移细胞数,Transwell实验检测细胞侵袭数,荧光定量PCR法和Western blotting法分别检测细胞TUSC5 mRNA、蛋白。结果 GEO数据库显示,骨肉瘤、癌旁组织中miR-639表达水平分别为11.2±0.4、7.1±0.6,两者比较,P <0.05;TCGA数据库显示,骨肉瘤、癌旁组织中miR-639表达水平分别为19.5±3.4、7.7±2.2,两者比较,P <0.05。miR-639高表达与骨肉瘤肿瘤直径、淋巴结转移、解剖部位有关(P均<0.05)。观察组、对照组集落形成数分别为(155±12)、(84±8)个,Ed U染色数分别为(14±3)、(6±2)个,迁移细胞数分别为(120±30)、(70±18)个,侵袭细胞数分别为(100±16)、(52±23)个,两组比较,P均<0.05。观察组和对照组TUSC5 mRNA分别为0.18±0.07、0.84±0.13,TUSC5蛋白分别为0.31±0.06、0.81±0.09,两组比较,P均<0.05。结论 miR-639在骨肉瘤组织中表达升高;miR-639 mimics转染促进了骨肉瘤细胞的增殖、迁移、侵袭能力,可能与其抑制TUSC5有关。

Objective To observe the expression of microRNA-639(miR-639)in osteosarcoma(OS)and the effects of miR-639 mimics transfection on the proliferation,migration,and invasion of human osteosarcoma cells U20S.Methods The expression level of miR-639 and the relationships between the miR-639 level and the clinicopathological characteristics of patients were analyzed by GEO database and TCGA database.We transfected miR-639 mimics and negative control of miR-639 mimics(miR-NC)into U2OS cells(which were taken as the observation group and the control group),and continued to culture for 24 h,after that,we collected the cells and performed the following experiments.Colony formation assay was used to detect the numbers of clones.Ed U assay was used to detect the staining numbers of Ed U in cells.Transwell migration and invasion assays were used to detect the numbers of migratory and invasive cells.RT-qPCR and Western blotting were used to detect the mRNA and protein levels of TUSC5.Results The database of GEO showed that the miR-639 level was 11.2±0.4 in the OS tissues and 7.1±0.6 in the adjacent tissues(P<0.05).The database of TCGA showed that the miR-639 level was 19.5±3.4 in the OS tissues and 7.7±2.2 in the adjacent tissues(P<0.05).The up-regulation of miR-639 was related to the tumor size,lymph node metastasis,and anatomic site.The numbers of colony formation in the observation group and the control group were 155±12 and 84±8(P<0.05).The Ed U staining numbers of observation group and the control group were 14±3 and 6±2(P<0.05).The numbers of migratory cells in the observation group and the control group were 120±30 and 70±18(P<0.05).The numbers of invasive cells in the observation group and the control group were 100±16 and 52±23(P<0.05).The TUSC5 mRNA levels of the observation group and the control group were 0.18±0.07 and 0.84±0.13(P<0.05),and the TUSC5 protein levels were 0.31±0.06 and0.81±0.09,with statistically significant difference(P<0.05).Conclusion The level of miR-639 is up-regulated in the OS tissues,and the miR-639 mimics transfection promotes the cell proliferation,migration,and invasion abilities,which may be related to the inhibition of TUSC5.