过/降表达miR-20a-5p的骨肉瘤细胞增殖、侵袭能力及Bax、Bcl-2、E2F5蛋白表达观察

Proliferation and invasion of osteosarcoma cells with over-/down-expression of miR-20a-5P and obser⁃vation of Bax,Bcl-2,and E2F5 protein expression

目的观察miR-20a-5p在骨肉瘤(OS)细胞中的表达变化,探讨miR-20a-5p表达对OS细胞增殖、凋亡及侵袭的影响及其分子机制。方法采用实时荧光定量PCR法检测正常人成骨细胞系hFOB1.19及OS细胞系(U-20S、143B、Saos-2)中的miR-20a-5p表达量。将OS细胞系143B随机分为miR-20a-5p过表达组、miR-20a-5p抑制表达组及阴性对照组,通过Lipofectamine^(TM)3000分别转染miR-20a-5p mimics、miR-20a-5p inhibit及scramble,转染24 h。采用CCK-8法检测三组培养0、24、48、72 h的的光密度值(OD值),流式细胞术检测细胞凋亡率,Transwell小室实验检测侵袭细胞数。在线数据库TargetScan预测结果显示E2F转录因子5(E2F5)存在与miR-20a-5p结合的潜在靶点,荧光素酶报告基因实验证实miR-20a-5p的靶基因为E2F5,采用Westen blotting法检测细胞Bax、Bcl-2、E2F5蛋白表达。结果OS细胞系U-20S、143B、Saos-2中miR-20a-5p相对表达量分别为1.94±0.35、1.05±0.13、0.35±0.06,均明显低于正常人成骨细胞系hFOB1.19中的3.89±0.53(P均<0.05)。随着培养时间的延长,三组OD值均呈升高趋势;培养24、48、72 h,相同培养时间下miR-20a-5p过表达组、阴性对照组、miR-20a-5p抑制表达组OD值依次升高,组间两两比较P均<0.05。miR-20a-5p过表达组、阴性对照组、miR-20a-5p抑制表达组细胞凋亡率、Bax蛋白相对表达量均依次降低,侵袭细胞数、Bcl-2、E2F5蛋白相对表达量均依次升高,组间两两比较P均<0.05。结论miR-20a-5p在OS细胞系中表达下调,过表达miR-20a-5p可抑制OS细胞增殖、侵袭并诱导细胞凋亡,其机制可能与下调E2F5、Bcl-2及上调Bax蛋白表达有关,降表达miR-20a-5p则具有相反的作用。

Objective To observe the expression changes of miR-20a-5p in the osteosarcoma(OS)cells,and to explore the effects of miR-20a-5p expression on the proliferation,apoptosis,and invasion of OS cells and its molecular mechanism.Methods Real-time quantitative PCR was used to detect the expression of miR-20a-5p in the normal human osteoblast cell line hFOB1.19 and OS cell lines(U-20S,143B,and Saos-2).The OS cell line 143B were randomly divided into the miR-20a-5p overexpression group,the miR-20a-5p inhibitory expression group and the negative control group,which were transfected with miR-20a-5p mimics,miR-20a-5p inhibit and scramble by Lipofectamine^(TM)3000 for 24 h,respectively.The optical density value(OD value)of the three groups at 0,24,48,and 72 h of culture was detected by CCK-8 method.The apoptosis rate was detected by flow cytometry.The number of invasive cells was detected by Transwell chamber assay.The prediction results of the online database TargetScan showed that E2F transcription factor 5(E2F5)had a potential target for binding to miR-20a-5p.The luciferase reporter gene assay confirmed that the target gene of miR-20a-5p was E2F5.Western blotting was used to detect the Bax,Bcl-2,and E2F5 protein expression.Results The relative expression levels of miR-20a-5p in OS cell lines U-20S,143B,and Saos-2 were 1.94±0.35,1.05±0.13,and 0.35±0.06,respectively,which were significantly lower than those in the normal human osteoblast cell line hFOB1.19 of 3.89±0.53(all P<0.05).Over the culture time,the OD values of the three groups showed an increasing trend.At 24,48,and 72 h of culture,the OD values increased in the miR-20a-5p overexpression group,the negative control group,and the miR-20a-5p inhibitory expression group under the same culture time in turn,with statistically significant difference between any two groups(all P<0.05).In the miR-20a-5p overexpression group,the negative control group,and the miR-20a-5p inhibitory expression group,the apoptosis rates and the relative expression of Bax protein decreased in sequence,and the number of invasive cells and the relative expression of Bcl-2 and E2F5 proteins increased in sequence,with statistically significant difference between any two groups(all P<0.05).Conclusions The expression of miR-20a-5p is downregulated in OS cell lines.Overexpression of miR-20a-5p can inhibit OS cells’proliferation and invasion,and induce apoptosis.The mechanism may be related to the down-regulation of E2F5,Bcl-2 and up-regulation of Bax protein expression.