miR-542-3p对骨肉瘤细胞侵袭能力的影响及其靶基因预测

Effect of miR-542-3p on invasion of osteosarcoma cells and the target genes prediction

目的明确miR-542-3p对骨肉瘤细胞系HOS和SAOS2细胞侵袭能力的影响,通过生物信息学建立miR-542-3p靶基因调控网络,并探讨其潜在作用靶点。方法使用miR-542-3p mimics转染HOS和SAOS2细胞,同时设立正常组(未转染)及阴性对照组(转染miR-neg mimics),Transwell小室试验检测转染后细胞的侵袭能力。通过美国国立生物信息中心(NCBI)基因表达共享数据库(GEO)联合GEO2R平台,获取并分析miRNA-542-3p与miR-neg转染的U2OS细胞的基因表达谱数据及其差异表达基因,进而使用miRNA预测数据库及Cytoscape软件,预测并建立miR-542-3p靶基因调控网络。实时荧光定量PCR验证miR-542-3p转染HOS和SAOS2细胞后,部分预测目标靶基因的mRNA表达水平变化。结果 miR-542-3p干预组HOS和SAOS2细胞的侵袭能力较正常组和阴性对照组明显降低(P均<0.05)。数据库差异基因分析表明,miR-542-3p与miR-neg转染的U2OS细胞的表达差异基因共有417个,其中185个基因表达下调。miRNA预测数据库预测的266个靶基因中,有14个与差异基因分析中得到的表达下调基因重合。实时荧光定量PCR分析得出,ILK、TBPL1、ETS1这3个预测靶基因在miR-542-3p干预组中的表达水平显著低于阴性对照组,差异均有统计学意义(P均<0.05)。结论 miR-542-3p通过下调ILK、TBPL1、ETS1显著抑制骨肉瘤细胞的侵袭能力。

Objective To testify the inhibitory effect of miR-542-3p on invasion of HOS and SAOS2 cells,and predict working model of its regulatory network via bioinformatic analysis.Methods HOS and SAOS2 cells were transfected with miR-542-3p mimics,and normal group and negative control group were set up.The invasion was detected by Transwell chambers.GEO database and GEO2R software were exercised to obtain and analyze the gene expression profile of U2OS cells transfected with miRNA-542-3p and miR-neg respectively.Target genes of miR-542-3p and its regulatory network were predicted and established by online miRNA predicting databases and Cytoscape software.The mRNA levels of interested target genes in miR-542-3p transfected cell group and negative control group were detected by real-time PCR.Results Invasion of HOS and SAOS2 cells was significantly inhibited by upregulating miRNA-542-3p as compared with negative control group(P<0.05 for all).Analysis of gene expression profiles from GEO database indicated that 417 genes were differently expressed in U2OS cells transfected with miRNA-542-3p or miR-neg,among which 185 genes were down-regulated.Of the 266 target genes predicted by miRNA predicting databases,14 were coincident with the down-regulated genes in the differentially expressed gene analysis.The results of Real-time PCR showed that mRNA levels of ILK,TBPL1 and ETS1 were significantly reduced by upregulating miRNA-542-3p as compared with negative control group(P<0.05 for all).Conclusion miR-542-3p can inhibit invasion of osteosarcoma cells by down-regulating ILK,TBPL1 and ETS1.