双功能基因APE1调控人骨肉瘤HOS细胞miRNAs的初步研究

Effect of a double functional gene APE1 on miRNAs expression in human osteosarcoma HOS cells

目的比较APE1-konckdown骨肉瘤HOS细胞miRNAs表达谱的差异,初步探讨APE1可能调控的miRNAs及其涉及的功能通路。方法利用Ad5/F35-APE1 siRNA腺病毒敲低骨肉瘤HOS细胞中APE1的表达,采用miRNA芯片检测敲低组和对照组miRNAs表达谱的改变,qRT-PCR验证,并利用生物信息学分析差异miRNAs涉及的功能。结果miRNAs芯片结果显示,APE1敲低组中有32个miRNAs表达显著改变;qRT-PCR验证有13个miRNAs的改变与芯片结果趋势一致,其中7个上调,6个下调;基因聚类分析发现,13个显著变化的miRNAs涉及的靶基因的功能主要定位在细胞内,与蛋白、DNA结合以及转录功能有关,参与调控发育和细胞代谢等生物过程;Pathway分析显示其靶基因主要涉及粘附连接、轴突导向、ECM-受体相互作用、肿瘤通路,以及TGF-β、MAPK、ErbB、Wnt、mTOR及p53等关键的细胞信号通路。结论 APE1能直接影响骨肉瘤细胞miRNAs的表达;生物信息学分析表明APE1通过调控miRNAs可能影响下游靶基因的表达,参与肿瘤发生、细胞生存、增殖、粘附等生物学过程和多个信号通路,与骨肉瘤的发生、发展、侵袭转移密切相关。

Objective To determine the endonuclease （APE1） knockdown HOS cells and differential expression profile between apurinic/apyrimidinic wild-type HOS cells, and to investigate the roles of these differential miRNAs in osteosarcoma cells. Methods After APE1 expression were down-regulated by APE1- siRNA adenovirus Ad5/F35-APE1 siRNA in HOS cells, microarray were used to confirm the change of microRNAs （miRNAs） in APEl-knockdown osteosarcoma HOS cells and wide-type cells. The results were confirmed by qRT-PCR. Co （GO） analysis and pathway mprehensive bioinformatics-based analysis, including target genes, gene ontology analysis were carried out to analyze these differential expressed miRNAs and their functions. Results miRNA microarray indicated that there were 32 miRNAs differentially expressed in APE1 knockdown HOS cells, qRT-PCR demonstrated that 13 miRNAs （coincidence with the results of microarray） were significantly changed （ 〉 2-fold ）, with 7 of them up-regulated and the other 6 down-regulated. Furthermore, GO analysis showed that these miRNAs and their target genes affected by APE1 expression were located in intra-ceUular and played roles in the protein and DNA binding, transcription, developmental process and cellular metabolism. Pathway analysis showed that these miRNAs targets were involved in adhere junction, axon guidance, ECM-receptor interaction, cancer pathway, as well as some key cell signaling pathways （ such as TGF-β, MAPK, EtbB, Wnt, mTOR and p53 signaling pathway）. Conclusion APE1 appears to have a direct effect on global miRNAs expression. Bioinformatics-based analysis indicated APE1 may regulate miRNAs to affect the expression of downstream target genes. These interactions may be involved in the tumor, progenesis, cell survival, proliferation and adhesion, and many cell signal pathways, especially with regard to the tumorigenesis, development, invasion and metastasis of osteosarcoma.