肝癌细胞HepG2中p53调控miRNA-3661的生物信息分析与功能验证

Bioinformatics Analysis and Functional Verification of p53 Regulating miRNA-3661 in Hepatoma Cell HepG2

对已在前期实验中通过Dox诱导肝癌细胞HepG2 DNA损伤发现的受p53调控的hsa-miR-3661进行生物信息学分析,并通过分子生物学实验对其功能进行了验证,为miR-3661在肝肿瘤中的调控机制的研究提供理论基础。获取miR-3661结构与序列信息;预测靶基因,使用DAVID进行miRNA靶基因功能富集分析;分析miR-3661的p53结合位点,通过基因间的相互作用构建调控网络;进行细胞增殖实验验证miR-3661抑制肿瘤功能。结果表明,miR-3661序列保守,启动子区存在p53结合位点,暗示p53与hsa-miR-3661存在直接调控;预测靶基因1 009个,369个显著富集于细胞周期调控、细胞增殖、细胞凋亡等肿瘤相关生物学过程(P<0.05),主要参与了癌症信号通路、MAPK信号通路与Erb B信号通路(P<0.05);通过268组基因间的相互作用数据构建了p53、hsa-miR-3661和靶基因的调控网络,从系统生物学角度分析了参与多个肿瘤生物进程的关键靶基因;在实验中证实过表达miR-3661可以显著抑制肝癌细胞HepG2的增殖过程(P-value=0.001 46)。miR-3661受p53直接调控,其靶基因显著富集于多种肿瘤相关生物进程与信号通路,过表达miR-3661可显著抑制肝癌细胞增殖。

The aims of this work are to have the bioinformatics analysis of hsa-miR-3661 regulated by p53,which was found in doxorubicin（Dox）inducing the DNA damages of hepatoma cell HepG2 in previous experiment,and to verify its function by molecular biologicalexperiment,providing theoretical basis for the regulating mechanism of miR-3661 in hepatoma cells. After acquiring the structure and sequenceinformation of miR-3661,we predicted the target genes and used DAVID to do the functional enrichment analysis of miRNA target gene. Wethen analyzed the binding sites of p53 and miR-3661,and built the regulatory network by the interaction among genes. Finally,multiplicationexperiment verified the functions of miR-3661 in restraining tumor. The results showed that miR-3661 sequence was conserved,and thepromoter region existed in the binding site of p53,suggesting that there was direct regulation between p53 and hsa-miR-3661. We predictedthat there were 1009 target genes,and 369 genes of them significantly enriched in the biological procedure related to tumor,such as cell cycleregulation,proliferation,apoptosis and so on（P〈0.05）,mainly involved in cancer pathway,MAPK signaling pathway,Erb B signalingpathway（P〈0.05）. Using the interactions of 2830 groups of genes,we constructed the regulatory network among p53,hsa-miR-3661 andtarget genes,and analyzed key target gene participating in several tumorous biological process from the perspective of system biology. It wasconfirmed in the experiment that the overexpression of miR-3661 significantly inhibited the proliferation of hepatoma cell HepG2（P-value=0.00146）. In conclusion,miR-3661 is directly regulated by p53 and its target genes significantly enrich in various biological processes andsignal pathways related to tumor. Moreover,the overexpression of miR-3661 significantly inhibits the proliferation of hepatoma cell.