低氧诱导的HIF-1α对结肠癌细胞生长代谢的影响及其调控机制

Hypoxia-induced HIF-1α down-regulates FUT4 expression under hypoxia in colon cancer via inducing hsa-miRNA-23a-3p

目的低氧环境下肿瘤细胞生长代谢异常,探讨低氧诱导因子(hypoxia inducible factor 1,HIF)对岩藻糖基转移酶Ⅳ(fucosyltransferase4,FUT4)的负性调控机制及病理生理意义。方法前期从基因表达数据库中筛选到符合研究条件的芯片GSE77606,结合京都基因与基因组百科全书通路富集分析,发现低氧环境下结肠癌(colon cancer)细胞生长代谢异常,FUT4表达明显下降;随后通过TargetScan数据库筛选出与FUT4信使RNA序列互补的微小RNA(miRNA),并结合定量RT-PCR(QRT-PCR)、荧光素酶报告基因实验及突变实验、短发夹RNA干扰实验和miRNA沉默和过表达实验等探索其具体的调控机制。结果QRT-PCR证实低氧培养HCT-116细胞明显低表达FUT4(P <0.001);TargetScan数据库筛选结合荧光素酶报告基因实验和突变实验证实hsa-miRNA-23a-3p可靶向FUT4信使RNA并下调其表达(P <0.05),同时QRT-PCR显示低氧环境可诱导hsa-miRNA-23a-3p的表达(P <0.05);短发夹RNA干扰实验证实hsa-miRNA-23a-3p的表达受到HIF-1α的正性调控(P <0.01);最后hsa-miRNA-23a-3p沉默或过表达实验进一步证实了hsa-miRNA-23a-3p可正反调控FUT4的表达水平(P <0.001)。结论结肠癌细胞在低氧环境中通过HIF-1α诱导has-miRNA-23a-3p下调FUT4的表达,影响代谢调控,降低细胞侵袭性。

ObjectiveThe growth and metabolism of tumor cells in hypoxia environment are abnormal.We aim to investigate the negative regulation mechanism of hypoxia inducible factor(HIF)on fucosyltransferase4(FUT4)and its pathophysiological significance.MethodsThe array datasets(GSE77606)was selected from the Gene Expression Omnibus(GEO)in the early stage,and analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG). We have found that the metabolic pathway wasenriched and the expression of FUT4 wassignificantlydecreased in the colon cancer cells cultured under hypoxia. MicroRNA(miRNA)that targets the messenger RNA(mRNA)sequence of FUT4 gene is screened by TargetScan. The mechanism by whichFUT4 is down-regulated un-der hypoxiais explored by quantitative reverse transcription PCR assay(QRT-PCR),luciferase reporter assays,mutagenesis assay,short hairpin RNA(shRNA)interference assay,miRNA silencing and over expression assay.ResultsQRT-PCR confirmed that the expression of FUT4 in HCT-116 cells cultured in hypoxia is significantlydecreased(P < 0.001). TargetScan database screening combined with luciferase reporter assays and mutagenesisassay confirmed that hsa-miRNA-23 a-3 p targeted the m RNA of FUT4 and down regulated its expression(P < 0.05),and QRT-PCR showed that the hypoxia environment could induce the expression of hsa-miRNA-23 a-3 p(P < 0.05).ShRNA interference experiment confirmed that the expression of hsa-miRNA-23 a-3 p was positively regulated byHIF-1 α(P < 0.01). Finally,hsa-miRNA-23 a-3 p silencing and over-expression assays further confirmed tha thsa-miRNA-23 a-3p negatively regulated the expression level of FUT4(P < 0.001).ConclusionThe expression of FUT4 was down-regulated in colon cancer cells through hsa-miRNA-23 a-3p that induced by HIF-1α in hypoxic environment.