目的:我们先前已经表征了IGF2BP1在HCC中的致癌特性,及其通过短非编码RNA(ncRNA)的调控。最近的证据表明IGF2BP1本身可以调节长ncRNAs(lncRNAs)。因此,本研究旨在探讨IGF2BP1与各种上游和下游ncRNAs之间的相互作用及其与HCC发病机制的联系。
方法:生物信息学分析用于鉴定与IGF2BP1相互作用的上游和下游ncRNAs。用针对IGF2BP1的siRNA和微小RNA模拟物转染Huh-7细胞。使用RTqPCR确定相对基因表达,并通过蛋白质印迹定量IGF2BP1蛋白。荧光素酶结合试验用于探索IGF2BP13'UTR的靶向性。用MTT法测定肝癌的肿瘤发生,BrdU掺入测定,集落形成试验,和划痕试验。
结果:生物信息学分析确定了三种致癌lncRNAs——即H19、FOXD2-AS1和SNHG3——可能受IGF2BP1调控。IGF2BP1的敲除降低了所有三种致癌lncRNAs的表达并抑制了恶性细胞行为。miR-186被揭示为IGF2BP1的可能的上游调节因子。miR-186模拟物降低IGF2BP1mRNA和蛋白水平。与5名健康对照相比,10名HCC患者的癌组织中miR-186显著降低,而IGF2BP1升高。此外,miR-186模拟物导致致癌lncRNAsH19,SNHG3和FOXD2-AS1的下调,并伴随细胞活力的降低,扩散,迁移,和克隆性。
结论:miR-186可能通过对IGF2BP1的作用抑制致癌lncRNAsH19、SNHG3和FOXD2-AS1,从而在HCC中发挥抑癌作用。
OBJECTIVE: We have previously characterized oncogenic properties of IGF2BP1 in HCC, and its regulation by short noncoding RNAs (ncRNAs). Recent evidence suggests that IGF2BP1 itself may regulate long ncRNAs (lncRNAs). Therefore, this study aimed at exploring the interplay between IGF2BP1 and various upstream and downstream ncRNAs and its link to HCC pathogenesis.
METHODS: Bioinformatic analysis was used to identify up- and downstream ncRNAs interacting with IGF2BP1. Huh-7 cells were transfected with siRNAs against IGF2BP1 and microRNA mimics. Relative gene expression was determined using RTqPCR and IGF2BP1 protein was quantified by western blot. Luciferase binding assay was used to explore the targeting of IGF2BP1 3\'UTR. HCC tumorigenesis was measured by MTT assay, BrdU-incorporation assay, colony-forming assay, and scratch assay.
RESULTS: Bioinformatic analysis identified three oncogenic lncRNAs - namely H19, FOXD2-AS1, and SNHG3 - potentially regulated by IGF2BP1. Knockdown of IGF2BP1 decreased the expression of all three oncogenic lncRNAs and inhibited malignant cell behaviors. miR-186 was revealed as a possible upstream regulator of IGF2BP1. miR-186 mimics decreased IGF2BP1 mRNA and protein levels. miR-186 was significantly lower while IGF2BP1 was elevated in cancerous tissues from ten HCC patients compared to five healthy controls. In addition, miR-186 mimics caused a downregulation of the oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 and a concomitant decrease in cell viability, proliferation, migration, and clonogenicity.
CONCLUSIONS: miR-186 may exert tumor suppressor effects in HCC by repressing oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 through its effect on IGF2BP1.