GSE32662 and GSE114068 were used for the identification of differentially expressed genes, eRNAs, enhancers and enhancer-regulated genes in MTC. Metascape and the transcription factor affinity prediction method were used for gene function enrichment and transcription factor prediction, respectively. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to assess the binding of histone H3 lysine 27 acetylation (H3K27ac)-enriched regions to anti- H3K27ac. RIP-qPCR was used to detect the binding between FOXQ1 and LINC00887. CCK8 and Transwell were performed to measure the proliferation and invasion of MTC cells, respectively. Intracellular reactive oxygen species (ROS) levels were quantified using a ROS assay kit.
Four eRNAs (H1FX-AS1, LINC00887, MCM3AP-AS1 and A1BG-AS1) were screened, among which LINC00887 was the key eRNA promoting the proliferation and invasion of MTC cells. A total of 135 genes controlled by LINC00887-regulated enhancers were identified; among them, BCL2, PRDX1, SFTPD, TPO, GSS, RAD52, ZNF580, and ZFP36L1 were significantly enriched in the \"ROS metabolic process\" term. As a transcription factor regulating genes enriched in the \"ROS metabolic process\" term, FOXQ1 could recruit LINC00887. Overexpression of FOXQ1 restored LINC00887 knockdown-induced downregulation of GSS and ZFP36L1 transcription in MTC cells. Additionally, FOXQ1 overexpression counteracted the inhibitory effects of LINC00887 knockdown on the proliferation and invasion of MTC cells and the promotion of intracellular ROS accumulation induced by LINC00887 knockdown.
LINC00887 was identified as a key eRNA promoting the malignant phenotype of MTC cells. The involvement of FOXQ1 was essential for LINC00887 to play a pro-tumorigenic role in MTC. Our findings suggest that the FOXQ1/LINC00887 axis is a potential therapeutic target for MTC.
方法:使用GSE32662和GSE114068鉴定差异表达基因,eRNAs,MTC中的增强子和增强子调节基因。将Metascape和转录因子亲和力预测方法用于基因功能富集和转录因子预测,分别。qRT-PCR用于检测基因转录水平。ChIP-qPCR用于评估组蛋白H3赖氨酸27乙酰化(H3K27ac)富集区与抗H3K27ac的结合。RIP-qPCR用于检测FOXQ1和LINC00887之间的结合。CCK8和Transwell检测MTC细胞的增殖和侵袭,分别。使用ROS测定试剂盒定量细胞内活性氧(ROS)水平。
结果:筛选出4种eRNAs(S1FX-AS1、LINC00887、MCM3AP-AS1和A1BG-AS1),其中LINC00887是促进MTC细胞增殖和侵袭的关键eRNA。总共鉴定了由LINC00887调节的增强子控制的135个基因;其中,BCL2,PRDX1,SFTPD,TPO,GSS,RAD52、ZNF580和ZFP36L1在“ROS代谢过程”术语中显著富集。作为一种转录因子,调节富含“ROS代谢过程”术语的基因,FOXQ1可以招募LINC00887。FOXQ1的过表达恢复了LINC00887敲低诱导的MTC细胞中GSS和ZFP36L1转录的下调。此外,FOXQ1过表达抵消了LINC00887敲低对MTC细胞增殖和侵袭的抑制作用以及LINC00887敲低诱导的细胞内ROS积累的促进。
结论:LINC00887被鉴定为促进MTC细胞恶性表型的关键eRNA。FOXQ1的参与对于LINC00887在MTC中发挥促肿瘤作用至关重要。我们的研究结果表明,FOXQ1/LINC00887轴是MTC的潜在治疗靶点。