Sp1-mediated up-regulation of lnc00152 promotes invasion and metastasis of retinoblastoma cells via the miR-30d/SOX9/ZEB2 pathway
Sp1-mediated up-regulation of lnc00152 promotes invasion and metastasis of retinoblastoma cells via the miR-30d/SOX9/ZEB2 pathway
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DOI：
10.1007/s13402-020-00522-8
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发表时间：
2020-06-01
影响因子：
6.6
通讯作者：
Zhang, Jun
中科院分区：
文献类型：
--
作者：
Gao, Yali;Luo, Xiaoling;Zhang, Jun
目的以前，我们发现长链非编码RNA（lncRNA）MEG 3可能在视网膜母细胞瘤中起肿瘤抑制剂的作用。然而，总体而言，对lncRNA在视网膜母细胞瘤中的作用知之甚少。本研究旨在探讨lnc 00152在视网膜母细胞瘤中的表达及其临床意义。方法利用GEO数据集筛选Lnc 00152及其下游靶点。使用RT-qPCR测定原始患者样品中lnc 00152的水平。采用Logistic回归分析计算侵袭和转移的比值比。采用考克斯回归分析评估无复发生存期。采用划痕法、transwell法和成瘤实验检测视网膜母细胞瘤细胞在体外和体内的迁移和侵袭能力。EMT相关蛋白的水平采用蛋白质印迹法测定。使用双荧光素酶报告基因和RNA下拉测定验证lnc 00152与其靶标之间的结合位点。使用ChIP测定法测定Lnc 00152激活转录因子。结果Lnc 00152在视网膜母细胞瘤组织中表达显著上调，是肿瘤侵袭、转移和复发的危险因素。Lnc 00152过表达的视网膜母细胞瘤细胞表现出向间充质细胞转化的趋势，具有显著增加的迁移和侵袭能力，显著降低的E-cadherin表达水平，以及显著增加的N-cadherin、SOX 9和ZEB 2表达水平。此外，我们发现lnc 00152，这是由Sp1激活，可以抑制miR-30 d作为一种内源性的miRNA“海绵”，从而调节SOX 9和ZEB 2的表达。结论Lnc 00152可能与视网膜母细胞瘤的侵袭、转移及预后有关。此外，我们认为Lnc 00152可被Sp1激活，通过miR-30 d/SOX 9/ZEB 2途径诱导EMT，从而促进视网膜母细胞瘤细胞的侵袭和转移。
Purpose Previously, we found that long non-coding RNA (lncRNA) MEG3 may act as a tumour suppressor in retinoblastoma. Overall, however, little is known about the role of lncRNAs in retinoblastoma. Here, we aimed to determine the expression and clinical significance of lnc00152 in retinoblastoma. Methods Lnc00152 and its downstream targets were selected using GEO datasets. The level of lnc00152 in primary patient samples was determined using RT-qPCR. Odds ratios of invasion and metastasis were calculated using logistic regression analysis. Recurrence-free survival was assessed using Cox regression analysis. Scratch wound healing, transwell and tumorigenesis assays were used to determine migration and invasion abilities of retinoblastoma cells in vitro and in vivo. Levels of EMT-related proteins were measured using Western blotting. Binding sites between lnc00152 and its targets were validated using dual-luciferase reporter and RNA pull-down assays. Lnc00152 activating transcription factors were determined using ChIP assays. Results We found that Lnc00152 was significantly up-regulated in retinoblastoma tumour tissues, and was a risk factor for tumour invasion, metastasis and recurrence. Lnc00152 overexpressing retinoblastoma cells exhibited a tendency to transform into mesenchymal cells, with significantly increased migration and invasion capacities, significantly decreased E-cadherin expression levels, and significantly increased N-cadherin, SOX9 and ZEB2 expression levels. In addition, we found that lnc00152, which was activated by Sp1, could inhibit miR-30d as an endogenous miRNA 'sponge', thereby regulating the expression of SOX9 and ZEB2. Conclusions Our data indicate that Lnc00152 may be associated with retinoblastoma invasion, metastasis and prognosis. In addition, we conclude that Lnc00152, which can be activated by Sp1, can induce EMT via the miR-30d/SOX9/ZEB2 pathway and, by doing so, promote the invasion and metastasis of retinoblastoma cells.