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.

中文翻译：

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Sp1 介导的 lnc00152 上调通过 miR-30d/SOX9/ZEB2 通路促进视网膜母细胞瘤细胞的侵袭和转移。

目的

以前，我们发现长链非编码 RNA (lncRNA) MEG3 可能在视网膜母细胞瘤中充当肿瘤抑制因子。然而，总的来说，人们对 lncRNA 在视网膜母细胞瘤中的作用知之甚少。在这里，我们旨在确定 lnc00152 在视网膜母细胞瘤中的表达和临床意义。

方法

Lnc00152 及其下游目标是使用 GEO 数据集选择的。使用 RT-qPCR 确定主要患者样本中 lnc00152 的水平。使用逻辑回归分析计算侵袭和转移的优势比。使用 Cox 回归分析评估无复发生存率。划痕愈合、transwell 和肿瘤发生测定用于确定视网膜母细胞瘤细胞在体外和体内的迁移和侵袭能力。使用蛋白质印迹法测量 EMT 相关蛋白的水平。使用双荧光素酶报告基因和 RNA 下拉分析验证了 lnc00152 与其靶标之间的结合位点。Lnc00152 激活转录因子是使用 ChIP 测定确定的。

结果

我们发现Lnc00152在视网膜母细胞瘤肿瘤组织中显着上调，是肿瘤侵袭、转移和复发的危险因素。过表达 Lnc00152 的视网膜母细胞瘤细胞表现出向间充质细胞转化的趋势，迁移和侵袭能力显着增强，E-cadherin 表达水平显着降低，N-cadherin、SOX9 和 ZEB2 表达水平显着增加。此外，我们发现被 Sp1 激活的 lnc00152 可以抑制 miR-30d 作为内源性 miRNA“海绵”，从而调节 SOX9 和 ZEB2 的表达。

结论

我们的数据表明 Lnc00152 可能与视网膜母细胞瘤的侵袭、转移和预后有关。此外，我们得出结论，可以被 Sp1 激活的 Lnc00152 可以通过 miR-30d/SOX9/ZEB2 途径诱导 EMT，从而促进视网膜母细胞瘤细胞的侵袭和转移。