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长链非编码RNA LINC01260通过NF-κB信号通路靶向CARD11抑制脊髓胶质瘤细胞的增殖、迁移和侵袭。

Long Non-Coding RNA LINC01260 Inhibits the Proliferation, Migration and Invasion of Spinal Cord Glioma Cells by Targeting CARD11 Via the NF-κB Signaling Pathway.

作者信息

Wu Dong-Mei, Han Xin-Rui, Wen Xin, Wang Shan, Wang Yong-Jian, Shen Min, Fan Shao-Hua, Zhuang Juan, Zhang Zi-Feng, Shan Qun, Li Meng-Qiu, Hu Bin, Sun Chun-Hui, Lu Jun, Zheng Yuan-Lin

机构信息

Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, China.

College of Health Sciences, Jiangsu Normal University, Xuzhou, China.

出版信息

Cell Physiol Biochem. 2018;48(4):1563-1578. doi: 10.1159/000492279. Epub 2018 Aug 2.

Abstract

BACKGROUND/AIMS: Spinal cord glioma is a highly aggressive malignancy that commonly results in high mortality due to metastasis, high recurrence and limited treatment regimens. This study aims to elucidate the effects of long non-coding RNA LINC01260 (LINC01260) on the proliferation, migration and invasion of spinal cord glioma cells by targeting Caspase recruitment domain family, member 11 (CARD11) via nuclear factor kappa B (NF-κB) signaling.

METHODS

The Multi Experiment Matrix (MEM) website was used for target gene prediction, and the DAVID database was used for analysis of the relationship between CARD11 and the NF-κB pathway. In total, 60 cases of glioma tissues and adjacent normal tissues were collected. Human U251 glioma cells were grouped into blank, negative control (NC), LINC01260 vector, CARD11 vector, siRNA-LINC01260, siRNA-CARD11, LINC01260 vector + CARD11 vector and LINC01260 + siRNA-CARD11 groups. A dual-luciferase reporter assay was conducted to verify the target relationship between LINC01260 and CARD11. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were employed to assess expression of LINC01260, E-cadherin, p53, CARD11, Ki67, N-cadherin, matrix metalloproteinase (MMP)-9, NF-κBp65 and NF-κBp50. MTT, flow cytometry, wound-healing and Transwell assays were performed to examine cell viability, the cell cycle, apoptosis, invasion and migration. Tumor growth was assessed through xenografts in nude mice.

RESULTS

CARD11 was confirmed to be a target gene of LINC01260 and was found to be involved in regulating the NF-κB pathway. Compared with adjacent normal tissues, glioma tissues showed reduced expression of LINC01260 and elevated expression of CARD11 and genes related to apoptosis, invasion and migration; activation of NF-κB signaling was also observed. In contrast to the blank and NC groups, an elevated number of cells arrested in G1 phase, increased apoptosis and reduced cell proliferation, invasion and number of cells arrested in S and G2 phases, as well as tumor growth were found for the LINC01260 vector and siRNA-CARD11 groups.

CONCLUSIONS

Our findings demonstrate that overexpression of LINC01260 inhibits spinal cord glioma cell proliferation, migration and invasion by targeting CARD11 via NF-κB signaling suppression.

摘要

背景/目的:脊髓胶质瘤是一种高度侵袭性的恶性肿瘤,由于转移、高复发率和有限的治疗方案,通常导致高死亡率。本研究旨在通过核因子κB(NF-κB)信号通路靶向半胱天冬酶募集结构域家族成员11(CARD11),阐明长链非编码RNA LINC01260(LINC01260)对脊髓胶质瘤细胞增殖、迁移和侵袭的影响。

方法

利用多实验矩阵(MEM)网站进行靶基因预测,并使用DAVID数据库分析CARD11与NF-κB通路之间的关系。共收集60例胶质瘤组织及相邻正常组织。将人U251胶质瘤细胞分为空白组、阴性对照组(NC)、LINC01260载体组、CARD11载体组、siRNA-LINC01260组、siRNA-CARD11组、LINC01260载体+CARD11载体组和LINC01260+siRNA-CARD11组。进行双荧光素酶报告基因检测以验证LINC01260与CARD11之间的靶向关系。采用逆转录定量聚合酶链反应(RT-qPCR)和蛋白质印迹分析评估LINC01260、E-钙黏蛋白、p53、CARD11、Ki67、N-钙黏蛋白、基质金属蛋白酶(MMP)-9、NF-κBp65和NF-κBp50的表达。进行MTT、流式细胞术、伤口愈合和Transwell检测以检测细胞活力、细胞周期、凋亡、侵袭和迁移。通过裸鼠异种移植评估肿瘤生长。

结果

证实CARD11是LINC01260的靶基因,并发现其参与调节NF-κB通路。与相邻正常组织相比,胶质瘤组织中LINC01260表达降低,CARD11及与凋亡、侵袭和迁移相关的基因表达升高;还观察到NF-κB信号通路的激活。与空白组和NC组相比,LINC01260载体组和siRNA-CARD11组中G1期停滞的细胞数量增加,凋亡增加,细胞增殖、侵袭减少,S期和G2期停滞的细胞数量减少,肿瘤生长也减少。

结论

我们的研究结果表明,LINC01260的过表达通过抑制NF-κB信号通路靶向CARD11,从而抑制脊髓胶质瘤细胞的增殖、迁移和侵袭。

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