LncRNA DLEU7-AS1通过调控膜突蛋白MSN的表达促进胃癌细胞增殖与迁移的作用机制

doi:10.19401/j.cnki.1007-3639.2023.04.003

摘要/Abstract

摘要：

背景与目的：越来越多的研究表明长链非编码RNA（long non-coding RNA, lncRNA）在肿瘤的发生、发展过程中发挥着重要作用，然而大多数lncRNA在胃癌中的作用和机制尚不明确。LncRNA DLEU7-AS1在胃癌中的作用和机制的研究鲜见报道。本文旨在研究DLEU7-AS1对胃癌恶性表型的影响并初步探讨其分子机制。方法：采用癌症基因组图谱（the Cancer Genome Atlas，TCGA）数据库分析DLEU7-AS1在胃癌组织中的表达及其对胃癌患者生存期的影响。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）验证DLEU7-AS1在胃癌组织及胃癌细胞系中的表达情况。采用5-氮杂-2’-脱氧胞苷（5-aza-2’-deoxycytidine，DAC）和曲古抑菌素A（trichostatin A，TSA）处理胃癌细胞株，分析表观遗传学调控对DLEU7-AS1表达的影响。采用小干扰RNA（small interfering RNA，siRNA）下调HGC-27、AGS细胞株中DLEU7-AS1的表达，采用重组质粒上调MGC-803和MKN-45中DLEU7-AS1的表达，采用RTFQ-PCR验证效果；采细胞计数试剂盒（cell counting kit-8，CCK-8）细胞增殖毒性实验、transwell小室迁移实验、平板克隆形成实验以及流式细胞术研究DLEU7-AS1对胃癌细胞增殖、迁移及凋亡和细胞周期的影响；采用RNA测序技术（RNA-sequencing，RNA-seq）分析沉默DLEU7-AS1后的下游信号转导通路的变化，并采用RTFQ-PCR和蛋白质印迹法（Western blot）进行验证；采用RNA免疫共沉淀实验（co-immunoprecipitation，RIP）探讨DLEU7-AS1对下游信号分子的调控机制。结果：TCGA数据库分析及RTFQ-PCR检测均证明 DLEU7-AS1在胃癌中表达升高。DLEU7-AS1的表达与胃癌患者生存期呈负相关。DAC和TSA处理胃癌细胞株后，DLEU7-AS1表达上调，说明其表达受表观遗传学调控。沉默DLEU7-AS1抑制胃癌细胞增殖、迁移，促进细胞凋亡；过表达DLEU7-AS1促进胃癌细胞增殖、迁移，抑制细胞凋亡。RNA-seq结果表明，DLEU7-AS1表达下调后会导致膜突蛋白（moesin，MSN）表达量的显著降低，RTFQ-PCR及Western blot的结果验证了这一结论。Rescue实验结果进一步证实，过表达MSN可部分回复干扰DLEU7-AS1对胃癌细胞增殖和迁移的抑制作用，提示MSN可能作为DLEU7-AS1下游效应分子。DLEU7-AS1主要定位在细胞核中，DLEU7-AS1与P300结合以及MSN启动子附近的H3K27的高度富集。结论：LncRNA DLEU7-AS1在胃癌中高表达并且其表达与胃癌患者生存期呈负相关，DLEU7-AS1可能通过招募P300调控MSN的转录进而促进胃癌的增殖和迁移等恶性表型。

关键词: 胃癌, DLEU7-AS1, 膜突蛋白, 表观调控, 增殖, 迁移

Abstract:

Background and purpose: An increasing number of studies have demonstrated that lncRNA plays a critical role in the occurrence and development of tumors. However, the function of lncRNA in human gastric cancer remains largely unknown. So far, the role and mechanism of lncRNA DLEU7-AS1 in gastric cancer have not been reported. This study aimed to investigate the effect of DLEU7-AS1on the tumorigenesis and progression of gastric cancer and its mechanism. Methods: The public database the Cancer Genome Atlas (TCGA) was used to analyze the expression of DLEU7-AS1 in gastric cancer tissues and the correlation between its expression and the survival of gastric cancer patients. Then real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was performed to verify the expression of DLEU7-AS1 in gastric cancer tissues and gastric cancer cell lines. Gastric cancer cell lines were treated with 5-aza-2’-deoxycytidine (DAC) and trichostatin A (TSA) to explore whether epigenetic regulation participated in DLEU7-AS1 transcription. SiRNA was used to down-regulate the expression of DLEU7-AS1 in HGC-27 and AGS cells, and recombinant plasmid was used to up-regulate the expression of DLEU7-AS1 in MGC-803 and MKN-45. The effect was verified by RTFQ-PCR. Cell biological experiments, such as cell counting kit-8 (CCK-8) cell proliferation toxicity test, transwell chamber assay, plate colony formation assay and flow cytometry were used to investigate the effect of DLEU7-AS1 on the proliferation, migration, apoptosis and cell cycle progression of gastric cancer cells. RNA sequencing (RNA-seq) was used to analyze downstream signal pathways after silencing DLEU7-AS1 and tested by RTFQ-PCR and Western blot. And RNA Co-immunoprecipitation (RIP) was used to explore the regulatory mechanism of DLEU7-AS1 on downstream signal molecules. Results: The results of public database analysis and RTFQ-PCR demonstrated that DLEU7-AS1 was up-regulated in gastric cancer tissues compared with normal tissues. DLEU7-AS1 expression was negatively correlated with the survival of gastric cancer patients. DLEU7-AS1 was up-regulated in gastric cancer cell lines treated with DAC and TSA, indicating that its expression was epigenetically regulated. DLEU7-AS1 downregulation inhibited gastric cancer cells proliferation and migration and promoted cell apoptosis, while overexpression of DLEU7-AS1 promoted cell proliferation and metastasis and inhibited cell apoptosis. The results of RNA-seq showed that the downregulation of DLEU7-AS1 expression led to a significant decrease in moesin (MSN) expression, which was confirmed by RTFQ-PCR and Western blot. Rescue experiment results further verified that MSN overexpression could partially restore the inhibition effect of knockdown of DLEU7-AS1 on the proliferation and migration of gastric cancer cells. Considering that DLEU7-AS1 mainly located in the nucleus, DLEU7-AS1 binding to P300 and H3K27 highly enriched near the MSN promoter, it was proposed that DLEU7-AS1 might regulate the expression of MSN by recruiting P300, thus contributing to the proliferation and migration of gastric cancer cells. Conclusion: LncRNA DLEU7-AS1 is abnormally up-regulated in gastric cancer and negatively correlated with survival of gastric cancer patients. DLEU7-AS1 may promote the proliferation and migration of gastric cancer by recruiting P300 to regulate the transcription of MSN, which provides a new idea for the diagnosis and treatment of gastric cancer..

Key words: Gastric cancer, DLEU7-AS1, Moesin, Epigenetic regulation, Proliferation, Migration

中图分类号:

R735.2

肖岚姝, 潘柳荻, 刘毅, 王洁, 陈惠. LncRNA DLEU7-AS1通过调控膜突蛋白MSN的表达促进胃癌细胞增殖与迁移的作用机制[J]. 中国癌症杂志, 2023, 33(4): 327-341.

XIAO Lanshu, PAN Liudi, LIU Yi, WANG Jie, CHEN Hui. LncRNA DLEU7-AS1 contributes to proliferation and migration of gastric cancer by regulating MSN transcription[J]. China Oncology, 2023, 33(4): 327-341.

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Gene	Forward primer (5' to 3')	Reverse primer (5' to 3')
DLEU7-AS1	AGGGGCCACATGGTTCTTTT	AGTTCTCCCTTGCTGCACTC
MSN	CCATGCCCAAAACGATCAGTG	ACCAAACTTCCCTCAAGCCA
GAPDH	GACAGTCAGCCGCATCTTCT	GCGCCCAATACGACCAAATC

SiRNA	Sense (5' to 3')	Antisense (5' to 3')
DLEU7-AS1-491	GCUGCCUACAUUUCAGUAATT	UUACUGAAAUGUAGGCAGCTT
DLEU7-AS1-1602	GCAAGGGAGAACUAAACUATT	UAGUUUAGUUCUCCCUUGCTT
NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT