慢性镉暴露下食管鳞状细胞癌细胞的lncRNA-mRNA共表达网络分析

doi:10.3969/j.issn.1004-616x.2023.04.004

摘要/Abstract

摘要： 目的：构建食管鳞状细胞癌(ESCC)细胞在慢性低浓度镉暴露诱导下的长链非编码RNA(lncRNA)和mRNA的共表达网络，探讨关键lncRNA和mRNA在镉促ESCC演进及放化疗抵抗中的潜在功能及分子机制。方法：EC109细胞持续暴露于5 μmol/L氯化镉培养12周，构建慢性镉暴露ESCC细胞模型CCT-EC109。采用Illumina NovaSeq 6000系统对暴露株CCT-EC109和亲本株EC109细胞进行全转录组测序，运用生物信息学方法分析差异lncRNA和mRNA表达谱，利用基因本体(GO)和京都基因与基因组大百科全书(KEGG)对差异lncRNA的靶基因进行功能分析，根据Pearson相关系数利用Cytoscape软件构建lncRNA-mRNA共表达网络，并对筛选的lncRNA和mRNA进行实时荧光定量PCR(qPCR)验证。结果：EC109细胞与CCT-EC109细胞存在差异表达lncRNA(DE-lncRNA) 2 794个，差异mRNA(DE-mRNA) 4 267个；DE-lncRNA的靶基因与DE-mRNA取交集，获得1 546个DE-lncRNA调控的镉暴露相关mRNA；GO分析显示这些基因主要富集在代谢过程、膜与膜封闭腔部分及催化反应等功能；KEGG分析提示Hippo信号通路是最显著富集项之一。差异表达最显著的前10个lncRNA的38个共表达mRNA构建共表达网络，经qPCR验证，与mRNA关联最多的lncRNA NONHSAT097388.2以及ECE1、EMP2、ORAT1、ZNF713在CCT-EC109中表达均下调(均为P<0.01)，而MFAP5和PGF表达均升高(均为P<0.05)。结论：lncRNA-mRNA共表达网络可能在慢性镉暴露诱导ESCC细胞演进和治疗抵抗机制中发挥重要作用，相关基因有望成为镉暴露ESCC患者预后判断的潜在生物标志物和治疗靶点。

关键词: 镉, 食管鳞状细胞癌, 长链非编码RNA, 信使RNA, 共表达网络

Abstract: OBJECTIVE： To investigate involvement of the long non-coding RNA (lncRNA) and messenger RNA (mRNA) co-expression network in chronic low-dose cadmium exposure in esophageal squamous carcinoma (ESCC) cells，and to explore molecular mechanisms of hub lncRNAs and mRNAs in the cadmium-associated cancer progression and chemo-and radio-resistance. METHODS： EC109 cells were continually treated with 5 μmol/L cadmium chloride over 12 weeks to construct the chronic cadmium treated (CCT)-ESCC cell model，named as CCT-EC109. The whole-transcriptome sequencing was performed by the Illumina NovaSeq 6000 system，and then bioinformatics methods were applied to identify the differentially expressed (DE)- lncRNA and DE-mRNA expression profiles. Functions of target genes of DE-lncRNA were then predicted based on gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) forecast analysis. The lncRNA-mRNA co-expression network was conducted based on the Pearson correlation coefficient and the screened lncRNAs and mRNAs were verified using quantitative real-time polymerase chain reaction (qPCR). RESULTS： A total of 2 794 DE-lncRNAs and 4 267 DE-mRNAs were identified in the EC109 and CCT-EC109 cells. Intersection of the target genes of DE-lncRNA and DE-mRNAs yielded 1 546 cadmium-related mRNAs targeted by corresponding DE-lncRNA. GO analysis showed these genes were mainly enriched in metabolic process，membrane components and membrane closed cavity，and catalytic reaction，etc. For KEGG analysis，Hippo was shown to be the most significant enrichment pathway. Thirty-eight mRNAs were identified to be co-expressed with the top ten DE-lncRNAs. The results of qPCR assay indicated that both the key lncRNA NONHSAT097388.2 and its co-expressed mRNAs including EMP2，ECE1，ORAI1，and ZNF713，were significantly downregulated in the exposed cell line (all P<0.01)，while the expressions of PGF and MFAP5 were found to be upregulated (both of P<0.05). CONCLUSION： LncRNA-mRNA co-expression networks may play important roles in the mechanisms of progression and resistance in ESCC cells chronically exposed to cadmium. Meanwhile，these hub RNAs may serve as prognostic biomarkers or therapeutic targets for the ESCC patients with cadmium exposure.

Key words: cadmium, esophageal squamous carcinoma cell, long non-coding RNA, messenger RNA, co-expression network

中图分类号:

R735.1

彭琳, 曾明钦, 陈炯玉, 张厦蓉, 朱芮, 黄裔腾. 慢性镉暴露下食管鳞状细胞癌细胞的lncRNA-mRNA共表达网络分析[J]. 癌变·畸变·突变, 2023, 35(4): 266-272,278.

PENG Lin, ZENG Mingqin, CHEN Jiongyu, ZHANG Xiarong, ZHU Rui, HUANG Yiteng. Integrative lncRNA-mRNA co-expression network analysis in the chronic cadmium-exposed esophageal squamous cell carcinoma cell[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2023, 35(4): 266-272,278.

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