Study on Differential Expression Profile of lncRNA between Concurrent Chemoradioresistant Nasopharyngeal Carcinoma Cell Lines and Parental Cell Lines
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摘要:
目的 分析鼻咽癌同期放化疗抵抗细胞株与亲本细胞株间差异表达的长链非编码RNA（long non-coding RNA，lncRNA），探索lncRNA在鼻咽癌同期放化疗抵抗中可能发挥的作用。 方法 体外构建鼻咽癌CEN1和CNE2细胞同期放化疗抵抗模型，诱导鼻咽癌细胞同期放化疗抵抗细胞株CEN1CRR和CNE2CRR，应用高通量测序筛选2组细胞株间差异表达的lncRNAs，对差异表达的lncRNAs的靶基因进行GO和KEGG分析。 结果 以Log2FC > 1或 < -1，FDR < 0.05为差异表达标准，和CNE1相比，CNE1CRR差异表达的lncRNAs2746个（358个上调，2063 个下调）；和CNE2相比，CNE2CRR差异表达的lncRNAs3475个（265个上调，520个下调）；此外，387个lncRNAs在CNE1CRR和CNE2CRR中表达同时下调，49个lncRNAs在CNE1CRR和CNE2CRR中同时上调。在GO分析中发现，2组细胞中差异表达lncRNAs的靶基因在生物过程（biological process，BP）、分子功能（molecular function，MF）、细胞成分（cellular component，CC）等方面均有参与。在KEGG分析结果中发现，CNE1组细胞中，差异lncRNAs的靶基因主要富集的信号通路有细胞凋亡、病毒致癌、代谢途径等。CNE2组细胞中，差异lncRNAs的靶基因主要富集的信号通路有代谢途径（硫辛酸、磷酸戊糖、花生四烯酸、醚脂质）、T细胞受体信号通路、核苷酸切除修复等（P < 0.05）。 结论 同期放化疗抵抗细胞株与亲本细胞株的lncRNA表达谱存在明显差异，这些差异表达的lncRNA可能参与了鼻咽癌的同期放化疗抵抗，并为其机制研究奠定基础。 -
关键词:
- 长链非编码RNA（lncRNA） /
- 表达谱 /
- 鼻咽癌细胞 /
- 同期放化疗抵抗 /
- 高通量测序
Abstract:Objective To analyze the long non coding RNA （lncRNA） differentially expressed between nasopharyngeal carcinoma cell lines resistant to concurrent radiotherapy and chemotherapy and their parent cell lines, and explore the possible role of lncRNA in nasopharyngeal carcinoma resistance to concurrent radiotherapy and chemotherapy. Methods The concurrent chemoradiotherapy resistance model of nasopharyngeal carcinoma CEN1 and CNE2 cells was constructed in vitro, and the concurrent chemoradiotherapy resistance cell lines CEN1CRR and CNE2CRR of nasopharyngeal carcinoma cells were induced. The differentially expressed lncRNAs between the two groups of cell lines were screened by high-throughput sequencing, and the target genes of the differentially expressed lncRNAs were analyzed by GO and KEGG. Results With Log2FC > 1 or < - 1 and FDR < 0.05 as the differential expression criteria, 2746 lncRNAs （358 up-regulated and 2063 down-regulated） were differentially expressed in CNE1 CRR compared with CNE1. Compared with CNE2, CNE2CRR differentially expressed 3475 lncRNAs （265 up-regulated, 520 down-regulated）. In addition, 387 lncRNAs were downregulated in CNE1CRR and CNE2CRR, and 49 lncRNAs were upregulated in CNE1CRR and CNE2CRR. GO analysis showed that the target genes differentially expressing lncRNAs in the two groups of cells were involved in biological process （BP）, molecular function （MF）, and cell component （CC）. In the KEGG analysis results, it was found that in CNE1 group cells, the main signal pathways for the enrichment of target genes of differential lncRNAs were apoptosis, viral carcinogenesis, metabolic pathways, etc. In CNE2 group, the target genes of differential lncRNAs were mainly enriched through metabolic pathways （lipoic acid, pentose phosphate, arachidonic acid, ether lipid）, T cell receptor signaling pathways, nucleotide excision repair, etc. （P < 0.05）. Conclusions There are significant differences in lncRNA expression profiles between concurrent chemoradiotherapy-resistant cell lines and parental cell lines. These differentially expressed lncRNAs may participate in concurrent chemoradiotherapy resistance of nasopharyngeal carcinoma, and lay a foundation for its mechanism research. -
图 1 生存曲线图
A： CNE1组细胞的生存曲线；B：CNE2组细胞的生存曲线。
Figure 1. Figures of survival curves
*P < 0.05。
图 2 母细胞株和放化疗抵抗细胞株差异表达lncRNAs的火山图
注：其中红点表示显著上调的lncRNAs，蓝点表示显著下调的lncRNAs，中间灰点表示无明显变化的lncRNAs。A：CNE1组细胞中lncRNAs的差异表达；B：CNE2组细胞中lncRNAs的差异表达。
Figure 2. Volcano plot of differentially expressed lncRNAs in parental and chemoradioresistant cell lines
图 3 母细胞株和放化疗抵抗细胞株差异表达lncRNAs的聚类热图
注：绿色区域表示低表达lncRNAs，红色区域表示高表达lncRNAs。
Figure 3. Clustering heat mapof differentially expressed lncRNAs in parental and chemoradioresistant cell lines
图 4 CNE1CRR与CNE1，CNE2CRR与CNE2中差异表达lncRNA韦恩图
A：CNE1细胞组差异表达；B：CNE2组细胞差异表达。
Figure 4. Venn diagram of differentially expressed lncRNAs in group CNE1CRR and CNE1，CNE2CRR and CNE2
图 5 GO功能富集分析柱状图
A：柱形图显示，CNE1组差异lncRNAs靶基因的GO功能富集情况；B：柱形图显示，CNE2组中的差异lncRNAs靶基因的GO功能富集情况。横轴表示功能富集显著性水平（P-Value）的-Log10P值，纵轴表示GOterm；BP: Biological Process; MF：Molecular Function；CC: Cellular Component。
Figure 5. Histogram of GO functional enrichment analysis
表 1 CNE1和CNE2放化疗抵抗细胞鉴别的放射生物学参数
Table 1. Radiobiological parameters to identify the chemoradioresistance of CNE1 andCNE2
分组 SF2 D0（Gy） Dq（Gy） CNE1 0.60 1.96 1.41 CNE1 CRR 0.65 2.30 1.53 CNE2 0.50 1.93 0.89 CNE2 CRR 0.68 2.55 1.57 
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表 2 CNE1CRR 及CNE2CRR中上调或下调前10位的lncRNAs
Table 2. Top 10 lncRNAs up-regulated and down-regulated in CNE1CRR and CNE2CRR
上调lncRNA Log2FC 下调lncRNA Log2FC CNE1CRR USP27X-AS1 10.13 LOC100505817 −9.96 LOC100131315 10.12 LOC102723739 −8.77 LINC00514 9.32 LINC01004 −8.71 LINC00466 9.16 LOC102724281 −8.51 XIST 8.77 CPS1-IT1 −8.44 COLCA1 8.62 CDKN2B-AS1 −8.41 LOC102724360 7.96 GABPB1-AS1 −8.23 LOC101929042 7.44 LOC100505771 −8.22 LOC101926931 7.06 SNHG10 −8.03 LOC101929173 6.47 AC091729.9 −7.96 CNE2CRR LOC100507103 4.84 LOC101928152 −5.82 DBH-AS1 4.63 LINC00239 −5.46 LOC101927037 4.52 LINC00992 −5.39 LOC102724255 4.26 LOC101929173 −5.39 LINC00470 4.02 LINC00638 −5.24 LOC113230 3.84 SEC24B-AS1 −5.07 LOC101928430 3.58 IL21-AS1 −4.89 LOC100996442 3.52 TMEM254-AS1 −4.87 LOC642366 3.39 LINC00968 −4.87 LRP4-AS1 3.39 LOC101928117 −4.77
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表 3 CNE1和CNE2组差异lncRNAs靶基因通路富集
Table 3. Pathway enrichment of differentially expressed lncRNAs target genes in CNE1 and CNE2 groups
通路编号 通路名称 富集指数 数量 FisherP CNE1组 04141 Proteinprocessingin endoplasmic reticulum 2.21 14 < 0.0001* 05162 Measles 2.17 11 0.01* 04210 Apoptosis 2.35 8 0.02* 05203 Viral carcinogenesis 1.79 14 0.02* 01212 Fatty acid metabolism 2.76 5 0.03* 05169 Epstein-Barr virus infection 1.71 13 0.04* 05166 HTLV-I infection 1.61 16 0.04* 00310 Lysine degradation 2.6 5 0.04* 01100 Metabolic pathways 1.25 55 0.04* 00900 Terpenoid backbone biosynthesis 3.78 14 0.04* CNE2组 00590 Arachidonic acid metabolism 4.81 4 0.01* 04660 T cell receptor signaling pathway 3.75 5 0.01* 00565 Ether lipid metabolism 5.84 3 0.01* 04141 Protein processing in endoplasmic reticulum 2.92 6 0.02* 03420 Nucleotide excision repair 5.22 3 0.02* 05014 Amyotrophic lateral sclerosis （ALS） 4.54 3 0.03* 00785 Lipoic acid metabolism 27.24 1 0.04* 00030 Pentose phosphate pathway 5.84 2 0.05 *P < 0.05。
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