长链非编码RNA PANDAR促进结直肠癌转移的作用和机制研究

doi:10.19401/j.cnki.1007-3639.2017.04.005

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)在肿瘤发生、发展过程中有重要作用。LncRNA CDKN1A反义链启动子DNA损伤激动RNA(promoter of CDKN1A antisense DNA damage activated RNA，PANDAR)与多种肿瘤的进展及预后相关，在结直肠癌转移中的作用尚未得到证实。该研究旨在探索lncRNA PANDAR在结直肠癌中的功能，并对其作用机制进行初步探索。方法：采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR)检测lncRNA PANDAR在结直肠癌细胞及组织中的表达，并分析其表达水平与结直肠癌临床病理特征的关系。构建lncRNA PANDAR沉默(HCT116-shPANDAR)和高表达(DLD1-PANDAR)及其对照(HCT116-shNC、DLD1-vector)稳定转染细胞系。通过Transwell和Matrigel实验检测lncRNA PANDAR对细胞迁移和侵袭能力的影响。采用RTFQ-PCR检测lncRNA PANDAR表达改变后介导细胞上皮-间质转化能力的主要调控基因表达情况，并对目的基因在介导lncRNA PANDAR调控结直肠癌细胞转移中的作用进行验证。结果：LncRNA PANDAR在结直肠癌细胞中的表达明显高于结直肠正常上皮细胞；LncRNA PANDAR在结直肠癌组织中的表达明显高于癌旁组织［(171.52±97.80)% vs (100.00±63.18)%，P<0.05］，且其表达水平与结直肠癌的TNM分期、淋巴结转移和远处转移相关(P<0.05)。在Transwell及Matrigel实验中，lncRNA PANDAR沉默能够明显减弱结直肠癌细胞的迁移［100.00% vs (42.08±4.77)%，P<0.05］和侵袭［100.00% vs (39.14±3.81)%，P<0.05］能力，lncRNAPANDAR高表达能够明显促进结直肠癌细胞的迁移［100.00% vs (194.12±9.33)%，P<0.05］和侵袭［100.00%vs (204.08±12.27)%，P<0.05］能力。采用RTFQ-PCR检测lncRNA PANDAR表达改变后介导结直肠癌细胞上皮-间质转化的基因表达情况发现，锌指E-盒结合同源异形盒(zinc-finger E-box binding homeobox 1，ZEB1)表达与lncRNA PANDAR表达呈显著正相关；在lncRNA PANDAR高表达的细胞中干扰ZEB1表达能够明显逆转lncRNAPANDAR高表达引起的细胞迁移和侵袭能力的增强。结论：LncRNA PANDAR能够通过调控ZEB1表达进而促进结直肠癌转移，lncRNA PANDAR可能成为结直肠癌新的诊断指标及治疗靶点。

关键词: 长链非编码RNA, CDKN1A反意义链启动子DNA损伤激动RNA, 结直肠癌, 转移

Abstract: Background and purpose: Accumulating evidence has revealed that long non-coding RNA (lncRNA) is correlated with carcinogenesis and tumor development. Recent literature suggested that lncRNA promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) was involved in the development of various cancers. However, the functional role of PANDAR in colorectal cancer (CRC) has not been elucidated yet. The present study aimed to explore the functional role of lncRNA PANDAR in promoting CRC metastasis and its mechanism. Methods: The expression of lncRNA PANDAR in CRC cell lines and tissues was detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR), and the correlation between lncRNA PANDAR expression and CRC clinicopathological characteristics was statistically analyzed. Then, lncRNA PANDAR stably silencing CRC cells (HCT116-shPANDAR), overexpression cells (DLD1-PANDAR) and control vector cells (HCT116-shNC and DLD1-vector) were established using lentiviral vectors. Moreover, Transwell assay and Matrigel assay were performed to investigate the function of lncRNA PANDAR in CRC migration and invasion. Furthermore, the expression of transcriptional factors mediating epithelial-mesenchymal transition of lncRNA PANDAR overexpression cells were monitored by RTFQ-PCR assay, and the function of the target gene in modulating lncRNA PANDAR mediated CRC metastasis was also explored. Results: The expression levels of lncRNA PANDAR in normal colorectal epithelial cells were much lower than in CRC cell. The levels of lncRNA PANDAR in tumor-adjacent tissues were verified to be much lower than in CRC tissues ［(171.52±97.80)% vs (100.00±63.18)%, P<0.05］. Moreover, the expression of lncRNA PANDAR was detected to be significantly correlated with CRC TNM stage, lymph node metastasis and distant metastasis (P<0.05). Besides, lncRNA PANDAR deficiency significantly reduced the migration ［100.00% vs (42.08±4.77)%, P<0.05］ and invasion ［100.00% vs (39.14±3.81)%, P<0.05］ capabilities in CRC cells, in contrast, the migration ［100.00% vs (194.12±9.33)%, P<0.05］ and invasion ［100.00% vs (204.08±12.27)%, P<0.05］ capabilities of CRC cells were obviously increased with lncRNA PANDAR overexpression. Furthermore, zinc-finger E-box binding homeobox 1 (ZEB1) expression was detected to be positively correlated with lncRNA PANDAR expression, and ZEB1 silencing could significantly reverse the increased migration and invasion capabilities induced by lncRNA PANDAR in CRC cells. Conclusion: LncRNA PANDAR could promote CRC metastasis by potentially targeting ZEB1. LncRNA PANDAR might be a promising diagnostic marker and therapeutic target for CRC patients.

Key words: Long non-coding RNA, Promoter of CDKN1A antisense DNA damage activated RNA, Colorectal cancer, Metastasis

刘　宁,成冬冬,姜金波. 长链非编码RNA PANDAR促进结直肠癌转移的作用和机制研究[J]. 中国癌症杂志, 2017, 27(4): 268-275.

LIU Ning, CHENG Dongdong, JIANG Jinbo. The functional role of long non-coding RNA PANDAR in promoting colorectal cancer metastasis and its mechanism[J]. China Oncology, 2017, 27(4): 268-275.

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长链非编码RNA PANDAR促进结直肠癌转移的作用和机制研究

The functional role of long non-coding RNA PANDAR in promoting colorectal cancer metastasis and its mechanism

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