Upregulated LncRNA ZEB1-AS1 by c-Myc promote the proliferatio-n of colorectal cancer cells
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摘要:
目的 探讨长链非编码RNA ZEB1-AS1在结直肠癌中高表达的机制及其在结直肠癌中的作用。 方法 实时荧光定量PCR检测结直肠癌细胞和组织中ZEB1-AS1的表达，分析其表达量与患者预后的相关性。生物信息学预测ZEB1-AS1转录因子结合位点，双荧光素酶报告基因进行转录因子验证。MTS方法检测ZEB1-AS1对结直肠癌细胞增殖能力的影响。 结果 ZEB1-AS1在结直肠癌细胞和组织中高表达，且与结直肠癌患者较差的预后正相关。转录因子c-Myc在ZEB1-AS1启动子上有一个结合位点。过表达c-Myc转录上调ZEB1-AS1的表达，反之亦然。MTS结果显示过表达ZEB1-AS1促进结直肠癌细胞增殖，而敲低ZEB1-AS1则抑制该细胞的增殖能力。 结论 c-Myc转录上调ZEB1-AS1，进而促进结直肠癌进程。c-Myc/ZEB1-AS1或许可作为结直肠癌治疗的潜在靶标。 Abstract:Objective To explore the function and mechanism of the high expression of LncRNA in colorectal cancer. Methods ZEB1-AS1 expression in colorectal cancer cells and tissues was detected by qRT-PCR, and the relationship between ZEB1-AS1 expression and the prognosis of patients with colorectal cancer was analyzed. Bioinformatics analysis was used to determine transcriptional factor binding sites of ZEB1-AS1, while dual luciferase reporter gene assay was applied to verify transcriptional factors. The influence of ZEB1-AS1 on the proliferation ability of colorectal cells were studied by MTS assay. Results It was suggested that ZEB1-AS1 was highly expressed in colorectal cancer cells and tissues and it was positively corelated with the poor prognoses of colorectal cancer patients. Bioinformatics analysis combined with dual luciferase reporter gene assay showed that ZEB1-AS1 upregulated by c-Myc could promote its activity and enhance its expression. MTS findings demonstrated that overexpressed ZEB1-AS1 accelerated the proliferation of colorectal cells, while the knocked-down ZEB1-AS1 oppressed the proliferation of colorectal cells. Conclusion c-Myc can upregulate ZEB1-AS1, thus promoting the colorectal cancer progression. Therefore, c-Myc/ZEB1-AS1 axis might be a patient target for colorectal cancer therapy in the future. -
Key words:
- long non-coding RNAs /
- ZEB1-AS1 /
- c-Myc /
- colorectal cancer /
- proliferation
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图 1 ZEB1-AS1在结直肠癌中的表达量及临床意义
A: 使用在线软件CPAT(http://lilab.research.bcm.edu/cpat/)和CPC(http://cpc.cbi.pku.edu.cn/)预测ZEB1-AS1蛋白质编码功能; B: 实时荧光定量PCR方法检测ZEB1-AS1在结肠上皮细胞和5种结直肠癌细胞中的表达量; C: 实时荧光定量PCR方法检测ZEB1-AS1在21例结直肠癌组织及配对癌旁组织中的表达量; D: 在线软件OncoLnc(http://www.oncolnc.org/)分析ZEB1-AS1表达量与结直肠癌患者预后的相关性.
图 2 c-Myc转录调控ZEB1-AS1的表达
A: 在线软件JASPAR(http://jaspar.genereg.net/)预测转录因子c-Myc在ZEB1-AS1启动子上有一个结合位点; B: 在NCM460细胞中转染c-Myc过表达质粒; C: 在LOVO细胞中转染靶向c-Myc的shRNAs, western blot检测c-Myc蛋白水平表达量, 实时荧光定量PCR检测ZEB1-AS1的表达量. sh-2#沉默效果最好，用于后续研究; D: 在NCM460细胞中, 同时共转染c-Myc过表达质粒和报告基因pGL4-wt-ZEB1-AS1/pGL4-del-ZEB1-AS1, 48 h后，行双荧光素酶报告基因实验检测.
图 3 ZEB1-AS1促进结直肠癌细胞增殖
A: 在NCM460细胞中转染ZEB1-AS1过表达质粒，实时荧光定量PCR检测ZEB1-AS1的表达，MTS方法检测该细胞的活性, *P<0.05, **P<0.01 vs 对照组; B: 在LOVO细胞中转染靶向ZEB1-AS1的shRNAs, 实时荧光定量PCR检测ZEB1-AS1的表达, MTS方法检测该细胞的活性, *P<0.05, **P<0.01 vs 对照组.
表 1 引物序列
名称 序列（5′-3′） 实时荧光定量PCR引物 ZEB1-AS1-F TCCCTGCTAAGCTTCCTTCAGTGT ZEB1-AS1-R GACAGTGATCACTTTCATATCC GAPDH-F ATTCCATGGCACCGTCAAGGCTGA GAPDH-R TTCTCCATGGTGGTGAAGACGCCA 报告基因引物 pGL4-wt-ZEB1-AS1-F-XhoⅠ CCGctcgagACCGGGGCGGCGCAGGCG pGL4-wt-ZEB1-AS1-R-HindⅢ CCGaagcttCGCGGCAGGCGGGCTGCG pGL4-del-ZEB1-AS1-F AGGTCTCGATCCCTCGGG pGL4-del-ZEB1-AS1-R GGTGGTTGCACAGTCGCCT 小写字母表示限制性内切酶位点. 
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