miR-497-5p Inhibits Proliferation of Pancreatic Cancer Cells by Targeting CCNE1 Gene
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摘要:目的
明确miR-497-5p在胰腺癌（PaCa）中的表达及临床意义，并探究其对PaCa细胞增殖的影响及机制。
方法实时荧光定量PCR实验检测miR-497-5p的表达，卡方检验和Kaplan-Meier生存法分析miR-497-5p的表达与临床病理特征及预后的关系；CCK-8实验和流式细胞术检测过表达miR-497-5p对Capan-2和PANC-1细胞增殖和周期的影响，Spearman相关性检验分析miR-497-5p表达与G1/S特异性细胞周期蛋白E1（cyclin E1, CCNE1）mRNA表达的关系；双荧光素酶报告基因实验和蛋白质印迹法验证miR-497-5p对CCNE1表达的调控作用。
结果miR-497-5p在癌组织中的表达显著低于癌旁正常组织（P < 0.001），T3+T4期患者癌组织中miR-497-5p的表达显著低于T1+T2期癌组织（P < 0.001）；低表达miR-497-5p与较高的T分期相关（P=0.003）；低表达miR-497-5p的患者5年总体生存率显著低于高表达者（P=0.036）。与对照组相比，miR-497-5p过表达组细胞增殖显著降低，G0/G1期比例增加，S期比例减少。CCNE1 mRNA在癌组织的表达显著高于癌旁正常组织（P < 0.001），且与miR-497-5p表达呈负相关（P < 0.001）。miR-497-5p可直接与CCNE1 mRNA 3’-UTR互补结合从而抑制CCNE1蛋白的表达。
结论miR-497-5p在PaCa中低表达，且与较高的T分期及不良预后相关；过表达miR-497-5p通过靶向调控CCNE1基因诱导细胞周期阻滞进而抑制PaCa细胞增殖。
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关键词:
- miR-497-5p /
- 胰腺癌 /
- 预后 /
- 增殖 /
- CCNE1
Abstract:ObjectiveTo determine miR-497-5p expression in pancreatic cancer (PaCa) and its clinical significance, and to explore its effects and mechanisms on the proliferation of PaCa cells.
MethodsThe expression levels of miR-497-5p were detected by real-time quantitative PCR experiment. The relation between miR-497-5p expression and clinicopathological features, the prognosis of PaCa patients were analyzed by Chi-square test and Kaplan-Meier method. The effects of miR-497-5p overexpression on the proliferation and cell cycle of Capan-2 and PANC-1 cells were detected by cell counting kit-8 (CCK-8) experiment and flow cytometry. The relation between miR-497-5p level and the cyclin E1 (CCNE1) mRNA expression was analyzed by Spearman correlation test. The regulatory effect of miR-497-5p on CCNE1 expression was confirmed by dual luciferase reporter experiment and Western blot.
ResultsmiR-497-5p expression in cancer tissues was significantly lower than that in adjacent normal tissues (P < 0.001), and its expression in T3+T4 stages cancer tissues were significantly lower than those in T1+T2 stages cancer tissues (P < 0.001); low expression of miR-497-5p was associated with advanced T stage (P=0.003); The 5-year overall survival rate of patients with low miR-497-5p expression was significantly lower than those with high miR-497-5p expression (P=0.036). Compared with the control group, miR-497-5p overexpression significantly reduced cell proliferation, increased the cell proportion in G0/G1 phase and decreased the cell proportion in S phase. The expression levels of CCNE1 mRNA in cancer tissues were significantly higher than those in adjacent normal tissues (P < 0.001), and its expression was negatively correlated with miR-497-5p expression (P < 0.001). miR-497-5p could bind directly to the 3'-UTR of CCNE1 mRNA and inhibit the expression of CCNE1 protein.
ConclusionmiR-497-5p expression is downregulated in PaCa cells and associated with relatively advanced T stage and poor prognosis; the overexpression of miR-497-5p induces the cell cycle arrest to inhibit the proliferation of PaCa cells by targeting CCNE1 gene.
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Key words:
- miR-497-5p /
- PaCa /
- Prognosis /
- Proliferation /
- CCNE1
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作者贡献姜达伟：标本收集，实验操作，统计分析，论文撰写与修改许浏：标本收集，统计分析王伟林：实验设计，论文撰写与修改
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图 1 实时荧光定量PCR实验分析miR-497-5p在癌与癌旁正常组织(A)及不同T分期癌组织(B)中的表达
Figure 1 miR-497-5p expression in cancer tissues, adjacent normal tissues(A) and different T stages cancer tissues(B) detected by real-time quantitative PCR
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图 2 Kaplan-Meier法分析miR-497-5p的表达与PaCa患者预后的关系
Figure 2 Relation between miR-497-5p expression and prognosis of PaCa patients detected by Kaplan-Meier method
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图 3 miR-497-5p模拟物促进miR-497-5p的表达(A)并抑制Capan-2(B)和PANC-1(C)细胞的增殖
Figure 3 miR-497-5p mimics promoted miR-497-5p expression(A) and inhibited proliferation of Capan-2(B) and PANC-1(C) cells
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图 4 miR-497-5p模拟物对Capan-2(A)和PANC-1(B)细胞周期的影响
Figure 4 Effect of miR-497-5p mimics on cell cycle of Capan-2(A) and PANC-1(B) cells
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图 5 生物信息学分析miR-497-5p对CCNE1的调控作用
Figure 5 Effect of miR-497-5P on CCNE1 bioinformatics analysis
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图 6 miR-497-5p对CCNE1基因的调控作用分析
Figure 6 Analysis of the regulatory effect of miR-497-5p on CCNE1 gene
表 1 miR-497-5p的表达与胰腺癌患者临床病理特征之间的关系
Table 1 Relation between miR-497-5p expression and clinicopathological characteristics of pancreatic cancer (PaCa) patients
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