miR-497-5p靶向调控CCNE1基因抑制胰腺癌细胞增殖

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。

miR-497-5p Inhibits Proliferation of Pancreatic Cancer Cells by Targeting CCNE1 Gene

Objective To 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. Methods The 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. Results miR-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. Conclusion miR-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.