miR-1269a调控HOXD10基因抑制胆管癌细胞侵袭的作用及其机制

杨海霞; 米建强; 梁军; 邵秋菊; 齐宇红; 王启明; 常浩; 李恩孝

doi:10.3971/j.issn.1000-8578.2018.18.0175

miR-1269a调控HOXD10基因抑制胆管癌细胞侵袭的作用及其机制

Role and Mechanism of miR-1269a on Targeted Regulation of HOXD10 in Invasion of Cholangiocarcinoma Cells

摘要

摘要:
目的探讨miR-1269a对HOXD10基因的调控作用及对胆管癌细胞侵袭能力的影响。
方法预测并筛选出调控HOXD10基因的miR-1269a作为研究对象; 转染miR-1269a模拟物(mimic)及抑制剂(inhibitor)至胆管癌细胞系后检测HOXD10mRNA及蛋白的表达变化, 并观察miR-1269a对胆管癌细胞侵袭能力的影响。双荧光素酶报告基因实验验证miR-1269a与HOXD10之间的靶向作用关系。
结果 miR-1269a在胆管癌组织中表达显著高于癌旁组织(P=0.0023);miR-1269a模拟物可显著降低胆管癌细胞中HOXD10 mRNA(Mz-CHA-1:P=0.0025;RBE:P=0.0038)及蛋白表达水平, 且细胞的侵袭能力较对照组显著增强(Mz-CHA-1:P=0.004;RBE:P=0.004)。miR-1269a抑制剂转染则出现相反的结果(QBC939:P=0.16;HCCC9810:P=0.13)。miR-1269a明显抑制野生型HOXD10-3’UTR的荧光素酶活性, 而对突变型质粒转染细胞的荧光素酶活性无影响。
结论 miR-1269a靶向负性调控HOXD10基因, 在胆管癌细胞侵袭过程中发挥重要作用。

Abstract:
Objective To explore the possible mechanism of microRNA-1269a(miR-1269a) on the targeted regulation of HOXD10 in the invasion of human cholangiocarcinoma (CCC) cells.
Methods We detected six miRNAs levels in human CCC samples and cells for screening miR-1269a as the research object.miR-1269a mimic and inhibitor were transfected into four CCC cells by Lipofectamine liposome respectively.The expressions of HOXD10 mRNA and protein were detected by real-time quantitative PCR (qPCR) and Western blot.The effect of miR-1269a on the invasion of CCC cells was observed.Double luciferase reporter assay was applied to verify the targeting relationship between miR-1269a and HOXD10.
Results miR-1269a expression was significantly upregulated in CCC tissues, compared with adjacent normal tissues (P=0.0023).The mRNA and protein levels of HOXD10 in miR-1269a mimic transfection group were lower than those in control group (Mz-CHA-1:P=0.0025;RBE:P=0.0038).miR-1269a mimic significantly elevated the invasion capacity of CCC cells (Mz-CHA-1:P=0.004;RBE:P=0.004), while miR-1269a inhibitor remarkably inhibited the invasion (QBC939:P=0.16;HCCC9810:P=0.13).Double luciferase reporter gene test showed that miR-1269a could significantly inhibit the luciferase activity of wild-type HOXD10-3'UTR, but had no effect on the luciferase activity of mutant plasmid transfected cells.
Conclusion miR-1269a may regulate the invasion of CCC cells by targeting HOXD10, and could be used as an effective target for the molecular therapy of CCC.

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