miR-1269a调控HOXD10基因抑制胆管癌细胞侵袭的作用及其机制

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。

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

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.