miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1，ELMO1）可以促进多种癌细胞的侵袭和转移，但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性，以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示，miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示，miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示，低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05），较MDA-231乳腺癌细胞高75%（P＜0.05），即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明，miR-145组ELMO1表达量低于阴性对照组，miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05），证明miR-145抑制ELMO1的表达。qRT-PCR显示，过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示，miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明，miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明，miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05），抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）；miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05），于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述，miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译，从而抑制乳腺癌的侵袭。

MiR-145 Inhibits Invasion of Breast Cancer Cells by Targeting ELMO1

Engulfment and cell motility protein 1 (ELMO1）can promote the invasion and metastasis of manykinds of cancer cells, but whether the expression of ELMO1 is regulated by microRNAs is rarely studies. The purpose of this study was to explore the correlation between miR-145 and ELMO1 expression, and the effect of miR-145 on the invasion of breast cancer by binding to ELMO1 mRNA. MiR-145 binding to ELMO1’s 3′UTR was predicted by TargetScan (http://www.targetscan.org/), and the luciferase reporter gene assays confirmed the complementary binding. Transwell invasion assay showed that the number of breast cancer cells that crossing the basement membrane decreased by 40% (P＜0.05) in the miR-145 expression group and decreased by 79% (P＜0.05) in the siELMO1+miR-145 group when compared with the negative control. However, the number of invaded cells in the siELMO1+miR-145 group and the siELMO1 group showed no significant difference (P>0.05), suggesting that miR-145 inhibited cells invasion by binding to the mRNA of ELMO1. Results from qRT-PCR found that miR-145 was significantly down-regulated in highly invasive breast cancer cell lines. Western blot data showed that ELMO1 expression in miR-145 group was lower than that in the negative control group, and ELMO1 expression in miR-145 inhibitor group was higher than that in inhibitor NC group, demonstrating that miR-145 inhibited ELMO1 expression. There was no significant difference in ELMO1 mRNA levels by qRT-PCR between the control group and the group with miR-145 overexpression (P>0.05), suggesting that the regulatory effect of miR-145 on ELMO1 was achieved by inhibiting its translation. F-actin polymerization experiments showed a significant difference between miR-145 group and control group at 20 s and 60 s (P＜0.05). Results from Western blot demonstrated that Rac1 activation was 60% lower in miR-145 group than control group, and 55% lower in inhibitor negative control group as compared with the miR-145 inhibitor group. The phosphorylated integrin β1 with miR-145 transfection was 42% and 31% lower at 15 min and 30 min respectively as compared with that of the negative control group (P<0.05). It was concluded that overexpression of miR-145 significantly promotes F-actin polymerization, Rac1 activation and integrin β1 phosphorylation in breast cancer cells. In summary, miR-145 inhibits the translation of ELMO1 by targeting ELMO1 mRNA, by inhibiting the invasion of breast cancer.