miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

Effects of miR-449a on proliferation and migration of human breast cancer cell line MCF-7

目的:通过敲低微小RNA(microRNA,miRNA)-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法:采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂(inhibitor),转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法(Western blot)实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果:分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组(Mock组),阴性对照组(negative control组,NC组)和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性(P<0.01)。结论:在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。

Objective： To study the effects of knockdown of miR-449a on the proliferation and migration of human breast cancer cell line Michigan Caneer Foundation-7 （MCF-7）. Methods： Using miRNA chip screening of the differential expressions of miRNA in human breast cancer cell MCF-7 and normal breast cells MCF 10A. title inhibitor d miR-449a was synthesized by chemical and detected by real-time PCR afiler transfeetion aimed to verify the expression. Cell Counting Kit-8 （CCK-8） assay was used to detect the ability of cell proliferation after transfection with mitl-449a inhibitor. Scratch assay was used to detect cell migration of MCF-7, and cell invasion ability was showed by transwell assay; The MCF-7 cell proliferation and migration related proteins, β-catenin and E-cadherin, were detected by Western blot. The potential target gene of miR-449a was predicted by bioinfformtics software, and Notch homolog I （ Notch 1 ） was proved to be the target gene of miR- 449a by luciterase assay. Results： MCF-7 and MCF-10a cells wele collected separately, and miRNA chip results showed that the level of miR- 449a in MCF-7 cells was significantly higher than that of MCF-10A. In this study, the ceils were divided into mock group, negative control group （NC group） and treatment gqoup, the MCF-7 cells were collected betbre and after treatment and CCK-8 results showed that knockdown of miR-449a decreased MCF-7 cell proliferation ability significantly. Scratch assay results showed that downregulated miR-449a was related to the deereased metastasis of MCF-7 cells. Transwell results showed that knocekdown of miR-449a inhibited the invasion of MCF-7 cells. Western blot showed Ihe expression of β-catenin was decreased and the expression of E-cadherin was increased alter knockdoown of miR-449a. Luciferase assay showed that miR-449a could signiticantly decrease the luciferase activity of Notch homolog 1- untranslated region （Notch 1-3 ＇-UTR） plasmid （P 〈 0.01 ）. Conclusion： Inhibition of miR-449a in brast cancer cell line MCF-7 can significantly inhibit the proliferation and nilgration of cancer cells, Milch may be achieved by decreasing the expression of Notch 1 protein.