miR-4728-3p通过PI3K/AKT信号通路调控DOT1L基因抑制乳腺癌细胞增殖、侵袭和迁移

miR-4728-3p regulates DOT1L gene through PI3K/AKT signaling pathway and inhibits proliferation, invasion and migration of breast cancer cells

目的:探讨miR-4728-3p通过调控类端粒沉默干扰体-1(disruptor of telomeric silencing 1-like, DOT1L)基因对乳腺癌细胞增殖、侵袭和迁移的影响。方法:使用乳腺癌细胞MCF-7、MDA-MB-231及人正常乳腺细胞MCF-10A。转染MCF-7和MDA-MB-231细胞随机分为NC组(转染miR-4728-3p-NC)和敲低组(转染miR-4728-3p-inhibitor)。利用RT-qPCR技术检测不同的细胞中miR-4728-3p和DOT1L的表达量改变情况。集落克隆形成实验和EDU实验可以同时检测体内各个细胞异常增殖的情况;TUNEL荧光标记检测法和流式细胞术实验可以同时检测不同组癌细胞的凋亡变化;划痕实验和Transwell实验可以同时检测到在各个细胞内因不同处理而可能产生的细胞迁移率和侵袭力的改变;Western blot检测与细胞凋亡状态相关细胞蛋白以及与细胞通路凋亡相关细胞蛋白p-AKT和p-PI3K相对表达的含量。结果:在乳腺癌细胞MCF-7、MDA-MB-231以及SKBR3中发现miR-4728-3p和DOT1L的表达高于人正常乳腺细胞MCF-10A(P<0.05)。转染细胞miR-4728-3p后,与NC组细胞进行实验比较,发现敲低组细胞中miR-4728-3p和DOT1L的表达量明显降低,且敲低组细胞的增殖能力明显减弱,细胞凋亡率明显升高,细胞迁移能力大大降低。敲低组的细胞凋亡活性蛋白相对表达明显发生变化。其中p-AKT与p-PI3K蛋白均被抑制激活。RT-qPCR和Western blot实验验证DOT1L是miR-4728-3p的下游靶向基因。结论:敲低miR-4728-3p可以下调DOT1L的表达从而促进乳腺癌细胞凋亡,抑制乳腺癌细胞增殖,同时使乳腺癌细胞侵袭和迁移能力减弱。

Objective:To investigate the effect of miR-4728-3 p on proliferation, invasion and migration of breast cancer cells by regulating disruptor of telomeric silencing 1-like(DOT1 L) gene.Methods:MCF-7 and MDA-MB-231 breast cancer cells and MCF-10 A normal breast cancer cells were used.Transfected MCF-7 cells and MDA-MB-231 cells were randomly divided into NC group(transfected with miR-4728-3 p-NC) and knockdown group(transfected with miR-4728-3 p-inhibitor).The expression levels of miR-4728-3 p and DOT1 L in different breast cancer cells were detected by RT-qPCR.Colony cloning assay and EDU assay can simultaneously detect the abnormal proliferation of each cell in vivo.TUNEL fluorescence labeling and flow cytometry could simultaneously detect the apoptosis of different groups of cancer cells.The scratch test and Transwell test could simultaneously detect the changes in cell migration and invasion due to different treatments in each cell.Western blot was used to detect the relative expression levels of cell proteins related to apoptosis state and cell pathway apoptosis, including p-AKT and p-PI3 K.Results:The expression levels of miR-4728-3 p and DOT1 L in MCF-7,MDA-MB-231 and SKBR3 cells were significantly higher than that in MCF-10 A cells(P<0.05).After transfection of miR-4728-3 p, compared with NC group, the expression levels of miR-4728-3 p and DOT1 L in the knockdown group were significantly decreased.The proliferative function of cells in knockdown group was obviously weakened.The apoptosis rate in knockdown group was significantly increased. The ability of cell migration in knockdown group was greatly reduced. The relative expression of apoptotic active protein in knockdown group was significantly changed. p-AKT and p-PI3K were both inhibited and activated. RT-q PCR and Western blot experiments confirmed that DOT1L was the downstream target gene of miR-4728-3p. Conclusion: Knockdown of miR-4728-3p can down-regulate the expression of DOT1L,promote apoptosis of breast cancer cells,inhibit cells proliferation,and weaken the invasion and migration ability of breast cancer cells.