外泌体介导的miR-18b-5p调控NEDD9对乳腺癌细胞增殖、迁移、侵袭的影响

Study on the exosome-mediated miR-18b-5p in promoting the proliferation,migration and invasion of breast cancer cells by regulating NEDD9

目的:探讨miR-18b-5p调控神经前体细胞表达下调蛋白9(NEDD9)对乳腺癌细胞侵袭、迁移的影响及其作用机制。方法:qRT-PCR检测人正常乳腺癌上皮细胞MCF-10A与2株乳腺癌细胞株(SKBR-3和SKBR-3/PR)中miR-18b-5p的表达水平;Western blotting检测SKBR-3/PR细胞来源外泌体的表面标志物;CCK8和Transwell实验检测SKBR-3/PR细胞转染miR-18b-5p inhibitor并提取的外泌体与SKBR-3细胞共培养后对SKBR-3细胞增殖和侵袭、迁移能力。生物信息学预测软件Target Scan预测miR-18b-5p与NEDD9的靶向结合位点。双荧光素酶实验分析miR-18b-5p与NEDD9的靶向调控关系。Transwell实验检测上调NEDD9对SKBR-3细胞侵袭迁移能力的影响。结果:miR-18b-5p在乳腺癌细胞株中相对于正常乳腺癌上皮细胞中高表达(P<0.01);SKBR-3/PR细胞来源的外泌体高表达CD63和TSG101等特异性蛋白(P<0.01),SKBR-3/PR细胞转染miR-18b-5p inhibitor提取外泌体后对SKBR-3细胞的增殖、侵袭和迁移显著被抑制(P<0.01)。生物信息学软件Target Scan预测结果显示,NEDD93′UTR上存在潜在与miR-18b-5p靶向结合的位点,miR-18b-5p能够负向调控NEDD9基因表达(P<0.01)。过表达NEDD9能抑制SKBR-3细胞侵袭能力(P<0.01)。结论:miR-18b-5p通过靶向NEDD9促进乳腺癌细胞侵袭、迁移。

Objective:To investigate the effects of miR-18b-5p on the invasion and migration of breast cancer cells by down-regulating the expression of neural precursor cell expressed developmentally down-regulated 9(NEDD9),and its mechanism.Methods:The expression levels of miR-18b-5p in human normal breast cancer epithelial cells MCF-10A and two breast cancer cell lines of SKBR-3 and SKBR-3/PR were detected using qRT-PCR.The surface markers of SKBR-3/PR cell-derived exosomes were detected using Western blotting.The CCK8 and Transwell assay were used to investigate the effects of the transfection of SKBR-3/PR cells with miR-18b-5p inhibitor and extraction of exosomes on the proliferation,invasion and migration of SKBR-3 cells.The targeted binding sites of miR-18b-5p to NEDD9 were predicted using Bioinformatics prediction software Target Scan.Dual luciferase assay was used to analyze the targeted regulation relationship between miR-18b-5p and NEDD9.The effects of up-regulation of NEDD9 on the invasion and migration of SKBR-3 cells were detected using Transwell assay.Results:The expression of miR-18b-5p in breast cancer cell line was higher than that in normal cancer cell line(P<0.01).The expression levels of CD63 and TSG101 specific proteins SKBR-3/PR in cell-derived exosomes were high(P<0.01).The proliferation,invasion and migration of SKBR-3 cells were significantly inhibited by the extractive exosomes of SKBR-3/PR cells transfected with miR-18b-5p inhibitor(P<0.01).The prediction results of bioinformatics software Target Scan showed that the NEDD93′UTR had potential binding sites with miR-18b-5p,and the miR-18b-5p could negatively regulate NEDD9 gene expression(P<0.01).The overexpression of NEDD9 could inhibit the invasion of SKBR-3 cells(P<0.01).Conclusions:The miR-18b-5p can promote the invasion and migration of breast cancer cells by targeting NEDD9.