miR-620通过靶向ING4调控乳腺癌MCF-7细胞放射敏感性

miR-620 regulates radiosensitivity of breast cancer MCF-7 cells by targeting ING4

目的:探讨miR-620对乳腺癌MCF-7细胞放射敏感性的影响及其机制。方法:收集2017年3月至2018年3月在海南省儋州市人民医院手术切除的21例乳腺癌患者的癌及癌旁组织标本,以及乳腺癌细胞MCF-7、BCaP-37和乳腺上皮细胞HBL-100,采用qPCR法检测癌组织和细胞中miR-620和生长抑制因子4(ING4)mRNA的表达。利用脂质体转染技术,分别将miR-620抑制剂(anti-miR-620)和抑制剂阴性对照(anti-miR-NC)、anti-miR-620和ING4小干扰RNA(si-ING4)、anti-miR-620和小干扰RNA阴性对照序列(si-NC)转染至MCF-7细胞,经放射处理后(依次记为IR+anti-miR-620组、IR+anti-miR-NC组、IR+antimiR-620+si-ING4组、IR+anti-miR-620+si-NC组),利用克隆形成实验、MTT法和FCM分别检测细胞放射敏感性、细胞增殖活力、细胞周期分布和凋亡率。双荧光素酶报告基因实验和WB法验证miR-620和ING4的靶向关系。结果:与癌旁组织和HBL-100细胞比较,乳腺癌组织和细胞中miR-620表达均显著升高(均P<0.01)、ING4 mRNA表达均显著降低(均P<0.01)。与IR+anti-miR-NC组比较,IR+anti-miR-620组MCF-7细胞增殖活力、S期细胞比例均显著降低(均P<0.01),细胞凋亡率、G0-G1期细胞比例、放射敏感性均显著升高(均P<0.01)。与IR+anti-miR-620+si-NC组比较,IR+anti-miR-620+si-ING4组MCF-7细胞增殖活力、S期细胞比例均显著升高(均P<0.01),细胞凋亡率、G0-G1期细胞比例和放射敏感性均显著降低(均P<0.01)。双荧光素酶报告基因实验证明ING4是miR-620的靶基因,miR-620靶向负性调控ING4表达。结论:敲减miR-620可能通过上调ING4表达抑制乳腺癌MCF-7细胞增殖,并促进细胞凋亡和放射敏感性。

Objective:To investigate the effect of miR-620 on the radiosensitivity of breast cancer MCF-7 cells and its mechanism.Methods:The cancer tissues and para-cancerous tissue specimens of 21 patients with breast cancer who had surgical resection in Danzhou City People’s Hospital of Hainan Province from March 2017 to March 2018 were collected.In addition,breast cancer MCF-7and BCaP-37 cells and breast epithelial HBL-100 cells were also cultured in vitro.The mRNA expression of miR-620 and inhibitor of growth 4(ING4)in breast cancer tissues and cells was detected by qRT-PCR.MCF-7 cells were respectively transfected with miR-620inhibitor(anti-miR-620),inhibitor negative control(anti-miR-NC),anti-miR-620 with ING4 small interfering RNA(si-ING4),and anti-miR-620 with small interfering RNA negative control(si-NC)using LipofectamineTM2000 lipofection technology before radiotherapy,namely IR+anti-miR-620 group,IR+anti-miR-NC group,IR+anti-miR-620+si-ING4 group,and IR+anti-miR-620+si-NC group,respectively.The cell radiosensitivity,cell proliferation activity,cell cycle distribution and apoptosis rate were detected by clone formation assay,MTT and FCM,respectively.The dual-luciferase reporter gene assay and Western blotting were used to verify the targeting relationship between miR-620 and ING4.Results:Compared with para-cancerous tissues and HBL-100 cells,the expression of miR-620 was significantly increased while the mRNA expression of ING4 was significantly reduced in breast cancer tissues and cells(all P<0.01).Compared with the IR+anti-miR-NC group,the proliferation activity and S phase cell proportion of MCF-7 cells in the IR+anti-miR-620 group were significantly decreased(all P<0.01),but the apoptosis rate,the proportion of G0-G1 phase cells,and the radiosensitivity were significantly increased(all P<0.01).Compared with the IR+anti-miR-620+si-NC group,the proliferation activity and the S-phase cell proportion of MCF-7 cells in the IR+anti-miR-620+si-ING4 group were significantly increased(all P<0.01),but the apoptosis rate,the proportion of G0-G1 phase cells,and the radiosensitivity were significantly decreased(all P<0.01).The dual-luciferase reporter gene assay showed that ING4 is a target gene of miR-620,and miR-620 targeted and negatively regulated the expression of ING4.Conclusion:Knockdown of miR-620 could inhibit the proliferation of MCF-7 cells but promote the apoptosis and radiosensitivity of MCF-7 cells by up-regulating ING4.