lncRNA HOTAIR通过miR-519d-3p/CCND1分子轴促进乳腺癌SKBR3细胞的恶性生物学行为

lncRNA HOTAIR promotes malignant biological behaviors of breast cancer SKBR3 cells through miR-519d-3p/CCND1 axis

目的:探究lncRNA HOTAIR/miR-519d-3p/CCND1分子轴对乳腺癌细胞增殖和转移的影响及其可能机制。方法:收集2017年3月至2019年2月南昌市第三医院乳腺外科手术切除的乳腺癌患者的乳腺癌组织及配对癌旁组织各50例,qPCR检测癌及癌旁组织中HOTAIR的表达水平,乳腺正常上皮细胞及乳腺癌细胞系中HOTAIR和miR-519d-3p的表达水平。将乳腺癌SKBR3细胞分为NC组、si-HOTAIR组、miR-519d-3p mimics组,miR-519d-3p mimics+pcHOTAIR组、miR-519d-3p mimics+pcCCND1组和si-HOTAIR+pcCCND1组,CCK-8法检测各组SKBR3细胞增殖能力、Transwell检测细胞侵袭和迁移能力、Western blotting检测SKBR3细胞中E-cadherin、N-cadherin、Vimentin以及CCND1表达水平。双荧光素酶报告基因检测HOTAIR和miR-519d-3p以及miR-519d-3p和CCND1的靶向关系。结果:HOTAIR在癌组织以及乳腺癌细胞系中呈高表达,且在SKBR3细胞系中表达最高。敲降HOTAIR可显著抑制SKBR3细胞增殖、侵袭和迁移,并显著增加E-cadherin的表达水平、降低N-cadherin和Vimentin的表达水平(均P<0.05)。双荧光素酶报告基因检测显示,HOTAIR靶向下调miR-519d-3p的表达,miR-519d-3p靶向下调CCND1的表达。敲降HOTAIR可增强miR-519d-3p对CCND1的下调作用,抑制SKBR3细胞EMT、增殖、侵袭和迁移能力(均P<0.05)。结论:敲降HOTAIR可抑制SKBR3细胞增殖和转移,其机制是通过调控miR-519d-3p/CCND1分子轴实现的。

Objective: To explore the effect of lncRNA HOTAIR/miR-519d-3p/cyclin D1(CCND1) axis on the proliferation and metastasis of breast cancer cells and its underlying mechanism. Methods: A total of 50 pairs of breast cancer tissues and corresponding para-cancer tissues resected from breast cancer patients in the Department of Breast Surgery, the Third Hospital of Nanchang from March 2017 to February 2019 were collected for this study. The expression level of HOTAIR in breast cancer tissues and paired paracancer tissues was detected by qPCR, in addition, the expressions of HOTAIR and miR-519d-3p in normal breast epithelial cells and breast cancer cell lines were also detected. Breast cancer SKBR3 cells were divided into NC group(without any treatment), si-HOTAIR group, mir-519d-3p mimics group, miR-519d-3p mimic+pcHOTAIR group, miR-519d-3p mimic+pcCCND1 group, and si-HOTAIR+pcCCND1 group. The proliferation ability of SKBR3 cells was detected by CCK-8. Invasion and migration of SKBR3 cells were detected by Transwell. The expression levels of E-cadherin, N-cadherin, Vimentin and CCND1 in SKBR3 cells were detected by Western blotting. The targeting relationship between HOTAIR and miR-519d-3p, miR-519d-3p and CCND1 was detected by Dualluciferase reporter gene system. Results: HOTAIR was highly expressed in breast cancer tissues and cell lines, with the highest expression in SKBR3 cells. HOTAIR knockdown significantly inhibited the proliferation, invasion and migration of SKBR3 cells, as well as increased the expression level of E-cadherin and decreased the expression levels of N-cadherin and Vimentin. Dual-luciferase reporter gene assay showed that HOTAIR targetedly down-regulated the expression of miR-519d-3p, and miR-519d-3p targetedly downregulated the expression of CCND1. Further studies showed that knockout of HOTAIR inhibited the EMT, proliferation, invasion and migration of SKBR3 cells through enhancing the inhibitory effect of miR-519d-3p on CCND1 expression(all P<0.05). Conclusion:HOTAIR knockdown inhibits proliferation and metastasis of SKBR3 cells by regulating the axis of miR-519d-3p/CCND1.