lncRNA SNHG6通过microRNA-26b-5p对三阴性乳腺癌细胞增殖、迁移、侵袭的作用机制研究

The effect of lncRNA SNHG6 on proliferation, migration and invasion of triple-negative breast cancer cells by microRNA-26b-5p

摘要:

目的 探究lncRNA SNHG6通过microRNA-26b-5p（miR-26b-5p）对三阴性乳腺癌（TNBC）细胞增殖、迁移、侵袭的作用机制。方法 体外培养人TNBC细胞系MDA-MB-231细胞，培养后分组转染。设置对照组（空载质粒转染），SNHG6敲减组（si-SNHG6慢病毒载体转染）、miR-26b-5p抑制组（空载质粒+miR-26b-5p-inhibitor转染）及共转染组（si-SNHG6慢病毒载体+miR-26b-5p-inhibitor转染）。实时荧光定量聚合酶链反应检测SNHG6及miR-26b-5p的表达，分别进行CCK-8实验、划痕实验及Transwell实验检测MDA-MB-231细胞增殖、迁移及侵袭能力，采用双荧光素酶报告实验检测SNHG6与miR-26b-5p的靶向作用。结果 与对照组比较，SNHG6敲减组SNHG6 mRNA表达下调（P <0.05），miR-26b-5p mRNA表达上调（P <0.05）；与对照组比较，miR-26b-5p抑制组SNHG6 mRNA表达上调（P <0.05），miR-26b-5p mRNA表达下调（P <0.05）；与SNHG6敲减组比较，miR-26b-5p抑制组和共转染组SNHG6 mRNA表达上调（P <0.05），miR-26b-5p mRNA表达下调（P <0.05）；与miR-26b-5p抑制组比较，共转染组SNHG6 mRNA表达下调（P <0.05），miR-26b-5p mRNA表达上调（P <0.05）。对照组、SNHG6敲减组、miR-26b-5p抑制组、共转染组24 h、48 h、72 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点OD值有差异（F =52.481，P =0.000）。②4组OD值有差异（F =50.336，P =0.000），与对照组比较，SNHG6敲减组及共转染组24 h、48 h及72 h的OD值降低（P <0.05），miR-26b-5p抑制组24 h、48 h及72 h的OD值升高（P <0.05）；与SNHG6敲减组比较，miR-26b-5p抑制组和共转染组24 h、48 h及72 h的OD值升高（P <0.05）；与miR-26b-5p抑制组比较，共转染组OD值降低（P <0.05）。③4组OD值变化趋势有差异（F =20.109，P =0.000）。与对照组比较，SNHG6敲减组和共转染组划痕愈合率降低，侵袭细胞数减少（P <0.05），miR-26b-5p抑制组划痕愈合率升高，侵袭细胞数增加（P <0.05）；与SNHG6敲减组比较，miR-26b-5p抑制组和共转染组划痕愈合率升高，侵袭细胞数增加（P <0.05）；与miR-26b-5p抑制组比较，共转染组划痕愈合率降低，侵袭细胞数减少（P <0.05）。且SNHG6与miR-26b-5p基因序列上存在结合位点。结论 敲减SNHG6通过靶向上调MDA-MB-231细胞中miR-26b-5p的表达，抑制TNBC增殖、迁移及侵袭。

Abstract:

Objective To explore whether long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6) regulates cell migration and invasion via microRNA-26b-5p in triple-negative breast cancer (TNBC).Methods Human TNBC MDA-MB-231 cell line was cultured in vitro, followed by being grouped and transfected with different vectors. Specifically, the control group, SNHG6 knockdown group, miR-26b-5p inhibition group and co-transfection group were transfected with empty plasmids, si-SNHG6 lentiviral vector, empty plasmids together with miR-26b-5p inhibitor, and si-SNHG6 lentiviral vector together with miR-26b-5p inhibitor, respectively. The expressions of SNHG6 and miR-26b-5p were detected via qRT-PCR. The proliferation, migration and invasion abilities of MDA-MB-231 cells were detected via CCK-8 assay, scratch assay and transwell assay, respectively. The interaction between SNHG6 and miR-26b-5p was detected by dual-luciferase reporter assay.Results Compared with the control group, the mRNA expression of SNHG6 was down-regulated and that of miR-26b-5p was up-regulated in the SNHG6 knockdown group, while the mRNA expression of SNHG6 was up-regulated and that of miR-26b-5p was down-regulated in the miR-26b-5p inhibition group (P < 0.05). Compared with the SNHG6 knockdown group, the mRNA expression of SNHG6 was up-regulated and that of miR-26b-5p was down-regulated in the miR-26b-5p inhibition group and the co-transfection group (P < 0.05). Compared with the miR-26b-5p inhibition group, the mRNA expression of SNHG6 was down-regulated and that of miR-26b-5p was up-regulated in the co-transfection group (P < 0.05). The OD values at 24 h, 48 h and 72 h were compared among the four groups, and the repeated-measures analysis of variance revealed that the OD values were different among the time points (F = 52.481, P = 0.000) and among the groups (F = 50.336, P = 0.000). Specifically, the OD values at 24 h, 48 h and 72 h in SNHG6 knockdown group and co-transfection group were lower than those in the control group (P < 0.05), while they were higher in the miR-26b-5p inhibition group than those in the control group (P < 0.05). Compared with the SNHG6 knockdown group, the OD values at 24 h, 48 h and 72 h in the miR-26b-5p inhibition group and co-transfection group were higher (P < 0.05). The OD values were lower in the co-transfection group than those in the miR-26b-5p inhibition group (P < 0.05). The change trends of OD values were different among the four groups (F = 20.109, P = 0.000). Compared with the control group, the scratch healing rate was lower and the number of invasive cells was decreased in the SNHG6 knockdown group and co-transfection group (P < 0.05), while the scratch healing rate was higher and the number of invasive cells was increased in the miR-26b-5p inhibition group (P < 0.05). Compared with the SNHG6 knockdown group, the scratch healing rate was higher and the number of invasive cells was increased in the miR-26b-5p inhibition and the co-transfection group (P < 0.05). Compared with the miR-26b-5p inhibition group, the scratch healing rate was lower and the number of invasive cells was decreased in the co-transfection group (P < 0.05). In addition, there was a binding site within miR-26b-5p for SNHG6.Conclusions Knockdown of SNHG6 inhibits the cell proliferation, migration and invasion in TNBC by targeting and up-regulating the expression of miR-26b-5p in MDA-MB-231 cells.