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

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)。(2)4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)。(3)4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。

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 q RT-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 m RNA expression of SNHG6 was down-regulated and that of miR-26b-5p was upregulated in the SNHG6 knockdown group, while the m RNA expression of SNHG6 was up-regulated and that of miR-26b-5p was down-regulated in the mi R-26b-5p inhibition group(P < 0.05). Compared with the SNHG6knockdown 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 and72 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 cotransfection 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 cotransfection 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 upregulating the expression of miR-26b-5p in MDA-MB-231 cells.