抑制miR-125b表达对TNF-α诱导下三阴性乳腺癌细胞迁移、侵袭能力的影响

Effects of inhibiting miR-125b expression on TNF-α-induced migration and invasion of TNBC cells

目的观察抑制miR-125b表达对三阴性乳腺癌(TNBC)细胞迁移、侵袭能力的影响,并探讨其机制。方法将TNBC细胞MDA-MB-468分为4组,抑制组和NC组分别转染miR-125b抑制剂(miR-125b inhibitor)、miRNC阴性对照48 h后加入TNF-α10 ng/m L干预72 h,TNF-α组加入TNF-α10 ng/m L干预72 h,正常组不干预。分别采用细胞划痕实验及Transwell实验观察细胞的迁移及侵袭能力,荧光定量PCR法检测细胞中的miR-125b,Western boltting法检测磷酸化核转录因子κB (NF-κB) p65蛋白及上皮间质转化(EMT)相关蛋白E-cadherin、Vimentin。结果细胞迁移率及穿膜细胞数NC组、TNF-α组>空白组>抑制组(P均<0. 05),miR-125b及磷酸化NF-κB p65、Vimentin蛋白相对表达量NC组、TNF-α组>空白组>抑制组(P均<0. 05),E-cadherin蛋白相对表达量NC组、TNF-α组<空白组<抑制组(P均<0. 05),NC组、TNF-α组各指标差异无统计学意义(P均> 0. 05)。结论抑制miR-125b表达可能通过干扰NF-κB活性,抑制细胞发生EMT,从而降低TNF-α诱导的TNBC细胞迁移和侵袭能力。

To investigate the effects of inhibiting the expression of miR-125b on the migration and invasion of the triple negative breast cancer(TNBC)cells and to explore its mechanism.Methods MDA-MB-468 cells were divided into four groups:the miR-125b inhibitor group,negative control group(NC group),blank control group,and TNF-αgroup.The miR-125b inhibitors(miR-125b inhibitor group)and unrelated sequences(NC group)were transfected into MDA-MB-468 cells,respectively.At 48 h after transfection,10 ng/mL TNF-αwas added to the miR-125b inhibitor group,NC group,and TNF-αgroup for 72 h,while the blank control group was given only PBS solution.Scratch test and Transwell assay was used to observe the migration and invasion abilities.Fluorescent quantitative PCR was used to detect the expression of miR-125b.Western blotting was used to detect phosphorylated nuclear factor-κB(NFκB)p65 protein and epithelial-mesenchymal transition(EMT)-related proteins E-cadherin and Vimentin.Results The migration rate and the number of penetrating cells were in the following order:TNF-αgroup>blank control group>miR-125b inhibitor group(all P<0.05).The expression of phosphorylated NF-κB p65 and Vimentin decreased by miR-125b inhibition:NC group and TNF-αgroup>blank control group>miR-125b inhibitor group(both P<0.05),while the expression of E-cadherin was increased:NC group and TNF-αgroup<blank control group<miR-125b inhibitor group(P<0.05),and no significant differences were found in the indexes between the NC group and TNF-α(all P>0.05).Conclusion Inhibiting miR-125b expression may inhibit TNF-α-induced cell migration and invasion by interfering with NF-κB activity and inhibiting EMT in cells.