长链非编码RNA RAD51-AS1通过ERK信号通路对乳腺癌细胞迁移侵袭的影响

Effects of long non-coding RNA RAD51-AS1 on migration and invasion of breast cancer cells through ERK signaling pathway

目的探讨RAD51反义RNA 1(RAD51-AS1)通过抑制细胞外信号调节激酶(ERK)信号调控乳腺癌细胞增殖、迁移和侵袭等生物学行为的潜在机制。方法采用实时荧光定量PCR(qPCR)检测55对乳腺癌组织和癌旁正常乳腺组织以及乳腺癌细胞(BT549、MCF7、SK-BR-3、BT474、MDA-MB-231)和正常乳腺上皮细胞(MCF-10A)中RAD51-AS1的表达情况。培养MDA-MB-231细胞并分为NC组(转染pcDNA3.1)、RAD51-AS1组(转染pcDNA3.1-RAD51-AS1以过表达RAD51-AS1)和RAD51-AS1+ERK激动剂组(同时转染pcDNA3.1-RAD51-AS1和ERK激动剂以过表达RAD51-AS1并激活ERK信号)。通过CCK-8法、划痕实验和Transwell实验分别评估细胞的增殖、迁移和侵袭能力。利用qPCR和Western blot检测细胞中ERK、p-ERK和基质金属蛋白酶9(MMP-9)的表达情况。结果乳腺癌组织的RAD51-AS1表达量低于癌旁组织(0.419±0.039 vs.1.000±0.063,P<0.05);同时乳腺癌细胞MCF7(0.712±0.025)、SK-BR-3(0.581±0.039)、BT474(0.519±0.034)和MDA-MB-231(0.390±0.036)的RAD51-AS1表达量低于MCF-10A细胞的1.000±0.073(P<0.05)。RAD51-AS1组MDA-MB-231细胞的增殖、迁移和侵袭能力均低于NC组(P<0.05)。RAD51-AS1组中p-ERK和MMP-9表达低于NC组(P<0.05)。另外,RAD51-AS1+ERK激动剂组MDA-MB-231细胞的增殖、迁移和侵袭能力均高于RAD51-AS1组(P<0.05)。RAD51-AS1+ERK激动剂组中p-ERK和MMP-9表达高于RAD51-AS1组(P<0.05)。结论RAD51-AS1通过下调ERK信号通路活性来抑制乳腺癌细胞增殖、迁移和侵袭过程,发挥肿瘤抑制因子的功效。

Objective To investigate the underlying mechanism of long non-coding RNA RAD51 antisense RNA 1(RAD51-AS1)regulating proliferation,migration and invasion processes of breast cancer cells through extracellular signal-regulated kinase(ERK)signaling pathway.Methods Real-time quantitative polymerase chain reaction(qPCR)was applied to access the expressions of RAD51-AS1 in 55 paired breast cancer tissues and adjacent non-cancer tissues as well as breast cancer cells(BT549,MCF7,SK-BR-3,BT474,MDA-MB-231)and normal breast epithelial cells(MCF-10A).MDA-MB-231 cells were cultured and divided into NC group(cells transfected with pcDNA3.1),RAD51-AS1 group(cells transfected with pcDNA3.1-RAD51-AS1 as to upregulate RAD51-AS1)and RAD51-AS1+ERK activator group(cells transfected with pcDNA3.1-RAD51-AS1 and ERK signal activator as to upregulate RAD51-AS1 and activate ERK).The CCK-8 kit,wound healing and Transwell assays were employed to detect the proliferation,migration and invasion abilities of cells.Additionally,qPCR and Western blot were applied to detect the expressions of ERK,p-ERK and matrix metallopeptidase-9(MMP-9).Results The RAD51-AS1 expression in breast cancer tissues was lower than that of non-cancer tissues(0.419±0.039 vs.1.000±0.063,P<0.05).RAD51-AS1 were downregulated in breast cancer cells MCF7(0.712±0.025),SK-BR-3(0.581±0.039),BT474(0.519±0.034)and MDA-MB-231(0.390±0.036)as compared with 1.000±0.073 of MCF-10A cells(P<0.05).The proliferation,migration and invasion abilities of MDA-MB-231 cell were decreased in RAD51-AS1 group as compared to NC group(P<0.05).The expressions of p-ERK and MMP-9 in RAD51-AS1 group were declined as compared to NC group.Meanwhile,the proliferation,migration and invasion abilities of MDA-MB-231 cells were increased in RAD51-AS1+ERK activator group as compared to RAD51-AS1 group(P<0.05).The expressions of p-ERK and MMP-9 in RAD51-AS1+ERK activator group were higher than those in RAD51-AS1 group.Conclusion RAD51-AS1 suppresses the proliferation,migration and invasion abilities of breast cancer cells through inhibiting ERK signaling pathway,and acts as a tumor suppressor.