miR-29b对神经母细胞瘤增殖、侵袭和转移的影响研究

Effect of mir-29b in the proliferation, invasion and migration of neuroblastoma cells

目的检测miR-29b在肾上腺和神经母细胞瘤组织中的表达情况,探讨miR-29b对神经母细胞瘤细胞增殖、侵袭和转移能力的影响。方法收集12例神经母细胞瘤患儿的肿瘤组织和癌旁肾上腺组织标本,实时定量PCR检测miR-29b的表达水平;miR-29相关核苷酸(mimics、inhibitor、NC)瞬时转入SK-N-SH神经母细胞瘤细胞后,CCK-8实验检测miR-29b对该细胞系增殖的抑制作用,Transwell小室、划痕实验进行侵袭、迁移能力实验,Western blot方法检测上皮转化相关蛋白(E-cadherin、Vimentin)的表达水平。结果与癌旁肾上腺组织比较,miR-29b在神经母细胞瘤组织的表达水平明显降低(P<0.05);miR-29相关核苷酸(mimics、inhibitor、NC)转染细胞后,miR-29b mimics组出现抑制细胞增殖的效应(P<0.05);Transwell小室实验显示miR-29b mimics组的穿膜细胞量(36.33±8.08)少于miR-29b inhibitor组(112.30±11.68)和NC组(112.30±11.68);划痕实验显示转染miR-29b mimics后,其愈合率(51.67±4.50)%较miR-29b inhibitor组(87.67±3.79)%和NC组(85.00±3.00)%明显下降,细胞的侵袭、迁移能力明显下降;Western blot实验显示,与NC组相比,miR-29b mimics组E-cadherin表达增高,Vimentin表达降低,而miR-29b inhibitor组中E-cadherin表达降低,Vimentin表达增高(P<0.05)。结论神经母细胞瘤组织中miR-29b表达水平下降,miR-29b过表达可以抑制细胞的增殖、侵袭和迁移能力,这种抑癌作用可能与抑制EMT有关。

Objective To detect the expression of miR-29b in adrenal gland and neuroblastoma and examine the effect of miR-29b on the proliferation,invasion and migration of neuroblastoma cells.Methods The expression of miR-29b was detected by real-time quantitative polymerase chain reaction(PCR)in neuroblastoma tissues and paracancerous adrenal tissues.And miR-29b related nucleotides(mimics/inhibitor/NC)were transiently transferred into neuroblastoma SK-N-SH cells.The growth inhibition of miR-29b on SK-N-SH was determined bv CCK-8 assay,cell migration and invasion ability was evaluated by Transwell invasion and Scratch test and the expressions of epithelial transformation related proteins(E-cadherin&vimentin)were detected by Western blot.Results miR-29b became down-regulated in neuroblastoma tissues compared with that in paracancerous adrenal tissues(P<0.05).After transfection with miR-29-related nucleotides(mimics/inhibitor/NC),miR-29b mimics exhibited the effect of inhibiting cell proliferation(P<0.05);Transwell tests showed that the migrated cells in miR-29b mimics group(36.33±8.08)were less than that in miR-29b inhibitor(112.30±11.68)and NC group(112.30±11.68);Scratch test showed that the healing rate(51.67±4.50)of mir-29b was significantly lower than that of mir-29b inhibitor group(87.67±3.79)and NC group(85.00±3.00).And migration and invasion ability of K-N-SH cells decreased significantly(P<0.05);Western blot showed that the expression of E-cadherin increased in miR-29b mimics group and the expression of Vimentin declined while the opposite result appeared in miRNA-29b inhibitor group(P<0.05).Conclusion miR-29b is down-regulated in neuroblastoma tissues.And an overexpression of miR-29b can inhibit cell proliferation,invasion and migration of neuroblastoma SK-N-SH cells.It may be related with an inhibition of epithelial mesenchymal transition(EMT).