长链非编码RNA MALAT1对神经母细胞瘤系细胞生物学特性的影响

Effects of long non-coding RNA MALAT1 on proliferation and invasiveness of neuroblastoma cells and its mechanism

目的探讨长链非编码RNA MALAT1对神经母细胞瘤系细胞生物学特性的影响及作用机制。方法体外培养神经母细胞瘤系SHEP2细胞,细胞分为Control、sh-MALAT1、miR-181a-5p inhibitor和sh-MALAT1+miR-181a-5p inhibitor组,其中sh-MALAT1组转染sh-MALAT1,miR-181a-5p inhibitor组转染miR-181a-5p inhibitor,sh-MALAT1+inhibitor组共同转染sh-MALAT1与miR-181a-5p inhibitor,Control组加入等量空载体。PCR检测mRNA水平;生物信息预测MALAT1与miR-181a-5p的靶向关系,荧光素酶实验鉴定;CCK8法检测细胞增殖能力;Hoechst法检测细胞凋亡;划痕实验测试细胞迁移;Transwell实验检测细胞侵袭;免疫印迹法检测蛋白表达。结果 sh-MALAT1明显降低MALAT1并提高miR-181a-5p在神经母细胞瘤细胞系SHEP2细胞mRNA水平。miR-181a-5p mimic明显降低MALAT1 wt荧光素酶活性。sh-MALAT1抑制SHEP2细胞增殖、侵袭及迁移,促进细胞凋亡;miR-181a-5p inhibitor促进细胞增殖、侵袭及迁移,抑制细胞凋亡,并减弱sh-MALAT1产生的影响。同时,sh-MALAT1抑制PI3K/Akt信号通路,而miR-181a-5p inhibitor可激活此信号通路并减弱sh-MALAT1的抑制作用。结论MALAT1靶向下调miR-181a-5p表达促进神经母细胞瘤细胞系SHEP2细胞增殖、迁移和侵袭。

Objective To investigate the effects of long non-coding RNA MALAT1 on survival and metastasis of neuroblastoma cells and their mechanisms.Methods The mRNA level of MALAT1 and miR-181a-5p in the neuroblastoma cell line and the cell transfected with sh-MALAT1 were measured by qRT-PCR.The targeted-relationship between MALAT1 and miR-181a-5p was predicted by bioinformatics and confirmed by luciferase reporter assay.The neuroblastoma cells proliferation,apoptosis,migration and invasion were detected respectively by CCK8,Hoechst,wound healing and transwell tests,and the levels of protein related to proliferation,invasion and PI3K/Akt signal pathway were detected by Western blot in the neuroblastoma cells after the MALAT1 and miR-181a-5p were silenced.Results Sh-MALAT1 declined the mRNA level of MALAT1 and enhanced mRNA level of miR-181a-5p in the neuroblastoma cells.MiR-181a-5p mimic decreased the luciferase activity of MALAT1 wt.Sh-MALAT1 inhibited neuroblastoma cells proliferation,migration,invasion,and promoted their apoptosis,and miR-181a-5p inhibitor promoted the cell proliferation,migration,invasion,inhibited apoptosis,and weakened the effect of sh-MALAT1.Meanwhile,sh-MALAT1 inhibited PI3K/Akt signal pathway,and miR-181a-5p inhibitor activated this pathway and weakened the inhibition effect of sh-MALAT1 on the neuroblastoma cells proliferation,migration and invasion.Conclusions MALAT1 promoted the proliferation,invasion and migration of the neuroblastoma cells by down regulation of miR-181a-5p expression.