miR-125a-3p靶向负调控PLK4抑制神经母细胞瘤细胞增殖及其机制探讨

The mechanism of miR-125a-3p inhibiting cell proliferation in neuroblastoma cells by targeting and negatively regulating PLK4

目的:研究miR-125a-3p对神经母细胞瘤细胞增殖的影响,并探讨其作用机制。方法:运用qRT-PCR法检测Hela、SH-SY-5Y、SHEP、SK-N-BE细胞中miR-125a-3p的表达;将miR-125a-3p组(转染miR-125a-3p mimics)、miR-NC组(未转染细胞)、inhibitor-NC组(转染空inhibitor)、miR-125a-3p inhibitor组(转染miR-125a-3p inhibitor)、siPLK4组(转染siPLK4)、miR-125a-3p+Vector组(miR-125a-3p mimics和pcDNA 3.1共转染)、miR-125a-3p+PLK4组(miR-125a-3p mimics和pcDNA 3.1-PLK4共转染)以脂质体法转染至SH-SY-5Y细胞;MTT法检测各组细胞的增殖情况;Western blot检测各组细胞中PLK4、PIK3CA、Akt、p-Akt蛋白的表达;双荧光素酶报告基因检测实验检测各组细胞的荧光活性。结果:与Hela细胞相比,SH-SY-5Y、SHEP、SK-N-BE细胞中miR-125a-3p的表达均显著降低(P<0.05);与Control组相比,miR-125a-3p组细胞的增殖显著降低,PLK4、PIK3CA、p-Akt蛋白的表达量均显著降低(P<0.05);PLK4为miR-125a-3p的靶基因。过表达PLK4可逆转miR-125a-3p对SH-SY-5Y细胞增殖及PI3K/Akt信号通路的抑制作用。结论:miR-125a-3p可抑制神经母细胞瘤细胞增殖,其作用机制与靶向负调控PLK4有关,将可为神经母细胞瘤的治疗提供新靶点。

Objective:To study the effect of miR-125 a-3 p on cell proliferation of neuroblastoma,and explore its mechanism.Methods:qRT-PCR was used to detect the miR-125 a-3 p expression in Hela,SH-SY-5 Y,SHEP and SK-N-BE cells.miR-125 a-3 p group(transfected miR-125 a-3 p mimics),miR-NC group(untransfected cells),inhibitor-NC group(transfected empty inhibitor),miR-125 a-3 p inhibitor group(transfected miR-125 a-3 p inhibitor),siPLK4 group(transfected siPLK4),miR-125 a-3 p+Vector group(co-transfected miR-125 a-3 p mimics and pcDNA 3.1),miR-125 a-3 p+PLK4 group(co-transfected miR-125 a-3 p mimics and pcDNA 3.1-PLK4) were all transfected into SH-SY-5 Y cells by liposome method. MTT assay was used to detect cell proliferation in each group. Western blot was used to detect the protein expression of PLK4,PIK3 CA,Akt and p-Akt in each cell. Dual luciferase reporter gene assay was used to detect the fluorescence activity in each group. Results: Compared with Hela cells,the expression of miR-125 a-3 p was significantly decreased in SH-SY-5 Y,SHEP and SK-N-BE cells( P < 0. 05). Compared with control group,the proliferation of the cells was significantly decreased,and the protein expression levels of PLK4,PIK3 CA and p-Akt were significantly decreased( P < 0. 05) in miR-125 a-3 p group. PLK4 was the target of miR-125 a-3 p. Overexpression PLK4 rescue the inhibitory effect of miR-125 a-3 p on SH-SY-5 Y cell proliferation and PI3 K/Akt signaling pathway. Conclusion: miR-125 a-3 p could inhibit the proliferation of neuroblastoma cells,and its mechanism may be related to the targeted negative regulation of PLK4,which will provide a new target for the treatment of neuroblastoma.