南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (2): 184-192.doi: 10.12122/j.issn.1673-4254.2021.02.04

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piRNA NU13对肾母细胞瘤细胞增殖、凋亡、迁移和侵袭的影响

张朝霞, 王 璋, 金黎明, 谭小军, 王钊颖, 沈炼桔, 魏光辉, 何大维   

  • 发布日期:2021-02-05

Effect of piRNA NU13 in regulating biological behaviors of human Wilms tumor cells in vitro

  • Published:2021-02-05

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摘要：

目的 探讨piwil2诱导的肿瘤样干细胞（piwil2-iCSCs）来源的差异piRNA NU13对肾母细胞瘤细胞（G401）生物学行为的影响。方法 RT-qPCR检测G401中piRNA NU13及NOP56的表达情况；通过脂质体转染技术，在肾母细胞瘤细胞株G401中过表达及抑制piRNA NU13，并用RT-qPCR技术验证转染后G401细胞中piRNA NU13的表达量；通过CCK8检测转染后G401细胞增殖情况，流式细胞术检测细胞凋亡能力，划痕实验和Transwell实验分别检测细胞迁移和侵袭能力的改变；通过双荧光素酶实验检测NOP56与piRNA NU13的结合情况；通过Western blot检测MMP2、MMP9、BAX、Bcl2、NOP56蛋白表达情况。结果 在人肾母细胞瘤细胞G401中piRNA NU13表达水平低于肾小管上皮细胞HK2（P<0.05）；NOP56在G401及肾母细胞瘤组织均高表达（P<0.05）；在G401中上调piRNA NU13表达能够抑制肿瘤细胞的增殖、迁移、侵袭，并促进肿瘤细胞凋亡（P<0.05）；过表达piRNA NU13可抑制MMP2、MMP9及Bcl2的表达水平，促进BAX表达（P<0.05）；piRNA NU13与预测靶基因NOP56非直接结合，但可间接调控NOP56的表达。结论 piRNA NU13在肾母细胞瘤中低表达，NOP56在肾母细胞瘤中高表达，piNU13可能间接调控NOP56表达从而抑制肿瘤细胞增殖、迁移、侵袭，促进其凋亡。

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Abstract:

Objective To investigate the role of the differential piRNA NU13 derived from piwil2-induced cancer stem-like cells (piwil2-iCSCs) in regulating biological behaviors of Wilms tumor cells (G401). Methods The expressions of piRNA NU13 and NOP56 were detected in Wilms tumor cell line G401 using RT-qPCR. G401 cells were transfected with piRNA NU13 mimics and inhibitor for its over-expression and inhibition, and the transfection efficiency was verified with RT-qPCR. The changes in proliferation of G401 cells after transfection were detected using CCK8 assay, and cell apoptosis was analyzed using flow cytometry. Wound healing assay and Transwell assay were performed to examine the changes in migration and invasion abilities of the transfected cells. The binding of NOP56 and piRNA NU13 was detected using dual luciferase experiment. The protein expressions of MMP2, MMP9, BAX, Bcl2, and NOP56 in the cells were detected with Western blotting. Results RT-qPCR showed that the expression of piRNA NU13 decreased significantly in human Wilms tumor G401 cells as compared with that in renal tubular epithelial cell line HK2 (P<0.05), and NOP56 was highly expressed in G401 cells and Wilms tumor tissues (P<0.05). Over-expression of piRNA NU13 significantly suppressed the proliferation, migration and invasion of G401 cells, promoted cell apoptosis (P<0.05), inhibited the expression of MMP2, MMP9 and Bcl2, and enhanced the expression of BAX (P<0.05). The results of dual luciferase experiment showed that piRNA NU13 did not bind to NOP56 directly but regulated the expression of NOP56 in an indirect manner. Conclusion piRNA NU13 is down-regulated and NOP56 is highly expressed in Wilms tumor. piNU13 may regulate the expression of NOP56 indirectly to inhibit the proliferation, migration and invasion and promote apoptosis of Wilms tumor cells in vitro.

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