MDM2剪接变体P2-MDM2-10和MDM2-∆5在乳腺癌细胞中的功能作用

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells.

作者信息

Huun Johanna, Gansmo Liv B, Mannsåker Bård, Iversen Gjertrud T, Sommerfelt-Pettersen Jan, Øvrebø Jan Inge, Lønning Per E, Knappskog Stian

机构信息

Section of Oncology, Department of Clinical Science, University of Bergen, Bergen, Norway.

Section of Oncology, Department of Clinical Science, University of Bergen, Bergen, Norway; Department of Oncology, Haukeland University Hospital, Bergen, Norway.

出版信息

Transl Oncol. 2017 Oct;10(5):806-817. doi: 10.1016/j.tranon.2017.07.006. Epub 2017 Aug 29.

DOI:10.1016/j.tranon.2017.07.006

PMID:28844019

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5576977/

Abstract

BACKGROUND

MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood.

METHODS

We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis).

RESULTS

In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P=4.3 × 10 and P=7.1 × 10, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P=.039 and P=.070, respectively) but also the proapoptotic gene PUMA (P=.010 and P=.033, respectively), accompanied by induction of apoptosis and repression of senescence.

CONCLUSION

We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells.

GENERAL SIGNIFICANCE

Alternative splicing may influence the oncogenic effects of the MDM2 gene.

摘要

背景

MDM2是p53的负调控因子，在多种人类癌症中上调。虽然在正常组织和恶性细胞中都观察到了不同的MDM2剪接变体，但其功能尚不清楚。

方法

我们通过在MCF-7细胞中过表达MDM2剪接变体，并分析下游基因的表达（qPCR和蛋白质印迹法）、亚细胞定位（免疫荧光）、细胞周期分析（Nucleocounter3000）、凋亡分析（膜联蛋白V检测）和衰老诱导（β-半乳糖苷酶分析）来评估其作用。

结果

在对MCF-7乳腺癌细胞中的MDM2剪接变体进行筛选，并结合健康白细胞的数据后，我们发现P2-MDM2-10和MDM2-Δ5是表达水平最高的剪接变体。与MDM2全长蛋白相比，我们发现P2-MDM2-10和MDM2-Δ5在正常组织中的表达水平在携带启动子SNP309TT基因型的个体中最高。虽然我们未检测到MDM2-Δ5编码的蛋白质产物，但P2-MDM2-10变体产生的蛋白质明显比MDM2-FL更稳定。两种剪接变体在应激细胞中均显著上调（分别为P = 4.3×10和P = 7.1×10）。值得注意的是，化疗处理以及P2-MDM2-10或MDM2-Δ5的过表达均导致内源性MDM2-FL的mRNA水平升高（分别为P = 0.039和P = 0.070），同时促凋亡基因PUMA的mRNA水平也升高（分别为P = 0.010和P = 0.033），并伴有凋亡诱导和衰老抑制。