Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion
Introduction
Breast cancer is the most common malignant tumor for women around the world, and its incidence rate is increasing in recent years. Like most cancers, breast cancer is a systemic disease, and many factors and biological active substances such as growth factors, cytokines and hormones are involved in the complex pathogenetic processes. The abnormality of those active substances-mediated signal transduction pathways can lead to excessive amplification of certain genes that cause normal cells to accept the signal of abnormal proliferation, differentiation and growth, and eventually result in cancerogenesis [1].
SET, also termed I2PP2A, PHAPII and TAF-1b, was first identified in 1992 in a study of leukemia [2]. SET is known as inhibitor-2 of protein phosphatase 2A (I2PP2A) and exists at elevated levels in the brains of those with Alzheimer’s disease relative to normal age-matched controls [3] and in leukemia cancer cells [4]. SET is overexpressed in many cancer cells including Wilm tumors [5], pancreatic tumors [6], prostate cancer [7], lung tumors [8], ovarian cancer [9] and testicular cancers [10]. It has also been demonstrated that SET is involved in neuronal apoptosis [11]. As an Endogenous inhibitor of PP2A, SET-mediated PP2A inhibition occurs via dephosphorylation of proteins, such as the protein kinase B (Akt) [12] and extracellular signal-regulated kinase (ERK) [13]. SET also regulate apoptosis, the cell cycle as well as cell motility through its functions of controlling histone acetylation, beta-adrenergic receptor phosphorylation and granzyme activity [14], [15], [16], [17].
Because SET has such a diversity of functions, we hypothesized that SET is involved in breast tumor. In this study, we described the construction and characterization of a stable SET siRNA breast cancer cells MDA-MB-231 and ZR-75-30 and explored the effects of SET knockdown on the breast tumor cells.
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Chemicals and reagents
The human breast cancer cells MDA-MB-231 and ZR-75-30 were obtained from the American Type Culture Collection. 293T cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences (Chinese Academy of Science, China). RPMI 1640 and culture medium, fetal bovine serum (FBS), penicillin–streptomycin and trypsin were purchased by Gibco/Invitrogen (Carlsbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit for cell viability
Construction of stable SET siRNA breast cancer cells
In order to silence SET expression in MDA-MB-231 and ZR-75-30 cells, we designed one pair of siRNA oligonucleotides targeting the SET gene. The oligonucleotides encoding the siRNAs were cloned into a self-inactivating lentivirus vector containing the human U6 promoter upstream of the siRNA multiple cloning sites, and the resulting constructs were verified by DNA sequencing.
To determine the efficiency of SET siRNAs of stably transfected siRNA MDA-MB-231 and ZR-75-30 cells, SET mRNA and protein
Discussion
As a lethal disease, 90% of human cancer deaths are caused by metastasis. Breast cancer has the highest incidence rate for cancer in women in industrialized countries and poor prognosis due to its strong metastatic ability [21]. SET-mediated PP2A inhibition is an important regulatory mechanism for a number of physiological and pathological processes, including proliferation, differentiation, apoptosis and carcinogenesis [22]. In vitro studies showed that up-regulation of SET was associated with
Acknowledgments
This work was supported by the NSFC (No. 81272180), the National Basic Reasearch Program of China (No. 2012CB518200), and the Upgrade Scheme of Shenzhen Municipal Key Laboratory (No. CXB201104220031A & ZDSY20120615085804889), The National Science Foundation of China (No. 21102094), The Project of Shenzhen Basic Research Plan (No. GJHS20120628151305456).
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