Original articleTPX2 siRNA regulates growth and invasion of esophageal cancer cells
Introduction
Targeting protein for xenopus kinesin-like protein 2 (Tpx2) is a microtubule-associated protein that regulates the formation of the spindle by promoting microtubule nucleation from the chromatin and stabilizes the spindle microtubules in a Ran-dependent manner. [1], [2] The expression of Tpx2 is tightly controlled by the cell cycle, exclusively expressed in proliferating cells from the G1/S transition until the end of cytokinesis [3], [4], [5]. Therefore, TPX2 is implicated in tumor cell proliferation [1], [2], [6], [7]. Accumulating evidence has indicated that upregulation of TPX2 gene along with increased gene copy numbers, is commonly observed in many human tumor types with including saliva gland cancer [8], lung squamous cell carcinoma [9], breast cancer [10] and esophageal cell carcinoma (ESCC) [11]. Moreover, TPX2 expression is positively correlated with tumor grade and stage, as well as the metastasis of lymph nodes. Furthermore, TPX2 is highly expressed in human tumors with chromosome instability (CIN) among a signature of genes deregulated. [12] ESCC is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Current investigations have demonstrated that TPX2 expression is correlated with cell proliferation and poor prognosis among patients with ESCC [13], [14], supporting that TPX2 acts as a tumor promoter in ESCC. Notably, suppression of TPX2 with RNA interference disrupts the formation of the two microtubule asters and spindle, leading to defects in microtubule organization during mitosis, which has never been reported in ESCC.
In this present study, we show that knock-down of TPX2 by siRNA blocked the proliferation, migration and invasion of EC9706 cells in vitro. Meanwhile, down regulation of TPX2 causes cell apoptosis, demonstrating that inhibition of TPX2 level could be a potential therapeutic strategy for patients with ESCC.
Section snippets
Cell line
Human esophageal cancer EC9706 cells were kindly provided by The State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences.
Reagents and apparatus
TPX2 rabbit anti-human polyclonal antibody was generously provided by Dr Ulrike Bauer from the Mattaj Lab, Germany. β-actin rabbit polyclonal antibody and goat anti-rabbit horseradish peroxidase labeled IgG (H + L) secondary antibody were purchased from TianGen Biotech (Beijing) Co., Ltd. RT-PCR kit and DNA Marker 1000 were
TPX2 mRNA decreased after TPX2 siRNA tranfection
Electrophoretic band results suggest that the expression of TPX2 mRNA in the TPX2 siRNA treated EC9706 cells decreased in the later time points, while TPX2 mRNA of either blank control or negative control group remained unchanged. GeneTools image system was used to analyze the ratio of TPX2 gene bands with reference bands of β-actin, which represents the relative strength of TPX2 mRNA content. H test results manifested that TPX2 mRNA level show a significant difference between TPX2 siRNA group
Discussion
The TPX2 gene first identified by Heidebrecht [15] in 1977 is a nuclear proliferation-related protein and is located on human chromosome 20q11.2 [16], [17]. The relative molecular mass of the protein is 100KD. The expression of TPX2 is strictly regulated by cell-cycle, it can only be detected in the G1-S phase junction, and then is undetected after the completion of cell division [18]. In the S and G2 phase, human TPX2 distributes to the nucleus and combines with the mitotic spindle closely in
Conclusion
In summary, siRNA could effectively inhibit the invasion and metastasis of EC9706 cells, promote the apoptosis of tumor cells and may become a new approach for treatment of esophageal carcinoma.
Contributors
H.C.L. and S.L.L. conceived and designed the experiments; G.H.Z, Y.H.L., J.F.M., L.S.S,. L.Z., and J.W.L. performed the experiments; H.C.L. and P.W. wrote the manuscript.
Disclosure of interest
The authors declare that they have no conflicts of interest concerning this article.
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