Original article
TPX2 siRNA regulates growth and invasion of esophageal cancer cells

https://doi.org/10.1016/j.biopha.2014.08.008Get rights and content

Abstract

Purpose

Observe how specific small RNA interference (siRNA) aimed at TPX2 gene suppresses TPX2 gene expression in esophageal cancer EC9706 cells and the effect on esophageal cancer cell growth and invasion ability.

Methods

Transfect TPX2 siRNA into EC9706 cells via lipofectamin 2000. The experiments were divided into three groups, a negative control, a blank control and an siRNA interference group (24 h, 48 h, 72 h, 96 h). We examined RNA and protein level alteration of the TPX2 gene after TPX2 siRNA transfection by RT-PCR and Western blot analysis. Detection of how TPX2 siRNA influences EC9706 cell proliferation was done by MTT, cell apoptosis monitored through Tunel assay, in vitro invasion ability via Boyden chamber and cell cycle change by flow cytometry.

Results

After effective siRNA transfection, TPX2 mRNA and protein expression level in siRNA interference group were (0.31 ± 0.08, 0.39 ± 0.12),72 h after transfection, significantly lower than blank control group (1.00 ± 0.01) and negative control group (0.98 ± 0.11), (F = 71.182, t1 = 8.17, t2 = 7.90, P < 0.05); MTT results demonstrated that cell growth and proliferation were inhibited and the inhibition rate was up to 35.4% (P < 0.05) compared with the control group. TUNEL results indicated that cell apoptosis index in siRNA interference group was 18.28 ± 0.35, higher than that in blank control group (4.07 ± 0.26)and negative control group (4.13 ± 0.22), (F = 244.5, t1 = 60.61, t2 = 53.32, P < 0.01). Boyden chamber results showed that the transmembrane cell number was 45.30 ± 8.08 in siRNA interference group, less than blank control group (121.90 ± 7.83), (F = 122.46, t1 = 11.81, t2 = 10.47, P < 0.01); besides, in siRNA interference group cell invasion inhibition rate was 71.42 ± 9.12, higher than negative control group (5.65 ± 3.55), (t = 14.256, P < 0.01). Flow cytometry results illustrated that more EC9706 cells went into apoptosis and cell cycle arrested in S phase. Similar results were obtained by in vivo transplantation, as TPX2 siRNA transfection significantly reduced tumor growth of the xenograft in nude mice.

Conclusion

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.

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.

References (33)

  • O.J. Gruss et al.

    Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in Hela cells

    Nat Cell Biol

    (2002)
  • M.R. Anderson et al.

    Met receptor signaling: a key effector in esophageal adenocarcinoma

    Clin Cancer Res

    (2006)
  • H. Shigeishi et al.

    Expression of TPX2 in salivary gland carcinomas

    Oncol Rep

    (2009)
  • D.M. Lin et al.

    TPX2 expression and its significance in squamous cell carcinoma of lung

    Zhonghua Bing Li Xue Za Zhi

    (2006)
  • D. Colak et al.

    Age-specific gene expression signatures for breast tumors and cross-species conserved potential cancer progression markers in young women

    PLoS One

    (2013)
  • H.C. Liu et al.

    Expression of TPX2 mRNA and its correlation with clinical pathology in esophageal cancer

    J Clin Exp Pathol

    (2010)
  • Cited by (21)

    • Knockdown of circ_0003340 induces cell apoptosis, inhibits invasion and proliferation through miR-564/TPX2 in esophageal cancer cells

      2020, Experimental Cell Research
      Citation Excerpt :

      Upregulation of TPX2 enhanced the malignancy of ESCC [29], and it is associated with poor prognosis after ESCC operative treatment [30]. However, downregulation of TPX2 induced apoptosis and arrested proliferation and invasion in EC9706 cells [31]. Additionally, it has been confirmed that the expression of TPX2 could be affected by miRNA [32,33], and in the present study we verified that miR-564 decreased TPX2 protein levels.

    • TPX2 promotes migration and invasion of human breast cancer cells

      2015, Asian Pacific Journal of Tropical Medicine
      Citation Excerpt :

      More and more evidences show that the abnormal expression of TPX2 is related to the invasion and development of tumors. In many tumor tissues, abnormally high expression of TPX2 has been confirmed [12,13], such as colon cancer [14], esophagus cancer [15,16], bladder cancer [17], and liver cancer [18]. In addition, TPX2 is also confirmed to be a molecular marker for unfavorable prognosis in patients.

    • Downregulation of CDK-8 inhibits colon cancer hepatic metastasis by regulating Wnt/β-catenin pathway

      2015, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Taken together, these observations suggested that therapeutic interventions that target CDK-8 of colon cancer may be of clinical value. RNA interference (RNAi), which has been proven to be a powerful tool for suppressing gene expression, may provide a promising way forward in making this pathway a particularly attractive target therapy [10–13]. In this study, we explored the role of CDK-8 in colon cancer and found that knockdown of CDK-8 inhibited cell metastasis and invasion in vitro and in vivo.

    View all citing articles on Scopus
    View full text