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Short hairpin RNA-mediated down-regulation of CENP-A attenuates the aggressive phenotype of lung adenocarcinoma cells

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Abstract

Background

Deregulation of centromere protein (CENP)-A, a centromere-specific histone variant, has in the past been linked to cancer initiation and progression. Additionally, our previous work has shown that CENP-A upregulation predicts a poor overall survival in patients with lung adenocarcinoma. The aim of this study was to uncover the biological role of CENP-A in lung adenocarcinoma growth and invasion, including its underlying molecular mechanisms.

Methods

CENP-A expression was knocked down in human lung adenocarcinoma A549 and PC-9 cells using a short hairpin RNA (shRNA) technology. Subsequently, the effects of this knock down on the proliferation, apoptosis, cell cycle progression, colony formation, migration, invasion and tumorigenicity were assessed. Additionally, Western blot analyses were performed to examine concomitant expression changes in key proteins involved in cell cycle regulation and apoptosis.

Results

We found that shRNA-mediated knock down of CENP-A significantly inhibited the in vitro proliferation and colony formation of A549 and PC-9 cells as compared to control shRNA-transfected cells. In addition, CENP-A down-regulation was found to induce G0/G1 cell cycle arrest and apoptosis, and to inhibit the in vitro migration and invasion of A549 and PC-9 cells. Down-regulation of CENP-A was also found to significantly suppress the in vivo growth of xenografted A549 cells. At the protein level, we found that the expression of p21, p27, CHK2 and Bax was markedly increased and that the expression of CCNG1, Skp2, Cks1 and Bcl-2 was markedly decreased in CENP-A down-regulated cells.

Conclusion

Based on our results we conclude that down-regulation of CENP-A may attenuate the aggressive phenotype of lung adenocarcinoma cells. As such, CENP-A may serve as a promising therapeutic target for lung adenocarcinoma.

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Acknowledgments

This work was supported by grants from the Fund of Zhejiang Provincial Health Office (No.2013KYB217) to Qing Wu, and from “085” first-class discipline construction of science and technology innovation in Shanghai University of Traditional Chinese Medicine (No. 085ZY1220).

Competing interests

The authors have declared that no competing interests exist.

Author information

Authors and Affiliations

  1. Department of Respiratory Medicine, Guangxing Hospital, Hangzhou Hospital of Traditional Chinese Medicine, Zhejiang University of Traditional Chinese Medicine, Hangzhou, People’s Republic of China

    Qing Wu

  2. Department of pathology, maternal and child care service centre, Xinjiang Uighur Autonomous Region, Urumqi, People’s Republic of China

    Yong-Feng Chen

  3. Department of Radiation Oncology, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, People’s Republic of China

    Jie Fu

  4. Department of Pathology, The First Hospital, Medical college, Zhejiang University, Hangzhou, People’s Republic of China

    Qi-Han You

  5. Department of Pathology, Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China

    Shou-Mei Wang, Xue Huang, Xiao-Jun Feng & Shu-Hui Zhang

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  1. Qing Wu
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  2. Yong-Feng Chen
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Corresponding authors

Correspondence to Qing Wu or Shu-Hui Zhang.

Additional information

Qing Wu, Yong-Feng Chen and Jie Fu have contributed equally to this research.

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Fig. S1

Measurement of the expression levels of CENP-A in A549 cells and non-malignant human lung epithelial cells (HBEC4 and LL 24). Real-time PCR analysis revealed a 6-8-fold over-expression of CENP-A in A549 cells compared to non-malignant human lung epithelial cells (HBEC4 and LL 24). (JPEG 507 kb)

Fig. S2

Effect of CENP-A silencing on expression of CENP-A in A549 cells. Compared to control shRNA-transfected cells, the delivery of CENP-A1 shRNA significantly reduced the mRNA and protein expression levels of CENP-A at 48 h after transfection. (JPEG 533 kb)

Fig. S3

The CENP-A expression level was comparable between CENP-A-silenced A549 lung adenocarcinoma cells and LL 24 normal lung epithelial cells. (JPEG 284 kb)

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Wu, Q., Chen, YF., Fu, J. et al. Short hairpin RNA-mediated down-regulation of CENP-A attenuates the aggressive phenotype of lung adenocarcinoma cells. Cell Oncol. 37, 399–407 (2014). https://doi.org/10.1007/s13402-014-0199-z

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