Skip to main content
SpringerLink
Log in

Downregulation of PEBP4, a target of miR-34a, sensitizes drug-resistant lung cancer cells

  • Research Article
  • Published:
Tumor Biology

Abstract

The aim of this study was to determine the relationship and underlying mechanisms between ectopic expression of phosphatidylethanolamine-binding protein 4 (PEBP4) and cisplatin (DDP)-induced cytotoxicity in the lung cancer cell line A549 to provide an experimental basis for future chemotherapeutic applications involving PEBP4 in human lung cancer. A recombinant plasmid, pcDNA3-PEBP4, and a PEBP4-targeting small hairpin RNA (shRNA) were transfected into the lung cancer cell line A549. The PEBP4 protein expression levels were determined for each group by Western blot, and after 48 h of cisplatin (DDP) treatment, the viability of cells in the treatment and control groups was determined by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis in each treatment group was determined using flow cytometry. Western blotting was used to examine expression of the p53 protein in A549 cells from each group. We employed a luciferase reporter-gene assay to confirm PEBP4 as a target gene of miR-34a. Western blotting was used to determine the effects of miR-34a on PEBP4 protein expression in A549 cells. Following transfection of A549 cells with either the recombinant plasmid pcDNA3-PEBP4 or a PEBP4-targeting shRNA, Western blotting analyses showed PEBP4 protein expression was significantly higher in the pcDNA3-PEBP4-transfected group compared with the control or PEBP4-shRNA-transfected groups (p < 0.01). Furthermore, PEBP4 protein expression was significantly reduced in the PEBP4-shRNA-transfected group (p < 0.01). After 48 h of DDP treatment, MTT assays indicated that A549 cell viability was significantly lower in the DDP-treated group compared with the control group (p < 0.01). The viability of A549 cells in the pcDNA3-PEBP4-transfected group was lower than that in the control group (p < 0.05) but higher than that in either the DDP-treated or PEBP4-shRNA-transfected groups (p < 0.05). Moreover, the viability of A549 cells in the PEBP4-shRNA-transfected group was significantly lower than that in either the control (p < 0.01) or DDP-treated (p < 0.05) groups. Flow cytometry and Western blotting analyses indicated that the number of apoptotic cells and p53 protein expression were significantly higher in the DDP-treated group compared with the control group (p < 0.01). In the pcDNA3-PEBP4-transfected group, the number of apoptotic cells and p53 protein expression level were higher than those in the control group (p < 0.05) but lower than those in the DDP-treated and PEBP4-shRNA-transfected groups (p < 0.05). The number of apoptotic cells and p53 protein expression level in the PEBP4-shRNA-transfected group were higher than those in the control (p < 0.01) and DDP-treated (p < 0.05) groups. The luciferase reporter-gene assay showed that the relative luciferase activity after transfection with a miR-34a mimic was significantly reduced compared with the control group (p < 0.01). Western blotting analysis demonstrated that PEBP4 protein expression was significantly decreased in A549 cells 48 h after transfection with a miR-34a mimic compared with the control group (p < 0.01). In conclusion, overexpression of PEBP4 reduced the sensitivity of A549 cells to DDP-induced cytotoxicity, mainly through the altered expression of the p53 protein or the modulation of miR-34a.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Marshall HM, Bowman RV, Yang IA, Fong KM, Berg CD. Screening for lung cancer with low-dose computed tomography: a review of current status. J Thorac Dis. 2013;5:S524–39.

    PubMed Central  PubMed  Google Scholar 

  2. Wakelee H. Management of early stage non-small cell lung cancer. Transl Lung Cancer Res. 2013;2:315.

    PubMed Central  PubMed  Google Scholar 

  3. Templeton AK, Miyamoto S, Babu A, Munshi A, Ramesh R. Cancer stem cells: progress and challenges in lung cancer. Stem Cell Investigation. 2014;1:99.

    Google Scholar 

  4. Chen W, Zheng R, Zhang S, Zhao P, Li G, Wu L, et al. Report of incidence and mortality in China cancer registries, 2009. Chin J Cancer Res. 2013;25:10–21.

    PubMed Central  CAS  PubMed  Google Scholar 

  5. Park BJ. Robotic lobectomy for non-small cell lung cancer (NSCLC): multi-registry study of long-term oncologic results. Ann Cardiothorac Surg. 2012;1(1):24–6.

    PubMed Central  PubMed  Google Scholar 

  6. Bar J, Urban D, Borshtein R, Nechushtan H, Onn A. EGFR mutation in lung cancer: tumor heterogeneity and the impact of chemotherapy. Chin Clin Oncol. 2013;2:2.

    PubMed  Google Scholar 

  7. Zhan P, Qian Q, Wan B, Yan TD, Yu LK. Prognostic value of TTF-1 expression in patients with non-small cell lung cancer: a meta-analysis. Transl Cancer Res. 2013;2:25–32.

    Article  Google Scholar 

  8. Bernier I, Jolles P. Purification and characterization of a basic 23 kDa cytosolic protein from bovine brain. Biochim Biophys Acta. 1984;790(2):174–81.

    Article  CAS  PubMed  Google Scholar 

  9. Garcia R, Grindlay J, Rath O, et al. Regulation of human myoblast differentiation by PEBP4. EMBO Rep. 2009;10(3):278–84.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Amaya I, Ratcliffe OJ, Bradley DJ. Expression of CENTRORADIALIS (CEN) and CEN-like genes in tobacco reveals a conserved mechanism controlling phase change in diverse species. Plant Cell. 1999;11(8):1405–18.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Erttmann KD, Gallin MY. Onchocerca volvulus: identification of cDNAs encoding a putative phosphatidyl-ethanolamine-binding protein and a putative partially processed mRNA precursor. Gene. 1996;174(2):203–7.

    Article  CAS  PubMed  Google Scholar 

  12. Wilson R, Ainscough R, Anderson K, et al. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994;368(6466):32–8.

    Article  CAS  PubMed  Google Scholar 

  13. Kikuchi R, Kawahigashi H, Ando T, et al. Molecular and functional characterization of PEBP genes in barley reveal the diversification of their roles in flowering. Plant Physiol. 2009;149(3):1341–53.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Odabaei G, Chatterjee D, Jazirehi AR, et al. Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis. Adv Cancer Res. 2004;91:169–200.

    Article  CAS  PubMed  Google Scholar 

  15. Zeng L, Imamoto A, Rosner MR. Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target. Expert Opin Ther Targets. 2008;12(10):1275–87.

    Article  CAS  PubMed  Google Scholar 

  16. Keller ET, Fu Z, Brennan M. The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein. J Cell Biochem. 2005;94(2):273–8.

    Article  CAS  PubMed  Google Scholar 

  17. Sagisaka T, Matsukawa N, Toyoda T, et al. Directed neural lineage differentiation of adult hippocampal progenitor cells via modulation of hippocampal cholinergic neurostimulating peptide precursor expression. Brain Res. 2010;1327:107–17.

    Article  CAS  PubMed  Google Scholar 

  18. Yeung K, Seitz T, Li S, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature. 1999;401(6749):173–7.

    Article  CAS  PubMed  Google Scholar 

  19. Corbit KC, Trakul N, Eves EM, et al. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem. 2003;278(15):13061–8.

    Article  CAS  PubMed  Google Scholar 

  20. Shemon AN, Heil GL, Granovsky AE, et al. Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands. PLoS One. 2010;5(5):e10479.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Yu GP, Huang B, Chen GQ, et al. PEBP4 gene expression and its significance in invasion and metastasis of non-small cell lung cancer. Tumour Biol. 2012;33(1):223–8.

    Article  CAS  PubMed  Google Scholar 

  22. Wang X, Li N, Liu B, et al. A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization. J Biol Chem. 2004;279(44):45855–64.

    Article  CAS  PubMed  Google Scholar 

  23. Liu H, Qiu J, Li N, et al. Human phosphatidylethanolamine-binding protein 4 promotes transactivation of estrogen receptor alpha (ERalpha) in human cancer cells by inhibiting proteasome-dependent ERalpha degradation via association with Src. J Biol Chem. 2010;285(29):21934–42.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Qiu J, Xiao J, Han C, et al. Potentiation of tumor necrosis factor-alpha-induced tumor cell apoptosis by a small molecule inhibitor for anti-apoptotic protein hPEBP4. J Biol Chem. 2010;285(16):12241–7.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Yu GP, Chen GQ, Wu S, et al. The expression of PEBP4 protein in lung squamous cell carcinoma. Tumour Biol. 2011;32(6):1257–63.

    Article  CAS  PubMed  Google Scholar 

  26. Zhang J, Wang L, Xing Z, et al. Status of bi- and multi-nuclear platinum anticancer drug development. Anticancer Agents Med Chem. 2011;10(4):272–82.

    Article  Google Scholar 

  27. Zhang J, Liu D, Li Y, et al. Status of non-classical mononuclear platinum anticancer drug development. Mini Rev Med Chem. 2009;9(11):1357–66.

    Article  CAS  PubMed  Google Scholar 

  28. Shen Z, Zhan G, Ye D, et al. MicroRNA-34a affects the occurrence of laryngeal squamous cell carcinoma by targeting the antiapoptotic gene survivin. Med Oncol. 2012;29(4):2473–80.

    Article  CAS  PubMed  Google Scholar 

  29. Link A, Kupcinskas J, Wex T, et al. Macro-role of microRNA in gastric cancer. Dig Dis. 2012;30(3):255–67.

    Article  PubMed  Google Scholar 

  30. Wong MY, Yu Y, Walsh WR, et al. microRNA-34 family and treatment of cancers with mutant or wild-type p53 (Review). Int J Oncol. 2011;38(5):1189–95.

    CAS  PubMed  Google Scholar 

  31. O'Day E, Lal A. MicroRNAs and their target gene networks in breast cancer. Breast Cancer Res. 2010;12(2):201.

    Article  PubMed Central  PubMed  Google Scholar 

  32. Cha YH, Kim NH, Park C, et al. MiRNA-34 intrinsically links p53 tumor suppressor and Wnt signaling. Cell Cycle. 2012;11(7):1273–81.

    Article  CAS  PubMed  Google Scholar 

  33. Balca-Silva J, Neves SS, Goncalves AC, et al. Effect of miR-34b overexpression on the radiosensitivity of non-small cell lung cancer cell lines. Anticancer Res. 2012;32(5):1603–9.

    CAS  PubMed  Google Scholar 

  34. Hui C, Yujie F, Lijia Y, et al. MicroRNA-34a and microRNA-21 play roles in the chemopreventive effects of 3,6-dihydroxyflavone on 1-methyl-1-nitrosourea-induced breast carcinogenesis. Breast Cancer Res. 2012;14(3):R80.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Zhang LN, Li JY, Xu W. A review of the role of Puma, Noxa and Bim in the tumorigenesis, therapy and drug resistance of chronic lymphocytic leukemia. Cancer Gene Ther. 2013;20(1):1–7.

    Article  PubMed  Google Scholar 

  36. Shimizu K, Okita R, Nakata M. Clinical significance of the tumor microenvironment in non-small cell lung cancer. Ann Transl Med. 2013;1:20.

    PubMed Central  PubMed  Google Scholar 

  37. Wang X, Li N, Li H, et al. Silencing of human phosphatidylethanolamine-binding protein 4 sensitizes breast cancer cells to tumor necrosis factor-alpha-induced apoptosis and cell growth arrest. Clin Cancer Res. 2005;11(20):7545–53.

    Article  CAS  PubMed  Google Scholar 

  38. Li P, Wang X, Li N, et al. Anti-apoptotic hPEBP4 silencing promotes TRAIL-induced apoptosis of human ovarian cancer cells by activating ERK and JNK pathways. Int J Mol Med. 2006;18(3):505–10.

    CAS  PubMed  Google Scholar 

  39. Ji X, Wang Z, Sarkar FH, et al. Delta-tocotrienol augments cisplatin-induced suppression of non-small cell lung cancer cells via inhibition of the Notch-1 pathway. Anticancer Res. 2012;32(7):2647–55.

    CAS  PubMed  Google Scholar 

  40. Yu G, Shen Z, Chen G, et al. PEBP4 enhanced HCC827 cell proliferation and invasion ability and inhibited apoptosis. Tumour Biol. 2013;34(1):91–8.

    Article  CAS  PubMed  Google Scholar 

  41. Kumar B, Yadav A, Lang J, et al. Dysregulation of microRNA-34a expression in head and neck squamous cell carcinoma promotes tumor growth and tumor angiogenesis. PLoS One. 2012;7(5):e37601.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Li L, Yuan L, Luo J, et al. MiR-34a inhibits proliferation and migration of breast cancer through down-regulation of Bcl-2 and SIRT1. Clin Exp Med. 2013;13(2):109–17.

    Article  PubMed  Google Scholar 

Download references

Conflicts of interest

None.

Author information

Authors and Affiliations

  1. Department of Cardiothoracic Surgery, The Affiliated Jiangyin Hospital of Southeast University Medical College, No. 163, Shoushan Rd, Jiangyin, 214400, China

    Guiping Yu, Guoqiang Chen, Bin Huang & Song Wu

  2. Department of Thoracic Surgery, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan, 215300, China

    Ning Zhong

Authors
  1. Guiping Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ning Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoqiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Song Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guoqiang Chen.

Additional information

Guiping Yu and Ning Zhong contributed equally to this study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, G., Zhong, N., Chen, G. et al. Downregulation of PEBP4, a target of miR-34a, sensitizes drug-resistant lung cancer cells. Tumor Biol. 35, 10341–10349 (2014). https://doi.org/10.1007/s13277-014-2284-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-014-2284-3

Keywords

Search

Navigation