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Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells

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Abstract

Oncogenic KRAS, an important target for antitumor therapy, contributes to pancreatic cancer tumorigenesis, progression and maintenance. However, intracellular compensation regulation can help cells to resist the targeted therapy. Gene knockdown method such as RNAi may help to understand this intracellular regulatory system and discover novel therapeutic approach. In this study, two stable transfected cell lines were constructed through lentivirus-mediated shRNA targeting KRAS of PANC-1 cells, to investigate cell response to the knockdown of KRAS. Human whole genome microarray was then used to compare the gene expression profile. As a result, ribosomal proteins L26 and L29 (RPL26 and RPL29) were dramatically upregulated by KRAS-shRNA specifically. To identify whether RPL26 or RPL29 was critical for PANC-1 cells, siRNAs against RPL26 and RPL29 were designed and transfected in vitro. The results showed that knockdown of RPL26 or RPL29 expression significantly suppressed cell proliferation, induced cell arrest at G0/G1 phase and enhanced cell apoptosis. Reactive oxygen species (ROS) assay indicated that silencing of RPL26 or RPL29 markedly decreased the intracellular ROS generation. Our findings imply that siRNA interference against RPL26 and RPL29 is of potential value for intervention of pancreatic cancer.

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Abbreviations

RNAi:

RNA interference

shRNA:

Short hairpin RNA

siRNA:

Small interfering RNA

ROS:

Reactive oxygen species

PC:

Pancreatic cancer

FBS:

Fetal bovine serum

ACDU:

Automated cell deposition unit

FCM:

Flow cytometry

NC:

Negative control

qPCR:

Quantitative real-time PCR

FITC:

Fluorescein isothiocyanate

PI:

Propidium iodide

PBS:

Phosphate-buffered saline

DCFH-DA:

2,7-Dichlorfluorescein-diacetate

DCF:

2,7-Dichlorfluorescein

MTT:

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

DMSO:

Dimethyl sulfoxide

GO:

Gene Ontology

PVDF:

Polyvinylidene fluoride

References

  1. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ (1998) Increasing complexity of Ras signaling. Oncogene 17:1395–1413

    Article  PubMed  CAS  Google Scholar 

  2. McCormick F (1989) ras GTPase activating protein: signal transmitter and signal terminator. Cell 56:5–8

    Article  PubMed  CAS  Google Scholar 

  3. Jaffee EM, Hruban RH, Canto M, Kern SE (2002) Focus on pancreas cancer. Cancer Cell 2:25–28

    Article  PubMed  CAS  Google Scholar 

  4. Jemal A, Murray T, Ward E et al (2005) Cancer statistics, 2005. CA Cancer J Clin 55:10–30

    Article  PubMed  Google Scholar 

  5. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M (1988) Most human carcinomas of the exocrine pancreas contain mutant cK-ras genes. Cell 53:549–554

    Article  PubMed  CAS  Google Scholar 

  6. Shi X, Liang Z, Ren X, Liu T (2008) Combined silencing of K-ras and Akt2 oncogenes achieves synergistic effects in inhibiting pancreatic cancer cell growth in vitro and in vivo. Cancer Gene Ther 16:227–236

    PubMed  Google Scholar 

  7. Zhao M, He H, Sun H, Ren K, Shao R (2009) Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells. Biochem Biophys Res Commun 387:239–244

    Article  PubMed  CAS  Google Scholar 

  8. Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nat Rev Cancer 5:275–284

    Article  PubMed  CAS  Google Scholar 

  9. Gottesman MM (2002) Mechanisms of cancer drug resistance. Annu Rev Med 53:615–627

    Article  PubMed  CAS  Google Scholar 

  10. Fleming JB, Shen GL, Holloway SE, Davis M, Brekken RA (2005) Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-C directed therapy. Mol Cancer Res 3:413–423

    Article  PubMed  CAS  Google Scholar 

  11. Hopfner M, Schuppan D, Scherubl H (2008) Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer. World J Gastroenterol 14:1–14

    Article  PubMed  Google Scholar 

  12. Moffat J, Grueneberg DA, Yang X et al (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283–1298

    Article  PubMed  CAS  Google Scholar 

  13. Kenneth JL, Thomas DS (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆ Ct method. Methods 25:402–408

    Article  Google Scholar 

  14. Jiang R, Cabras G, Sheng W, Zeng Y, Ooka T (2009) Synergism of BARF1 with Ras induces malignant transformation in primary primate epithelial cells and human nasopharyngeal epithelial cells. Neoplasia 11:964–973

    PubMed  CAS  Google Scholar 

  15. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  PubMed  CAS  Google Scholar 

  16. Ruggero D, Pandolfi PP (2003) Does the ribosome translate cancer? Nat Rev Cancer 3:179–192

    Article  PubMed  CAS  Google Scholar 

  17. Amsterdam A, Sadler KC, Lai K, Farrington S, Bronson RT, Lees JA, Hopkins N (2004) Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol 2:690–698

    Article  CAS  Google Scholar 

  18. Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8:579–591

    Article  PubMed  CAS  Google Scholar 

  19. DeNicola GM, Karreth FA, Humpton TJ et al (2011) Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475:106–109

    Article  PubMed  CAS  Google Scholar 

  20. Fang J, Seki T, Maeda H (2009) Therapeutic strategies by modulating oxygen stress in cancer and inflammation. Adv Drug Deliv Rev 61:290–302

    Article  PubMed  CAS  Google Scholar 

  21. Friday BB, Adjei AA (2005) K-ras as a target for cancer therapy. Biochim Biophys Acta 1756:127–144

    PubMed  CAS  Google Scholar 

  22. Ji Z, Mei FC, Lory PL, Gilbertson SR, Chen Y, Cheng X (2009) Chemical genetic screening of KRAS-based synthetic lethal inhibitors for pancreatic cancer. Front Biosci 14:2904–2910

    Article  PubMed  CAS  Google Scholar 

  23. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553

    Article  PubMed  CAS  Google Scholar 

  24. Lee NS, Dohjima T, Bauer G et al (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20:500–505

    PubMed  CAS  Google Scholar 

  25. Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P (2006) A versatile tool for conditional gene expression and knockdown. Nat Methods 3:109–116

    Article  PubMed  CAS  Google Scholar 

  26. Brummelkamp TR, Bernards R, Agami R (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243–247

    Article  PubMed  CAS  Google Scholar 

  27. Links M, Brown R (1999) Clinical relevance of the molecular mechanisms of resistance to anti-cancer drugs. Expert Rev Mol Med 1:1–21

    Google Scholar 

  28. Naito S, Yokomizo A, Koga H (1999) Mechanisms of drug resistance in chemotherapy for urogenital carcinoma. Int J Urol 6:427–439

    Article  PubMed  CAS  Google Scholar 

  29. Warner JR, McIntosh KB (2009) How common are extraribosomal functions of ribosomal proteins? Mol Cell 34:3–11

    Article  PubMed  CAS  Google Scholar 

  30. Takagi M, Absalon MJ, McLure KG, Kastan MB (2005) Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell 123:49–63

    Article  PubMed  CAS  Google Scholar 

  31. Wang Y, Cheong D, Chan S, Hooi SC (1999) Heparin/heparan sulfate interacting protein gene expression is up-regulated in human colorectal carcinoma and correlated with differentiation status and metastasis. Cancer Res 59:2989–2994

    PubMed  CAS  Google Scholar 

  32. Liu JJ, Huang BH, Zhang J, Carson DD, Hooi SC (2006) Repression of HIP/RPL29 expression induces differentiation in colon cancer cells. J Cell Physiol 207:287–292

    Article  PubMed  CAS  Google Scholar 

  33. Liu JJ, Zhang J, Ramanan S, Julian JA, Carson DD, Hooi SC (2004) Heparin/heparan sulfate interacting protein plays a role in apoptosis induced by anticancer drugs. Carcinogenesis 25:873–879

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr Jianzhong Xi (Peking University, Department of Biomedical Engineering College of Engineering, Beijing, China) for lentiviruses production and critical reading of the manuscript. National New Drug Research and Development Project (Grant 2010ZX09401-403) supported this work.

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Authors and Affiliations

  1. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China

    Chaodong Li & Daijie Chen

  2. Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai, 200240, People’s Republic of China

    Chaodong Li, Mei Ge, Yu Yin & Minyu Luo

  3. Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China

    Mei Ge & Yu Yin

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  1. Chaodong Li
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  2. Mei Ge
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  3. Yu Yin
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  4. Minyu Luo
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  5. Daijie Chen
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Correspondence to Daijie Chen.

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Li, C., Ge, M., Yin, Y. et al. Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells. Mol Cell Biochem 370, 127–139 (2012). https://doi.org/10.1007/s11010-012-1404-x

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  • DOI: https://doi.org/10.1007/s11010-012-1404-x

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