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MiR-608 regulating the expression of ribonucleotide reductase M1 and cytidine deaminase is repressed through induced gemcitabine chemoresistance in pancreatic cancer cells

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Abstract

Purpose

Gemcitabine resistance is the main problem in pancreatic adenocarcinoma patients. Hence, we aimed to identify the correlation between expression of RRM1 and CDA as the resistance genes and their predicted targeting miR-608 in the resistant pancreatic cancer cell lines to gemcitabine.

Methods

Dual luciferase assay was performed to determine whether both RRM1 and CDA are targeted by miR-608 in 293T and pancreatic cancer cell lines. AsPC-1 and MIA PaCa-2 cell lines became gradually resistant to gemcitabine by exposing to the increasing doses of gemcitabine. After RNA and miRNAs extraction and cDNA conversion, the expressions of RRM1, CDA and miR-608 in all cell lines were studied by quantitative PCR. Pre-miR-608 transfection to the cell lines was done by calcium phosphate method. MTT assay was performed for analyzing the chemo sensitivity of different cell lines to gemcitabine.

Results

Luciferase assays showed that miR-608 targeted RRM1 and CDA genes in 293T, AsPC-1 and MIA PaCa-2 cell lines. Compared to parental cell line, resistant MIA PaCa-2 and AsPC-1 cells demonstrated increased expression of RRM1 and CDA. On the other hand the expression of miR-608 in resistant MIA PaCa-2 and AsPC-1 cells was lower than parental cells. Furthermore, transfection of MIA PaCa-2 and AsPC-1 cells by miR-608 lead to decreased expression of RRM1 and CDA and lowered viability of the cells in comparison with scrambled microRNA transfected cells.

Conclusion

During resistance induction in pancreatic cancer cells, miR-608 which is targeting RRM1 and CDA is downregulated which leads to upregulation of these genes.

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Abbreviations

PDAC:

Pancreatic adenocarcinoma

GEM:

Gemcitabine

CDA:

Cytidine deaminase

RRM1:

Ribonucleotide reductase M1

dNTP:

Deoxynucleoside triphosphate

miR:

miRNA, microRNA

dFdU:

2′2′-Difluorodeoxyuridine

IC:

Inhibitory concentration

MTT:

3-(4,5-Di methylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide

References

  1. Long J, Zhang Y, Yu X, Yang J, LeBrun DG, Chen C, Yao Q, Li M (2011) Overcoming drug resistance in pancreatic cancer. Expert Opin Ther Targets 15(7):817–828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Valsecchi ME, Holdbrook T, Leiby BE, Pequignot E, Littman SJ, Yeo CJ, Brody JR, Witkiewicz AK (2012) Is there a role for the quantification of RRM1 and ERCC1 expression in pancreatic ductal adenocarcinoma? BMC Cancer 12(1):1

    Article  Google Scholar 

  3. Fujita H, Ohuchida K, Mizumoto K, Itaba S, Ito T, Nakata K, Yu J, Kayashima T, Souzaki R, Tajiri T (2010) Gene expression levels as predictive markers of outcome in pancreatic cancer after gemcitabine-based adjuvant chemotherapy. Neoplasia 12(10):807–808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hagmann W, Jesnowski R, Löhr JM (2010) Interdependence of gemcitabine treatment, transporter expression, and resistance in human pancreatic carcinoma cells. Neoplasia 12(9):740–747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rajabpour A, Rajaei F, Teimoori-Toolabi L (2016) Molecular alterations contributing to pancreatic cancer chemoresistance. Pancreatology 17(2):310–320

    Article  PubMed  Google Scholar 

  6. Rukov JL, Shomron N (2011) MicroRNA pharmacogenomics: post-transcriptional regulation of drug response. Trends Mol Med 17(8):412–423

    Article  CAS  PubMed  Google Scholar 

  7. Bhutia YD, Hung SW, Patel B, Lovin D, Govindarajan R (2011) CNT1 expression influences proliferation and chemosensitivity in drug-resistant pancreatic cancer cells. Can Res 71(5):1825–1835

    Article  CAS  Google Scholar 

  8. Ueno H, Kiyosawa K, Kaniwa N (2007) Pharmacogenomics of gemcitabine: Can genetic studies lead to tailor-made therapy? Br J Cancer 97(2):145–151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nakano Y, Tanno S, Koizumi K, Nishikawa T, Nakamura K, Minoguchi M, Izawa T, Mizukami Y, Okumura T, Kohgo Y (2007) Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells. Br J Cancer 96(3):457–463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Souglakos J, Boukovinas I, Taron M, Mendez P, Mavroudis D, Tripaki M, Hatzidaki D, Koutsopoulos A, Stathopoulos E, Georgoulias V (2008) Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine. Br J Cancer 98(10):1710–1715

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jordheim LP, Dumontet C (2013) Do hENT1 and RRM1 predict the clinical benefit of gemcitabine in pancreatic cancer? Biomarkers 7(4):663–671

    Article  CAS  Google Scholar 

  12. Jordheim LP, Sève P, Trédan O, Dumontet C (2011) The ribonucleotide reductase large subunit (RRM1) as a predictive factor in patients with cancer. Lancet Oncol 12(7):693–702

    Article  CAS  PubMed  Google Scholar 

  13. Elnaggar M, Giovannetti E, Peters GJ (2012) Molecular targets of gemcitabine action: rationale for development of novel drugs and drug combinations. Curr Pharm Des 18(19):2811–2829

    Article  CAS  PubMed  Google Scholar 

  14. Carpi FM, Vincenzetti S, Ubaldi J, Pucciarelli S, Polzonetti V, Micozzi D, Mignini F, Napolioni V (2013) CDA gene polymorphisms and enzyme activity: genotype–phenotype relationship in an Italian-Caucasian population. Pharmacogenomics 14(7):769–781

    Article  CAS  PubMed  Google Scholar 

  15. Sugiyama E, Kaniwa N, Kim S-R, Kikura-Hanajiri R, Hasegawa R, Maekawa K, Saito Y, Ozawa S, J-i Sawada, Kamatani N (2006) Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism. J Clin Oncol 25(1):32–42

    Article  Google Scholar 

  16. Funamizu N, Okamoto A, Kamata Y, Misawa T, Uwagawa T, Gocho T, Yanaga K, Manome Y (2010) Is the resistance of gemcitabine for pancreatic cancer settled only by overexpression of deoxycytidine kinase? Oncol Rep 23(2):471

    CAS  PubMed  Google Scholar 

  17. Rukov JL, Wilentzik R, Jaffe I, Vinther J, Shomron N (2013) Pharmaco-miR: linking microRNAs and drug effects. Brief Bioinform 15:648–659

    Article  PubMed  PubMed Central  Google Scholar 

  18. Hashimoto Y, Akiyama Y, Yuasa Y (2013) Multiple-to-multiple relationships between microRNAs and target genes in gastric cancer. PLoS ONE 8(5):e62589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang XJ, Ye H, Zeng CW, He B, Zhang H, Chen YQ (2010) Dysregulation of miR-15a and miR-214 in human pancreatic cancer. J Hematol Oncol 3(1):1

    Article  Google Scholar 

  20. Hwang J-H, Voortman J, Giovannetti E, Steinberg SM, Leon LG, Kim Y-T, Funel N, Park JK, Kim MA, Kang GH (2010) Identification of microRNA-21 as a biomarker for chemoresistance and clinical outcome following adjuvant therapy in resectable pancreatic cancer. PLoS ONE 5(5):e10630

    Article  PubMed  PubMed Central  Google Scholar 

  21. Ji Q, Hao X, Zhang M, Tang W, Yang M, Li L, Xiang D, DeSano JT, Bommer GT, Fan D (2009) MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells. PLoS ONE 4(8):e6816

    Article  PubMed  PubMed Central  Google Scholar 

  22. Li Y, VandenBoom TG, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH (2009) Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Can Res 69(16):6704–6712

    Article  CAS  Google Scholar 

  23. Park J-K, Lee EJ, Esau C, Schmittgen TD (2009) Antisense inhibition of microRNA-21 or-221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas 38(7):e190–e199

    Article  CAS  PubMed  Google Scholar 

  24. Nakahira S, Nakamori S, Tsujie M, Takahashi Y, Okami J, Yoshioka S, Yamasaki M, Marubashi S, Takemasa I, Miyamoto A (2007) Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer. Int J Cancer 120(6):1355–1363

    Article  CAS  PubMed  Google Scholar 

  25. Azizi M, Teimoori-Toolabi L, Arzanani MK, Azadmanesh K, Fard-Esfahani P, Zeinali S (2014) MicroRNA-148b and microRNA-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines. Cancer Biol Ther 15(4):419–427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gheidari F, Bakhshandeh B, Teimoori-Toolabi L, Mehrtash A, Ghadir M, Zeinali S (2014) TCF4 silencing sensitizes the colon cancer cell line to oxaliplatin as a common chemotherapeutic drug. Anticancer Drugs 25(8):908–916

    Article  CAS  PubMed  Google Scholar 

  27. Davidson JD, Ma L, Flagella M, Geeganage S, Gelbert LM, Slapak CA (2004) An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines. Can Res 64(11):3761–3766

    Article  CAS  Google Scholar 

  28. Bengala C, Guarneri V, Giovannetti E, Lencioni M, Fontana E, Mey V, Fontana A, Boggi U, Del Chiaro M, Danesi R (2005) Prolonged fixed dose rate infusion of gemcitabine with autologous haemopoietic support in advanced pancreatic adenocarcinoma. Br J Cancer 93(1):35–40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu X, Wang W, Lin L, Song S (2010) Expression of Gemcitabine-resistance-related gene and polymorphism of ribonucleotide reductase M1 gene promoter in Gemcitabine-resistant A549/Gem and NCI-H460/Gem cell lines. Zhonghua Zhong Liu Za Zhi [Chin J Oncol] 32(1):17–21

    Google Scholar 

  30. Wang J, Chen J, Chang P, LeBlanc A, Li D, Abbruzzesse JL, Frazier ML, Killary AM, Sen S (2009) MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease. Cancer Prev Res 2(9):807–813

    Article  CAS  Google Scholar 

  31. Zheng J, Deng J, Xiao M, Yang L, Zhang L, You Y, Hu M, Li N, Wu H, Li W (2013) A sequence polymorphism in miR-608 predicts recurrence after radiotherapy for nasopharyngeal carcinoma. Can Res 73(16):5151–5162

    Article  CAS  Google Scholar 

  32. Ma X-P, Yu G, Chen X, Xiao Q, Shi Z, Zhang L-Y, Chen H, Zhang P, Ding D-L, Huang H-X (2016) MiR-608 rs4919510 is associated with prognosis of hepatocellular carcinoma. Tumor Biol 37(7):9931–9942

    Article  CAS  Google Scholar 

  33. Ryan BM, McClary AC, Valeri N, Robinson D, Paone A, Bowman ED, Robles AI, Croce C, Harris CC (2012) rs4919510 in hsa-mir-608 is associated with outcome but not risk of colorectal cancer. PLoS ONE 7(5):e36306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Huang A-J, Yu K-D, Li J, Fan L, Shao Z-M (2012) Polymorphism rs4919510: C > G in mature sequence of human microRNA-608 contributes to the risk of HER2-positive breast cancer but not other subtypes. PLoS ONE 7(5):e35252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Alves R, Antunes F, Salvador A (2006) Tools for kinetic modeling of biochemical networks. Nat Biotechnol 24(6):667–672

    Article  CAS  PubMed  Google Scholar 

  36. Wang S, Raghavachari S (2011) Quantifying negative feedback regulation by micro-RNAs. Phys Biol 8(5):055002

    Article  PubMed  PubMed Central  Google Scholar 

  37. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135(2):216–226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pedraza JM, van Oudenaarden A (2005) Noise propagation in gene networks. Science 307(5717):1965–1969

    Article  CAS  PubMed  Google Scholar 

  39. Bhardwaj V, Bhushan A, Lai JC, Tadinada SM (2012) Failure of pancreatic cancer chemotherapy: consequences of drug resistance mechanisms. INTECH Open Access Publisher, Rijeka

    Google Scholar 

  40. Azmi AS, Beck FW, Bao B, Mohammad RM, Sarkar FH (2011) Aberrant epigenetic grooming of miRNAs in pancreatic cancer:a systems biology perspective. Epigenomics 3(6):747–759

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Achiwa H, Oguri T, Sato S, Maeda H, Niimi T, Ueda R (2004) Determinants of sensitivity and resistance to gemcitabine: the roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non-small cell lung cancer. Cancer Sci 95(9):753–757

    Article  CAS  PubMed  Google Scholar 

  42. Samulitis BK, Pond KW, Pond E, Cress AE, Patel H, Wisner L, Patel C, Dorr RT, Landowski TH (2015) Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors. Cancer Biol Ther 16(1):43–51

    Article  CAS  PubMed  Google Scholar 

  43. Kasinski AL, Slack FJ (2011) MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nat Rev Cancer 11(12):849–864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This research was funded by Qazvin University of Medical Sciences (Grant Number: 28/20/10563) and Pasteur Institute of Iran (Grant Number: 94/0201/1405).

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Authors and Affiliations

  1. Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

    Azam Rajabpour & Farzad Rajaei

  2. Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran

    Azam Rajabpour, Ali Afgar & Ladan Teimoori-Toolabi

  3. Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

    Azam Rajabpour & Farzad Rajaei

  4. Cancer Institute of Iran, Imam Khomeini Medical Complex, Tehran University of Medical Sciences, Tehran, Iran

    Habibollah Mahmoodzadeh

  5. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

    Jalal-e-Din Radfar

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  1. Azam Rajabpour
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Correspondence to Farzad Rajaei or Ladan Teimoori-Toolabi.

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Conflict of interest

Azam Rajabpour declares that she has no conflict of interest. Ali Afgar declares that he has no conflict of interest. Habibollah Mahmoodzadeh declares that he has no conflict of interest. Jalal-e-Din Radfar declares that he has no conflict of interest. Farzad Rajaei declares that he has no conflict of interest. Ladan Teimoori-Toolabi declares that she has no conflict of interest.

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Rajabpour, A., Afgar, A., Mahmoodzadeh, H. et al. MiR-608 regulating the expression of ribonucleotide reductase M1 and cytidine deaminase is repressed through induced gemcitabine chemoresistance in pancreatic cancer cells. Cancer Chemother Pharmacol 80, 765–775 (2017). https://doi.org/10.1007/s00280-017-3418-2

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