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BMC Cancer

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Open Access 01-12-2015 | Research article

Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer

Authors: Alexey Popov, Arpad Szabo, Václav Mandys

Published in: BMC Cancer | Issue 1/2015

Abstract

Background

RT-qPCR quantification of miRNAs expression may play an essential role in pancreatic ductal adenocarcinoma (PDAC) diagnostics. RT-qPCR-based experiments require endogenous controls for the result normalization and reliability. However, expression instability of reference genes in tumors may introduce bias when determining miRNA levels.

Methods

We investigated expression of 6 miRNAs, isolated from FFPE samples of pancreatic adenocarcinomas. Four internal controls were utilized for RT-qPCR result normalization: artificial miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91.

Results

We found miR-21, miR-155 or miR-217 expression values in tumors may differ up to several times, depending on selected internal controls. Moreover, different internal controls can produce controversial results for miR-96, miR-148a or miR-196a quantification. Also, expression of our endogenous controls varied significantly in tumors. U6 demonstrated variation from −1.03 to 8.12-fold, miR-16 from −2.94 up to 7.38-fold and the U91 from −3.05 to 4.36-fold respectively. On the other hand, the most stable gene, determined by NormFinder algorithm, was U91. Each miRNA normalized relatively to the spike or U91, demonstrated similar expression values. Thus, statistically significant and insignificant differences between tumors and normal tissues for miRNAs were equal for the spike and the U91. Also, the differences between the spike and U91 were statistically insignificant for all of miRs except miR-217. Among three endogenous controls, U91 had the lowest average expression values and standard deviation in cancer tissues.

Conclusions

We recommend U91 as a new normalizer for miRNA quantification in PDACs.

Hidalgo M. Pancreatic cancer. The New England journal of medicine. 2010;362(17):1605–17. doi:10.1056/NEJMra0901557.CrossRefPubMed

Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. P Natl Acad Sci USA. 2008;105(30):10513–8. doi:10.1073/pnas.0804549105.CrossRef

Sassen S, Miska EA, Caldas C. MicroRNA - implications for cancer. Virchows Arch. 2008;452(1):1–10. doi:10.1007/s00428-007-0532-2.CrossRefPubMed

Shenouda SK, Alahari SK. MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metast Rev. 2009;28(3–4):369–78. doi:10.1007/s10555-009-9188-5.CrossRef

Zhang BH, Pan XP, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol. 2007;302(1):1–12. doi:10.1016/j.ydbio.2006.08.028.CrossRefPubMed

Lynam-Lennon N, Maher SG, Reynolds JV. The roles of microRNA in cancer and apoptosis. Biol Rev. 2009;84(1):55–71. doi:10.1111/j.1469-185X.2008.00061.x.CrossRefPubMed

Lee EJ, Gusev Y, Jiang JM, Nuovo GJ, Lerner MR, Frankel WL, et al. Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer. 2007;120(5):1046–54. doi:10.1002/Ijc.22394.CrossRefPubMedPubMedCentral

du Rieu MC, Torrisani J, Selves J, Al Saati T, Souque A, Dufresne M, et al. MicroRNA-21 Is Induced Early in Pancreatic Ductal Adenocarcinoma Precursor Lesions. Clin Chem. 2010;56(4):603–12. doi:10.1373/clinchem.2009.137364.CrossRefPubMed

Moriyama T, Ohuchida K, Mizumoto K, Yu J, Sato N, Nabae T, et al. MicroRNA-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion, and chemoresistance. Mol Cancer Ther. 2009;8(5):1067–74. doi:10.1158/1535-7163.Mct-08-0592.CrossRefPubMed

10.

Ryu JK, Hong SM, Karikari CA, Hruban RH, Goggins MG, Maitra A. Aberrant MicroRNA-155 Expression Is an Early Event in the Multistep Progression of Pancreatic Adenocarcinoma. Pancreatology. 2010;10(1):66–73. doi:10.1159/000231984.CrossRefPubMedPubMedCentral

11.

Habbe N, Koorstra JBM, Mendell JT, Offerhaus GJ, Ryu JK, Feldmann G, et al. MicroRNA miR-155 is a biomarker of early pancreatic neoplasia. Cancer Biol Ther. 2009;8(4):340–6. doi:10.4161/Cbt.8.4.7338.CrossRefPubMedPubMedCentral

12.

Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is regulated by the let-7 MicroRNA family. Cell. 2005;120(5):635–47. doi:10.1016/j.cell.2005.01.014.CrossRefPubMed

13.

Yu SN, Lu ZH, Liu CZ, Meng YX, Ma YH, Zhao WG, et al. miRNA-96 Suppresses KRAS and Functions as a Tumor Suppressor Gene in Pancreatic Cancer. Cancer Res. 2010;70(14):6015–25. doi:10.1158/0008-5472.Can-09-4531.CrossRefPubMed

14.

Zhao WG, Yu SN, Lu ZH, Ma YH, Gu YM, Chen J. The miR-217 microRNA functions as a potential tumor suppressor in pancreatic ductal adenocarcinoma by targeting KRAS. Carcinogenesis. 2010;31(10):1726–33. doi:10.1093/carcin/bgq160.CrossRefPubMed

15.

Hanoun N, Delpu Y, Suriawinata AA, Bournet B, Bureau C, Selves J, et al. The Silencing of MicroRNA 148a Production by DNA Hypermethylation Is an Early Event in Pancreatic Carcinogenesis. Clin Chem. 2010;56(7):1107–18. doi:10.1373/clinchem.2010.144709.CrossRefPubMed

16.

Mardin W, Mees ST. MicroRNAs: Novel Diagnostic and Therapeutic Tools for Pancreatic Ductal Adenocarcinoma? Ann Surg Oncol. 2009;16(11):3183–9. doi:10.1245/s10434-009-0623-1.CrossRefPubMed

17.

Peltier HJ, Latham GJ. Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues. Rna. 2008;14(5):844–52. doi:10.1261/Rna.939908.CrossRefPubMedPubMedCentral

18.

Xiang MQ, Zeng Y, Yang RR, Xu HF, Chen Z, Zhong J, et al. U6 is not a suitable endogenous control for the quantification of circulating microRNAs. Biochem Bioph Res Co. 2014;454(1):210–4. doi:10.1016/j.bbrc.2014.10.064.CrossRef

19.

Hansen CN, Ketabi Z, Rosenstierne MW, Palle C, Boesen HC, Norrild B. Expression of CPEB, GAPDH and U6snRNA in cervical and ovarian tissue during cancer development. Apmis. 2009;117(1):53–9. doi:10.1111/j.1600-0463.2008.00015.x.CrossRefPubMed

20.

Qi LQ, Bart J, Tan LP, Platteel I, van der Sluis T, Huitema S et al. Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma. Bmc Cancer. 2009;9. doi: 10.1186/1471-2407-9-163.

21.

Gironella M, Seux M, Xie MJ, Cano C, Tomasini R, Gommeaux J, et al. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc Natl Acad Sci U S A. 2007;104(41):16170–5. doi:10.1073/pnas.0703942104.CrossRefPubMedPubMedCentral

22.

Greither T, Grochola LF, Udelnow A, Lautenschlager C, Wurl P, Taubert H. Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int J Cancer. 2010;126(1):73–80. doi:10.1002/ijc.24687.CrossRefPubMed

23.

Szafranska AE, Davison TS, John J, Cannon T, Sipos B, Maghnouj A, et al. MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene. 2007;26(30):4442–52. doi:10.1038/sj.onc.1210228.CrossRefPubMed

24.

Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. Jama. 2007;297(17):1901–8. doi:10.1001/jama.297.17.1901.CrossRefPubMed

25.

Szafranska AE, Doleshal M, Edmunds HS, Gordon S, Luttges J, Munding JB, et al. Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues. Clin Chem. 2008;54(10):1716–24. doi:10.1373/clinchem.2008.109603.CrossRefPubMedPubMedCentral

26.

Huang F, Tang J, Zhuang X, Zhuang Y, Cheng W, Chen W, et al. MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha. PloS one. 2014;9(2):e87897. doi:10.1371/journal.pone.0087897.CrossRefPubMedPubMedCentral

27.

Zhang Y, Li M, Wang H, Fisher WE, Lin PH, Yao Q, et al. Profiling of 95 microRNAs in pancreatic cancer cell lines and surgical specimens by real-time PCR analysis. World journal of surgery. 2009;33(4):698–709. doi:10.1007/s00268-008-9833-0.CrossRefPubMedPubMedCentral

28.

Link A, Becker V, Goel A, Wex T, Malfertheiner P. Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer. PloS one. 2012;7(8):e42933. doi:10.1371/journal.pone.0042933.CrossRefPubMedPubMedCentral

29.

Jamieson NB, Morran DC, Morton JP, Ali A, Dickson EJ, Carter CR, et al. MicroRNA molecular profiles associated with diagnosis, clinicopathologic criteria, and overall survival in patients with resectable pancreatic ductal adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18(2):534–45. doi:10.1158/1078-0432.CCR-11-0679.CrossRef

30.

Wang J, Chen J, Chang P, LeBlanc A, Li D, Abbruzzesse JL, et al. MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease. Cancer prevention research. 2009;2(9):807–13. doi:10.1158/1940-6207.CAPR-09-0094.CrossRefPubMed

31.

Hong TH, Park IY. MicroRNA expression profiling of diagnostic needle aspirates from surgical pancreatic cancer specimens. Annals of surgical treatment and research. 2014;87(6):290–7. doi:10.4174/astr.2014.87.6.290.CrossRefPubMedPubMedCentral

32.

Kent OA, Mullendore M, Wentzel EA, Lopez-Romero P, Tan AC, Alvarez H, et al. A resource for analysis of microRNA expression and function in pancreatic ductal adenocarcinoma cells. Cancer Biol Ther. 2009;8(21):2013–24.CrossRefPubMedPubMedCentral

33.

Feng J, Yu JB, Pan XL, Li ZL, Chen Z, Zhang WJ, et al. HERG1 functions as an oncogene in pancreatic cancer and is downregulated by miR-96. Oncotarget. 2014;5(14):5832–44.CrossRefPubMedPubMedCentral

34.

Ma MZ, Kong X, Weng MZ, Cheng K, Gong W, Quan ZW, et al. Candidate microRNA biomarkers of pancreatic ductal adenocarcinoma: meta-analysis, experimental validation and clinical significance. J Exp Clin Canc Res. 2013;32:71. doi:10.1186/1756-9966-32-71.CrossRef

35.

Zhang R, Li M, Zang W, Chen X, Wang Y, Li P, et al. MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35(1):837–44. doi:10.1007/s13277-013-1115-2.CrossRef

36.

Schultz NA, Werner J, Willenbrock H, Roslind A, Giese N, Horn T, et al. MicroRNA expression profiles associated with pancreatic adenocarcinoma and ampullary adenocarcinoma. Modern Pathol. 2012;25(12):1609–22. doi:10.1038/modpathol.2012.122.CrossRef

37.

Wang J, Raimondo M, Guha S, Chen JY, Diao LX, Dong XQ, et al. Circulating microRNAs in Pancreatic Juice as Candidate Biomarkers of Pancreatic Cancer. J Cancer. 2014;5(8):696–705. doi:10.7150/Jca.10094.CrossRefPubMedPubMedCentral

38.

Nakata K, Ohuchida K, Mizumoto K, Kayashima T, Ikenaga N, Sakai H, et al. MicroRNA-10b is overexpressed in pancreatic cancer, promotes its invasiveness, and correlates with a poor prognosis. Surgery. 2011;150(5):916–22. doi:10.1016/j.surg.2011.06.017.CrossRefPubMed

39.

Sun M, Estrov Z, Ji Y, Coombes KR, Harris DH, Kurzrock R. Curcumin (diferuloylmethane) alters the expression profiles of microRNAs in human pancreatic cancer cells. Mol Cancer Ther. 2008;7(3):464–73. doi:10.1158/1535-7163.MCT-07-2272.CrossRefPubMed

40.

Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004;64(11):3753–6. doi:10.1158/0008-5472.CAN-04-0637.CrossRefPubMed

41.

Gee HE, Buffa FM, Camps C, Ramachandran A, Leek R, Taylor M, et al. The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis. Brit J Cancer. 2011;104(7):1168–77. doi:10.1038/sj.bjc.6606076.CrossRefPubMedPubMedCentral

42.

Appaiah HN, Goswami CP, Mina LA, Badve S, Sledge GW, Liu YL, et al. Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients. Breast Cancer Res. 2011;13(5):R86. doi:10.1186/Bcr2943.CrossRefPubMedPubMedCentral

43.

McDermott AM, Kerin MJ, Miller N. Identification and Validation of miRNAs as Endogenous Controls for RQ-PCR in Blood Specimens for Breast Cancer Studies. PloS one. 2013;8(12):e83718. doi:10.1371/journal.pone.0083718.CrossRefPubMedPubMedCentral

44.

Liu JQ, Gao J, Du YQ, Li ZS, Ren Y, Gu JJ, et al. Combination of plasma microRNAs with serum CA19-9 for early detection of pancreatic cancer. Int J Cancer. 2012;131(3):683–91. doi:10.1002/Ijc.26422.CrossRefPubMed

45.

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22. doi:10.1373/clinchem.2008.112797.CrossRefPubMed

Title: Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer
Authors: Alexey Popov
Arpad Szabo
Václav Mandys
Publication date: 01-12-2015
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2015
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-015-1785-9

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