Direct Assay of miR-223, miR-145, and miR-16 by a Novel Method of Mimic PCR in Serum of Breast Cancer Patients

Document Type: Research Article


1 Department of Medicine Biotechnology, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Biology, Faculty of Science, Lorestan University, Khoramabad, Iran

3 Department of Biology, Faculty of Science, Payame Noor University, PO Box 19395-3697, Tehran, Iran


The role of microRNAs (miRs) is shown as a biomarker whose expression level changes have been associated with cancer development and progression. Circulating miRNAs have been postulated as biomarkers for breast cancer. Detection and diagnosis of miRNA markers could provide an improved and sensitive method in the clinical application. In this study, we applied and improved the effective approach by coupling a deoxyuridine-incorporated RT oligonucleotide with a secondary structure and a mimic PCR for assessment and direct detection of circulating miRs in serum. We designed mut miR-16, mut miR-145, and mut miR-223 as mimic miRs. These mimic miRs were used in RT-PCR for detection and quantification assay. As normalization of these mut miRs was performed with themselves, our mimic construct with 80 bp of these miRs shows the best normalization for this method in blood samples. This assay was tested in serum from 15 patients with different stages of breast cancer and 10 healthy female donors. According to our results, using a combination of miR-16 and miR-145 could represent as one of the best biomarker (p


Benes V, Castoldi M. 2010. Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50: 244-249.

Berindan-Neagoe I, Monroig Pdel C, Pasculli B, Calin GA. 2014. MicroRNAome genome: a treasure for cancer diagnosis and therapy. CA Cancer J Clin 64: 311-336.

Bonci D, De Maria R. 2016. miR-15/miR-16 loss, miR-21 upregulation, or deregulation of their target genes predicts poor prognosis in prostate cancer patients. Mol Cell Oncol 3: e1109744.

Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC. 2005. miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 204: 280-285.

Brooks M. 2009. Breast cancer screening and biomarkers. Methods Mol Biol 472: 307-321.

Cheng Y, Zhang X, Jiao X, Wang Y, Zhang Y. 2009. Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew Chem Int Ed Engl 48: 3268-3272.

Davoren PA, McNeill RE, Lowery AJ, Kerin MJ, Miller N. 2008. Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol Biol 9: 76.

Duffy MJ. 2012. Tumor markers in clinical practice: a review focusing on common solid cancers. Med Princ Pract 22: 4-11.

Gu Y, Wang XD, Lu JJ, Lei YY, Zou JY, Luo HH. 2015. Effect of mir-16 on proliferation and apoptosis in human A549 lung adenocarcinoma cells. Int J Clin Exp Med 8: 3227-3233.

Han Q, Zhang HY, Zhong BL, Wang XJ, Zhang B, Chen H. 2016. MicroRNA-145 inhibits cell migration and invasion and regulates epithelial-mesenchymal transition (EMT) by targeting connective tissue growth factor (CTGF) in esophageal squamous cell carcinoma. Med Sci Monit 22: 3925-3934.

Hossienzadeh Colagar A, Moradi Firouzjah H, Halalkhor S. 2015. Vitamin D receptor poly (A)-microsatellite polymorphism and serum levels of 25-hydroxyvitamin D: association with susceptibility to breast cancer. J Breast Cancer 18(2):119-125.

Hui AB, Lenarduzzi M, Krushel T, Waldron L, Pintilie M, Shi W, Perez-Ordonez B, Jurisica I, O'Sullivan B, Waldron J, Gullane P, Cummings B, Liu FF. 2010. Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas. Clin Cancer Res 16: 1129-1139.

Hunt EA, Goulding AM, Deo SK. 2009. Direct detection and quantification of microRNAs. Anal Biochem 387: 1-12.

Iorio MV, Croce CM. 2012. MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 4: 143-159.

Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P, Taccioli C, Volinia S, Liu CG, Alder H, Calin GA, Ménard S, Croce CM. 2007. MicroRNA signatures in human ovarian cancer. Cancer Res 67: 8699-8707.

Kazarian A, Blyuss O, Metodieva G, Gentry-Maharaj A, Ryan A, Kiseleva EM, Prytomanova OM, Jacobs IJ, Widschwendter M, Menon U, Timms JF. 2017. Testing breast cancer serum biomarkers for early detection and prognosis in pre-diagnosis samples. Br J Cancer 116: 501-508.

Kodahl AR, Lyng MB, Binder H, Cold S, Gravgaard K, Knoop AS, Ditzel HJ 2014. Novel circulating microRNA signature as a potential non-invasive multi-marker test in ER-positive early-stage breast cancer: a case control study. Mol Oncol 8: 874-883.

Lan H, Lu H, Wang X, Jin H. 2015. MicroRNAs as potential biomarkers in cancer: opportunities and challenges. Biomed Res Int 2015: 125094.

Liang H, Yan X, Pan Y, Wang Y, Wang N, Li L, Liu Y, Chen X, Zhang CY, Gu H, Zen K. 2015. MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3. Mol Cancer 14: 58.

Loberg M, Lousdal ML, Bretthauer M, Kalager M. 2015. Benefits and harms of mammography screening. Breast Cancer Res 17: 63.

Ma L, Teruya-Feldstein J, Weinberg RA. 2007. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449: 682-688.

Ma L. 2010. Role of miR-10b in breast cancer metastasis. Breast Cancer Res 12: 210.

Marabita F, de Candia P, Torri A, Tegnér J, Abrignani S, Rossi RL. 2016. Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Brief Bioinform 17: 204-212.

Mathe A, Scott RJ, Avery-Kiejda KA. 2015. MiRNAs and other epigenetic changes as biomarkers in triple negative breast cancer. Int J Mol Sci 16: 28347-28376.

Moon PG, Lee JE, Cho YE, Lee SJ, Jung JH, Chae YS, Bae HI, Kim YB, Kim IS, Park HY, Baek MC. 2015. Identification of developmental endothelial locus-1 on circulating extracellular vesicles as a novel biomarker for early breast cancer detection. Clin Cancer Res 22: 1757-1766.

Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K. 2006. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 25: 2537-2545.

Patnaik S, Patnaik, Sai Yendamuri, Eric Kannisto, John C. Kucharczuk, Sunil Singhal, Anil Vachani. 2012. MicroRNA expression profiles of whole blood in lung adenocarcinoma. PLoS One 7: e46045.

Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. 2005. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11: 1737-1744.

Renjie W, Haiqian L. 2015. MiR-132, miR-15a and miR-16 synergistically inhibit pituitary tumor cell proliferation, invasion and migration by targeting Sox5. Cancer Lett 356: 568-578.

Schummer M, Thorpe J, Giraldez MD, Bergan L, Tewari M, Urban N. 2015. Evaluating serum markers for hormone receptor-negative breast cancer. PLoS One 10: e0142911.

Sharbati-Tehrani, S, Kutz-Lohroff B, Bergbauer R, Scholven J, Einspanier R. 2008. miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol Biol 9: 34.

Shimomura A, Shiino S, Kawauchi J, Takizawa S, Sakamoto H, Matsuzaki J, Ono M, Takeshita F, Niida S, Shimizu C, Fujiwara Y, Kinoshita T, Tamura K, Ochiya T. 2016. Novel combination of serum microRNA for detecting breast cancer in the early stage. Cancer Sic 107: 326-334.

Sprague BL, Stout NK, Schechter C, van Ravesteyn NT, Cevik M, Alagoz O, Lee CI, van den Broek JJ, Miglioretti DL, Mandelblatt JS, de Koning HJ, Kerlikowske K, Lehman CD, Tosteson AN. 2015. Benefits, harms, and cost-effectiveness of supplemental ultrasonography screening for women with dense breasts. Ann Intern Med 162: 157-166.

Stearns V. 2016. Novel biomarkers in the continuum of breast cancer. Anticancer Res 36: 3224.

Stout NK, Stout NK, Lee SJ, Schechter CB, Kerlikowske K, Alagoz O, Berry D, Buist DS, Cevik M, Chisholm G, de Koning HJ, Huang H, Hubbard RA, Miglioretti DL, Munsell MF, Trentham-Dietz A, van Ravesteyn NT, Tosteson AN, Mandelblatt JS. 2014. Benefits, harms, and costs for breast cancer screening after US implementation of digital mammography. J Natl Cancer Inst 106: dju092.

van Schooneveld E, Wildiers H, Vergote I, Vermeulen PB, Dirix LY, Van Laere SJ. 2015. Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management. Breast Cancer Res 17: 21.

Vilquin P, Donini CF, Villedieu M, Grisard E, Corbo L, Bachelot T, Vendrell JA, Cohen PA. 2015. MicroRNA-125b upregulation confers aromatase inhibitor resistance and is a novel marker of poor prognosis in breast cancer. Breast Cancer Res 17: 13.

Wan G, Lim QE, Too H.P. 2010. High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers. RNA 16: 1436-1445.

Yang M, Chen J, Su F, Yu B, Su F, Lin L, Liu Y, Huang JD, Song E. 2011. Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells. Mol Cancer 10: 117.

Yao B, Li J, Huang H, Sun C, Wang Z, Fan Y, Chang Q, Li S, Xi J. 2009. Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification. RNA 15: 1787-1794.

Zhao FL, Dou YC, Wang XF, Han DC, Lv ZG, Ge SL, Zhang YK. 2014. Serum microRNA-195 is down-regulated in breast cancer: a potential marker for the diagnosis of breast cancer. Mol Biol Rep 41: 5913-5922.

Zheng M, Dou YC, Wang XF, Han DC, Lv ZG, Ge SL, Zhang YK. 2016. MicroRNA-145 inhibits growth and migration of breast cancer cells through targeting oncoprotein ROCK1. Tumour Biol 37: 8189-8196.

Zhu Y, Guo M, Zhang L, Xu T, Wang L, Xu G. 2016. Biomarker triplet NAMPT/VEGF/HER2 as a de novo detection panel for the diagnosis and prognosis of human breast cancer. Oncol Rep 35: 454-462.