DOI 10.1515/cclm-2013-0950 Clin Chem Lab Med 2014; aop Stamatia-Maria Rapti, Christos K. Kontos, Iordanis N. Papadopoulos and Andreas Scorilas* Enhanced miR-182 transcription is a predictor of poor overall survival in colorectal adenocarcinoma patients Abstract Background: Colorectal cancer is the second most fre- quent cause of cancer-related death in the developed world. Recent studies have tried to associate colorectal cancer with the aberrant expression of several microR- NAs. The aim of the present study was the development of a highly sensitive quantitative real-time PCR which can be used to evaluate the miR-182 expression levels in colorec- tal adenocarcinoma and adjacent non-cancerous tissue specimens and associate them with several clinicopatho- logical characteristics, aiming to examine the prognostic potential of miR-182. Methods: Total RNA was isolated from 116 malignant colo- rectal adenocarcinoma specimens and 60 paired non-can- cerous tissues. Then, polyadenylation of 2 μg total RNA by poly(A) polymerase and reverse transcription with suita- ble oligo-dT-adapter followed. miR-182 levels were quanti- fied by real-time PCR based on SYBR Green chemistry. The results were analyzed by the comparative quantification cycle method and by extensive biostatistical analysis. Results: miR-182 was found to be significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p < 0.001). miR-182 expression increases as the histological grade increases (p= 0.013). miR-182 overexpression is associated with high depth of tumor invasion, positive regional lymph node status, and advanced TNM stage of patients. Therefore, miR-182 is an unfavorable prognostic marker in colorectal adenocarci- noma, predicting poor overall survival (p= 0.007). Most importantly, miR-182 expression retained its unfavorable prognostic significance among patients with well- or mod- erately differentiated colorectal adenocarcinoma (p= 0.006) and among metastasis-free patients (p= 0.025). Conclusions: The increased levels of the oncogene-like miR-182 increase the risk for disease progression and pre- dict poor overall survival for colorectal adenocarcinoma patients. Keywords: colorectal cancer; microRNAs (miRNAs); molecular tumor markers; prognostic biomarkers; quan- titative real-time PCR (qPCR). *Corresponding author: Andreas Scorilas, Department of Biochemistry and Molecular Biology, University of Athens, Panepistimiopolis, 15701 Athens, Greece, Phone: +30 2107274306, Fax: +30 2107274158, E-mail: ascorilas@biol.uoa.gr Stamatia-Maria Rapti and Christos K. Kontos: Department of Biochemistry and Molecular Biology, University of Athens, Athens, Greece Iordanis N. Papadopoulos: Fourth Surgery Department, University of Athens, University General Hospital “Attikon”, Athens, Greece Introduction According to WHO 2008 report [1], colorectal cancer (CRC) amounts to 9.4% of cancer cases worldwide, with a yearly incidence of about 1.26 million patients. In 2008, 608,000 patients died of this disease [2]. Furthermore, CRC occurs most frequently in the developed world, as it constitutes the second most common cancer-related cause of death in Europe and the US [3, 4]. Only a minority of CRC cases have an inherited genetic background, while its main causes are modern lifestyle, diet, and increased life expectancy. Colorectal cancer is highly curable, by surgically removing the pathological tissue, provided that the disease is detected at an early stage. However, CRC is often diagnosed at an advanced stage, when the disease has already developed distant metastases; therefore, the prognosis for these patients is rather poor. In many cases, metastasis constitutes the main cause of death among CRC patients. Consequently, it is very important to dis- cover novel biomarkers in order to achieve early diagno- sis, accurate staging, and follow-up of the progression of the disease. A lot of research has been conducted, espe- cially during the last 5 years, highlighting the importance of microRNAs (miRNAs) as promising biomarkers of most cancer types, including CRC [5]. miRNAs appear to be differentially expressed at differ- ent stages of development [6]. They also have a tissue-spe- cific expression pattern, which is deregulated in different cancer types [7]. miRNAs are involved in tumor progres- sion and metastasis through regulation of cell migra- tion, invasion, proliferation, epithelial-to-mensenchymal Brought to you by | Columbia University Authenticated | 156.111.216.125 Download Date | 3/18/14 6:37 PM