br inappropriate for screening large asymptomatic patients T
inappropriate for screening large asymptomatic patients. Therefore, detection of CRC is challenging owing to the lack of a specific non-invasive markers. The discovery of microRNAs (miRNAs) found in plasma or serum has opened a new pathway for diagnosing CRC.
miRNAs are small noncoding (19–25 nucleotides in length), en-dogenous, and single-stranded RNAs that play important roles in reg-ulating gene expression; they are also associated with numerous im-portant pathways, including developmental and oncogenic pathways . Several recent studies indicated diﬀerential CRC miRNA T-5224 profiles in plasma or serum versus healthy individuals, such as miR-141, miR-21, miR-142-3p, miR-26a-5p, miR-1914*, and miR-1915 [7–10], which provide the foundation for studying the potentially di-agnostic roles of miRNAs in CRC.
Genome-wide miRNA and mRNA expression analyses have been used to identify the functional involvement of miRNAs in the
Corresponding author at: Translational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, No. 1017 Dongmen North Road, Shenzhen, Guangdong, 518020, China. Corresponding author at: Department of Surgery, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, No. 1017 Dongmen North Road, Shenzhen, Guangdong, 518020, China. E-mail addresses: [email protected] (F.-R. Li), [email protected] (X.-F. Yu).
1 Contributed equally.
progression of cancers . Possible miRNA–mRNA interactions can be predicted using new computer algorithms and techniques [12–14], which enabled detailed analysis and prediction  and improved knowledge on cancer development and pathogenesis.
Currently, two major qPCR-based tools are used for miRNA quan-tification assay, namely, the poly(A) [16–19] and stem-loop methods [20–22]. Although the poly(A) method can determine miRNA expres-sion in a high-throughput manner, the approach is less specific than others due to the nonspecific reverse transcription (RT). The stem-loop method requires individual miRNA-specific hydrolytic Taqman probes and is thus too costly for high-throughput miRNA expression profiling . Moreover, given the limited number (usually six) of bases guiding the binding of the 3′ end of the step-loop primer to the target miRNAs, the eﬃciency of the stem-loop method is relatively low even with a pulse RT reaction . In the present study, we first performed a powerful qPCR-based assay, the S-Poly(T) Plus real-time PCR assay, which exhibits superior sensitivity, specificity, and eﬃciency , to select and validate diﬀerentially expressed plasma miRNAs from a sample set including 101 CRC patients, 20 patients with colorectal noncancerous polyps (NCP), and 134 healthy controls. After the two-phase selection and validation process, we identified a miRNA panel (miR-144-3p, miR-425-5p, and miR-1260b) with high CRC diagnostic eﬃciency. Furthermore, we integrated predicted or validated targets of the three miRNAs and analyzed their overrepresented pathways by using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome database. Therefore, we demonstrated a plasma 3-miRNA panel that may serve as novel noninvasive biomarker to diagnose CRC and may be related to CRC development.
2. Materials and methods
2.1. Patients and control subjects
This study was approved by the Institutional Ethics Committees at Shenzhen People’s Hospital (Shenzhen, China) and Sun Yat-sen University Cancer Center, and written informed consent was obtained from all study participants. A total of 255 participants were enrolled at the Shenzhen People’s Hospital and Sun Yat-sen University Cancer Center from October 2014 to December 2015, including 101 CRC pa-tients, 20 patients with NCP, and 134 healthy controls. The subjects in this study were newly diagnosed with CRC or NCP prior to receiving any treatments. Histological typing was performed according to the World Health Organization criteria. Staging was confirmed according to the tumor–node–metastasis (TNM) staging system. For the selection of the samples in the healthy controls, we used asymptomatic and ap-parently healthy volunteers without a previous history of cancer or other benign diseases. These healthy controls were matched to the CRC patients according to age and gender. The clinical characteristics of the study subjects are presented in Table 1. No significant diﬀerence was observed in the distribution of age and sex between the screening and validation datasets for both healthy and CRC groups (P > 0.05). Overall, 62.4% (63/101) of the patients were diagnosed with stages IeII CRC, while 37.6% (38/101) of the patients presented stage IIeIV. All CRC cases in this study were adenocarcinomas.