Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer worldwide, with an estimation of 1.2 million new cases per year and more than 600,000 deaths [1‐3]. The risk of CRC increases with age, whereby most cases are diagnosed in individuals aged 50 and above [4]. Incidence rate of CRC has been increasing in Asian countries. In Malaysia, it ranks the second after lung cancer and breast cancer in men and women, respectively [5]. The 5-year survival rate exceeds 90% when CRC is detected at an early, localized stage [6]. However, most cases are diagnosed at late stages due to inconvenient settings of current CRC screening tests and low population compliance. Colonoscopy has significant contribution in the detection of neoplastic lesions, but the requirements of bowel preparation, sedation and invasive nature have hindered its widespread application as a screening tool [7]. Other structural tests such as computed tomographic colonography and double contrast barium enema are limited by the concern of radiation exposure and cost [8]. Fecal-based analyses such as occult blood, immunochemical and stool DNA tests are common noninvasive screening tests [9]. Nevertheless, they are confined by low sensitivity and specificity against detection of premalignant lesions [10, 11]. Although stool DNA test utilizes molecular markers such as KRAS, APC and TP53 genes as detection targets, it is not favorably recommended by the US Preventive Service Task Force due to high cost, uncertainty of test performance and labor-intensive handling [12‐14]. Thus, there is an imperative need for other noninvasive biomarkers to complement and improve current diagnostic and prognostic tools in CRC.

MicroRNAs (miRNAs) are short (19–22 nucleotides), non-coding RNA molecules that act as regulators of gene expression [15]. Although their main mechanism of action is through mRNA degradation or translational inhibition, they can also induce gene activation [16, 17]. The pioneer discovery of miRNAs was initiated by Lee and colleagues in 1993 when they found lin-4, a small RNA molecule that was capable of controlling the larval development of Caenorhabditis elegans[18]. Since then, a vast number of miRNAs have been reported. To date, there are more than 2000 entries of human miRNAs in the miRNA database, miRBase 19.0, constituting around 1% of total genes that can regulate up to one third of human genome [19]. MiRNAs are evolutionary conserved across species and expressed in a tissue-specific manner [20]. They have been determined to play important roles in cancer pathophysiology such as cell proliferation, differentiation, apoptosis and metastasis [21‐23]. MiRNA genes are frequently located at fragile sites and genomic regions of deletion and amplification implicated in cancers [24, 25]. They can confer both oncogenic and tumor suppressive roles, depending upon their downstream targets [26‐28]. MiRNAs are detectable in tissues, blood, feces and other body fluids such as saliva, tears and urine [29‐32]. The presence of miRNAs in the circulation system is mediated via exosome transfer to the surrounding microenvironment [33, 34]. Blood-based miRNA profiling has become a favorable area of research for the identification of noninvasive biomarkers in cancers and other diseases [35‐38]. In addition, a study by Heneghan et al. reported a strong preference of using whole blood to serum or plasma for systemic miRNAs detection and quantification [39]. Circulating blood miRNAs are generally Ago-bound and protected from endogenous RNases that enable them to serve as stable blood biomarkers [40, 41].

An important concern for utilization of miRNAs as biomarkers is whether the dysregulated miRNAs are related to CRC alone or as a general mechanism in histologic progression to cancer [29, 42]. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of CRC.

MiRNA profiling studies in CRC and other diseases have been vastly reported [35‐38]. For instance, a recent tissue miRNA profiling study by Piepoli et al. revealed a cluster of dysregulated miRNAs (miR-195, miR-1280, miR-140-3p and miR-1246) that could be used to distinguish between colorectal and pancreatic cancers [49]. An investigation by Hamfjord et al. using high throughput sequencing in paired cancer tissues has reported 16 novel miRNAs (miR-490-3p, miR-628-3p/-5p, miR-1297, miR-3151, miR-3163, miR-3622a-5p, miR-3656, miR-105, miR-549, miR-1269, miR-1827, miR-3144-3p, miR-3177, miR-3180-3p and miR-4326) that have not been previously described in CRC [50]. For blood-based miRNA profiling, Ahmed et al. have identified a huge panel of differentially expressed miRNAs in sporadic colon cancer patients [51]. Our data corresponded to theirs whereby an overlap of certain markers (miR-130a, miR-150, miR-18b, miR-338, miR-342 and miR92a) was observed in our blood array study. Apart from that, Ng et al. have evaluated a panel of 95 miRNAs using real-time PCR-based array and plasma miR-17-3p and miR-92 were shown to be significantly expressed between the CRC cases and controls [52]. The main aspect of our study is to determine whether the circulating blood miRNAs are reflective of those in the tissues. Therefore, we have investigated the correlation of miRNA expressions between paired cancer tissue and whole blood of CRC patients. The levels of miR-193a-3p, miR-23a and miR-338-5p were significantly up-regulated and positively correlated in both the tissue and blood samples. The triple miRNA classifier identified in this study was of high sensitivity (80.0%), specificity (84.4%) and accuracy (83.3%) in defining CRC. The findings have collectively implicated the potentiality of circulating blood miRNAs as noninvasive biomarkers.

Mir-193a-3p is part of the miR-193 family, together with miR-193a-5p and miR-193b. Nevertheless, the information on miR-193a-3p is limited and its molecular mechanism and role in carcinogenesis remain largely unknown. To our knowledge, the present paper is the first report on the up-regulation of miR-193a-3p in CRC. Although miR-193a-3p has not been mentioned in CRC, its dysregulation has been observed in pleural mesothelioma, ostersarcoma and oral cancer, suggesting its impact in tumorigenesis [53‐55]. Clearly, the role of miR-193a-3p in cancer needs to be robustly investigated.

The role of miR-23a has been reported with varying conclusions. Recently, Jahid et al. revealed the function of miR-23a in promoting the migration and invasion of CRC cells and stem cells by down-regulating metastasis suppressor 1 (MTSS1) gene [56]. The finding is consistent with our study whereby there is an increasing expression of miR-23a in the more advanced tumors. A deep sequencing project using serum samples has demonstrated a common up-regulation of miR-23a in CRC and lung cancer patients [57]. An in vitro study by Zhu et al. using gastric adenocarcinoma cell line (MGC803) revealed a growth-promoting function of miR-23a via regulation of interleukin-6 receptor (IL-6R) gene [58]. Besides its individualistic function, miR-23a has been shown to possess cooperative functions with miR-27a and miR-24 [59]. The three miRNAs are derived from a single primary transcript, located on chromosome 9q22. These miRNAs have been bioinformatically predicted to regulate cell proliferation and tissue development via Wnt signaling pathway [60]. Wnt pathway is associated with advancement of dysplasia in aberrant crypt foci and acts as ‘gatekeeper’ in the initiation of adenoma-carcinoma sequences in CRC. In addition, Rogler et al. found miR-23b, a paralog of miR-23a to have an amplification effect with miR-23a in regulating transforming growth factor beta signaling by targeting SMAD genes [61]. This is in support with our microarray and validation studies whereby mir-23a and miR-23b were discovered to be significantly up-regulated in both tissue and blood samples. MiR-23a, together with miR-23b have been reported to play certain roles in glutamine catabolism, cell cycle regulation and glucose metabolism via regulation of c-Myc gene [59, 62]. On the contrary, miR-23a expression has also been found to be repressed in several malignancies, including oral squamous cell carcinoma and acute promyelocytic leukemia [55, 63]. The findings unveiled the possibility of diverse miRNA functions in different cell types and diseases.

MiR-338-3p and miR-338-5p are isoforms of miR-338. In general, over-expression of miR-338 has been observed in many cancers, namely CRC, hepatocellular carcinoma and head and neck/oral cancer [64‐66]. A tissue miRNA profiling by Schetter et al. revealed 37 miRNAs, including miR-338, to be differentially expressed in CRC tissues when compared with paired noncancerous tissues [64]. In a recent investigation on CRC recurrence-related miRNAs by Ju et al., miR-338-5p was found to be significantly up-regulated and positively correlated with cancer metastasis [67]. Our findings corresponded to their study whereby mir-338-5p expression was shown to be significantly higher in the more advanced tumors.

An individual miRNA is definitely not an appealing marker in discriminating CRC. The fact that miRNAs could act as cell proliferating factors in certain cancers and apoptotic factors in another has evoked the necessity of studying their cooperative functions in order to provide a holistic picture of miRNA regulations in cancer biology and pathogenesis.

Dysregulations in circulating blood miRNAs are reflective of those in colorectal tissues. The triple miRNA classifier of miR-193a-3p, miR-23a and miR-338-5p appears to be a potential blood biomarker for early detection of CRC. Our study serves as an exploratory basis for further investigation in larger prospective and randomized clinical studies with higher number of samples from healthy controls and patients of advanced adenoma and various stages of CRC. A noninvasive miRNA screening assay using the triple miRNA classifier could then be developed to identify asymptomatic individuals with colorectal neoplasia prior to more invasive colonoscopy examination.