Research Article: Identification and Validation of Potential Biomarkers for the Detection of Dysregulated microRNA by qPCR in Patients with Colorectal Adenocarcinoma

Date Published: March 24, 2015

Publisher: Public Library of Science

Author(s): Xiaobing Wu, Xuehu Xu, Shuling Li, Shangbiao Wu, Rong Chen, Qingping Jiang, Haibo Liu, Yan Sun, Yong Li, Yuandong Xu, Anthony W.I. Lo.

http://doi.org/10.1371/journal.pone.0120024

Abstract

Colorectal cancer represents a lethal disease that has raised concern and has attracted significant attention. Adenocarcinoma is the most common type of colorectal cancer (CRC). MicroRNAs are thought to be potential biomarkers of CRC. Many researchers have focused on the expression pattern of miRNAs in CRC. However, previous studies did not pay particular attention to the effects of the degree of differentiation of the cancer with respect to the miRNA expression profile. First, this study compared the expression level of 1547 miRNAs by qRT-PCR in Colorectal adenocarcinoma tissues to that in paired normal tissues. In all, 93 miRNAs were identified that were significantly dysregulated in Colorectal adenocarcinoma relative to normal tissues (P<0.05). Then, we analyzed their potential as cancer biomarkers by ROC analysis, and the result revealed that three miRNAs with high sensitivity and specificity are suitable as biomarkers for the diagnosis of CRC (the value of the AUC was greater than 0.7). Interestingly, previous reports of 23 of these miRNAs have been scarce. Furthermore, we wanted to analyze the difference between well- and moderately differentiated cancers, and as expected, 58 miRNAs showed significant dysregulation. Importantly, 32 miRNAs were able to not only distinguish cancer tissues from normal tissues, but they were also able to identify well- and moderately differentiated cancers. In conclusion, the degree of differentiation has an important influence on the miRNA expression pattern. To avoid misdiagnoses and missed diagnoses, tumors of different degrees of differentiation should be treated differently when miRNAs are used as cancer biomarkers.

Partial Text

Colorectal cancer is a heavy health burden and is estimated to be the third most common cancer worldwide in both sexes [1–3]. In fact, it has been estimated that CRC caused more than 500,000 deaths worldwide in 2013 alone [4]. Current screening tools for CRC are still unsatisfactory because of low sensitivity and specificity. Therefore, many researchers have attempted to identify novel biomarkers for the detection of CRC, and many studies have found that microRNAs (miRNAs) are dysregulated in many cancer types include CRC [5,6]. Therefore, miRNAs have the potential to serve as diagnostic biomarkers for CRC [7].

Compared with normal tissues, a total of 93 miRNAs that were significantly dysregulated were identified by non-paired t-test, of which 82 were downregulated and 11 were upregulated (Table 2). With the exception of miR-150, miR-181a, miR-130b, and let-7i, which demonstrated less than a 2-fold change relative to the normal controls, the fold changes of the other 89 dysregulated miRNAs were greater than 2-fold (Table 2). Furthermore, among the dysregulated miRNAs, the fold changes of miR-1, miR-145 and miR-145* were downregulated more than 10-fold. Others that were downregulated more than 5-fold included miR-137, miR-133a, miR-4770, miR-143, miR-363, miR-490–5p, miR-133b, let-7d, miR-4510, miR-9, miR-144*, miR-99b, miR-99a, miR-4469 and miR-451. Out of the miRNAs that were upregulated, the fold change of miR-135b, miR-96 and miR-141 was more than five-fold (Table 2). Interestingly, to our knowledge, 23 of these miRNAs have never been reported to be dysregulated in Colorectal adenocarcinoma (Table 3); among these miRNAs, the fold changes of miR-99b and miR-4469 were downregulated more than 5-fold.

Colorectal adenocarcinoma represents a lethal disease that has attracted substantial attention, especially because the current screening methods for Colorectal adenocarcinoma are still limited by unsatisfactory sensitivity and specificity. MicroRNAs have been shown to directly impact tumor growth, tumor cell proliferation, and tumor invasion [10], and they have also been demonstrated to be potential biomarkers of Colorectal cancer. Many dysregulated miRNAs were identified in colorectal cancer specimens. However, no research has been conducted on a comparison of the miRNA profiles among different classifications of tumors. Additionally, previous studies have reported that miRNA profiles can contribute to the diagnostic and prognostic classification of human malignancies [14–16]. CRC contains a variety of histologic tumor types, for example: (1) adenocarcinoma; (2) gland scale cancer; (3) small cell carcinoma; (4) carcinoid; (5) anaplastic carcinoma; and (6) squamous cell carcinoma, among others. According to reports from the 1980s to the 1990s, the proportion of Colorectal adenocarcinoma has increased from 82.1% to 85.6% in China [17]. Therefore, Colorectal adenocarcinoma was the cancer type selected for our study. In the present study, we first compared the expression level of 1547 miRNAs in Colorectal adenocarcinoma tissues with that of paired normal tissues and identified 93 miRNAs that were significantly dysregulated in Colorectal adenocarcinoma relative to normal tissues (P<0.05). Of these, miR-1, miR-145 and miR-145* were downregulated more than ten-fold, and this result was consistent with the results of previous studies. Bao Y and his colleagues reported that miR-145 was significantly downregulated in colorectal cancer tissues [18] and that the targets of miR-145 are involved in cell cycle and neuregulin pathways [19]. In addition, they showed that the upregulation of miR-145 could improve the ability of colorectal cancer cells to migrate and invade; the mechanism of the promotion of migration and invasion is associated with the stabilization of Hsp-27, a protein that plays an important role in the promotion of metastasis [20]. Furukawa S and his colleagues demonstrates that miR-1 is downregulated in colorectal tumors and that miR-1 has the potential to suppress NOTCH3 expression through direct binding to its 3'-UTR region. It is also known that NOTCH signaling plays critical roles in colorectal tumorigenesis [21]. Downregulated miR-1 can cause tumor suppressor effects in colorectal cancer by the direct downregulation of the MET oncogene both at the RNA and the protein level. Re-expression of miR-1 leads to a MET-driven reduction of cell proliferation and motility, which suggests that the downregulation of miR-1 is one of the events that might enhance colorectal cancer progression [22]. For the most part, this result was verified by an independent sample test (Table 6). Moreover, we analyzed the potential of miRNAs as cancer biomarkers by ROC analysis, and the result revealed that all three miRNAs are suitable as diagnostic biomarkers, as their sensitivity, specificity, and the value of AUC are all greater than 0.7 [23]. In addition, 23 of these miRNAs have been scarcely reported until now (Table 3) with the exception of miR-4770 and miR-3195, which have been mentioned in our previous research [12]. Of these miRNAs, miR-4510, miR-99b and miR-4469 were downregulated more than 5-fold. Thus, we analyzed the sensitivity and specificity of these miRNAs by ROC analysis, and the results showed that they are capable of distinguishing cancer patients from normal controls. Next, we wanted to analyze the difference between well- and moderately differentiated cancers, and we found that expectedly, 58 miRNAs showed differential dysregulation (Table 4). Furthermore, to find miRNAs that could not only distinguish cancer patients from normal controls but that could also distinguish well-differentiated cancers from moderately differentiated ones, we formed a connection between the two results mentioned above. Surprisingly, 32 miRNAs met this criteria (Table 5). As shown, miR-145 and miR-99b were a part of this relationship. Their expression levels changed more than 5-fold, and the ROC analysis showed that miR-145 and miR-99b were able to distinguish well-differentiated cancers from moderately differentiated ones. However, the other 4 miRNAs (miR-1, miR-145, miR-4510 and miR-4469) were also dysregulated, but the differences were not statistically significant (Fig. 5).   Source: http://doi.org/10.1371/journal.pone.0120024