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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 7  |  Page : 1572-1577

Serum miR-20a and miR-486 are potential biomarkers for discriminating colorectal neoplasia: A pilot study


1 Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, China
2 National Center of Colorectal Surgery, The 3rd Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, China
3 Department of Anorectal Surgery, The 2rd Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, China

Date of Web Publication19-Dec-2018

Correspondence Address:
Heiying Jin
Department of Anorectal Surgery, The 2rd Affiliated Hospital of Nanjing University of Traditional Chinese Medicine Nanjing 210001
China
Yimei Fan
Department of Medical Genetics, Medical School, Nanjing University, Nanjing 210093
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1198_16

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 > Abstract 


Aim: Recent advances in circulating microRNAs (miRNAs) as noninvasive biomarkers have provided promising prospect in detecting colorectal cancer (CRC). However, the capability of miRNAs for detecting colorectal neoplasia (CRN, including precancerous lesions and curable stage CRCs) remains unclear. This study aimed to identify the potential of serum miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) selected from the literature for discriminating CRN patients.
Materials and Methods: The serum samples from 46 CRN patients and 33 healthy controls were analyzed with quantitative reverse transcription-polymerase chain reaction.
Results: Serum miR-20a and miR-486 were significantly downregulated in CRN patients compared to that of in healthy controls (fold change = 0.697 and 0.696, P = 0.01 and 0.05, respectively). The serum level of miR-92a was not significantly different between two groups, while miR-135b level in serum was too low to be accurately quantified. In addition, serum miR-486 level was much more downregulated in tubulovillous adenoma and high-grade intraepithelial neoplasia patients than that of in healthy controls. For miR-20a and miR-486, the area under the receiver operating characteristic curve for discriminating CRN patients were 0.676 and 0.629, respectively, while their combined value was 0.698. No significant correlation was observed between miR-20a and miR-486 serum levels with age, gender, location, or lesion size.
Conclusion: The results suggested that serum miR-20a and miR-486 could be potential noninvasive biomarkers for identifying CRN patients.

Keywords: Colorectal neoplasia, microRNA, potential biomarker, serum


How to cite this article:
Yang Q, Wang S, Huang J, Xia C, Jin H, Fan Y. Serum miR-20a and miR-486 are potential biomarkers for discriminating colorectal neoplasia: A pilot study. J Can Res Ther 2018;14:1572-7

How to cite this URL:
Yang Q, Wang S, Huang J, Xia C, Jin H, Fan Y. Serum miR-20a and miR-486 are potential biomarkers for discriminating colorectal neoplasia: A pilot study. J Can Res Ther [serial online] 2018 [cited 2019 Jun 25];14:1572-7. Available from: http://www.cancerjournal.net/text.asp?2018/14/7/1572/247713




 > Introduction Top


Colorectal cancer (CRC) has been a leading cause of cancer-associated death worldwide. Both incidence and mortality of CRC have been increased rapidly in the last decades due to the changes of dietary patterns and physical activities.[1] The mortality of CRC could be reduced by the early detection and removal of colorectal neoplasia (CRN, including precancerous lesions and curable stage CRCs).

The conventional screening guidelines of CRC included fecal occult blood test and colonoscopy, which have been available for years. However, none of these tests was ideal screening method due to the low sensitivity, high cost, and low compliance.[2] Thus, it is particularly necessary to develop new biomarkers for early detection.

MicroRNAs (miRNAs) are noncoding RNA molecules with the length of ~22 nucleotides. MiRNAs regulated mRNA expression and played the roles of oncogenic or tumor suppressor in the multistep processes of carcinogenesis.[3] Circulating miRNAs have been reported to be promising biomarkers for CRC detection because of their stability in circulating blood.[4],[5],[6]

In previous studies, the expression levels of miR-20a, miR-135b, and miR-92a were significantly different between CRC tissues and the matched adjacent normal tissues.[7],[8] Increased expression of miR-92a and miR-135b has also been demonstrated in adenoma tissues.[9],[10] Plasma miR-20a, miR-135b, and miR-92a could be used to discriminate CRC/adenoma patients from healthy controls.[6],[11],[12] MiR-486 could be involved for identifying CRC according to KRAS status.[13]

In this study, we aimed to identify above four miRNAs could discriminate CRN patients from healthy controls. In addition, the associations between validated miRNAs and clinicopathologic features were also clarified.


 > Materials and Methods Top


Patients and samples

Serum samples were collected from 46 CRN patients and 33 healthy controls. All the participants were recruited from the National Center of Colorectal Surgery, the 3rd Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. Samples were collected from all patients before any treatment. Details regarding the following information were summarized [Table 1]: age of onset, gender, tumor location, lesion size, and histology. The mean age of the CRN group was older than that of the healthy control (P < 0.001), and there were more males in the CRN group than that of in healthy control (P = 0.018). CRN was defined as tubular adenomas, tubulovillous adenomas (TVA), high-grade intraepithelial neoplasia (HGIN), and tumor-node-metastasis Stage I–III cancer. This study was approved by the Ethics Committee of the 3rd Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. The signed informed consent has been obtained from all participants.
Table 1: Clinicopathological characteristics of subjects

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The candidate biomarkers

Four miRNAs were selected as follows: miR-20a, miR-92a, miR-135b, and miR-486. Three miRNAs (miR-20a, miR-92a, and miR-135b) had been evaluated in both CRC tissues and blood specimens, furthermore, in more than three independent articles. The miR-486 was a novel biomarker for CRN diagnosis. It was only identified to be associated with CRC by KRAS status, without being evaluated in blood specimens.

RNA extraction

RNA was extracted from 250 μL serum using the mirVana PARIS kit (Ambion, USA) according to the manufacturer's protocol. For minimizing sample-to-sample variation during the RNA extraction, 25 fmol of synthetic Caenorhabditis elegans miRNA cel-miR-39 was added to each denatured sample for normalization.[14],[15] The concentration of total extracted RNA was measured by NanoDrop 1000 UV spectrophotometer (Thermo Scientific, USA).

Quantitative reverse transcription-polymerase chain reaction

Serum levels of the four miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) and cel-miR-39 were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with TaqMan miRNA Assays (Applied Biosystems; catalog NO.4427975). A volume of 2.5 μL of total RNA was reversely transcribed with TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Real-time PCR reactions were performed in a 10 μL of reaction volume, including TaqMan Universal Master Mix II with No Uracil-N glycosylase (Applied Biosystems), miRNA-specific assay, and RT product. The thermal cycling conditions were performed with ABI StepOnePlus real-time PCR system (Applied Biosystems): 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s, and 60°C for 1 min. The expression levels of each serum miRNA were normalized against that of cel-miR-39 and then calculated with the 2−ΔΔCt method.

Statistical analysis

Mann–Whitney U-test was applied for analyzing the differences in serum miRNA levels between two groups. For the receiver operating characteristic (ROC) curves analysis, the values of area under curve (AUC) were computed to reflect the discriminating performance of miRNAs. All P values were two-sided and P < 0.05 was considered statistically significant.


 > Results Top


Serum levels of miR-20a and miR-486 were significantly down-regulated in colorectal neoplasia patients

The four candidate miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) were selected from the literature. The serum levels of the four miRNAs were quantitatively determined in 46 CRN patients and 33 healthy controls. It aimed to identify the potential biomarkers for CRN patients. From the results, serum levels of both miR-20a and miR-486 were downregulated in CRN patients compared to that of healthy controls [fold change = 0.697 and 0.696, P = 0.01 and 0.05, respectively, [Figure 1]. No significant difference was observed in serum level of miR-92a between two groups [P = 0.15, [Figure 1]. The serum level of miR-135b was too low to be accurately quantified.
Figure 1: Serum levels of miR-20a, miR-486, and miR-92a expression in healthy controls and colorectal neoplasia patients

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The CRN patients were further divided into two groups: cancer group (CRC group) and adenoma group. Serum level of miR-20a was downregulated in CRC group (fold change = 0.604, P = 0.006) and slightly decreased in adenoma group, without statistical significance (fold change = 0.818, P = 0.115) [Figure 2]a. Serum level of miR-486 was downregulated in both adenoma (fold change = 0.483, P = 0.022) and CRC groups; however, the difference was not statistically significant in CRC group (fold change = 0.860, P = 0.272) [Figure 2]b.
Figure 2: Serum levels of miR 20a and miR 486 expression among healthy controls, patients with colorectal cancer and adenoma. (a) miR 20a; (b) miR 486

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Next, ROC curves were plotted to evaluate the discriminating performance of miR-20a and miR-486 as serum biomarkers for identifying CRN. The AUC values of serum miR-20a and miR-486 for discriminating CRN from healthy controls were 0.676 (P = 0.008) and 0.629 (P = 0.05), respectively. Combination of two miRNAs showed an improvement in AUC (AUC = 0.698, P = 0.003) for CRN [Figure 3].
Figure 3: Receiver operating characteristic curves for miR-20a, miR-486, and combination in discriminating colorectal neoplasia patients from healthy controls

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Associations between serum levels of miR-20a, miR-486 and colorectal neoplasia characteristics

The association between serum levels of miR-20a and miR-486 and CRN characteristics were explored, including the lesion size, histological subtypes, age, gender, and locations.

The correlations between serum miR-20a and miR-486 levels and lesion size were analyzed. From the results [Figure 4], neither serum levels of miR-20a (P = 0.237) nor miR-486 (P = 0.989) showed significant correlation with the lesion size.
Figure 4: Correlations between serum microRNAs levels and the lesion size

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Considering the different expression levels of miR-486 in adenoma patients and healthy controls, the correlation between serum miR-486 level and histologic subtypes was further evaluated. Significantly downregulated serum miR-486 levels were observed in TVA and HGIN patients, compared with that of in healthy controls [P = 0.03 and 0.05, [Figure 5]. As shown in [Figure 6], the expression level of miR-20a was significantly downregulated in both Stages I–II CRC (P = 0.018) and Stage III CRC (P = 0.039).
Figure 5: Serum miR-486 levels according to histologic subtypes of adenoma patients

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Figure 6: Serum miR-20a levels according to the stage of colorectal cancer

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For CRN patients, the correlations between serum miR-20a and miR-486 levels and other clinicopathologic factors were also examined. No significant correlation was observed between serum miR-20a or miR-486 level and age (P = 0.15, P = 0.35), gender, (P = 0.32, P = 0.34), or location (P = 0.49, P = 0.52) which is consistent with previous study.[16]


 > Discussion Top


Many previous reports have focused on the identification of CRC. Studies on diagnostic value of miRNAs, especially for early stages or colorectal adenomas, are limited. In this study, four miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) were selected as study candidates, which had been reported to be abnormally expressed in CRC tissues. The serum levels of the four candidate miRNAs were determined. We found that serum levels of miR-20a and miR-486 could discriminate CRN patients from healthy controls, with an AUC values of 0.676 (P = 0.008) and 0.629 (P = 0.05), respectively. Moreover, serum miR-20a and miR-486 levels were downregulated in CRN patients, suggesting the potential function of two miRNAs as tumor suppressors. In addition, this study demonstrated that diagnostic value was improved after the combination of two miRNAs.

Interestingly, downregulated levels of serum miR-486 were observed in patients with adenoma, which was statistically significant. This result suggested that the early stage of colorectal carcinogenesis could be indicated with the decreased serum levels of miR-486. It was well known that various histologic subtypes of CRN presented different molecular alterations. It was essential to consider the relevance of different miRNAs as biomarkers with each type of lesion. In our data, serum miR-486 level was down-regulated in TVA and HGIN patients, compared to healthy controls, suggesting the diagnostic potential of serum miR-486 in detecting this precancerous neoplasia. Previous study reported that abnormal expression of miR-486 was associated with the presence of the KRAS mutation, with an incidence of 35%–45% in all CRC.[13] Therefore, this study further confirmed the potential of miR-486 in early detection of CRN. However, Yamada et al. reported that the expression of miR-20a in serum samples varied between CRN patients and healthy controls.[17] The sample size of their study was smaller than ours, with the participants from various populations.

The miR-92a was reported to be overexpressed in serum of CRC patients.[11],[18] However, no significant difference was observed the serum miR-92a levels between CRN patients and healthy controls in our study. The contradictory results deserved comments. First, unlike tissue or cellular miRNAs, there was no consensus for normalizing the expression level of circulating miRNAs in healthy controls.[19] In previous studies, miR-16 or RNU6B was selected for normalization. In our study, a reliable control was involved for normalization, which was spiked-in synthetic cel-miR-39. Second, SYBR Green-based miRNA qRT-PCR was applied in previous studies, with less accuracy than ours (TaqMan miRNA qRT-PCR). A recent study applied TaqMan miRNA qRT-PCR for serum miR-92a levels detection and cel-miR-39 for normalization, which provided consistent results with ours.[17]

In our study, serum miR-135b levels were too low to be accurately quantified. Some studies have reported increased levels of miR-135b in CRC and adenoma tissues.[9],[20] In stool samples, miR-135b level was significantly higher in patients with CRC or adenomas.[21] For the serum samples, previous study has reported consistent results with ours.[22] Therefore, in our study, serum miR-135b was excluded as biomarker for detecting CRC.

For CRC, the occurrence of molecular alterations may be earlier than histomorphology variations; therefore, the assay involving serum miRNAs may be earlier approach for lesion detection before advanced malignance. In addition, the blood sample was easily available and less invasive, so serum miRNA could be a promising molecular biomarker for discriminating CRN in clinical applications.

The limitations of this study were as follows. First, this study was just a pilot experiment, and the larger-scale, multi-center trials involving different ethnic populations were required for further validation. In addition, more male participants were included in the CRN group, and they were also older than the healthy controls. Second, we did not validate whether serum miR-20a or miR-486 expression was regarded as a specific biomarker for detecting CRN or if this was a common phenomenon in the development of any cancer due to perturbations in the host immune response.[23]


 > Conclusion Top


Four miRNAs selected from the literature were analyzed. The results validated that serum circulating miR-20a and miR-486 could be applied for discriminating CRN patients from healthy controls. Another interesting finding was potential of serum miR-486 in detecting precancerous colorectal lesion. These results provided rationale for the potential clinical applications of miRNAs-based molecular biomarker in noninvasive and early CRC detection.

Acknowledgments

This study was supported by the National Nature Science Foundation of China (No. 30572447) and Natural Science Foundation of Jiangsu, China (BK2012724).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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