miR-124 Functions as A Marker in Diagnosing Colorectal Cancer

Background: MicroRNA-124 (miR-124) regulates cell growth, angiogenesis, invasion, and apoptosis of many human tumors. Our objective was to evaluate the levels of miR-124 in serum samples of colorectal cancer (CRC) patients to explore whether miR-124 in serum can be used as a biomarker for the detection of CRC. Methods: Serum miR-124 level was measured in 85 patients with CRC and 60 healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). The correlations between miR-124 levels and clinicopathologic factors were analyzed. Diagnostic performance of serum miR-124 level was calculated by using the receiver operating characteristic (ROC) curve. Results: The expression level of miR-124 in serum samples was signicantly lower in CRC patients than in healthy controls (P<0.001). A positive correlation between miR-124 level and tumor size (P=0.010), invasion depth (P=0.021), lymph node metastasis (P=0.015), and TNM stage (P=0.004) was observed. The serum miR-124 yielded an AUC (areas under the ROC curve) of 0.832 with a sensitivity of 80.0% and a specicity of 81.7%, and the optimal cutoff point of miR-124 was 1.61. Conclusions: Serum miR-124 may have a potential as a novel biomarker for the detection of CRC.


Background
Colorectal cancer (CRC) is the most common cancer worldwide, and it is the second cause of cancer death just behind lung cancer [1,2]. It causes 655,000 deaths worldwide every year [3]. Although the true etiology of CRC is still not clear, but there is an in-depth study for the risk factors of CRC, including environment, diet, and the synergy of lifestyle and genetic factors. Despite great progresses have been made in therapy of CRC, 30-40% patients still died of relapse and metastasis, among which liver metastasis is the leading cause of death [4][5][6]. Several CRC screening tests, including fecal occult-blood testing and colonoscopy, have been available for years and have aided in reducing the mortality associated with this disease [7][8][9][10]. However, the prognosis effect of patients with CRC is not ideal. Therefore, it is urgent to develop an effective noninvasive assay to screen patients of CRC at early stage.
MiRNAs are single-strand small RNA molecules that are between 19 and 22 nucleotides in length. They participate in various biological processes, such as cell proliferation, differentiation, apoptosis, metabolism and tumorigenesis through inhibition of RNA translation or degradation of target messenger RNA by binding to 3' untranslated region (UTR) of the target genes [11]. Studies con rmed that miRNAs play an important adjustment function in the process of the tumor development by regulating protooncogenes or tumor suppressor genes [12]. Recent studies have shown that miRNAs abnormally expressed in peripheral blood of CRC patients. Besides, miRNAs can participate in the progress of CRC, and have the potential function that serve as biomarkers for human cancers [13][14][15]. MicroRNA-124 (miR-124), a brain-enriched miRNA, was rst found to be involved in stem cell regulation and neurodevelopment [16,17]. Studies showed that miR-124 expression is down-regulated in a variety of cancers, including breast cancer, non-small cell lung cancer, and colorectal cancer [18][19][20]. However, the role of miR-124 in the diagnosis and prognosis of CRC has not been reported.
In the present study, we determined the serum miR-124 expression in CRC specimens, investigated the correlations between miR-124 expression and clinicopathological parameters, and further detected the diagnostic value of miR-124 for CRC.

Methods
Patients and sample collection.
The study was approved by the Ethics Committee of Chinese PLA General Hospital, and all the patients provided written informed consent in advance. Serum sample collection was performed between Chinese PLA General Hospital. 85 patients with CRC and 60 healthy controls were included in this study. None of the healthy controls had formerly been diagnosed with any malignancy. The clinicopathological parameters of patients were summarized in Table 1. Blood samples were obtained prior to surgery. Serum was separated after centrifugation (3,000 rpm, 10 min) and stored at -80˚C.

Results
Serum miR-124 down-regulation in blood samples of CRC.
The expression levels of serum miR-124 in CRC samples and healthy samples were detected by qRT-PCR. The results indicated that serum miR-124 expression in the CRC samples was signi cantly downregulated in comparison to that in the healthy control samples (P < 0.001, Figure. 1).
Relationship between miR-124 and clinicopathological characteristics of CRC patients  Figure. 2).
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from serum samples using MicroMini Kit (Qiagen) according to manufacturer's protocol. The RNA concentration and purity were controlled by UV spectrophotometry using a Nanodrop ND-1000 (Thermo Scienti c). The RNA specimens were stored at -80℃ until reverse transcription. The reverse transcription reaction was carried out with miScript Reverse Transcription Kit (Qiagen). For synthesis of cDNA, the reaction mixtures were incubated at 37℃ for 60 min, at 95℃ for 5 min and then held at 4℃. The cDNA specimens were stored at -20℃ until PCR. The amounts of miRNAs were quanti ed by qPCR using the miScript SYBR Green PCR kit (Qiagen). Quantitative PCR was run on a 7500 Real-Time PCR system (Applied Biosystems). The reaction mixtures were incubated at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s, 56℃ 30 s and 72℃ 35 s. The relative expression level (fold change) of miR-124 was determined by the 2 −ΔΔCt method using U6 as the endogenous control to normalize the data.

Statistical analysis
Statistical analyses were performed using SPSS 21.0 software (SPSS, Inc, Chicago, Illinois). Data were expressed as mean ± SD. The independent sample t-test was utilized to determine the statistical difference between CRC patients and healthy controls. The relationship between clinicopathologic parameters and miR-124 levels was examined using the χ 2 test. Sensitivity, speci city and area under curve (AUC) for serum miR-124 levels were determined using receiver operator characteristic (ROC) analysis. P-values of < 0.05 were considered to represent statistical signi cance.

Discussion
Colorectal cancer remains a signi cant cause of mortality worldwide. The CRC incidence and mortality are actually increasing despite recent advances in surgery, radiotherapy and chemotherapy [21]. And despite curative surgical resection of the primary tumor, 40-50% of the patients ultimately die of metastasis [22]. Due to fact that CRC usually appear obvious symptoms until it progresses to advanced stages, so the implementation of screening programs aimed at early detection is essential to reduce incidence and mortality rates. Currently, The most common clinical imaging screening methods is colonoscopy. However, there were some limitations for this method such as invasion character, and causing abdominal pain [23]. In addition, the sensitivity and speci city of the laboratory detection methods, such as stool test, occult blood immunology and tumor markers detection are poor [24].
Therefore, looking for a novel biomarker for CRC non-invasive examination is of important clinical signi cance.
Recent studies indicated that miRNAs, which are non-protein-coding small RNAs, are involved in cancer progression and metastasis. MiRNA expression signatures have been shown to be promising biomarkers for understanding the tumorgenesis of a wide array of human cancers [25,26]. Accordingly, studies speculated that some tumors secretion circulation miRNAs in serum or plasma could be molecular markers for clinical diagnosis of CRC. Ng et al. found that the high expression of miR-17-3p and miR-92a was closely related to CRC, and the expression levels of plasma miR-17-3p and miR-92a decreased signi cantly after surgery [27]. The results con rmed abnormal expression of miRNAs in the plasma circulation. Huang et al. further con rmed the diagnosis potential of miR-92a, indicated that the plasma of patients with CRC contains high levels miR-92a, and its diagnostic sensitivity and speci city are consistent with the results of Ng et al. [28].
Previous research con rmed that miR-124 was a tumor suppressor in various types of cancers, including glioblastoma, hepatocellular carcinoma, medulloblastoma, and gastric cancer [29,30]. Xie et al. [30] reported that miR-124 was down-regulated in gastric cancer cells and specimens and it inhibited cancer cell proliferation and induced apoptosis by targeting enhancer of zeste homolog 2 in gastric cancer. The expression level and mechanism of miR-124 have also been investigated in breast cancer. Han et al. [31] found that miR-124 played a critical role in inhibiting the invasive and metastatic potential of breast cancer cells. Moreover, the previous study also reported the function of miR-124 in colorectal cancer, it was signi cantly downregulated in CRC and interacted with ROCK1 to regulate the tumor cell proliferation [20]. However, the diagnostic value of miR-124 in CRC was not studied.
This study is the rst to demonstrate the potential role of serum miR-124 in the early detection of CRC.
The data showed that patients with CRC had signi cantly lower serum miR-124 levels compared with healthy control subjects. To evaluate the relationship between serum miR-124 and CRC progression, we further analyzed the correlation between miR-124 expression and clinical pathological features of CRC. The results demonstrated that there is a strong correlation between serum miR-124 levels and tumor size, invasion depth, lymph node metastasis, and TNM stage. These observations suggested a correlation between increased expression of miR-124 and clinical progression in CRC. Furthermore, we established the ROC curve to evaluate the diagnostic value of miR-124 in CRC. These results indicate that downregulation of serum miR-124 may occur in the early stage of CRC and can serve as a potential biomarker of early diagnosis in CRC.

Conclusions
In conclusion, the present study provides evidence that serum levels of miR-124 could be used to predict CRC disease progression. However, further investigations to determine the clinical values of miR-124 as a predictor for the patients with CRC are needed. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  Serum miR-124 expression in CRC patients (n=85) and healthy volunteers (n=60). MiR-124 expression was signi cantly lower in patients with CRC than that in healthy controls (**, P<0.001).