MicroRNA-124 alone might represent an indicator signifying cholangiocarcinoma prognosis

Background: Previous studies have demonstrated that microRNAs (miRNAs) played a crucial role in various diseases, including cancers. The aim of the study was to evaluate the clinical signicance of miR-124 in patients with cholangiocarcinoma (CCA). Methods: The expression pattern of miR-124 was detected in CCA tissues using quantitative reserve transcription polymerase chain reaction (qRT-PCR). The correlation of miR-124 expression with clinicopathological features and overall survival of patients were explored using chi-square test, Kaplan-Meier methods and Cox regression analyses. Results: The miR-124 expression level was strong down-regulated in CCA tissues compared with normal para-cancerous tissues (P<0.001). Moreover, aberrant miR-124 expression was signicantly associated with differentiation (P=0.045) and lymph node metastasis (P=0.040). In addition, Kaplan-Meier method and log-rank test revealed that patients with low miR-124 expression has a poorer overall survival compared with those with high miR-124 expression (P=0.002). Furthermore, multivariate analysis conrmed that miR-124 expression (P=0.006; HR=2.006; 95%CI: 1.224-3.289) was an independent prognostic indicator in CCA. Conclusions: Collectively, our results dened miR-124 expression plays important roles in CCA patients. MiR-124 expression might used as a valuable prognostic biomarker for patients with CCA.


Background
Cholangiocarcinoma (CCA) is a devastating malignancy derived from bile duct epithelial cells, and the mortality associated with CCA is currently increasing worldwide [1]. According to the statistics, the incidence of CCA changes widely among different geographic regions; the highest number of cases occurs in southeast Asia while the lowest number occurs in Australia [2]. Anatomically, CCA is categorized as intrahepatic cholangiocarcinomas (IHCCs) and extrahepatic cholangiocarcinoma (EHCCs), the latter being further divided into perihilar CCAs and distal CCAs [3]. CCA is di cult to diagnosis in early-stage due to its lack of early symptoms [4], and it is featured by high frequency of recurrence and metastasis. What's more, there is no effective chemoprevention or treatment, leading to an unfavorable prognosis [5].
MicroRNAs (miRNAs) are a type of endogenous coding small molecular RNAs with 18-25 nucleotides in length that could negatively control the translational inhibition of target mRNAs through base-pairing with their 3'-untranslated region (3'-UTR) [6,7]. MicroRNAs are known to play an important role in diverse biological processes, such as apoptosis, cell proliferation, and differentiation [8], which displays their functionality in carcinogenesis as tumor suppressor genes or oncogenes [9][10][11].
miR-124 is one of the cancer-related miRNAs. It is a brain-enriched miRNA and was rst discovered to be involved in stem cell regulation and neuro-development [12,13]. Previous studies indicated that miR-124 is down-regulated in various cancers. For example, in colorectal cancer, miR-124 is strongly decreased, and promotes apoptosis of colorectal cells [14]. In addition, Li et al. revealed that miR-124 is reduced and negatively related to lymph node metastasis in breast cancer [15]. The above studies indicated miR-124 play a crucial role in tumorigenesis and progression. However, the clinical signi cance of miR-124 remains unknown in CCA.
In the present study, we measured the relative expression level of miR-124 in CCA tissues via qRT-PCR, and analyzed the relationship of miR-124 level with clinicopathological features. We also explored the prognostic performance of miR-124 in CCA.

Patients and tissues samples
The use of tissues for this study has been approved by the Ethics Committee of the PLA Rocket Force Characteristic Medical Center. At the time of initial diagnosis, all patients had provided consent in the sense that their tumor samples could be used for investigation purposes. Written informed consents were received from all participants involved in the study.
In total, 119 frozen cancerous samples from CCA patients undergoing surgery from at our department were collected in this study. In addition, 119 matched para-cancerous samples were collected. All the specimens were diagnosed by two pathologists separately. None of the patients recruited in this study had undergone preoperative chemotherapy or radiotherapy. The clinical features of the patients, including age, gender, tumor size, location, distant metastasis, differentiation, TNM stage, and lymph node metastasis were listed in Table 1 was measured in triplicate, and the relative amount of miR-124 to U6 was calculated using the 2 -△△CT method.

Statistical analysis
All statistical analyses were preformed with the SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and graphs were generated via GraphPad Prism 5.0 (GraphPad Software Inc., USA). Data are shown as the mean ± standard deviation (SD). The Independent Student t test was used to compare the differences between groups. The relationship of miR-124 expression with clinicopathological factors was analyzed using Chi-square test. Patients survival and their differences were determined by the Kaplan-Meier method and log-rank test. A Cox's regression model was used for univariate and multivariate analysis. P<0.05 was considered to indicated a statistically signi cant result.

Results
Aberrant expression of miR-124 in CCA tissues Firstly, we compared the miR-124 expression in 119 CCA tissues and para-cancerous tissues with qRT-PCR assay. As indicated in Figure 1, the miR-124 level was signi cantly lower in CCA tissues than paracancerous samples (P<0.001), suggesting that expression of miR-124 was down-regulated in CCA tissues.
Association of miR-124 expression with clinicopathological characteristics in CCA The correlation between miR-124 expression and clinicopathological parameters was examined in 119 cases ( predictor of poor survival in breast cancer [21]. Gilje et al. showed that miR-124 is a potential new biomarker for prediction of neurological prognosis following cardiac arrest [22]. Han et al. demonstrated that miR-124 had a low expression in osteosarcoma tissues and low miR-124 expression was an unfavorable prognostic factor for overall survival [23]. Zhang et al. proved that miR-124 expression level was signi cantly decreased in lung cancer tissues and miR-124 was an independent prognostic factor for overall survival in lung cancer [24]. However, to our knowledge, the clinical signi cance of miR-124 in CCA has not been reported. Therefore, we investigated the feasibility of miR-124 as a new potential biomarker of prognosis for CCA.
In the present study, our data showed that miR-124 expression was signi cantly decreased in CCA tissues in comparisons with normal para-cancerous tissues. The relationship between miR-124 expression and various clinicopathological characteristics of CCA patients was investigated. The results indicated that abnormal miR-124 expression was dramatically correlated with differentiation and lymph node metastasis. It demonstrated that miR-124 might be involved in tumor metastasis and progression of CCA.
Furthermore, the overall survival rate of low miR-124 group was signi cantly shorter than that of high miR-124 expression group. Finally, in a multivariate Cox model, we found that miR-124 expression was an independent poor prognostic indicator for overall survival of CCA patients. Our results were in line with previous studies. For example, Tian et al. showed that miR-124 was reduced in CCA tissues and negatively related to lymph node involvement and distant metastasis [25].
In conclusion, our results showed that miR-124 expression was remarkably reduced in CCA tissues. The expression of miR-124 was closely associated with the progression of CCA patients. More importantly, the down-regulation of miR-124 is an independent poor indicator of prognosis in CCA patients. We believed that it is just the beginning of a long and still unexplored avenue of a highly promising investigation of miR-124. A more in-depth and larger-scale study remains to identify the role of miR-124 in CCA.

Consent for publication
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding
Funding information is not applicable.

Competing interests
The authors declare that they have no competing interests.  The relative expression of miR-124 was examined in CCA tissues via qRT-PCR assay. Results showed that miR-124 level was signi cantly lower in CCA tissues compared with normal para-carcinoma tissues (P<0.001).

Figure 2
Kaplan-meier analysis of the impact of miR-124 expression on overall survival. Patients with low miR-124 expression had a shorter overall survival rate than those with high miR-124 expression (P=0.002).