Serum miR‐92a‐1 is a novel diagnostic biomarker for colorectal cancer

Abstract Colorectal cancer (CRC) is one of the most common cancers worldwide, with high mortality. Abnormally expressed microRNAs (miRNAs) are considered novel biomarkers in cancer diagnosis. The aim of this study was to investigate the diagnostic value of miR‐92a‐1 in patients with CRC. Serum samples were collected from 148 patients pathologically diagnosed with CRC and 68 gender‐ and age‐matched healthy volunteers. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to measure serum miR‐92a‐1 level. Relationship between miR‐92a‐1 and clinicopathological features of CRC cases was analysed via chi‐square test. Receiver operating characteristic (ROC) curve was plotted to estimate the diagnostic value of miR‐92a‐1 in CRC. Serum miR‐92a‐1 was significantly up‐regulated in CRC patients compared with healthy individuals (P < .001). Moreover, miR‐92a‐1 expression was correlated with TNM stage (P = .02), histological stage (P = .003), lymph node metastasis (P = .003) and distant metastasis (P < .001). ROC analysis showed that the area under the ROC curve (AUC) was 0.914, suggesting high diagnostic accuracy of miR‐92a‐1 in ROC. The optimal cut‐off value was 1.485, with a sensitivity of 81.8% and a specificity of 95.6%. MiR‐92a‐1 is increased in CRC patients and correlated with aggressive clinical characteristics. Serum miR‐92a‐1 may be a potential diagnostic biomarker for CRC.

MiR-17-92 cluster includes four families: miR-17, miR-18, miR-19 and miR-92a. 20 MiR-92a family includes four members: miR-25, miR-92a-1, miR-92a-2 and miR-363. The members of miR-92a family play significant roles in the development and progression of CRC, and might serve as biomarkers for the cancer. A study carried out by Li et al demonstrated that the expression of miR-25 was up-regulated in CRC tissues and that its overexpression predicted poor prognosis for the patients. 2 In the study of Xu et al, the expression profile of miR-363 was confirmed to be a novel diagnostic biomarker for CRC. 21 MiR-92a has ever been reported to be involved in the development and metastasis of CRC and serve as an effective biomarker for the cancer diagnosis and prognosis. [22][23][24] Therefore, we speculated that the expression of miR-92a-1 might also act as an indicator in early detection of CRC.
In this study, we sought to investigate the expression level of miR-92a-1 in CRC patients, and its association with clinical characteristics.
In addition, we explored the diagnostic value of miR-92a-1 in CRC.

| Patients and sample collection
The present study was a retrospective investigation. A total of 148 CRC patients were recruited from Cangzhou Central Hospital.  (Table 1). This study was approved by the ethics committee of the hospital. All participants provided written informed consents for this research.

| RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were extracted from serum samples adopting TRIzol reagent kit (catalogue number: 15596026, Invitrogen) as per the manufacturer's protocols. The ratio of OD260/OD280 (1.9-2.0) was used to evaluate the purity of isolated RNAs. Total RNA was used for reverse transcription reaction employing TaqMan miRNA reverse transcription kit (catalogue number: 4366596, Applied Biosystems).

| Statistical analysis
Continuous data were present as mean ± SD and compared adopting Student's t test. Chi-square test was used to examine the correlation of miR-92a-1 with clinicopathological features of CRC patients.
Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic value of miR-92a-1 in CRC. All data analyses were performed in SPSS 21.0 statistical software. P-value less than .05 was considered as significant level.

| Expression level of miR-92a-1
According to qRT-PCR, the expression of miR-92a-1 was significantly increased in CRC patients, compared with healthy controls (Figure 1).

| Association of miR-92a-1 with clinicopathological characteristics of CRC
In the current study, CRC patients were divided into high (n = 86) and low (n = 62) expression groups, according to their median expression of miR-92a-1 in CRC tissues. Chi-square test was used to analyse the relationship between miR-92a-1 expression and clinicopathological profiles of CRC. Analysis results indicated that the overexpression of miR-92a-1 was correlated with advanced TNM stage (P = .02), poor histological stage (P = .003), positive lymph node metastasis (P = .003) and distant metastasis (P < .001). There was no significant relationship between miR-92a-1 and age, gender, or tumour size or location (all P > .05, Table 1).

| Diagnostic value of miR-92a-1 in CRC
To explore diagnostic value of miR-92a-1 in CRC, ROC analysis was performed and showed an area under the ROC curve (AUC) of 0.914, suggesting its high diagnostic accuracy in CRC. The cut-off value of miR-92a-1 for CRC diagnosis was 1.485, with a sensitivity of 81.8% and a specificity of 95.6% (Figure 2).

| D ISCUSS I ON
CRC is a prevalent malignant tumour in the world. 26 Despite significant improvements in surgical, neoadjuvant chemotherapy and radiotherapy, the survival rates of patients with CRC are still dismal. 27,28 Early diagnosis is essential for survival in CRC patients. 29 Until now, the gold standard for CRC diagnosis is colonoscopy, but its invasive nature limits its wide application in clinical practices. 30 Commonly used serum biomarkers for CRC diagnosis include CEA and CA19-9.
However, both of them only show low accuracy in CRC diagnosis. 31  Consequently, novel biomarkers with high sensitivity and specificity are urgently required for CRC diagnosis.
MiRNAs, short non-coding RNAs, play important roles in physiological and pathological conditions. Their expression profiles show close association with tumour development, progression and treatment response, suggesting their indicative functions in human malignancy. 32 In addition, the expressions of miRNAs are stable and can be easily detected in archived tissue specimens and body fluids. 33 According to existing documents, miRNAs may provide promising approaches for cancer diagnosis. In previous studies, several miR-NAs have been confirmed to play predictive roles in the processes of CRC. Wang et al reported that circulating miR-210 level was significantly different between CRC patients and healthy individuals, which showed diagnostic and prognostic capability for the cancer. 34 MiR-223, as another example, was reportedly up-regulated in CRC tissues, and its elevated expression was correlated with positive metastasis and poor prognosis in the patients. 35 In a word, abnormally expressed miRNAs are involved in the aetiology of CRC and might serve as indicators for the cancer.
As a member of miR-17-92 cluster, miR-92a was correlated with the progression of CRC. [22][23][24] Therefore, we speculated that miR-92a-1, the precursor of miR-92a, might play a crucial role in the progression of CRC.
In the current study, we investigated serum level of miR-92a-1 in CRC In summary, miR-92a-1 is up-regulated in CRC patients and correlated with malignant tumour development and progression. Serum miR-92a-1 may be a novel diagnostic biomarker for CRC.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
YS conceived and designed the experiments; ZL conceived and performed the experiments; and YS prepared figures. YS and ZL wrote the main manuscript text. All authors reviewed the manuscript. With the approval of Cangzhou Central Hospital Ethics Committee, written informed consent was obtained from every patient.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.