The early diagnostic value of combined detection of plasma miR-181b, miR-196a and miR-210 in pancreatic cancer

Background This study aimed to investigate the effect of combination of plasma miR-181b, miR-196a and miR-210 on early diagnosis of pancreatic cancer (PC).Methods In our study, the plasma was isolated from patients with PC and healthy individuals, respectively. The expressions of miR-181b, miR-196a and miR-210 were detected by qRT-PCR. Moreover, the level of carbohydrate antigen 199 (CA199) was measured by electrochemiluminescence (ECL) assay. Furthermore, the area under the receiver operating characteristic (ROC) curve (AUC) was used to analyze the diagnostic efficacy of miR-181b, miR-196a, miR-210 and CA199, as well as the combination of thses miRNAs in early PC patients and healthy individuals.Results The expressions of miR-181b, miR-196a and miR-210 were significantly upregulated in PC patients. In addition, the level of CA199 was also significantly upregulated in the plasma of PC patients. The expressions of miR-181b, miR-196a and miR-210 were closely associated with lymph nodes metastasis, clinical stage and vascular invasion, but had no correlation with the patient's age, gender and tumor size. Moreover, miR-181b, miR-196a and miR-210 have lower AUC than CA199 in PC patients. The combinations miR-181b + miR-196a, miR-181b + miR-210, miR-196a + miR-210 also have lower AUC than CA199 in PC patients. It is worth noting that the combinations miR-181b + miR-196a + miR-210 have higher AUC than CA199 in PC.Conclusions Our study demonstrated that the combination of plasma miR-181b, miR-196a and miR-210 had good value for PC early diagnosis.

The early diagnostic value of combined detection of plasma miR-181b, miR-196a and miR-210 in pancreatic cancer CURRENT 11 It is reported that fecal miR-181b, miR-196a and miR-210 are highly expressed in PC patients, suggesting that those miRNAs may be used as biomarkers for PC diagnosis. 12 However, the diagnostic value of the combinations of miR-181b, miR-196a and miR-210 in the plasma of PC has not been fully elucidated.
In this study, the effects of plasma miR-181b, miR-196a and miR-210 in the early diagnosis of PC were investigated. The expressions of plasma miR-181b, miR-196a and miR-210 were measured by qRT-PCR. The level of classic tumor markers including carbohydrate antigen 199 (CA199) was detected by electrochemiluminescence assay. In addition, the receiver operating characteristic curve (ROC) was used to reveal the effect of plasma miR-181b, miR-196a and miR-210 in clinical diagnose value of PC. We hoped to explore the detail molecular mechanism of plasma miR-181b, miR-196a and miR-210 in 4 the early diagnosis of PC.

Clinical samples
From  Table 1. This study was approved by the Ethics Committee of our hospital and it was performed in accordance with the Decla ration of Helsinki. Written informed consent was obtained from each patient and volunteers.

Real-Time fluorogenic PCR assays
Total RNA from plasma of PC patients and healthy volunteers was extracted using TRIZOL (Invitrogen, USA). Then, cDNA was synthesized from total RNA using Revert Aid First

Electrochemiluminescence assay
The plasma of PC patients and healthy volunteers was collected. The level of CA199 were measured by using electrochemical luminescence apparatus (CobasE601, Roche, Sweden).
The detection threshold of CA199 was 27 U/mL.

Statistical analysis
All statistical analyses were performed using SPSS 22.0 statistical software (Chicago, IL).
The results were presented in the form of mean ± standard deviation (SD). The two-tailed t test was used for comparison between two groups, while one-way ANOVA was selected for comparison among multiple groups. The chi-square test was used to compare the counting data groups. The clinical diagnostic value of miRNA detection was analyzed by the area under the ROC curve (AUC). P < 0.05 was considered to be statistically significant.

The levels of miR-181b, miR-196a, miR-210 and CA199 are upregulated in PC patients
The results of qRT-PCR showed that the levels of miR-181b, miR-196a and miR-210 in the plasma of PC patients were higher than those in healthy volunteers (P < 0.001) ( Figure   1A-C). In addition, we also found that CA199 level in the plasma of PC patients was markedly higher than that in healthy volunteers (P < 0.001) ( Figure 1D). All the results suggested that the levels of miR-181b, miR-196a, miR-210 and CA199 are upregulated in PC patients.

Discussion
In recent years, PC has been a therapeutic challenge due to difficulties and delays in diagnosis, which leads to its poor prognosis. Up to now, the main treatment methods for PC are surgical operation, chemotherapy and radiotherapy. Nevertheless, The median survival time of PC is about 5 to 8 months, and the 5-year survival rate is only about 4%. 5 In this study, we explored the early diagnostic value of plasma miR-181b, miR-196a and miR-210 in PC patients and found that combined detection of plasma miR-181b, miR-196a and miR-210 has early diagnostic value for PC.
Accumulating evidence has reported that a variety of miRNAs could play important roles in PC. [13][14][15][16] For example, miR-381, a tumor suppressor, inhibits cell proliferation, migration 7 and invasion, and induces apoptosis in PC. 17 Zhou et al. have reported that miR-340 is downregulated in PC and could suppress PC cell growth and reduce tumor size. 18 Xu et al. 19 have indicated that miR-143 is lowly expressed in PC and could regulate PC process by promoting PC cell apoptosis and inhibiting migration and invasion. In addition, Xu et al. 19 have also confirmed that miR-143 expression is closely associated with the tumor size, clinical staging and lymph nodes metastasis. Previous researches have indicated that the expressions of miR-196a and miR-210 are upregulated in the plasma of PC patients, indicating miR-196a and miR-210 could serve as potential biomarker for PC diagnosis. 20 Ren et al. 12 have reported that fecal miR-181b, miR-196a and miR-210 are highly expressed in PC patients, suggesting that those miRNAs may be used as biomarkers for PC diagnosis. In the present study, we found that the expressions of miR-181b, miR-196a and miR-210 were closely associated with lymph nodes metastasis, clinical stage and vascular invasion, but had no correlation with the patient's age, gender and tumor size. Therefore, we speculate that the combinations of miR-181b, miR-196a and miR-210 may be used as biomarkers for PC diagnosis.