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  • br For the second screening phase we divided the


    For the second screening phase, we divided the 53 CRC patients according to the TNM staging system into two groups, namely, 33 pa-tients with stage IeII CRC (Group 1) and 20 patients with stage III–IV CRC (Group 2). The 16 candidate miRNAs were tested in the three plasma groups from the above two CRC groups and the group composed of 87 healthy controls (Table S3). A total of nine candidates, which were significantly altered in all three groups (P < 0.05), were selected for further detection in the validation phase (marked in red in Table S3). Out of the 9 miRNAs, 8 miRNAs, namely, miR-27a-3p, miR-143-3p, miR-144-3p, miR-148a-3p, miR-423-3p, miR-424-5p, miR-483-5p, and miR-1260b, presented significantly lower AR-13324 levels in the CRC group compared with the healthy group. By contrast, the CRC group showed significantly higher expression levels of miR-425-5p compared with the healthy group.
    Fig. 2. Relative expression level of plasma miRNAs in the validation phase. miRNAs were quantified in the plasma of 48 CRC patients, 20 patients with NCP, and 47 healthy controls. miRNA levels were normalized to spiked-in cel-miR-54-5p and represented in scatter plots. Data are shown as mean ± SD. The longest line in the middle represents the mean value. CRC: colorectal cancer.
    3.2. Validating the identified miRNAs
    To verify the expression of 9 miRNAs for potential use as plasma biomarkers in CRC diagnosis, we further assessed these miRNAs in additional 48 CRC patients, 20 patients with NCP, and 47 healthy controls (validation phase). Comparison between the CRC patients group and healthy controls group was performed. The trend of miRNA expression alteration was generally concordant between the screening phases and the validation phase, and 8 out of the 9 miRNAs, except miR-483-5p (P = 0.918), were significantly downregulated. To de-termine the validity of the 8 miRNAs, we further analyzed the expres-sion differences among the CRC patients, patients with NCP, and healthy controls. As shown in Fig. 2, five miRNAs (miR-27a-3p, miR-144-3p, miR-148a-3p, miR-424-5p, and miR-1260b) were significantly downregulated in both CRC patients and patients with NPC as com-pared with those in healthy controls. miR-143-3p was significantly downregulated in CRC patients and upregulated in patients with NCP compared with healthy controls. miR-425-5p did not show significantly differential expression between the patients with NCP and healthy controls (P = 0.0818) but was significantly downregulated in CRC patients. Considering the disability to distinguish CRC patients from the patients with NCP (P = 0.0899), we excluded miR-423-3p from vali-dation miRNAs. We finally selected seven miRNAs, which were sig-nificantly down-regulated in CRC patients compared with patients with NCP and healthy controls, with considerable potential as plasma bio-markers for CRC diagnosis.
    3.3. Diagnostic performance of plasma miRNAs for CRC
    3.4. Relationship between plasma miRNAs expression and clinical parameters
    To investigate the clinical significance of the seven plasma miRNAs, we explored the correlation of the miRNA expression levels with  Cancer Epidemiology 60 (2019) 67–76
    demographic and clinical factors using Student’s t-test or one-way ANOVA. In this analysis, samples from the validation set were used to conduct the AR-13324 calculation. No obvious differences were observed when the CRC patients were stratified by age, location, tumor size, lymphatic metastasis, distant metastasis, CEA expression level, CA19-9 expression level, or TNM stage. By contrast, the expression levels of five plasma miRNAs (miR-27a-3p, miR-143-3p, miR-148a-3p, miR-424-5p, and miR-425-5p) were correlated with the gender of CRC patients (P < 0.05). As shown in Table S5, the expression levels of miR-27a-3p, miR-143-3p, miR-148a-3p, miR-424-5p, and miR-425-5p in 31 male cases were significantly lower than those in 17 female cases. In addi-tion, we found that the miR-144-3p expression level in FOBT positive patients were significantly lower than those in negative patients (P < 0.05).