Diagnostic and prognostic value of miR-106a in colorectal cancer

We sought to systematically evaluate the diagnostic and prognostic value miR106a in patients with colorectal cancer (CRC). An original study was conducted to explore correlations between tissue miR106a levels and outcomes for 138 patients diagnosed with CRC. To explore the diagnostic performance of miR106a, eligible studies were identified from medical databases from China and abroad. Based on these results, 15 studies (including our original study) were pooled and included in a meta-analyses. The pooled sensitivity, specificity, and diagnostic odds ratios of miR106a were 0.53 (95% confidence interval (CI): 0.49–0.57), 0.85 (95% CI: 0.82–0.88), and 7.22 (95% CI: 3.17–16.44) for diagnosis of CRC, and the area under the curve (AUC) for miR106a when diagnosing CRC was 0.72. Patients with higher expression of tissue miR106a had poor overall survival (pooled hazard ratio (HR): 1.50; 95% CI: 1.02–2.20), but not disease-free survival (pooled HR: 1.03; 95% CI: 0.40–2.65). Overexpression of miR106a may predict superior metastasis-free survival (pooled HR: 0.65; 95% CI: 0.33–1.27), but the effect was not significant in this study (p = 0.21).


SUPPLEMENTARY METHODS FOR ORIGINAL STUDY Tissue processing
This study was approved by the local Human Investigations Committees of the Second Affiliated Hospital of Nanchang University, Nanchang, and all patients were enrolled with informed consent under institutional review board-approved protocols. CRC tissue and corresponding normal tissues were obtained from 138 patients by surgical resection in the our hospital between January 2008 and October 2015. Normal tissues were validated by histopathology, and were taken from the surgical margins, at least 10 cm away from tumor. After these procedures, all tissues samples were immediately frozen in liquid nitrogen and kept at -80˚C until RNA extraction. No patients had received adjuvant treatment including radiotherapy or chemotherapy prior to surgery and diagnosis.

RNA isolation and qRT-PCR
Total RNA was extracted from tissue and plasma samples using Mini RNeasy Kits for tissues (QIAGEN, CA, USA) and TRIzol LS Reagent (Invitrogen, CA, USA) for plasma, followed with DNase I digestion using the RNase-Free DNase Set (QIAGEN, CA, USA) to exclude genomic DNA contamination. Mature miR106a and internal control U6 were detected by stem-loop real-time RT-PCR analysis using Taman Human MicroRNA Assay kits (Applied Biosystems, Foster City, CA, USA).

Heat map for gene differential expression
Heat map analysis, also known as cluster analysis was used to judge clustering mode under different experimental conditions by R-heatmap.2 (http://www. inside-r.org/packages/cran/gplots/docs/heatmap.2). To classify different expression by hierarchical clustering or K-means and other methods, and different colors represent different cases of the cluster group. In my heat map, all fold-change calculations from CT values using the ΔΔCT method of relative quantification were input, minValue and maxValue were found. Gradient legend is a pane of blended colors derived from the color range definitions. A linear scale is drawn with two drag-able pointers. Each color defined for a numeric range blends with the next color, thus forming a gradient strip.

Statistical analysis
To determine the diagnostic performance of serum miR106a level in colorectal cancer, receiver operating characteristic (ROC) analysis was performed and the area under the curve (AUC) value was calculated. The optimal cutoff threshold was determined at the point on the ROC curve at which (sensitivity+apecificity-100%) was maximal. Sensitivity and specificity were calculated with this cutoff value.
Survival analyses were conducted using the Kaplan-Meier method. Univariate Cox's proportional hazard regression analyses were applied to estimate hazard ratios (HRs) of death according to tissue miR106a expression levels. And multivariate models were used to adjust potential confounding factors for death, including age, sex, TNM stage, pathological differentiation and side of the tumor (left or right).

Supplementary methods for literature search and study selection
The following keywords were used in the searches: ("colorectal" OR "colon" OR "rectal") AND ("cancer" OR "tumor" OR "carcinoma" OR "adenocarcinoma") AND ("micro RNA" OR "micro rna" OR "miR" OR "micro RNA106a"OR "miR106a"). References of relevant articles and reviews were also scanned to include possible missed articles. Titles and abstracts were first scanned, and then full papers of potential eligible studies were reviewed. Meeting abstracts were excluded because of the limited data. Articles as full papers in English and Chinese were evaluated for eligibility. The retrieved studies were carefully examined to exclude potential duplicates or overlapping data.
Articles were included if they met all the following criteria: (1) study should evaluate diagnostic or prognostic value of blood or tissue miR106a level in CRC patients.
(2) for diagnostic studies, histologic assessment should be applied as reference standard for CRC; and (3) for studies analyzed the diagnostic value of miR106a, absolute number of true-positive (TP), false-positive (FP), truenegative (TN) and false-negative (FN) were reported or could be calculated; for prognostic studies, hazard ratio (HR) or risk ratio (RR) values with 95% confidence intervals (95% CI) were provided or could be calculated. Quality assessment of the included studies by QUADAS-2. It summarized "risk of bias'' and ''applicability concerns'' through judging each domain for each included study. It shows the major biases concentrated upon the ''patient selection'' and ''index text''. B. Publication bias from Deeks' test is shown by funnel plots. Every point represents one study and the line is the regression line. It shows no publication bias exists. C. The overall distribution of studies is summarized in the likelihood matrix. Each point corresponds to a study. One studies, Li. J et al was on the left side of the matrix, indicating a sensitive "rule out" test. However, it reported reasonable sensitivity with incorporation bias from knowledge of a desaturation study outcome. D. Fagan's nomogram describes the possibility miR-106 assay to confirm or exclude cancer patients. In detail, for any people with a pre-test probability of 20% to have cancers, if the miR-106a test in cancer detection was positive, the post-test probability to have cancer would rise to 55%; while a negative result of miR-106 assay meaning the post-test probability would drop to 12% for the same people. Hence, miR-106a assay may play an important role as initial screening method for cancer.