Igarashi H. miR-31 and anti-EGFR therapy in CRC.
SUPPLEMENTARY METHODS
Colon cancer cell line and RNA extraction
In this study, eight colon cancer cell lines (CaCO2, DLD1, HCT116, HT29, Lovo, RKO, SW48,
and SW480) were utilized. Total RNA was extracted from cell pellets using the TRIzol Reagent
(Invitrogen by Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.
RNA extraction from paraffin-embedded (FFPE) tissues and quantitative reverse
transcription-PCR of microRNA
Total RNA was extracted from FFPE tissues using the miRNeasy FFPE Kit (Qiagen, Valencia,
CA, USA) according to the manufacturer’s instructions. microRNA-31 (miR-31)-5p or -3p
expression were analyzed by quantitative reverse transcription-PCR as previously described.1
ΔCT was calculated by subtracting the CT values of U6 from the CT values of the gene of interest.
Expression of miR-31-5p or -3p in the samples were calculated using the equation 2−ΔCT, where ΔCT
= (CT miRNA − CT U6). microRNA (miRNA) expression levels were quantified in colorectal cancer
(CRC) specimens and paired normal mucosa specimens. To calculate the relative expression levels
of miRNA in each CRC, 2−ΔCT of the cancer tissue was divided by 2−ΔCT of the paired normal tissue.
The distribution of the relative miR-31-5p expression level in the 102 CRC specimens was as
follows: mean, 12.3; median, 4.6; standard deviation (SD), 22.7; range, 0.02–124.8; interquartile
range, 1.3–12.6. In our previous study of miR-31-5p expression in 721 CRC specimens, we defined
patients with CRC whose relative miR-31-5p expression level was ≥23.4 as “miR-31-5p highexpression group” and with <23.4 as “miR-31-5p low-expression group.” Since the high-expression
group consisted of a distinct group 1, we used this cut-off value for statistical analysis in the present
Igarashi H. miR-31 and anti-EGFR therapy in CRC.
study as well. Thus, 11 patients were in the high-expression group and 91 were in the low-expression
group.
By contrast, the distribution of the relative miR-31-3p expression level in the 102 CRC specimens
was as follows: mean, 0.71; median, 0.18; standard deviation (SD), 1.28; range, 0.00002–6.28;
interquartile range, 0.04–0.74. Cases with miR-31-3p expression were then divided into quartiles for
further analysis: Q1 (<0.04), Q2 (0.04–0.18), Q3 (0.19-0.73), and Q4 (≥0.74). We defined patients
with CRC whose relative miR-31-3p expression level was ≥ 0.74 (Q4) as “miR-31-3p highexpression group” and patients whose level was < 0.74 (Q1, Q2, and Q3) as “miR-31-3p lowexpression group.” Thus, 25 patients were in the high-expression group and 77 were in the lowexpression group.
DNA extraction and pyrosequencing of KRAS, NRAS, and BRAF
Genomic DNA was extracted from FFPE tissues of colorectal tumors using QIAamp DNA FFPE
Tissue Kit (Qiagen) according to the manufacturer’s instructions. Using extracted genomic DNA,
PCR and targeted pyrosequencing were performed for KRAS (codons 61 and 146) 2, NRAS (codons
12, 13, and 61) 3, and BRAF (V600E) 4 as previously described.
Igarashi H. miR-31 and anti-EGFR therapy in CRC.
REFERENCES (SUPPLEMENTARY METHODS)
1.
Nosho K, Igarashi H, Nojima M, et al. Association of microRNA-31 with BRAF mutation,
colorectal cancer survival and serrated pathway. Carcinogenesis. 2014;35:776-83.
2.
Imamura Y, Lochhead P, Yamauchi M, et al. Analyses of clinicopathological, molecular, and
prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer:
cohort study and literature review. Mol Cancer. 2014;13:135.
3.
Irahara N, Baba Y, Nosho K, et al. NRAS mutations are rare in colorectal cancer. Diagn Mol
Pathol. 2010;19:157-63.
4.
Liao X, Morikawa T, Lochhead P, et al. Prognostic role of PIK3CA mutation in colorectal
cancer: cohort study and literature review. Clin Cancer Res. 2012;18:2257-68.