Gender effects of single nucleotide polymorphisms and miRNAs targeting clock-genes in metastatic colorectal cancer patients (mCRC)

The circadian system is composed of a set of clock-genes including PERIOD, CLOCK, BMAL1 and CRY. Disrupting this system promotes cancer development and progression. The expression levels of miR-206, miR-219, miR-192, miR-194 and miR-132 regulating clock-genes and three functional polymorphisms rs11133373 C/G, rs1801260 T/C, rs11133391 T/C in CLOCK sequence were associated with the survival of 83 mCRC patients (50 males and 33 females). Longer overall survival (OS) was observed in women compared to men, 50 versus 31 months. This difference was associated with rs11133373 C/C genotype (p = 0.01), rs1801260 T/C+C/C genotype (p = 0.06) and rs11133391 T/T genotype (p = 0.06). Moreover women expressing high levels (H) of miR-192 (p = 0.03), miR-206 (p = 0.003), miR-194 (p = 0.02) and miR-219 (p = 0.002) had a longer OS compared to men. In women longer OS was reinforced by the simultaneous presence of two or more H-miR, 58 months versus 15 months (p = 0.0008); in this group of women an OS of 87 months was reached with the additional presence of rs11133391T/T genotype (p = 0.02). In this study we identified a subgroup of female patients who seems to have a better prognosis. Personalized medicine should prospectively take into account both genetic and gender differences.


SUPPLEMENTARY
. Gender-related survival for rs11133373 C/G, rs1801260 T/C and rs11133391 T/C polymorphisms PFS    Log-rank test*

DNA extraction and genotyping
gDNA was extracted by means of QIAmp DNA Blood kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. gDNA purity and concentration was measured by Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE). Pyrosequencing technique has been used to detect rs11133373 C/G, rs1801260 T/C and rs11133391 T/C polymorphisms.
Primers for PCR amplification and pyrosequencing were chosen by pyromark software (Biotage) and are listed in supplementary Table S4.
All PCR reactions were carried out for 40 cycles in a total volume of 25µl containing 10ng of gDNA, 0.2 μM of each primer (forward and reverse), 12.5 μl PCR Master Mix (Diatheva, Fano, Italy) and 0.625 U HotStarTaq polymerase (Diatheva). Reaction parameters were 95°C for 10 min followed by 40 cycles of 95°C for 30 s, 59°C for 20 s and 72°C for 30 min. A final extension at 72°C was carried out for 3 min. Successful and specific amplification of the region of interest was verified by visualizing 5 μl of the PCR product on a 2% agarose gel electrophoresis. The Pyrosequencing technique was performed on a PSQ 96MA instrument (Biotage) using PyroGold reagents (Qiagen) following the protocol suggested by the manufacturers and the determination of rs11133373 C/G, rs1801260 T/C and rs11133391 T/C polymorphisms were made by using PyroMark™ ID program (Qiagen).

miRNAs extraction and Quantitative Real-Time Polymerase Chain Reaction (q-RT-PCR)
Three to five 10-μm sections from FFPE specimens were obtained from the primary tumor. Representative areas from FFPE tumor blocks were evaluated by pathologists. Before cutting sections for miRNAs isolation, one slide was prepared for hematoxylin and eosin staining to select only representative samples with almost complete tumor infiltration. All assays were performed by investigators who were blinded to the clinical data of the sample cohort.
Total cellular RNA was isolated from human FFPE specimens using the miRNeasy FFPE Kit (Qiagen) according to the manufacturer's instructions.
The extracted RNA was quantified and its purity was evaluated by the NanoDrop 1000 spectrophotometer (Nanodrop Technologies) and 250 ng of total RNA was reverse transcribed using the miScript II RT Kit (Qiagen) according to the manufacturer's instructions. Conditions for the reverse transcription (RT) reaction were as follows: 37°C for 60 minutes and 95°C for 5 minute. Obtained cDNA was diluted 1:11 and used as template in the q-RT-PCR, mixed with QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer (Qiagen) and loaded into each well of a Custom miScript miRNA PCR Arrays made to include the set of miScript Primer Assay (Qiagen)of miR-192, miR-206, miR-132, miR-194 and miR-219 according to the manufacturer's instructions. The analysis was performed on an ABI-PRISM 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using the manufacturer's recommended program. Data were quantified using the SDS 2.1 software and normalized using RNU6-2 as endogenous control. The cycle threshold (Ct) value, which was calculated relatively to the endogenous control (ΔCt), was used to evaluate the relative changes in miRNA expression levels. The expression levels of each miRNA was expressed as value obtained from the Δct equation with respect to RNU-6-2 reference gene (Δct=Cttarget -Ctreference).