Trizol reagent

Cell or tissues were lysed by the Trizol reagent (Roche) and total RNA was isolated. TaqMan MicroRNA Reverse Transcription Kit (ABI) was used for miRNA reverse transcription. ReverTra Ace qPCR RT Kit (Toyobo) was applied for mRNA reverse transcription. TransStart Top Green qPCR SuperMix (TransGen Biotech) was used for quantification of miRNA/mRNA level. GAPDH (for mRNA) and U6 (for miRNA) were used respectively as positive control and the 2^-ΔΔCt algorithm was applied for relative quantification of mRNA and miRNA levels. Primers used for qRT-PCR were listed in Supplementary Table 2.

Total rebonucleic acid (RNA) was extracted from tumor, polyp and normal colorectal biopsy samples using Trizol reagent (Roche Applied Science, Indianapolis, IN, USA) and reverse transcribed to obtain complementary DNA (cDNA) with First Strand cDNA synthesis kit (Roche Applied Science, Indianapolis, IN, USA), respectively, in accordance with the manufacturer’s instructions. PCR primers and Taqman hydrolysis probes for MMP-7, COX-2, TIMP-1 and β-actin genes were obtained from Roche Applied Science (Roche Applied Science, Indianapolis, IN, USA). β-actin was used as a housekeeping gene. Real Time PCR was performed using Light Cycler 480 Probes Master kit (Roche Applied Science, Indianapolis, IN, USA), which is a ready-to-use reaction mix that contains FastStart Taq DNA Polymerase, under the following conditions: an initial denaturation for 10 minutes at 95°C, 40 cycles of denaturation for 10 seconds at 95°C, annealing for 30 seconds at 60°C and elongation for 10 seconds at 72°C. LightCycler 480 (V1.5.0) Software (Roche Applied Science, Indianapolis, IN, USA) was used to measure the CT values and relative mRNA expression levels were calculated with comparative 2-AACT method.

Total RNA from TCs was extracted using the TRIzol reagent (Roche) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of RNA using the AB High‐Capacity cDNA synthesis kit (Thermo Fisher Scientific) with random hexamers. Real‐time PCR was performed using SYBR green master mix on LightCycler^® 96 System (Roche Diagnostics Ltd). The sequences of the primers used were given in Table 1. qPCR was carried out using QuantiTect SYBR Green master mix (Qiagen) and an AB StepOne plus thermal cycler (Applied Biosystems). The PCR parameters used were a 10 seconds denaturation cycle at 95°C, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. PCR efficiencies were detected using a relative standard curve derived from diluted cDNA reaction mixture (a 2‐fold dilution series with five measuring points). The R2 values for all standard curves generated ranged between 0.997 and 0.999 and PCR efficiencies were between 90% and 110%. The β‐actin as an inner control was applied to normalize the mRNA expression results using 2‐ΔΔCt method.

Total RNA from HASMCs was extracted with the TRIzol reagent (Roche, Germany). Then, cDNA was synthesized from 2 μg of RNA using oligo (DT) primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). RT-PCR was carried out using a LightCycler 480 and SYBR Green Master Mix (Roche, Germany) according to the manufacturer's instructions. The results were quantified and normalized against GAPDH expression. The details of the primers are presented in Table 1.

Total RNA was extracted from cells with TRIzol reagent (Roche Diagnostics, Mannheim, Germany) and reverse transcribed into cDNA with MMLV reverse transcriptase (Invitrogen). The reactions were incubated at 37°C for 50 min, 72°C for 10 min, and 4°C for 5 min. qRT- PCR was performed using a Roche LightCycler 480 qRT-PCR system. All reactions were carried out in triplicate.