Trlzol reagent

Total RNA was extracted from Gc tissues and cells using Trlzol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the RT reagent kit (Takara Biotechnology co., ltd.) or the miRNA reverse Transcription kit (Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed using SYBr Premix ex Taq (Takara Biotechnology co., ltd.). The following primer pairs were used for qPCR: miRNA-34a-5p forward, 5′-UGGCAGUGUCUUAGCUGGUUGU-3′ and reverse, 5′-AACCAGCUAAGACACUGCAAUU-3′; SOX4 forward, 5′-GTGAGCGAGATCTCGGG-3′ and reverse, 5′-CAGGTTGGAGATGCTGGACTC-3′; GAPDH forward, 5′-CACATCGCT CAGACACCATG-3’ and reverse, 5′-TGACGGTGCCAT TGGAATTT-3′; The amplification parameters were as follows: Denaturation at 95 °C for 10 minute, followed by 40 cycles of denaturation at 95 °C for 30 second, annealing at 60 °C for 30 second and extension at 72 °C for 1 minute. mRNA and miRNA expression levels were quantified using the 2−ΔΔcq method^{[27 ]} and normalized to the internal reference genes GAPDH and u6, respectively.

Total RNA from cells or samples was extracted using TRlzol^® reagent (Thermo Fisher Scientific, Inc.). The samples were reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using a SYBR-Green kit (QuantiNova SYBR Green RT-PCR kit, cat. no. 208152; Qiagen AB). The thermocycling conditions were: Initial denaturation at 95°C for 8 min; denaturation at 95°C for 25 sec, annealing at 60°C for 1 min (37 cycles); and final extension at 72°C for 10 min. GAPDH was used as an endogenous control and the calculation of the relative gene expression was performed using the 2^−ΔΔCq method (34 (link)). Primers are as follows: DPP4 (rat) forward, 5′-ATTCCGTACCCAAAGGCAGG-3′ and reverse, 5′-AGGCCACGTCACACAAGTAG-3′; DPP4 (human) forward, 5′-TCTGCTGAACAAAGGCAATGA-3′ and reverse, 5′-CTGTTCTCCAAGAAAACTGAGC-3′; CREB forward, 5′-TAGTGCCCAGCAACCAAGT-3′ and reverse, 5′-ACATGTTACCATCTTCAAACTGAC-3′; CYP19A1, forward, 5′-ACTTATCCTATCAGGACGGAAGGT-3′ and reverse, 5′-AGGGGGCAATTTAGAGTCGC-3′; CYP11A1 forward, 5′-GCTGAAGTGGAGCAGGTACA-3′ and reverse, 5′-CTTTGACCAGGACTGAGCGT-3′; GAPDH (rat) forward, 5′-GCATCTTCTTGTGCAGTGCC-3′ and reverse, 5′-TACGGCCAAATCCGTTCACA-3′; GAPDH (human) forward, 5′-AATGGGCAGCCGTTAGGAAA-3′ and reverse, 5′-GCGCCCAATACGACCAAATC-3′.

Total RNA was extracted from GC tissues and cells using TRlzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the RT Reagent kit (Takara Biotechnology Co., Ltd.) or the miRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The following primer pairs were used for qPCR: NNT-AS1 forward, 5′-AGTTCCACCAAGTTTCTTCA-3′ and reverse, 5′-AGGTTTTGCCAGCATAGAC-3′; SOX4 forward, 5′-GTGAGCGAGATCTCGGG-3′ and reverse, 5′-CAGGTTGGAGATGCTGGACTC-3′; GAPDH forward, 5′-CACATCGCTCAGACACCATG-3′ and reverse, 5′-TGACGGTGCCATTGGAATTT-3′; U6: Forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; and miR-142-5p: Forward, 5′-AACTCCAGCTGGTCCTTAG-3′ and reverse, 5′-TCTTGAACCCTCATCCTGT-3′. The amplification parameters were as follows: Denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. mRNA and miRNA expression levels were quantified using the 2^−ΔΔCq method (27 (link)) and normalized to the internal reference genes GAPDH and U6, respectively.