Quantitative Analysis of Gene Expression

The total RNA was extracted using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, United States). Reverse transcription of miRNA was performed using a transcription kit (Shanghai GeneChem Co., Ltd., Shanghai, China) with RNA U6 as an internal control. mRNA was detected using a reverse transcription kit (Takara Biotechnology Ltd., Dalian, Liaoning, China) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence as an internal reference for the normalization of RT-PCR. SYBR Green quantitative PCR analysis was performed using a 7500 real-time fluorescence quantitative PCR system.
The expression levels of target genes were then estimated using the 2^−ΔΔCT method. ^{17 (link)
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Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).
The sequence of primers is presented in table 1.