Trizol reagent

Total RNA was extracted from transfected H9c2 cells using TRIzol reagents (Beyotime Biotechnology, Shanghai, China) and reversely transcribed to cDNA with TaqMan one-step reverse transcription (Applied Biosystems, USA). QRT-PCR experiment was carried out on an ABI Prism 7500 (Applied Biosystems, USA) according to the manufactures’ instructions. Relative mRNA expression levels of miR-539-5pand IRAK3were calculated using 2^−ΔΔCt method. β-actin and U6 were used as an internal standard. The specific primers were as follows: miR-539-5p forward, 5’-CCAAAGGAGCATCAGA GCAGA-3’, and reverse: 5’-AAGGGCTCGACAGAATTGGG-3’, IRAK3 forward: 5’-CAGGGGAAG TGAAGCGGATT-3’, and reverse: 5’-GGTCCCTTGGCTGTACTCAC-3’, U6 forward, 5’-CTCGCTT CGGCAGCACA, and reverse, 5’-AACGCTTCACGAATTTGCGT-3’, β-actin forward, 5’-ATCACTG CCACCCAGAAGAC-3’, and reverse, 5’-TTTCTAGACGGCAGGTCAGG-3’.

Total RNA was extracted from mouse livers, HepG2 and AML12 cells using Trizol reagents (R0016, Beyotime Biotechnology, Shanghai, China). cDNA were transcribed according to the instructions in the reverse transcription kit (A5001, Promega, United States). The cDNA was using real-time quantitative polymerase chain reaction (RT-qPCR) performed with the SYBR Green PCR Master Mix (11201E03, YEASEN, Shanghai, China) and gene-specific primers. The results were normalized with β-actin as control RT-qPCR was performed using a CFX-96 Real Time System (Bio-Rad, United States). Polymerase chains reaction primers are listed in Supplementary Table S1.

Total RNA in PC tissues and cells was separated using TRIZOL reagents (Beyotime Biotechnology, Hangzhou, China) and diluted into DNase/RNase-free water. After quantification, total RNA (2µg per sample) was reversely transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA). Finally, quantitative real-time PCR (qRT-PCR) was conducted to determine the expression level of target genes using SYBR™ Green PCR Master Mix (Solarbio, Wuhan, China). β-actin was used as the internal control. The primer sequences in our study were purchased from Tianyi Huiyuan (Wuhan, China) and shown in Supplementary Table 1.

Quantitative RT-PCR was performed as previously described, with some modifications [23 (link)]. Briefly, total RNA was extracted using the Trizol reagent (Beyotime) and immediately synthesized to cDNA using a PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). PCR primers were as follows: CCT3, forward: 5ʹ-CCTCCAGGTATCTTTTCCACTCT-3ʹ; reverse: 5ʹ-TCAGTCGGTGGTCATCTTTGG-3ʹ; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5ʹ-GACAGTCAGCCGCATCTTCT-3ʹ; reverse: 5ʹ-TTAAAAGCAGCCCTGGTGAC-3ʹ. Quantitative RT-PCR was performed in a StepOnePlus™ Real-time PCR system (Life Technologies, Carlsbad, CA) with the SYBR Green Master Mix (TaKaRa). Thermocycling conditions were as follows: 94°C for 2 min, 94°C for 5 sec, 60°C for 15 sec, 72°C for 30 sec (40 cycles). The 2^−ΔΔCt method was used to calculate fold changes in the mRNA expression of CCT3 normalized to GAPDH.

After treatment, total RNAs were collected using the TRIzol reagent (Beyotime, Shanghai, China) according to the manufacturer’s instructions. RNAs were reversely transcribed to cDNAs by using cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The sequences of the primers are listed as follows: forward primer for CDKN2B-AS1: 5’ -GAAGATCTGGAGCAGGAACCAC-3’; reverse primer for CDKN2B-AS1: 5’-GTCAATCAGAGCAAACTGCA GTG-3’; forward primer for miR-98-5p: 5’-CCCGGGTGAGGTAGTAAGTTG-3’; reverse primer for miR-98-5p: 5’-CTCAACTGGTGTCGTGGAGTC-3’; forward primer for U6: 5’-CTCGCTTCGGCAGCACAT-3’; reverse primer for U6 5’-AACGCTTCACGAATTTGCGT-3’; forward primer for SOCS1: 5’-CAACGGAACTGCTTCTTCGC-3’; reverse primer for SOCS1 5’-CTCGAAAAGGCAGTCGAAGG-3’; forward primer for GAPDH: 5’-TGAAGGGTGGAGCCAAAAG-3’; reverse primer for GAPDH 5’-AGTCTTCTGGGTGGCAGTGAT-3’. Reaction conditions: 5 min 95℃ followed by 40 cycles of 15 s 95℃, 15 s 58℃ and 32 s 72℃. qRT-PCR analysis was performed using QuFast SYBR Green PCR Master Mix (ELK Biotechnology, China) on an ABI 7500 Real-Time PCR System. The expression of targeted genes was calculated using 2^−ΔΔCt method, in which U6 and GAPDH, whose stability was determined by an online software RefFinder (Xie et al. 2023 (link)).

Total RNA was extracted using the TRIzol reagent (Beyotime). Then RNA was reversely transcribed to complementary DNA (cDNA) by PrimeScript™ RT Master Mix kit (Beyotime). The qRT-PCR was conducted by SYBR Green PCR Master Mix (Beyotime) and data were analyzed using 2^−ΔΔCt method. β-actin and U6 were introduced as the inner references. Primers used in this study:
NEAT1 (forward 5′-GTAATTTTCGCTCGGCCTGG-3′, reverse 5′-TACCCGAGACTACTTCCCCA-3′); miR-370-3p (forward, 5′-GCCTGCTGGGGTGGAACCTGGT-3′, reverse 5′-CTCAACTGGTGTCGTGGA-3′); Irak2 (forward, 5′-CATGGCTTGCTACATCTACC-3′, reverse 5′-ACGTTTGTCTGTCCAGTTGA-3′); β-actin (forward 5′-GCACCACACCTTCTACAATG-3′, reverse, 5′-TGCTTGCTGATCCACATCTG-3′); U6 (forward, 5′-TCCGGGTGATGCTTTTCCTAG-3′, reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′).