Trizol

Total RNA was extracted with TRIzol (Beyotime, Shanghai, China). For mRNA and miRNA, the cDNA was synthesized by the HiFiScript cDNA synthesis kit (Life Technologies, CA, USA) and cDNA synthesis kit (Sangon, Shanghai, China), respectively. Then, SYBR (Thermo Fisher Scientific) was employed for RT-qPCR assay. GAPDH and U6 were used as the reference genes for mRNA and miRNA, respectively. Data were analyzed with 2^−ΔΔCT method. The primers used in the study were listed as follows (5’-3’):
MALAT1 (F): TGCGAGTTGTTCTCCGTCTA
MALAT1 (R): TATCTGCGGTTTCCTCAAGC
miR-137 (F): ACACTCCAGCTGGGTTATTGCTTAAGAATAC
miR-137 (R): TGGTGTCGTGGAGTCG
BCL11A (F): ATGCGAGCTGTGCAACTATG
BCL11A (R): CAACACTCGATCACTGTGCC
DNMT1 (F): CCGACTACATCAAAGGCAGC
DNMT1 (R): AGGTTGATGTCTGCGTGGTA
CD44 (F): CTGCCGCTTTGCAGGTGTA
CD44 (R): CATTGTGGGCAAGGTGCTATT
ALDH (F): CACCTCGCTGGAGTACGGA
ALDH (R): CCATTCACATAGTGGCCCAAG
U6 (F): CGCTTCGGCAGCACATATAC
U6 (R): AAATATGGAACGCTTCACGA
GAPDH (F): TCAAGAAGGTGGTGAAGCAGG
GAPDH (R): TCAAAGGTGGAGGAGTGGGT

RNA-protein or RNA-RNA complex were isolated using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore). A549 cells (2×10⁵ cells) were disrupted using RIP lysis buffer for 5 min on ice. The antibodies, including anti-Argonaute 2 (anti-Ago2; cat. no. MABE253; EMD Millipore) at the dilution of 1:50 and anti-Immunoglobulin G (anti-IgG; cat. no. TS-1L-BK; EMD Millipore) at the dilution of 1:100 were incubated with protein A/G beads for 1 h at 4°C. Then, the A549 cell lysate was mixed with the beads to incubate for 4 h at 4°C. Beads were washed twice using PBS buffer (Sangon Biotech Co., Ltd.), and the mixture was centrifuged at 21,000 × g for 10 min at 4°C. RNA was extracted using TRIzol^® (Beyotime Institute of Biotechnology), and the immunoprecipitated RNA was detected by RT-qPCR.

Total RNA from A549 cells was extracted using TRIzol^® (Beyotime Institute of Biotechnology). The reverse transcription of CASC2 and the mRNA of TAB2 was performed using BeyoRT™ First Strand cDNA Synthesis Kit (Beyotime Institute of Biotechnology) at 42°C for 60 min, whereas the cDNA of miR-27b was obtained with One Step miRNA RT kit (HaiGene) at 37°C for 60 min followed by 85°C for 5 sec. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal references for miR-27b, CASC2 and TAB2. The expression of miR-27b, TAB2 and CASC2 was measured using the 2^−ΔΔCq method (19 (link)). The PCR reaction was conducted using SYBR-Green [Roche Diagnostics (Shanghai) Co., Ltd.] and an ABI 7500 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following primers: CASC2 forward, 5′-GCACATTGGACGGTGTTTCC-3′ and reverse, 5′-CCCAGTCCTTCACAGGTCAC-3′; miR-27b forward, 5′-AAAAGTCGACCGAAGATGCTCACCAGCCCTT-3′ and reverse, 5′-AAAAGTCGACGGCAGTGGCCTCTGCCTGGC-3′; TAB2 forward, 5′-AGTACAAGATATCTTTATGG-3′ and reverse, 5′-TGCTGTCTGTGGCTCCTGCT-3′); U6 forward, 5′-ATGGGTCGAAGTCGTAGCC-3′ and reverse, 5′-TTCTCGGCGTCTTCTTTCTCG-3′; and GAPDH forward, 5′-ATGTTCCAGTATGACTCCACTCACG-3′ and reverse, 5′-GAAGACACCAGTAGACTCCACGACA-3′.