Reverse Transcription Quantitative PCR (RT-qPCR)
Following total RNA extraction using TRIzol reagent (Invitrogen), cDNA was synthesized using Mir-X miRNA First-Strand Synthesis Kit (Takara Holdings Inc., Kyoto, Japan) with extracted total RNA (2 μg). The miRNA expression was measured by qPCR with SYBR Advantage qPCR Premix (Takara) on CFX133a Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression was determined with a U6 control through the 2
−ΔΔCt method. The primers used are depicted in Table 1.
Table 1 Primer Sequences
Cell Culture, Transfection and Infection
Human lung cancer cell lines, including H522, H460, SK-MES-1, and A549, and a human bronchial epithelial cell line BEAS-2B, were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cell lines H522, H460, and A549 were cultivated in RPMI-1640 medium (Gibco; Thermo Fisher Scientific), while SK-MES-1 cells were in minimal medium (Gib