Mir 133b

Total RNAs were isolated using an RNAzol reagent (Life Technology, San Francisco, CA, USA) based on the manufacturer's instructions. Briefly, complementary DNA was reverse transcribed from 1 μg of total RNA using an M-MLV reverse transcriptase (Promega, Madison, WI, USA) in a 25-μl reaction mixture. qRT-PCR was carried out in the ABI 7300 reverse transcription RT-PCR system with the SYBR Green Reverse transcription PCR Master Mix (TOYOBO, Osaka, Japan). The primers used in the reaction for miR-205, miR-133b, miR-200, miR-129, miR-137 and miR-21 were purchased from RiboBio (Guangzhou RiboBio Co., Guangzhou, China). The expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as internal control. In addition, following primers were also used:
CCNJ: F 5′-cctgcgcgagaaggaactg-3′ R 5′-cgttgtagcgatccatgaagtg-3′. HOTAIR: F 5′-ggtagaaaaagcaaccacgaagc-3′ R 5′-acataaacctctgtctgtgagtgcc-3′. GAPDH: F 5′-cgaccactttgtcaagctca-3′ R 5′-aggggtctacatggcaactg-3′.

The TSCC cell lines (SCC25 and CAL27) were purchased from American Type Culture Collection (Manassas, VA, USA). The cisplatin resistant cell lines (SCC25/CDDP and CAL27/CDDP) were established by stimulating sensitive cells with escalating doses of cisplatin (Sigma, St. Louis, MO, USA) as previously reported [16 (link)]. All cells were cultivated in Dulbecco’s Modified Eagle Medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37 °C and 5% CO₂.
Small interfering RNA (siRNA) targeting TUG1 (si-TUG1), siRNA targeting CXCR4 (si-CXCR4), siRNA negative control (si-NC), pcDNA and pcDNA-TUG1 overexpression vector (TUG1) were synthesized by Genepharma (Shanghai, China). The miRNA mimic or inhibitor targeting miR-133b (miR-133b or in-miR-133b) and their corresponding negative control (miR-NC or in-miR-NC) were purchased from RIBOBIO (Guangzhou, China). These oligonucleotides or vectors were transfected into SCC25/CDDP and CAL27/CDDP cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The cells were harvested at 24 h after the transfection, and the following experiments were conducted.