Mir 10a 5p mimic

TBX5 3′UTR segment was cloned by PCR (Forward primer: GCG GAG CTC GAA ATG AAA CCC AGC ATA; reverse primer: GCG AAG CTT AGC CTC ACA TCT TAC CCT), and then inserted into downstream of the luciferase gene between SacI and HindIII sites within the pMIR‐Report™ luciferase vector (Ambion, Austin, USA). The pRL‐TK vector (Promega, Fitchburg, USA) was used as a control. All plasmid vectors were extracted with the EZNA™ Endo‐free Plasmid Maxi Kit (Omega BioTek, Norcross, USA). The constructed vectors were then sent to the company (GenScript Company, Nanjing, China) for the verification of sequence integrity.
HeLa cells were cultured in a 48‐well plate containing DMEM high‐glucose medium (HyClone, USA) supplemented with 10% FBS (HyClone) 24 hrs before transfection. Then cells (0.5 × 10⁵ cells per well) were transfected with firefly pMIR‐Report™ luciferase (Ambion) and Renilla pRL‐TK (Promega) vectors (90 ng:10 ng per well) and at the same time with 50 nM miRNA control mimic or 50 nM miR‐10a‐5p mimic (GenePharma, China). The Renilla luciferase reporter was used for normalization. After 24 hrs of transfection, the cells were lysed, and luciferase activity was detected using Dual‐Luciferase^® Reporter 1000 Assay System (Promega) by a plate‐reading luminometer (Tecan).

The melanoma cell lines A375 (human), B16-F10 (mouse) and the normal human epidermal melanocyte HeMa-Lp were achieved from the American Type Culture Collection (Manassas, VA). In addition, mouse melanocytes (melan-a) were established in Alpaca Biological Engineering Laboratory, Shanxi Agricultural University, Taigu, China. Melanocytes were cultured in MelM (ScienCell, Carlsbad, CA, USA). The total cells were maintained in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% streptomycin-penicillin in a humidified chamber with 5% CO2 at 37°C.
The miR-10a-5p mimic, the mimics negative control (miR-NC) and miR-10a-5p inhibitor were produced and supplied by GenePharma (Shanghai, China). The pcDNA (Vector) and pcDNA-LIN28B overexpression (LIN28B-OE) plasmids were designed by GenePharma (Shanghai, China). The pcDNA (Vector) and pcDNA-TCF21 overexpression (TCF21-OE) plasmids were designed by GenePharma (Shanghai, China). The miR-10a-5p mimic (50 nM), miR-10a-5p inhibitors (50 nM) as well as recombinant plasmids (50 nM) were introduced into the melanoma cells by employing Lipofectamine 2000 (Invitrogen) on the basis of the manufacturer’s protocols. Finally, the transfection efficiency was assessed according to qRT-PCR assay after 48 h of transfection at 37°C.