Geneart site directed mutagenesis plus system

miR-200c experiments: Negative, scrambled control (4464058, Thermofisher Scientific, Waltham, MA, USA) or miR-200c (4464066, MC11714, Thermofisher Scientific, Waltham, MA, USA) mimics were used at a final concentration of 50 nmol/L. All transfections were performed using either RNAi Max or Lipofectamine 3000 (Thermo Fisher Scientific) and experiments were conducted following the manufacturer’s protocols.
Luciferase assay: 50 nM of scramble mimic negative control or miR-200c-3p mimic was co-transfected with 1 μg of Pmir-glo (E1330, Promega) fused with NPC1-3’UTR containing seed regions of miR-200c binding site or mutant. Cells were lysed after 48 h of transfection. The luciferase activities were measured by the Dual-Luciferase^® Reporter Assay system (E1910, Promega) according to the manufacture instruction with internal Renilla control. Site-direct mutagenesis of the seed region was performed utilizing GeneArt™ Site-Directed Mutagenesis PLUS system (A14604, Invitrogen, Waltham, MA, USA). The primers used are listed in Supplementary Table S1.
NPC1 siRNA silencing: Transient siRNA transfections were performed using 30–60 µM NPC1 siRNA from Ambion/Thermofisher (catalog AM16704, ID 106016), in combination with Lipofectamine RNAi Max (ThermoFisher) and conducted according to the manufacturer’s protocols.

The 3′-UTR of STAT3 containing let-7i binding sites were amplified by PCR and inserted into pMIR-REPORT™ (Thermo Fisher Scientific) to construct STAT3 wild-type reporter vector (STAT3-WT). The mutant type (STAT3-MUT) was made with GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific). STAT3-WT or STAT3-MUT and let-7i mimic, inhibitor or their negative controls were transfected into ADSCs using Lipofectamine 3000 (Thermo Fisher Scientific). The luciferase activity was examined using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) according the protocols. The transfections were performed three in independent experiments.

Promoter plasmid pGL4.1-MIR143HG was used as template for site-directed mutagenesis. GeneArt® Site-Directed Mutagenesis PLUS System (A14604, Thermo Fisher Scientific) was applied according to manufacturer’s protocol to obtain the mutated versions of the HRE I, II and III sites on the MIR143HG promoter fragment. All the primers sequences are listed below:

-113M-FP: 5′-CAAGGCAAGGTAGTCCATTGGGGGGTGCCTGGG-3′;

-113M-RP: 5′-CCCAGGCACCCCCCAATGGACTACCTTGCCTTG-3′;

-1009M-FP: 5′-GTAAGATAGGCACATCATTGGCGAGTCCGAAGC-3′;

-1009M-RP: 5′-GCTTCGGACTCGCCAATGATGTGCCTATCTTAC-3′;

-1157M-FP: 5′-GCCGAGGCCTGGTTCCATTGATCCCTTGATTTG-3′;

-1157M-RP: 5′-CAAATCAAGGGATCAATGGAACCAGGCCTCGGC-3′.

Mutations are underlined. All the mutations were confirmed by DNA sequencing.

The binding site between miR-200b-3p and CXCL12 mRNA 3ʹUTR was predicted by StarBase database, and wild-type (WT) CXCL12 sequence was amplified and inserted into pmirGLO dual-luciferase miRNA target expression vectors (Promega Corp., Madison, WI, USA) to construct the reporter vector pmirGLO-CXCL12-WT (WT CXCL12). A GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific, Inc., MA, USA) was used to mutate the binding site miR-200b-3p and CXCL12 mRNA 3ʹUTR. Next, the mutant (MUT) sequence was inserted into the pmirGLO vector to construct reporter vector pmirGLO-CXCL12-MUT (MUT CXCL12). The above vectors were co-transfected with mimics NC, miR-200b-3p mimics and inhibitors NC, miR-200b-3p inhibitors into AGS and SNU-1 cells, respectively. 48 h later, the luciferase activity in each group was determined. The ratio of the luminescence intensity of Renilla luciferase to that of firefly luciferase was used to reflect the binding intensity of miR-200b-3p to CXCL12 3ʹUTR.