Mir 93 5p mimic

The HGF overexpression (OE) vector (OE-HGF), HGF small interfering RNA (si; si-HGF), miR-93-5p mimic, miR-93-5p inhibitor and their corresponding negative controls (NCs) were purchased from Shanghai GenePharma Co., Ltd. (Table SI). pcDNA3.1 empty vector was used as the NC for OE-HGF. HepG2 cells were seeded (2×10⁴ cells/well) into a 6-well plate. Subsequently, cells were transfected with OE-HGF (2.5 µg), si-HGF (2.5 µg), miR-93-5p mimic (50 nM), miR-93-5p inhibitor (50 nM) or the corresponding NCs (50 nM) using Lipofectamine^® 3000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. The transfection efficiency and the subsequent assays were performed 48 h after transfection. Cells in the NC group were transfected with mimic-NC and inhibitor-NC, or empty vector and si-NC due to the similar transfection efficiency of their NC (Figs. S1 and S2).

For miR-93-5p overexpression, the miR-93-5p mimic or corresponding NC (miR-NC) was purchased from GenePharma (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were transfected with either the miR-93-5p mimic or miR-NC at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were then used for miR-93-5p expression analyses or for other experiments after 48 hr of transfection.
For the overexpression of TLR4 and Atg7, TLR4 and Atg7 cDNA with the 3′ UTR were cloned into the pMSCV-hygro vector. The primers corresponded to the National Center for Biotechnology Information reference sequence and were as follows: TLR4 (forward, 5′-CAGAGCTCGGAGTACAAAACTC-3′ and reverse, 5′-GGTCTAGAAAAGTGCTTTTTGCAAG-3′); Atg7 (forward, 5′-CAGAGCTCGTGCCTCATTGGGTC-3′ and reverse, 5′-GGTCTAGACAGTAATAAAGTGC-3′). The TLR4 or Atg7 cDNA was inserted into the pMD18-T Simple Vector (Takara, Otsu, Japan) to form the pMD18-T-TLR4 and pMD18-T-Atg7 vectors, respectively. Following sequencing, the recombinant segment of the correct clone was incised by BamHI and XbaI (Takara). The recombinant segment was inserted into pMSCV-hygro, which was incised by the same two restriction endonucleases. The clones were sequenced, and the correct clones were amplified and identified before transfection into H9c2 cells.