Alteration of gene expressions in RCC cell lines co-cultured with HGECs with down-regulated INSR

RCC cell lines (786-O cell line and Caki-1 cell line)were co-cultured with human renal glomerular endothelial cells (HGECs) treated with si-RNA of INSR (HGEC-siINSR). Gene expression alteration was analyzed using microarray. 786-O cell line and Caki-1 cell line were co-cultured with HGECs with down-regulated INSR. Small interfering RNA oligonucleotides were used to knock down INSR in HGEC. Non-silencing RNA duplex was used as a negative control. Transfection with 10 nM of small interfering RNA of INSR to HGECs was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) with the forward transfection method. Seventy-two hours after transfection, HGECs were seeded into 6-well ThinCert™ cell culture inserts, and 2.5 ml of Caki-1 cell suspension containing 8x104 cells/ml in CS-C Complete Medium was placed into a 6-well plate. The inserts were placed in each well of the 6-well plate, and co-cultures were maintained for 24 h.