Si kifc1

The small interference RNA (siRNA) sequences were designed for KIFC1: sense 5′-UCG AAA UGA GAA AUC UCG GAG-3′, antisense 5′-CCG AGA UUU CUC AUU UCG AAU-3′. The negative control siRNA (siRNA-NC) were random sequences that have no homology with any known mammalian gene. KIFC1 siRNA (si-KIFC1) and negative control (siRNA-NC) were synthesized by GenePharma Company (Shanghai, P.R. China). To overexpress miR-338-3p, miR-338-3p mimic and mimic negative control (miR-NC) were purchased from GenePharma. 786-O, 769-P, and OS-RC-2 cells were seeded at 1 × 10⁵ cells per well in six-well plates and incubated overnight at 37°C with 5% CO₂. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transiently transfect cells with si-KIFC1, siRNA-NC, miR mimic, or miR-NC (GenePharma). The efficiency of transfection was evaluated by real-time quantitative PCRRT-qPCR and Western blot analysis after 48 h of transfection.

Human GC cell lines AGS, BGC-823, MKN-28, MKN-45, MGC-803, and SGC-7901 and human embryonic kidney 293 (HEK-293) cells were purchased from the Shanghai Institutes for Biological Sciences (Shanghai, People’s Republic of China) and routinely cultured in Roswell Park Memorial Institute 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained in a humidified incubator containing 5% carbon dioxide at 37°C.
The miR-135a mimics, miR-135a inhibitors, small-interfering RNAs targeting KIFC1 (siKIFC1), short-hairpin RNA expression vectors targeting KIFC1 (shKIFC1) and their respective controls were obtained from GenePharma (Shanghai GenePharma Co. Ltd, Shanghai, People’s Republic of China). The cells were seeded into six-well plates, incubated for 24 hours, and then transfected with miR-135a mimics, inhibitors, siKIFC1, or shKIFC1 by using Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer’s suggestions. At 48 hours posttransfection, cells were harvested and used in subsequent experiments.