Sirna2

Negative control (NC) and small interfering RNAs (siRNAs) of CDCA2 were constructed by GenePharma Corporation (Shanghai, China). The siRNA sequences of CDCA2 were as follows: siRNA1, 5′-CACCUGCCUUUCUAAAUAUTT-3′; siRNA2, 5′-GGGCAAAGGAUCAAGUGAUTT-3′; siRNA3, 5′-CUGCCUUGGAAAGGAUUGATT-3′. Transfection was performed with Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. The assays were performed 48 h after transfection to assess the knockdown efficiency. A CDCA2 inhibitor lentivirus (shCDCA2) was then constructed according to siRNA1. To upregulate the expression of CDCA2 in DLD-1 cells, mammalian expression plasmids (pReceiver-M02-CDCA2) designed to specifically express CDCA2 were obtained from GeneCopoeia (Rockville, MD, USA).

Two pairs of small-interfering RNAs (siRNA1 and siRNA2) were synthesized by Invitrogen Life Technology (Invitrogen). siRNA2 was employed to knock-down hepcidin levels and siRNA1 was injected s the negative control. Cultured human cardiomyocytes were transfected with each siRNA (20 μM) using Lipofectamine RNAiMAX (Invitrogen) as per the manufacturer’s instructions. After 48 h, protein was extracted for western blot analysis.

PinX1 was cloned into the pcDNA3.1 (+) vector (Invitrogen, USA); the plasmid pcDNA3.1-PinX1 and empty vector were prepared using the QIAGEN Plasmid Midi Kit (QIAGEN, Germany). Then reconstructed and empty vector were transfected separately into the breast cancer cells by Lipofectamine 2000 (Invitrogen, USA) and a fresh cell culture medium containing 700 μg/mL, 1200 μg/mL, and 600 μg/mL of G418 (Amresco, USA) was applied 24 hours after transfection to MCF-7, MDA-MB-231, and SK-BR-3 accordingly. 3 weeks after the G418 screening, the cell clones were harvested using an Eclipse Ti-s (Nikon, Japan) microscope. The cell clones of pcDNA3.1-PinX1 group and empty vector group were maintained in the culture medium with 300 μg/mL of G418, and those with a high expression level of PinX1 were selected for later uses. Three different PinX1 siRNA fragments and siRNA NC were designed and synthesized by Ribbio (Guangzhou, China); the sequences were as follows: siRNA1, sense 5′-GGAGCCACAGAUCAUAUUA dTdT-3′, antisense 3′-dTdT CCUCGGUGUCUAGUAUAAU-5′; siRNA2, sense 5′-GGAGUAAUGACGAUUCCAA dTdT-3′, antisense 3′-dTdT CCUCAUUACUGCUAAGGUU-5′; siRNA3, sense 5′-GGACGCUACACUAGAAGAA dTdT-3′, antisense 3′-dTdT CCUGCGAUGUGAUCUUCUU-5′. MCF-10A cells were transfected separately with the three siRNAs and siRNA NC by Lipofectamine 2000 (Invitrogen, USA) to knockdown the PinX1.