The gene sequence of albumin and C/EBPα was selected for designing short-activating RNA molecules for its specific activation
using the parameters previously described. HepG2 is a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents selleck chemicals and expresses genes involved in a wide variety of liver-specific metabolic functions. HepG2 cells were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 100 units/mL penicillin, 0.1 mg/mL streptomycin, 2 mmol/L glutamine (Sigma), and 10% fetal bovine serum (Labtech International). For C/EBPα-saRNA transfection, cells were grown to 60% confluency in 24-well plates prior to transfection of 5, 10, and 20 nmoles of saRNA using Nanofectamine (PAA, UK) following the manufacturer’s protocol. This process was repeated three times at 16-hour intervals before cells were harvested for isolation of total
RNA for messenger RNA (mRNA) analysis. Rat liver epithelial cells and HepG2 cells were cultured in phenol-red free RPMI media in the presence of charcoal-stripped fetal calf serum (FCS). Following three sets of saRNA transfections at 8 hours, 16 hours, and 24 hours, the culture media was collected for total murine albumin ELISA (Assay Max, Albumin ELISA, Assay Pro USA) following the manufacturer’s instructions. Cell proliferation was quantified at 16, 24, and 96 hours following C/EBPα-saRNA transfection by mitochondrial dehydrogenase expression analysis, AZD0530 using WST-1 reagent following the manufacturer’s guideline (Roche,
UK). Briefly, the WST-1 reagent was used at 1:100 dilution to plates and incubated for 1 hour. The enzymatic reaction was measured at 450 nm using the Bio-Tek ELISA reader. Total RNA extraction from cell lines was performed using the RNAqueous-Micro kit (Ambion, UK) following the manufacturer’s instructions. Briefly, the cells were gently centrifuged followed by three pulses of sonication at Output 3 in Lysis buffer (Ambion, UK). The cell lysates were then processed through an RNA binding column, followed by multiple washes and elution. selleck chemical The total RNA isolated was quantified by a Nanodrop 2000 spectrophotometer. 500 ng of total extracted RNA was processed for elimination of genomic DNA followed by reverse transcription using the QuantiTect Reverse Transcription kit from Qiagen. We used a clinically relevant rat liver tumor model previously described. For in vivo therapy C/EBPα-saRNA was reconstituted with 100 μL of RNase/Dnase free H2O; 50 μL of 20 nM saRNA oligonucleotide and 50 μL of (TEA) core PAMAM dendrimer, previously described.