Energy metabolism function and gene expression reprogramming of hepatic steatosis by using high fat load NAFLD cell model
We established a hepatocyte steatosis model by subjecting the human hepatocellular carcinoma cell line (Huh7) to a high load of saturated (palmitate) and unsaturated (oleate) fatty acids for 24 hours. Huh7 was cultured in Dulbecco's modified eagle medium (DMEM) with high glucose (Sigma) supplemented with 10% fetal bovine serum (FBS) (Biowest), 2 mM L-Glutamine (Sigma), 10,000 U/mL penicillin and 10 mg/mL streptomycin (Sigma) at 37 °C under 5% CO2, in a 95% humidified atmosphere. Cells were exposed for 24 h to 1200 µM exogenous fatty acid mixture containing palmitic (P0500, Sigma) and oleic acid (O1008, Sigma). The mixture was freshly prepared before each experiment. In short, fatty acid stock solution was added to prewarmed complete medium (molar ratio of palmitic acid : oleic acid = 1:2, complexed with 300 µM fatty acid-free BSA (A8806, Sigma) to induce NAFLD at the stage of steatohepatitis.  Total RNA from control (n=3) and model (n=3) Huh7 cells were isolated by TRIZOL reagent (Thermo Scientific), and the RNA concentration, purity and integrity were assessed by the Synergy H1 Hybrid Multi-Mode Reader (BioTek) and 2100 Bioanalyzer (Agilent Technologies). Total RNA samples were stored at -80 °C until further analysis. The construction and deep sequencing of RNA libraries were accomplished with the assistance of BGI Europe Co., Ltd. Briefly, by oligo(dT) selection, mRNA was enriched and purified from total RNA, followed by RNA fragmentation, reverse transcription, end repair, add A, and adaptor ligation. After PCR amplification, the double-stranded PCR products were subjected to heat separation, cyclization (circularized by the splint oligo sequence), DNA nano ball synthesis. The single-stranded circular DNAs were formatted as the final library for library quality assessment and subsequent PE100 (paired-end 100) sequencing. The mRNA-sequencing was performed on a well-established DNBSEQ/MGISEQ2000 Technology Platform.