ABSTRACT: This study was designed to investigate the modulation of GBE on cellular cholesterol metabolism which has been suggested in recent studies. To explore the molecular mechanisms gain insights into the molecular network, we used microarray to high-throughput measurement of genes concerning both spatial and temporal levels to understand the complexity and dynamics of cells in response to GBE and Lovastatin, the genes expression pattern were similar between GBE and Lovastatin treatment in HepG2 cells, the major changed genes were involved in biosynthesis of steroids and cell cycle. Cholesterol metabolism related genes were further examined by RT-PCR and western blot, showing that the cholesterogenic genes were upregulated in response to the reduction of cholesterol and were consistent with the microarray findings. Keywords: time course HepG2 cells were maintained in Minimum Essential Medium supplemented with 10% FBS in humidified atmosphere with 5% CO2 at 37°C. For experiments, cells were seeded in 6-well plates at a density of 4×105 cells per well, when the cells were grown into a 70 % confluence, the medium was replaced with 2%FBS medium for later drug treatment, cells were incubated with 200μg/mL of GBE or 0.5μM Lovastatin for various time periods (2, 6, 12, 24hours). The control groups were treated with 0.1% DMSO that contained in drug as vehicle at most time point. The cells were collected and RNA were used for transcript profiling analysis. Label protocol:Total RNA were primed with T7 Promoter Primer (from the Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color) at 65 ℃ for 10 min, then reversed transcribed at 40℃ for 2 h in the presence of 1ul MMLV-RT (Agilent), and 10 mM dNTP, and RNase OUT (Agilent) and labeled with either Cy3-dCTP or Cy5-dCTP. Equal amounts of the RNA from control sample were mixed, and the mixture was labeled with Cy5, while the RNA from each GBE treated HepG2 cells was labeled with Cy3. Hybridization protocol: After mixed with hybridization buffer(GE healthcare), samples were applied to microarray slide, cover the slip with care. Place the slide in a wet box.Incubate in a hybridization chamber at 42℃ for 16-18 hours, avoid light exposure. After hybridization, slides were washed sequentially with 1 SSC/0.2 SDS(50°C), 0.1 SSC/0.2%SDS(50°C) and 0.1 SSC(room temperature) before scanning. Scan protocol: Axon GenePix 4000B Description: Total RNA was prepared by using TRIzol reagent (Life Technologies, USA), further purified with an RNEasy column (Qiagen, Germany), and quantified by using gel electrophoresis and spectrophotometer followed by reverse transcription labeling. Each RNA sample was reverse-transcribed into cDNA primed with oligo(dT) and labeled with either Cy3-dCTP or Cy5-dCTP by using Superscript II reverse transcriptase (Life Technologies, USA). After hybridization under a standard protocol, data acquisition was performed by using a laser scanner (Agilent, USA), and the image data were analyzed using ImaGene 4.2 (Biodiscovery, Santa Monica, CA, USA). Data processing: LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.
