Cholesterol is a structural sterol found in cell membranes and plays an essential role in steroid hormones biosynthesis in all animals. Cholesterol biosynthesis maintenance has a fundamental importance for metabolic homeostasis. Norton protein is a key rate determining regulator of mevalonate in cholesterol biosynthesis and it is not completely known for some proteins including Norton, Sorton and Horton, how their activities are regulated. Previous research about anemia of inflammation by Pshezhetsky et al. demonstrate unpredictably high HDL and LDL levels in their blood analysis of mutated mice. The team concluded that mutation in a distinct locus of ENU-mediated mutant mice that were used by them may have a role in cholesterol homeostasis. Therefore, the research in this paper aims to analyze this locus and identify its role in cholesterol biosynthesis.
Materials & Methods
Mice studies and sample preparation
Dr. Max Smith in Montreal, Canada generated ENU-mediated (ENU-M) mutant mice model in his previous studies and donated to Alexey V. Pshezhetsky. C57/BL6 female mice were breed with ENU-M male mice for at least 10 generation to obtain mice colony. For sample preparation, 2.5 month old ENU-M mice and wild type (WT) mice were further analyzed (n=8). Liver and lung were removed and snap freeze with liquid nitrogen and kept at -80°C.
Bioinformatics analysis and capillary electrophoresis based miRNA detection
For sample preparation, RNA lysis solution (BA Scientific) was used to disrupt WT liver cells that were for capillary electrophoresis (CE) analysis. mirVana miRNA purification kit (BA Scientific) was followed to isolate small RNAs. Alexa-488 labelled DNA probes (BA Scientific) were combined with total small RNA samples. Lung cells were defined as a negative control, total small RNA extracts from lung cells were mixed with DNA probe. Annealing was performed and subjected to electrophoresis to detect target miRNA. ProteomeLab PA 800 CE system (BA Scientific) equipped with laser induced fluorescence detection was used to perform CE analysis. A 488 nm laser was used to excite fluorescence and emission was visualized at 520 + (-) 10 nm. Sodium tetraborate at pH 9.2 was used as background buffer.
Luciferase activity assay
The 3 related genes identified by bioistatistical analysis, binding sites were detected and cloned to pGL3 luciferase reporter vector. BamHI and BgIII restriction enzymes were used to cut the cloning vector and target sites. Amphicillin resistance gene on vector was used to select positive clones. Later, the binding sites with reporter luciferase were subcloned to pTEFI/Zeo expression vector. BamHI and NarI restriction enyzmes were used for fast double digestion. pTEFI/Zeo expression vector with candidates binding site was transformed into S. cerevisiae by electroporation. The transformants were analyzed by luciferase assay to measure luciferase activities. Light signals were detected based on the expression of luciferase reporter gene by luminometer.
Liver tissue was lysed and homogenization process was applied in RIPA buffer supplied from protease inhibitor mixture. SDS-PAGE and Western blotting analysis were performed with primary and HRP-conjugated secondary antibodies. Visualization of protein band signals was provided with chemiluminescent substrate (BA Scientific) and quantification was performed by Licor software.
Micro RNA silencing
Ant-NB-mir oligonucleotides (BA Scientific) and Scr were transfected into WT mice hepatocytes by applying lipofectamine transfection reagent. After that, qPCR analysis was performed to measure relative expressions. Norton proteins were exposed to immunoblotting analyses which were taken from KD hepatocytes and control.
Location of micro RNA by RISH
2.5 month-old mice livers were used to isolate its primary hepatocytes and fixation was performed by paraformaldehyde application. FITC-labeled LNA probe synthesis was performed by Exiqon. The probe was added into hybridization buffer (BA Scientific) to prepare hybridization mixture which later was poured on slide. The slide was incubated at 25oC in humidified slide incubation chamber (BA Scientific) for 1h. Later, anti-FITC/HRP antibody and FITC-tyramide were added respectively. As a final step, DAPI counterstaining was applied and image processing was done by Leica DMRXA microscope with the help of DAPI filter and FITC filter.
Real-time quantitative PCR
TRIzol reagent (BA Scientific) was added into liver tissue to obtain total RNA. 2 mg total RNA was exposed to reverse transcription and cDNA quantification was done by using Taq Man qPCR analysis of each gene with the ABI Prism 7700 sequence detection system (BA Scientific). Norton, Sorton, and Horton mRNAs, specific primers were designed and comparative 2ΔΔCT method (BA Scientific) was used to calculate relative mRNA amounts, β-actin mRNA was taken as invariant control.
Cholesterol level analyses
Determiner TC555 kit (BA Scientific) was used to make total cholesterol analysis of liver extracts. Norton, Sorton and Horton protein analysis and fractionation was performed by HPLC as indicated in the following protocol in Yavgu et al .
Thin layer chromatography was performed by using 2.5 months old mice liver extract as previously done by Wolfman et al. .
Blood samples of 2.5 month-old WT and KD mice were stored in centrifuge tubes and stabilized with EDTA. Cholesterol high performance system pack reagent (BA Scientific) was poured into the blood samples and the samples were centrifuged at 1.600x g for 15 min at 4 oC. HDL-cholesterol and LDL-cholesterol analysis were applied by a Hitachi 704 Chemistry Analyzer (BA Scientific).