siRNA-mediated silencing of SASH1
Transfection of HAECs was performed using RNAiMAX (Life Technologies, 13778). Briefly, using RNAiMAX transfection reagent (Life technologies, 13778-100), two different siRNA targeting SASH1 (Ambion, S23572 and S23574) at a concentration of 20 nM were applied separately and compared to control siRNA (Ambion, 4390843). The medium was changed after 24h, total RNA and proteins were harvested 48h after transfection.
Efficiency of siRNA transfection and SASH1 knockdown was assessed by RT-qPCR and western blotting as described below.
Wound healing, proliferation and angiogenesis assays
Transfected HAECs were cultured for 48h to reach 90% confluence and were treated with 20 μM mitomycine-C (Sigma-Aldrich, M4287) for 90 min. Two linear scratches per well were performed in the cell monolayer using a 200 μl pipette tip and were incubated at 37°C and 5% CO2 for next 24h. Images of the gap were obtained at time 0h and 24h with fully motorized inverted microscope (Nikon Eclipse TiS) using bright field and 4X magnification. The wounded area was analysed using ImageJ software (NIH) by quantification of the surface of wounded area at 24h as compared to time 0h.
HAECs proliferation was evaluated using a colorimetric tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2H-5-tetrazolio]-1-3-benzene disulfonate) assay, based on the conversion of WST-1 into formazine by mitochondrial dehydrogenase enzyme in viable cells. After 48h of transfection, cells (5,000 cells/well) were seeded in 96-well plates and cultured for further 24h in 100 μl medium. Then 10 μl of WST-1 solution were added to each well, followed by 3h incubation. Absorbance was measured at 450 nm in a microtiter plate reader.
HAECs transfected with siRNA targeting SASH1 or control siRNA were cultured on Matrigel (BD Bioscience, 354234) and tubular formation network was evaluated at 24h. Briefly, 10 μl of Matrigel were pipetted into 15 wells slides (Ibidi, 81506) and brought to 37°C for 30 min to induce polymerization of the matrix. Subsequently, cells were seeded at a density of 50,000 cells per well. Tubular structures were quantified by manual counting on imageJ.
RNA extraction, reverse transcription and quantitative polymerase chain reaction (PCR)
Total RNA was extracted using the mirVana kit (Life Technologies,AM1560) and RNA quality was assessed by a 2100 Bioanalyzer (Agilent Technologies). Synthesis of cDNA was carried out using the Super Script II Reverse Transcriptase (Life Technologies, 18064-014). Real-time PCR was performed using Mx3005P QPCR System (Agilent Technologies) and SYBR Green (Thermo Scientific, AB-1158/B). The amplification program was: 95 °C for 15 min, 40 cycles of 95°C for 30 s and one cycle of 95°C for 1 min, 60°C for 30 sec and 95°C for 30 sec. Data were analyzed using MxPro® software using the ΔΔCT method143 and normalized to the ubiquitin or GAPDH control genes. The sequence of primers is listed in Table 1.
Genome-wide expression analysis and pre-processing of expression data
Total RNA was extracted from HAECs using the mirVANA kit (Life Technologies, AM1560). Transcriptome analysis of total RNA was performed using the Illumina HT-12 v4 BeadChip (http://www.Illumina.com). Briefly, RNA samples were processed in batches of 24 samples. 250 ng of total RNA was reverse transcribed, amplified and biotinylated using the IlluminaTotalPrep RNA Amplification Kit (Ambion/Applied Biosystems, AMIL1791). Each biotinylatedcRNA (750 ng) was hybridized to a single BeadChip at 58°C for 16–18h. BeadChips were scanned using the IlluminaHiscan array.
The summary probe-level data delivered by the Illumina scanner (mean and SD computed over all beads for a particular probe) was loaded in Genomestudio. The pre-processing was done by the Illumina software, at the level of the scanner and by Genomestudio included: correction for local background effects, removal of outlier beads, computation of average bead signal and SD for each probe and gene, calculation of detection P-values using negative controls present on the array, quantile normalization across arrays, check of outlier samples using a clustering algorithm, check of positive controls. Analyses were carried out on the mean level for all probes in each gene. To stabilise variance across expression levels, we applied an arcsinh transformation to the expression data. Compared to a log transformation, this transformation has the advantage not to discard negative expression values which can occur in Illumina data. A gene was declared significantly expressed in the dataset, i.e. expressed above background (as measured by the negative controls present on each array), when the detection p-value calculated by genome studio was <0.05 in more that 5% of the samples. Microarray analysis was performed using the Illumina HT-12 v4 BeadChip. To limit batch effect, each samples treated with the SASH1 siRNA was processed on the same beadchip as its siRNA control.
SASH1 protein localization in human aortic endothelial cells
The subcellular distribution of SASH1 in HAECs, determined using SASH1 antibody and confocal microscopy analysis, was shown to be mainly cytoplasmic, with a weak nuclear staining (Figure 3A). Immunostaining was performed with four different commercial antibodies that gave similar results (Suppl Fig 1) and was confirmed using fractionated lysis of HAECs followed by western blotting which showed a strong signal at the expected 180kDa size for SASH1 in the cytoplasm and a weaker signal in the nucleus. The specificity of this signal was validated using two different siRNA targeting SASH1.Transfection of both siRNAs reduced the expression of SASH1 protein to a similar extent (Figure 3B).
SASH1 mRNA expression is increased in carotids from smokers
To determine if the increase of SASH1 mRNA expression observed in monocytes of smokers in our previous study131 was also present in the arterial wall, SASH1 expression was tested in human carotid plaques from smokers (n=11), ex-smokers (n=9) and non-smokers (n=25). SASH1 mRNA expression was significantly increased in carotids from smokers when compared to non-smokers (p<0.01) and ex-smokers (p<0.001) (Figure 4). No difference was observed between non-smokers and ex-smokers (p=0.38), suggesting that cigarette smoke-induced increase of SASH1 expression can be reverted.
SASH1 siRNA-silencing affects cell cycle in human aortic endothelial cells
In order to determine the molecular pathways potentially linking SASH1 to vascular pathology, a transcriptomic analysis was performed using SASH1 siRNA-silenced HAECs from ten different individuals. The cells were transfected either with siSASH1 S23572 (si1), siSASH1 S23572 (si2) or a control siRNA (siCtrl). Analysis of the results revealed that 182 genes were differentially expressed (FDR<0.10) in both si1- and si2-silenced HAECs as compared to siCtrl-transfected cells (Suppl. data Table 1). Among these genes, CYP1A1, a major player in the cigarette smoke metabolism147, was shown to be down-regulated upon SASH1 knockdown; this decrease was confirmed independently by the RT-qPCR experiments (Figure 5A). Pathway analysis using the Ingenuity Pathway Analysis tool (IPA) performed on the 182 differentially expressed genes, showed that pathways involved in regulation of the cell cycle were over-represented (Table 2). IPA analysis prediction of established transcription factor activity, based on the list of differentially expressed genes upon SASH1 knockdown, showed a potential inhibition of the tumor suppressor TP53 (Suppl Fig 3), and in parallel an activation of FOXM1; the latter is strongly associated with cell proliferation148. Taken together, those results suggest an implication of SASH1 in the regulation of the cell cycle.
SASH1 siRNA-silencing increases proliferation and migration of human aortic endothelial cells
Our transcriptomic results suggested an implication of SASH1 in the cell cycle regulation. Therefore, we further investigated the effect of SASH1 on proliferation and migration of HAECs by using WST-1 and cell migration assay (wound healing assay, using mitomycin C to block proliferation). Two different siRNAs were used to silence SASH1 in HAECs as described above and the wound area or formazan formation was compared to that of HAECs treated with control siRNA. The closure of the wound and formazan concentration were significantly higher in both SASH1 siRNA-silenced HAECs than in control HAECs(Figure 6A and 6B), indicating that SASH1 represses migration and proliferation.
Table of contents :
I Introduction/Scientific background
I.1.a Physiopathology of atherosclerosis
I.1.b cell types involved in the plaque progression
I.2 High-Throughput technologies and atherosclerosis
I.3 Smoking and atherosclerosis
I.3.a The pathophysiology linking smoking and atherosclerosis
I.3.b Molecular pathways linking smoking to atherosclerosis
I.3.c Modeling cigarette smoke exposure in vitro
I.4.a SASH1 gene
I.4.b SASH1 protein
I.4.c SASH1 in the literature, the quest for SASH1 function
I.5 Thesis project: from Genome Wide Analysis in the Gutenberg Health Study to in vitro experiments
I.6 Aim of the thesis and experimental strategy
II Article: SASH1 a new link between cigarette smoke and atherosclerosis
II.3 SASH1 ongoing experiments and unpublished results
II.3.a Materials and methods
II.3.b Detecting the SASH1 protein in HAECs
II.3.c SASH1 expression in vascular cells
II.3.d Testing the effect SASH1 knockdown on the SASH1 network
II.3.e Effect of HAECs stimulation with inflammation related compounds on SASH expression and its sub cellular localization
II.3.f Transcriptomic analysis of cigarette smoke condensate stimulated HAECs
II.3.g In silico study of SASH1 methylation
II.4 Searching for SASH1 partner proteins
II.4.a Yeast two hybrid (Y2H)
II.4.c Mass spectrometry
Annex 1: SLC39A8 (ZIP8) and cigarette smoking, genesis of a new project
I.1 SLC39A8 gene organisation
I.2 SLC39A8 protein (ZIP8)
I.3 Zip8 in the literature
I.4 Material and methods
II.1 ZIP8 expression in vascular cells and in the vascular wall
II.2 ZIP8 expression and the plaque formation
II.3 ZIP8 expression in human carotid plaque according to the smoking status
II.4 Investigating the interdependence of ZIP8 and Gas6 expression in human macrophages
II.5 Methallothionein and ZIP8 expression
Annex 2. Highlights for the ZIP8 project
Annex 3. Hightlights of the SASH1 project
Annex 4. Verdugo et al 2013
Annex 5. Touat et al (publication in preparation)