Silencing Of FABP3Promotes Apoptosis And Induces Mitochondrion Impairment In Embryonic Carcinoma Cells

FABP3(fatty acid binding protein3) also known as H-FABP (heart fatty acidbinding protein) gene, is a low molecular weight protein with distinct tissuedistribution, which may play an important role in fatty acid transport, cell growth,cellular signaling, and gene transcription. Bioinformatics analysis revealed that theFABP3gene is located on chromosome1p33-p32and is composed of four exons. TheFABP3derives from an mRNA of1097bp with a402bp open reading frame (ORF),which encodes a133-amino acid protein.In previous studies, using suppress subtractive hybridization, we identifed genesthat differentially expressed in VSD and normal ventricular septum myocardium.Wefound that FABP3is more highly expressed in VSD myocardium than in normalmyocardium. Further studies by our group revealed that the expression levels ofFABP3mRNA and protein were up-regulated initially and then gradually decreasedwith P19cell differentiation.We found that overexpression of FABP3inhibitedproliferation, promoted apoptosis of embryonic myocardial cells, and affected thedifferentiation of cardiac precursors into mature cardiomyocytes. Therefore, weselected this protein for further analysis.We used P19cells because they can undergomyocardial cell differentiation after treatment with1%dimethylsulfoxide (DMSO).Under this condition, P19cells may mimic some behaviors of myocardial cells.In the present study, we aimed to elucidate the effects of FABP3on cardiocytesdifferentiation, proliferation, cell apoptosis, and mitochondrial function in P19cellsthrough silencing its expression using small interfering RNAs (siRNAs). Part IEffect of FABP3silencing on differentiation,proliferation and apoptosis of embryonic carcinoma cellsObjective: To investigate the effects of fatty acid binding protein-3genesilencing on proliferation, apoptosis and differentiation of mouse embryoniccarcinoma P19cells in vitro.Methods: The expression lentiviral plasmid for short hairpin RNA(shRNA) or anegative control (NC) of mouse was constructed by inserting a fragment of DNA forshRNA expression into a pGLV-U6-Puro expression vector. HEK-293T cells weretransfected with an expression lentiviral plasmid with a packing (pGag/Pol),VSV-G-expressing (pVSV-G) and a Rev (pRev)-expressing plasmid. The mediumcontaining lentiviruses was collected and filtered after42h and72h of transfection.P19cells were infected with this medium supplemented with5μg/ml polybrene.Stably transduced P19cells were selected using puromycin, adding the minimumconcentration of puromycin required to kill untransduced P19cells. The efficiency ofknockdown was detected by Real-time quantitative polymerase chain reaction(qPCR). Total RNA extracted from P19cells during the process of differentiation atvarious time points. The expression levels of cTnT, alpha-MHC, GATA4, MEF2c,BNP, NKX2.5gene mRNA were evaluated by qPCR. The growth of FABP3knockdown P19cells and control cells assessed using a cell viability CCK-8assay.We also performed fow cytometry analysis of the cell cycle distributions in thesestable sublines. Apoptotic cells were quantifed by fow cytometric analysis afterstaining with Annexin V-FITC and measurement of caspase-3activity.Results: We successfully constructed a shRNA expression vector toknockdown endogenous FABP3expression. The FABP3mRNA level of thepGLV-FABP3-shRNA-infected cells was24%that of the negative control cells. Theresult of the CCK8assay indicated that FABP3knockdown can prevent proliferationand cell cycle analysis showed a decrease in the percentage of cells in S-phase; FABP3knockdown can promote apoptosis induced by serum deprivation with theanalysis of annexin V-FITC and caspase-3activity.Conclusion: FABP3knockdown might have the potential to modulate cellgrowth, apoptosis.Part IIEffect of FABP3silencing on mitochondrial function inembryonic carcinoma cellsObjective: To explore the effects of FABP3knockdown on the mitochondrialfunction of embryonic carcinoma cells using small interfering RNA (siRNA).Methods: P19cells infected with a FABP3-shRNA expression lentivirus or anegative control (NC) expression lentivirus were differentiated into myocardial cellwith1%DMSO. The mitochondrial morphology was examined by transmissionelectron microscope. The mitochondrial DNA (mtDNA) copy number was evaluatedby real-time quantitative PCR. Cellular ATP production was determined usingluciferase-based luminescence assay. The mitochondrial membrane potential wasdetected by MitoTracker Red and reactive oxygen species (ROS) by DCFDA（2’,7’-Dichlorofluorescein diacetate，DCFDA）with confocal laser microscopy anda FACScan flow cytometer using Cell Quest software.Results:1) The mitochondria in FABP3knockdown cells were smaller withcondensed abnormal morphology. Signs of mitochondrial damage ranged fromswelling and reduced density to virtually hollow mitochondria with broken doublemembranes.2) There was no significant difference in mtDNA copy number betweenthe two groups.3) Suppression of FABP3expression substantially reduced celluarATP production.4) FABP3gene silencing significantly increased the ROS levels inmyocardial cells and slightly increased the mitochondrial membrane potential.Conclusion: Gene silencing of FABP3resulted in a certain degree of damage in mitochondrial function, which suggested that FABP3might play some role inmaintain normal function of mitochondrion.