title: Ablation of Stat3 by siRNA alters gene expression profiles in JEG-3 cells: a systems biology approach.
abstract: Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.
authors.affiliation: Department of Pediatrics, Pediatric Rheumatology Research, University of Oklahoma College of Medicine, Oklahoma City, OK 73104, USA. kaiyu-jiang@ouhsc.edu
authors.forename: K
authors.initials: K
authors.lastname: Jiang
authors.forename: L C
authors.initials: LC
authors.lastname: Krous
authors.forename: N
authors.initials: N
authors.lastname: Knowlton
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: M B
authors.initials: MB
authors.lastname: Frank
authors.forename: C
authors.initials: C
authors.lastname: Cadwell
authors.forename: M
authors.initials: M
authors.lastname: Centola
authors.forename: J N
authors.initials: JN
authors.lastname: Jarvis
mesh_terms: D000704:Analysis of Variance; D045744:Cell Line, Tumor; D002469:Cell Separation; D017077:Culture Media, Conditioned; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008431:Maternal-Fetal Exchange; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011247:Pregnancy; D034622:RNA Interference; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D012984:Software; D049490:Systems Biology / Q000379:methods; D014162:Transfection; D014327:Trophoblasts / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D018836:Inflammation Mediators; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor