Unknown,Transcriptomics,Genomics,Proteomics

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Global gene expression in response to genetic and growth factor manipulation of TWIST1 expression and function

ABSTRACT: In addition to their stem/progenitor properties, mesenchymal stem cells (MSCs) also exhibit various effector functions potent effector (angiogenic, anti-inflammatory, immune-modulatory) functions that are largely paracrine in nature. It is widely believed that effector functions underlie most of the therapeutic potential of MSCs and are independent of their stem/progenitor properties. Here we demonstrate that stem/progenitor and effector functions are coordinately regulated at the cellular level by the transcription factor Twist1 and specified within populations according to a hierarchical model. We further show that manipulation of Twist1 levels by genetic approaches or by exposure to widely used culture supplements including fibroblast growth factor 2 (Ffg2) and interferon gamma (IFN-gamma) alters MSC efficacy in cell-based and in vivo assays in a predictable manner. Thus, by mechanistically linking stem/progenitor and effector functions our studies provide a unifying framework in the form of an MSC hierarchy that models the functional complexity of populations. Using this framework, we developed a Clinical Indications Prediction (CLIP) scale that predicts how donor-to-donor heterogeneity and culture conditions impact the therapeutic efficacy of MSC populations for different disease indications. We used microarrays to detail the global gene expression in response to genetic and growth factor manipulation of TWIST1 expression and function. Mesenchymal stem cells at subconfluent culture in growth media (CONTROL), or supplemented with FGF2 (20 ng/ml) (FGF2), trasfected with scrambled (SCRAMBLED) or TWIST1 siRNA (TWIST1) were used for RNA extraction and hybridization on Affimentrix microarrays. We pooled three independently extracted RNA samples per group.

ORGANISM(S): Homo sapiens

SUBMITTER: Siddaraju Boregowda

PROVIDER: E-GEOD-76158 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC) valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive ECM molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify direct transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs) containing E-box consensus sequences that are potential Twist1 binding sequences. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1 responsive regulatory sequences. Murine MC3T3-E1 preosteoblast cells were treated with siScrambled control or siTwist1 to achieve knockdown of Twist1. Both siScr and siTwist1 were performed in triplicate. Isolated RNA was analyzed with Affymetrix muring MOE 430 2.0 gene chip microarray.

Project description:Mesenchymal stem cell (MSC) therapy improves liver function in patients with liver cirrhosis. However, the therapeutic mechanism underlying MSC therapy remains unclear. Therefore, this study aimed to elucidate the therapeutic mechanism underlying MSC therapy by analyzing changes in the modification and expression of proteins 1-month post-treatment with MSCs. This prospective study included 11 patients with cirrhosis who received MSC injection in the First Hospital of Lanzhou University. The laboratory indexes before and after treatment were collected to evaluate the clinical treatment effect of MSCs, and peptide segments were extracted from liver tissue before and after treatment to revealed the protein expression and lactylation modification. Meanwhile, weighted gene co-expression network analysis was used to analyze co-expression protein modules and their relationship with clinical features. The patients with liver cirrhosis showed an improvement trend after receiving MSC treatment, specifically, the liver protein synthesis function was significantly improved and coagulation function was also significantly improved. Proteomics combined with lactic acid proteomics revealed 160 Lysine lactylation (Kla) sites of 119 proteins. The downregulated lactylated proteins were associated with metabolic processes. The upregulated lactylated proteins were involved in biological processes. The protein-based co-expression module was significantly related to alkaline phosphatase, total bilirubin (TBIL), indirect bilirubin (IBIL), direct bilirubin (DBIL), and ALB, and 10 proteins significantly related to ALB and 10 proteins significantly related to bilirubin were screened. The changes in clinical indexes before and after treatment indicate that the clinical effects of MSC treatment are related to cell metabolism. Overall, this study is the first study to comprehensively and systematically reveal the changes of lactylated proteins and expression after treatment with MSC by the self-control of patients, and lays a foundation for understanding the functions of lactylation modification in MSC therapy.

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Global gene expression in response to genetic and growth factor manipulation of TWIST1 expression and function

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