Whole transcriptome analysis by RNA-Seq, a state-of-the-art technique to uncover complex biological processes. The blackberry example.
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Presented by: Miguel Perez-Aso
Introduction:
At the dawn of the transcriptomic era in cosmetic science, we are beginning to understand its potential. The transcriptome, the total amount of RNA transcribed by specific cells and tissues in an organism at specific physiological condition, can be analyzed by several technologies, among which RNA sequencing (RNA-Seq) has obvious advantages, such as good repeatability, high sensitivity, and wide detection range. Therefore, RNA-Seq has gradually replaced other technologies and is widely used for transcriptomic analysis. However, RNA-Seq faces several bioinformatic challenges, including computational analyses of big data, a cumbersome task.
In the present work we described, for the first time to our knowledge, that the blackberry (Rubus fruticosus fruit extract, RFE) elicits both pro-adipogenic and anti-lipolytic activities in adipocytes. We therefore sought to analyze the transcriptomic program of adipogenesis. To identify potential mechanisms and functional pathways involved in RFE’s activity, the transcriptome of adipocytes stimulated with RFE was also analyzed.
Methods:
The impact of RFE on adipogenesis and lipolysis in 3T3-L1 differentiated adipocytes was analyzed by Oil Red staining and glycerol measurement, respectively. To follow, 3T3-L1 pre-adipocytes and differentiated adipocytes in the presence or absence of RFE were grown and RNA was extracted. The transcriptome was then sequenced by RNA-Seq, data was subsequently processed with the PIGx bioinformatic pipeline and differential expression statistical analysis was performed using edgeR.
Results:
RFE elicits a strong adipogenic effect, as shown by an increase of 29 % (n=3, p<0.01) in Red Oil staining, as well as anti-lipolytic activity, detected by a decrease of 51 % (n=3, p<0.05) on glycerol release. Furthermore, whole transcriptomic analysis by RNA-Seq showed that RFE significantly regulated the expression of 4904 genes. A comprehensive bioinformatic analysis revealed that RFE strongly enhances the adipogenic program since it facilitated the regulation of 3500 out of the 6964 genes triggered by adipocyte differentiation (i.e., 50.3%). Moreover, functional profiling and molecular signature analysis revealed expected, i.e. fatty acid and triglyceride biosynthesis, as well as unexpected routes modulated by RFE. Of particular interest was the finding that pathways regulating the hypodermal extracellular matrix (ECM) were repressed by RFE.
Discussion:
In the present work, we present compelling data showing, for the first time to our knowledge, that RFE induces adipogenesis and inhibits lipolysis. Transcriptome sequencing by RNA-Seq and analysis by state-of-the-art bioinformatic tools, strongly indicate that RFE induces lipid accumulation by facilitating most of the pro-adipogenic program at the cellular level. Whole transcriptome analysis also revealed unpredicted pathways that fell outside of our initial hypothesis. Among them, that RFE significantly represses ECM constituents. Since it has been described that excessive fat accumulation causes an excess of deposition of ECM components such as collagens and that cellulite, the orange peel-type dimpling of the skin on the thighs and buttocks, is a disorder of the subcutaneous fat layer characterized by increased accumulation of ECM, our results indicate that, while RFE potentiates adipose tissue enlargement, it also prevents the formation of cellulite.
Conclusion:
In the present work, we found that the blackberry significantly promotes adipogenesis and inhibits lipolysis in 3T3-L1 adipocytes. RNA-Seq followed by a comprehensive transcriptomic analysis revealed that RFE modulates the expression of 50.3 % out of the 6964 genes regulated during the adipogenic program triggered by adipocyte differentiation. Unexpected pathways regulated by RFE were also found, such as that RFE inhibits the expression of ECM components, therefore indicating that RFE induces adipogenesis while preventing cellulite.
Our work illustrates that full comprehensive bioinformatic analysis following whole transcriptome sequencing by RNA-Seq is of great interest not only to delineate foreseeable mechanisms of action, but also to reveal unanticipated pathways triggered by cosmetic products.
At the dawn of the transcriptomic era in cosmetic science, we are beginning to understand its potential. The transcriptome, the total amount of RNA transcribed by specific cells and tissues in an organism at specific physiological condition, can be analyzed by several technologies, among which RNA sequencing (RNA-Seq) has obvious advantages, such as good repeatability, high sensitivity, and wide detection range. Therefore, RNA-Seq has gradually replaced other technologies and is widely used for transcriptomic analysis. However, RNA-Seq faces several bioinformatic challenges, including computational analyses of big data, a cumbersome task.
In the present work we described, for the first time to our knowledge, that the blackberry (Rubus fruticosus fruit extract, RFE) elicits both pro-adipogenic and anti-lipolytic activities in adipocytes. We therefore sought to analyze the transcriptomic program of adipogenesis. To identify potential mechanisms and functional pathways involved in RFE’s activity, the transcriptome of adipocytes stimulated with RFE was also analyzed.
Methods:
The impact of RFE on adipogenesis and lipolysis in 3T3-L1 differentiated adipocytes was analyzed by Oil Red staining and glycerol measurement, respectively. To follow, 3T3-L1 pre-adipocytes and differentiated adipocytes in the presence or absence of RFE were grown and RNA was extracted. The transcriptome was then sequenced by RNA-Seq, data was subsequently processed with the PIGx bioinformatic pipeline and differential expression statistical analysis was performed using edgeR.
Results:
RFE elicits a strong adipogenic effect, as shown by an increase of 29 % (n=3, p<0.01) in Red Oil staining, as well as anti-lipolytic activity, detected by a decrease of 51 % (n=3, p<0.05) on glycerol release. Furthermore, whole transcriptomic analysis by RNA-Seq showed that RFE significantly regulated the expression of 4904 genes. A comprehensive bioinformatic analysis revealed that RFE strongly enhances the adipogenic program since it facilitated the regulation of 3500 out of the 6964 genes triggered by adipocyte differentiation (i.e., 50.3%). Moreover, functional profiling and molecular signature analysis revealed expected, i.e. fatty acid and triglyceride biosynthesis, as well as unexpected routes modulated by RFE. Of particular interest was the finding that pathways regulating the hypodermal extracellular matrix (ECM) were repressed by RFE.
Discussion:
In the present work, we present compelling data showing, for the first time to our knowledge, that RFE induces adipogenesis and inhibits lipolysis. Transcriptome sequencing by RNA-Seq and analysis by state-of-the-art bioinformatic tools, strongly indicate that RFE induces lipid accumulation by facilitating most of the pro-adipogenic program at the cellular level. Whole transcriptome analysis also revealed unpredicted pathways that fell outside of our initial hypothesis. Among them, that RFE significantly represses ECM constituents. Since it has been described that excessive fat accumulation causes an excess of deposition of ECM components such as collagens and that cellulite, the orange peel-type dimpling of the skin on the thighs and buttocks, is a disorder of the subcutaneous fat layer characterized by increased accumulation of ECM, our results indicate that, while RFE potentiates adipose tissue enlargement, it also prevents the formation of cellulite.
Conclusion:
In the present work, we found that the blackberry significantly promotes adipogenesis and inhibits lipolysis in 3T3-L1 adipocytes. RNA-Seq followed by a comprehensive transcriptomic analysis revealed that RFE modulates the expression of 50.3 % out of the 6964 genes regulated during the adipogenic program triggered by adipocyte differentiation. Unexpected pathways regulated by RFE were also found, such as that RFE inhibits the expression of ECM components, therefore indicating that RFE induces adipogenesis while preventing cellulite.
Our work illustrates that full comprehensive bioinformatic analysis following whole transcriptome sequencing by RNA-Seq is of great interest not only to delineate foreseeable mechanisms of action, but also to reveal unanticipated pathways triggered by cosmetic products.