Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis

Advanced Proteomic and Epigenetic Characterization of Ethanol-Induced Microglial Activation

Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes and have been implicated in several diseases and disorders of the central nervous system. Here, we described a method optimized for sensitive and rapid quantitative proteomic analysis of microglia that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a microglial cell count expected from an individual mouse brain (~300K) while also simultaneously providing the first comprehensive proteomic characterization of a novel adult-derived mouse microglial cell line. This enhanced method was used throughout all subsequent works and was especially necessary when we attempted to fully characterize ethanol-exposed primary microglia and microglia exposed to the pro-inflammatory stimulus, LPS. This global study yielded the deepest proteome coverage to date of microglia after acute ethanol exposure and showed clear and distinct differences between ethanol induced alternative activation of microglia and the pro-inflammatory activation of microglia induced by LPS. The proteomic dataset generated from this study helped to further support that acute ethanol exposure produces an alternative activation phenotype in microglia that does not fully align with the full activation that can be detrimental to neuronal function. Several predicted pathways and downstream functions could be considered targets for mechanistic understanding of ethanol-induced microglial activation including inhibition of pathways such as cell migration and phagocytosis. Finally, we applied both the methods and novel pathways to investigate a more mechanistic hypothesis behind ethanol-induced microglial activation. Novel predicted downstream functional outcomes such as inhibition of phagocytosis and cell migration were identified. Additionally, previous data from our lab, which suggests the possible role of histone methylation, in particular H3K4me3 through the activity of the histone demethylase KDM5B, was hypothesized as an upstream regulator associated with microglial activation. Taken together, these pathways could be responsible for the phenotypic changes found in ethanol-induced microglial activation. Therefore, we aimed to determine a possible mechanism for ethanol-induced microglial activation and how ethanol affects microglial response to a pro-inflammatory stimulus. Specifically, we investigated the role of H3K4me3 in LPS- and ethanol-treated mouse primary microglia and determined genes associated with this mark through ChIP-seq analysis and how enrichment of this mark is altered based on these treatments. Overlap of gene/gene promoters identified by ChIP-seq analysis with differentially expressed proteins identified by proteomics provided insight into the transcriptional regulation associated with H3K4me3 in the context of LPS- and ethanol-induced microglial activation phenotype. Lastly, in terms of downstream functional outcomes associated with ethanol-treated microglia, we focused on ethanol effects on phagocytosis and inflammatory response predicted from our proteomic data and the possible role of TREM2 (an important regulator of microglial phagocytosis and immune response)

Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult‐Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2-h gradient on a 75-cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label-free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006

Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2-h gradient on a 75-cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label-free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006

Cornus Officinalis Promotes IGFBP2 and Autophagy in Human 1.1B4 Pancreatic Cell Line as Revealed by Employing a Global Proteomic Approach via Mass Spectrometry

Type 1 diabetes (T1D) results in the loss of pancreatic beta cells and subsequent loss of insulin production. Exogenous insulin is the only effective treatment, but there is still no cure or interventional therapy available to inhibit progression of T1D. Successful T1D interventional therapy must protect pancreatic beta cells from autoimmunity while enhancing beta cell survival and function. Our data suggests Cornus officinalis (CO) may be a candidate for interventional therapy to protect pancreatic beta cells from autoimmune attack and increase their function. CO has been used in traditional Chinese medicine (TCM) for over 2,000 years and has shown characteristics of anti-diabetic effects in vitro and in vivo but never examined in the application of T1D. Our prior publication (Mol. and Cell. Endo.2019:494:110491), has shown increased proliferation and protection against Th1 cytokine attack upon CO treatment using a human pancreatic beta cell line, 1.1B4. From this, we sought to define precise molecular mechanism by employing a global and phosphorylation mass spectrometry (MS) approach. We applied CO to 1.1B4 cells for 2, 6, 12, and 24h then collected the cell lysates for MS analysis. Our global MS analysis revealed a 12-fold increase in beta cell functional regulator, IGFBP2, at multiple time points. IGFBP2 has been shown to display a T2D protective effect and regulate glucose metabolism. The ingenuity pathway analysis program (IPA) predicted an increase in insulin starting at 2h and the NRF2-mediated oxidative stress pathway at 12h and 24h. Furthermore, NRF2 is an upstream regulator of P62 which was significantly hyperphosphorylated at multiple timepoints from our MS analysis. Nrf2 is responsible for activating antioxidant enzymes upon oxidative stress, which is caused by proinflammatory cytokines in T1D. P62 aids in this pathway by targeting proteins for autophagy upon oxidative stress in order to keep cellular homeostasis within beta cells rather than cells progressing through apoptosis. Autophagy is critical for beta cell function and survival as it promotes survival under beta cell stress which would otherwise lead to cell death. The recovery and protection of autophagy in beta cells of patients in the pre-diagnosed stages of T1D could provide a beneficial interventional therapy in order to delay or inhibit the onset of T1D. Altogether, our proteomic analysis revealed an increase in IGFBP2 and predicted an increase in the NRf2-mediated oxidative stress pathway upon CO induction. Further analysis will examine the IGFBP2 and Nrf2-mediated oxidative pathway as a mechanism of CO induced protective and proliferative effects in pancreatic beta cells

Cornus Officinalis Promotes IGFBP2 and Autophagy in Human 1.1B4 Pancreatic Cell Line as Revealed by Employing a Global Proteomic Approach via Mass Spectrometry

Type 1 diabetes (T1D) results in the loss of pancreatic beta cells and subsequent loss of insulin production. Exogenous insulin is the only effective treatment, but there is still no cure or interventional therapy available to inhibit progression of T1D. Successful T1D interventional therapy must protect pancreatic beta cells from autoimmunity while enhancing beta cell survival and function. Our data suggests Cornus officinalis (CO) may be a candidate for interventional therapy to protect pancreatic beta cells from autoimmune attack and increase their function. CO has been used in traditional Chinese medicine (TCM) for over 2,000 years and has shown characteristics of anti-diabetic effects in vitro and in vivo but never examined in the application of T1D. Our prior publication (Mol. and Cell. Endo.2019:494:110491), has shown increased proliferation and protection against Th1 cytokine attack upon CO treatment using a human pancreatic beta cell line, 1.1B4. From this, we sought to define precise molecular mechanism by employing a global and phosphorylation mass spectrometry (MS) approach. We applied CO to 1.1B4 cells for 2, 6, 12, and 24h then collected the cell lysates for MS analysis. Our global MS analysis revealed a 12-fold increase in beta cell functional regulator, IGFBP2, at multiple time points. IGFBP2 has been shown to display a T2D protective effect and regulate glucose metabolism. The ingenuity pathway analysis program (IPA) predicted an increase in insulin starting at 2h and the NRF2-mediated oxidative stress pathway at 12h and 24h. Furthermore, NRF2 is an upstream regulator of P62 which was significantly hyperphosphorylated at multiple timepoints from our MS analysis. Nrf2 is responsible for activating antioxidant enzymes upon oxidative stress, which is caused by proinflammatory cytokines in T1D. P62 aids in this pathway by targeting proteins for autophagy upon oxidative stress in order to keep cellular homeostasis within beta cells rather than cells progressing through apoptosis. Autophagy is critical for beta cell function and survival as it promotes survival under beta cell stress which would otherwise lead to cell death. The recovery and protection of autophagy in beta cells of patients in the pre-diagnosed stages of T1D could provide a beneficial interventional therapy in order to delay or inhibit the onset of T1D. Altogether, our proteomic analysis revealed an increase in IGFBP2 and predicted an increase in the NRf2-mediated oxidative stress pathway upon CO induction. Further analysis will examine the IGFBP2 and Nrf2-mediated oxidative pathway as a mechanism of CO induced protective and proliferative effects in pancreatic beta cells

Polyphenol Supplementation Reverses Age-Related Changes in Microglial Signaling Cascades

Microglial activity in the aging neuroimmune system is a central player in aging-related dysfunction. Aging alters microglial function via shifts in protein signaling cascades. These shifts can propagate neurodegenerative pathology. Therapeutics require a multifaceted approach to understand and address the stochastic nature of this process. Polyphenols offer one such means of rectifying age-related decline. Our group used mass spectrometry (MS) analysis to explicate the complex nature of these aging microglial pathways. In our first experiment, we compared primary microglia isolated from young and aged rats and identified 197 significantly differentially expressed proteins between these groups. Then, we performed bioinformatic analysis to explore differences in canonical signaling cascades related to microglial homeostasis and function with age. In a second experiment, we investigated changes to these pathways in aged animals after 30-day dietary supplementation with NT-020, which is a blend of polyphenols. We identified 144 differentially expressed proteins between the NT-020 group and the control diet group via MS analysis. Bioinformatic analysis predicted an NT-020 driven reversal in the upregulation of age-related canonical pathways that control inflammation, cellular metabolism, and proteostasis. Our results highlight salient aspects of microglial aging at the level of protein interactions and demonstrate a potential role of polyphenols as therapeutics for age-associated dysfunction

Presentation_1_Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.pptx

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.</p

Table_1_Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.xlsx

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.</p

Table_6_Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.xls

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.</p