Response Gene To Complement 32 Research Articles

RGC32 promotes the progression of ccRCC by activating the NF-κB/SHP2/EGFR signaling pathway.

The role and clinical significance of the response gene to complement 32 (RGC32) in various cancers have been documented, yet its implications in clear cell Renal Cell Carcinoma (ccRCC) remain underexplored. This study investigated RGC32's diagnostic and prognostic relevance in ccRCC using bioinformatics methods with data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). The impact of RGC32 on ccRCC progression was assessed through nude mouse tumor assays. Immunohistochemistry evaluated RGC32 levels in ccRCC and adjacent normal tissues, while cell proliferation, migration, and invasion capabilities were analyzed using CCK-8, monoclonal proliferation assays, Transwell, and wound healing assays, respectively. Western blotting measured relevant protein expressions. Bioinformatics analysis highlighted RGC32's significant role in ccRCC pathogenesis. Elevated RGC32 expression in ccRCC tissues was linked to disease progression. Functionally, RGC32 was found to enhance the expression of proteins such as p-PI3K, CyclinA1, CyclinD1, p-STAT3, MMP2, MMP3, MMP9, p-SMAD2/3, Snail, Slug, and N-Cadherin via the NF-κB/SHP2/EGFR pathway, while decreasing E-cadherin levels. Moreover, RGC32 facilitated ccRCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). RGC32 is a pivotal factor in ccRCC development, primarily through the activation of the NF-κB/SHP2/EGFR signaling pathway.

Response gene to complement-32 serves as a functional dyad in smooth muscle cells: Cell cycle activator and mediator of cellular differentiation

Background and Aims : Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) and SMC phenotypic plasticity are critical processes in the development of atherosclerosis. We have previously shown that the sublytic C5b-9 effector Response Gene to Complement-32 (RGC-32) regulates EC proliferation, migration, and cytoskeletal reorganization. Our goal was to explore the effect of RGC-32 on cell cycle activation and TGF-β induced differentiation of SMC and the extracellular matrix (ECM) production.

RGC-32′ dual role in smooth muscle cells and atherogenesis

Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-β-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-β-induced ECM production.

RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis.

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.

RGC-32 Facilitates Reactive Astrocytosis in Experimental Autoimmune Encephalomyelitis (EAE) by Maintaining a Mature Phenotype

Abstract We previously showed that lack of Response Gene to Complement 32 (RGC-32) decreases the severity of clinical scores of mice with EAE and renders spinal cord astrocytes a progenitor phenotype reminiscent of radial glia. In this study, we used RNA-sequencing to investigate whether RGC-32 deferentially regulates biological processes related to astrocyte maturation and activation. We purified neonatal astrocytes from WT and RGC-32 knock-out (KO) mice, stimulated them with TGF-β and then performed RNA-seq (Genewiz, S. Plainfield, NJ). Spinal cords from WT and RGC-32 KO mice with EAE were harvested at day 0 and at the peak of disease (day 14) and subjected to immunohistochemistry for astrocytes lineage marker GFAP, as well as radial glial marker HOPX, neural stem cell marker CD133, and proliferation marker Ki-67. Pathway enrichment analysis revealed that the genes deferentially expressed in WT but not RGC-32 KO astrocytes belonged to GO categories related to brain development, axonal guidance, cell migration and extracellular matrix (ECM) regulation. Real-Time PCR confirmed that Ephrin A7, bone morphogenetic protein 1 and polycystin 1, other genes related to brain development and ECM were significantly up-regulated in WT astrocytes after TGF-β stimulation. We found that spinal cords of RGC-32 KO mice displayed a higher number of HOPX+ cells at day 14. Similarly, the number of CD133+ cells with a radial glial morphology was higher in RGC-32 KO mice. In addition, we found that RGC-32 KO astrocytes had a higher proliferative index at the peak of EAE, as shown by their increased expression of Ki-67. These data suggest that RGC-32 is an important regulator of astrocyte maturation and reactive astrocyte formation during EAE

RGC-32 Regulates Generation of Reactive Astrocytes in Experimental Autoimmune Encephalomyelitis.

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.

RGC-32 regulates astrocyte differentiation during experimental autoimmune encephalomyelitis

Abstract Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of response gene to complement 32 (RGC-32) alters astrocyte morphology in spinal cords during experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In addition, we found that RGC-32 regulates TGF-β-induced extracellular matrix production and growth factors expression. We investigated if the lack of RGC-32 alters the expression of the glial fibrillary acidic protein (GFAP) and of astrocytes progenitor markers vimentin and fatty acid binding protein 7 (FABP7) during EAE. Immunohistochemical analysis of spinal cords during the acute phase of EAE (day 14 post EAE induction) showed that the elongated, radial glial cell-like astrocytes from RGC-32 knock-out (KO) mice expressed higher levels of vimentin and FABP7 as compared to wild type (WT) mice, confirming their immature phenotype. In addition, we found that the density of white matter GFAP+ astrocytes was higher in WT as compared to KO mice (p=0.02). Using double staining immunohistochemistry for GFAP and connective tissue growth factor (CTGF), known to be involved in astrocyte differentiation, we found a lower number of CTGF+ astrocytes during EAE in spinal cords of RGC-32 KO when compared with WT mice (p=0.002). We next purified neonatal astrocytes from WT and RGC-32 KO mice, and we found significantly lower levels of GFAP and CTGF mRNA and protein and higher levels of vimentin in RGC-32 KO astrocytes when compared with WT astrocytes. The expression pattern of astrocytic progenitor markers and that of CTGF suggests that RGC-32 is an important regulator of astrocyte differentiation during EAE.

Response gene to complement 32 expression in macrophages augments paracrine stimulation-mediated colon cancer progression

M2-polarized tumor associated macrophages (TAMs) play an important role in tumor progression. It has been reported that response gene to complement 32 (RGC-32) promotes M2 macrophage polarization. However, whether RGC-32 expression in macrophages could play a potential role in tumor progression remain unclear. Here we identified that increasing RGC-32 expression in colon cancer and tumor associated macrophages was positively correlated with cancer progression. In vitro studies confirmed that colon cancer cells upregulated RGC-32 expression of macrophages via secreting TGF-β1. RGC-32 expression promoted macrophage migration. In addition, stimulation of HCT-116 cells with the condition mediums of RGC-32-silienced or over-expressed macrophages affected tumor cell colony formation and migration via altered COX-2 expression. In an animal model, macrophages with RGC-32 knockdown significantly decreased the expression of COX-2 and Ki67 in the xenografts, and partly inhibited tumor growth. Together, our results provide the evidences for a critical role of TGF-β1/RGC-32 pathway in TAMs and colon cancer cells during tumor progression.

Role of Complement and Complement-Related Adipokines in Regulation of Energy Metabolism and Fat Storage.

Adipose tissue releases many cytokines and inflammatory factors described as adipokines. In obesity, adipokines released from expanding adipose tissue are implicated in disease progression and metabolic dysfunction. However, mechanisms controlling the progression of adiposity and metabolic complications are not fully understood. It has been suggested that expanding fat mass and sustained release of inflammatory adipokines in adipose tissue lead to hypoxia, oxidative stress, apoptosis, and cellular damage. These changes trigger an immune response involving infiltration of adipose tissue with immune cells, complement activation and generation of factors involved in opsonization and clearance of damaged cells. Abundant evidence now indicates that adipose tissue is an active secretory source of complement and complement-related adipokines that, in addition to their inflammatory role, contribute to the regulation of metabolic function. This article highlights advances in knowledge regarding the role of these adipokines in energy regulation of adipose tissue through modulating lipogenic and lipolytic pathways. Several adipokines will be discussed including adipsin, Factor H, properdin, C3a, Acylation-Stimulating Protein, C1q/TNF-related proteins, and response gene to complement-32 (RGC-32). Interactions between these factors will be described considering their immune-metabolic roles in the adipose tissue microenvironment and their potential contribution to progression of adiposity and metabolic dysfunction. The differential expression and the role of complement factors in gender-related fat partitioning will also be addressed. Identifying lipogenic adipokines and their specific autocrine/paracrine roles may provide means for adipose-tissue-targeted therapeutic interventions that may disrupt the vicious circle of adiposity and disease progression. © 2019 American Physiological Society. Compr Physiol 9:1411-1429, 2019.

Sublytic C5b-9 induces proliferation of glomerular mesangial cells via ERK5/MZF1/RGC-32 axis activated by FBXO28-TRAF6 complex.

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b‐9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy‐1 nephritis (Thy‐1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC‐32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal‐regulated kinase 5 (phosphorylated ERK5, p‐ERK5), F‐box only protein 28 (FBXO28) and TNF receptor‐associated factor 6 (TRAF6) were all markedly up‐regulated both in the renal tissues of rats with Thy‐1N (in vivo) and in the GMCs upon sublytic C5b‐9 stimulation (in vitro). Further in vitro experiments revealed that up‐regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63‐ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1‐dependent RGC‐32 up‐regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1‐binding element within RGC‐32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 genes respectively markedly reduced GMC proliferation and ECM production in Thy‐1N rats. Together, these findings indicate that sublytic C5b‐9 induces GMC proliferative changes in rat Thy‐1N through ERK5/MZF1/RGC‐32 axis activated by the FBXO28‐TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.

Highlight on Endothelial Activation and Beyond.

Highlight on Endothelial Activation and Beyond.

Loss of Response Gene to Complement 32 (RGC-32) in Diabetic Mouse Retina Is Involved in Retinopathy Development.

Diabetic retinopathy (DR) is a severe and recurrent microvascular complication in diabetes. The multifunctional response gene to complement 32 (RGC-32) is involved in the regulation of cell cycle, proliferation, and apoptosis. To investigate the role of RGC-32 in the development of DR, we used human retinal microvascular endothelial cells under high-glucose conditions and type 2 diabetes (T2D) mice (+Leprdb/ + Leprdb, db/db). The results showed that RGC-32 expression increased moderately in human retinal endothelial cells under hyperglycemic conditions. Histopathology and RGC-32 expression showed no significant changes between T2D and control mice retina at 16 and 24 weeks of age. However, RGC-32 expression was significantly decreased in T2D mouse retina compared to the control group at 32 weeks of age, which develop features of the early clinical stages of DR, namely reduced retinal thickness and increased ganglion cell death. Moreover, immunohistochemistry showed that RGC-32 was predominantly expressed in the photoreceptor inner segments of control mice, while the expression was dramatically lowered in the T2D retinas. Furthermore, we found that the level of anti-apoptotic protein Bcl-2 was decreased (approximately 2-fold) with a concomitant increase in cleaved caspase-3 (approximately 3-fold) in T2D retina compared to control. In summary, RGC-32 may lose its expression in T2D retina with features of DR, suggesting that it plays a critical role in DR pathogenesis.

Response Gene to Complement 32 Maintains Blood Pressure Homeostasis by Regulating α-Adrenergic Receptor Expression.

Hypertension prevalence is much higher among children and adolescents with low birth weight and greater postnatal weight gain than in individuals with normal birth weight. However, the cause and molecular mechanisms underlying this complication remain largely unknown. Our previous studies have shown that RGC-32 (response gene to complement 32)-deficient (RGC-32-/-) mice are born significantly smaller but grow faster than their WT (wild type) controls, which allows adult RGC-32-/- mice to attain body weights similar to those of control mice. The objective of this study is to determine whether RGC-32-/- mice develop hypertension, and if so, to elucidate the underlying mechanisms. By using a radiotelemetry system, we found that RGC-32-/- mice exhibit higher mean arterial pressure than WT mice (101±4 versus 119±5 mm Hg), which enabled us to use RGC-32-/- mice to study the mechanisms underlying low birth weight-related hypertension. The increased blood pressure in RGC-32-/- mice was associated with increased vascular tone and decreased distensibility of small resistance arteries. The increased vascular tone was because of an increase in the relative contribution of sympathetic versus parasympathetic activity and was linked to increased expression of AT1R (angiotensin II type I receptor) and α1-AdR (α1-adrenergic receptor) in arterial smooth muscles. Mechanistically, RGC-32 regulated AT1R gene transcription by interacting with Sp1 (specificity protein 1) transcription factor and further blocking its binding to the AT1R promoter, leading to suppression of AT1R expression. The attenuation of AT1R leads to reduction in α1-AdR expression, which was critical for the balance of sympathetic versus parasympathetic control of vascular tone. Of importance, downregulation of RGC-32 in arterial smooth muscles was also associated with low birth weight and hypertension in humans. Our results indicate that RGC-32 is a novel protein factor vital for maintaining blood pressure homeostasis, especially in individuals with low birth weight.

Response Gene to Complement 32 in Vascular Diseases.

Response gene to complement 32 (RGC32) is a protein that was identified in rat oligodendrocytes after complement activation. It is expressed in most of the organs and tissues, such as brain, placenta, heart, and the liver. Functionally, RGC32 is involved in various physiological and pathological processes, including cell proliferation, differentiation, fibrosis, metabolic disease, and cancer. Emerging evidences support the roles of RGC32 in vascular diseases. RGC32 promotes injury-induced vascular neointima formation by mediating smooth muscle cell (SMC) proliferation and migration. Moreover, RGC32 mediates endothelial cell activation and facilitates atherosclerosis development. Its involvement in macrophage phagocytosis and activation as well as T-lymphocyte cell cycle activation also suggests that RGC32 is important for the development and progression of inflammatory vascular diseases. In this mini-review, we provide an overview on the roles of RGC32 in regulating functions of SMCs, endothelial cells, and immune cells, and discuss their contributions to vascular diseases.

Response gene to complement 32 interacts with SMAD3 to promote TGF-β-induced extracellular matrix production in multiple sclerosis

Abstract We have previously shown that response gene to complement 32 (RGC-32) mediates transforming growth factor-β (TGF-β)-induced astrocyte reactivity and extracellular matrix production. However, the molecular mechanisms underlying RGC-32 expression and function remain to be elucidated. In the present study, we found that TGF-β induced expression and nuclear translocation of both RGC-32 and SMAD3 in astrocytes. Using specific pathways inhibitors, we have found that RGC-32 induction is mediated by SMAD-dependent and independent mechanisms. We investigated whether RGC-32 interacts with SMAD2 or SMAD3 in astrocytes and if this association plays a role in extracellular matrix synthesis. Co-immunoprecipitation showed that SMAD3, but not SMAD2 physically interacts with RGC-32 in astrocytes. SIS3, an inhibitor of SMAD3 phosphorylation, significantly reduced RGC-32 translocation to the nucleus, indicating that SMAD3 phosphorylation is required for RGC-32 nuclear translocation. On the other hand, silencing RGC-32 with siRNA had no effect on SMAD3 nuclear translocation. Y27632, a selective ROCK inhibitor was also able to significantly inhibit RGC-32 nuclear translocation, while the SMAD3 nuclear translocation was unaffected. The inhibition of SMAD3 phosphorylation also blocked the RGC-32-induced collagen type I expression, indicating a synergistic action between RGC-32 and SMAD3 to induce the expression of collagen type I. Taken together, our data demonstrate for the first time that RGC-32 interacts with SMAD3 to mediate the extracellular matrix production in astrocytes. These data suggest that a similar mechanism might be involved in mediation of gliosis seen in chronic multiple sclerosis lesions.

Abstract 168: Response Gene to Complement 32 Suppresses Adipose Tissue Thermogenic Genes through Inhibiting Beta3 Adrenergic Receptor mTORC1 Signaling

Our previous study have shown that response gene to complement 32 (RGC-32) deficiency (Rgc32-/-) protects mice from diet-induced obesity and increases thermogenic gene expression in adipose tissues. However, the underlying mechanisms by which RGC-32 regulates thermogenic gene expression remain to be determined. In the present study, we found that RGC-32 expression in white adipose tissue (WAT) was suppressed during cold exposure-induced WAT browning. Rgc32-/- significantly increased thermogenic gene expression in the differentiated stromal vascular fraction (SVF) of inguinal WAT (iWAT). Interestingly, Rgc32-/- and cold exposure regulated a common set of genes in iWAT as shown by RNA sequencing data. Pathway enrichment analyses showed that RGC-32 deficiency downregulated PI3K/Akt signaling-related genes. Consistently, Akt phosphorylation was also decreased in Rgc32-/- iWAT, which led to an increase in β3-adrenergic receptor expression and subsequent activation of mTORC1. β3-adrenergic receptor antagonist SR 59230A and mTORC1 inhibitor rapamycin blocked the Rgc32 deficiency-induced thermogenic gene expression in iWAT both in vitro and in vivo. These results indicate that RGC-32 suppressed iWAT thermogenic gene expression through down-regulation of β3-AR expression and mTORC1 activity via a PI3K/Akt-dependent mechanism.

RGC32 Promotes Bleomycin-Induced Systemic Sclerosis in a Murine Disease Model by Modulating Classically Activated Macrophage Function.

Systemic sclerosis (SSc) is a multisystem autoimmune disorder that is characterized by inflammation and fibrosis in the skin and internal organs. Previous studies indicate that inflammatory cells and cytokines play essential roles in the pathogenesis of SSc; however, the mechanisms that underlie the inflammation-driven development of SSc are not fully understood. In this study, we show that response gene to complement 32 (RGC32) is abundantly expressed in mouse macrophages in the early stage of bleomycin-induced SSc. Importantly, RGC32 is required to induce the inflammatory response during the onset of SSc, because RGC32 deficiency in mice significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammatory mediators inducible NO synthase (iNOS) and IL-1β in macrophages. RGC32 appears to be a novel regulator for the differentiation of classically activated macrophages (M1 macrophages). IFN-γ and LPS stimulation induces RGC32 expression in primary peritoneal macrophages and bone marrow-derived macrophages. RGC32 deficiency impairs the polarization of M1 macrophages and attenuates iNOS and IL-1β production. Mechanistically, RGC32 interacts with NF-κB proteins and promotes iNOS and IL-1β expression by binding to their promoters. Collectively, our data reveal that RGC32 promotes the onset of SSc by regulating the inflammatory response of M1 macrophages, and it may serve as a promising therapeutic target for treating SSc.

Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32.

Response gene to complement-32 (RGC-32) activates cyclin-dependent kinase 1, regulates the cell cycle and is deregulated in many human tumours. We previously showed that RGC-32 expression is upregulated by the cancer-associated Epstein-Barr virus (EBV) in latently infected B cells through the relief of translational repression. We now show that EBV infection of naïve primary B cells also induces RGC-32 protein translation. In EBV-immortalised cell lines, we found that RGC-32 depletion resulted in cell death, indicating a key role in B cell survival. Studying RGC-32 translational control in EBV-infected cells, we found that the RGC-32 3′untranslated region (3′UTR) mediates translational repression. Repression was dependent on a single Pumilio binding element (PBE) adjacent to the polyadenylation signal. Mutation of this PBE did not affect mRNA cleavage, but resulted in increased polyA tail length. Consistent with Pumilio-dependent recruitment of deadenylases, we found that depletion of Pumilio in EBV-infected cells increased RGC-32 protein expression and polyA tail length. The extent of Pumilio binding to the endogenous RGC-32 mRNA in EBV-infected cell lines also correlated with RGC-32 protein expression. Our data demonstrate the importance of RGC-32 for the survival of EBV-immortalised B cells and identify Pumilio as a key regulator of RGC-32 translation.

Hypoxia-inducible factor 1α participates in hypoxia-induced epithelial-mesenchymal transition via response gene to complement 32.

The aim of the present study was to explore the function of response gene to complement 32 (RGC-32) in hypoxia-induced epithelial-mesenchymal transition (EMT) in pancreatic cancer. Three kinds of hypoxia-inducible factor 1α (HIF-1α) small interfering (si)RNA were synthesized and the different effects on the expression of HIF-1α were detected by western blotting. In human pancreatic cancer BxPC-3 cells, HIF-1α levels were diminished using siRNA transfection or HIF-1α inhibitor pretreatment, and the expression levels of RGC-32 and EMT-associated proteins were analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, the protein levels of epithelial marker, E-cadherin, and mesenchymal marker, vimentin, were determined by western blotting. Results demonstrated that HIF-1α-Homo-488 siRNA and HIF-1α-Homo-1216 siRNA diminished the protein level of HIF-1α. Compared with normoxia, hypoxia induced the levels of HIF-1α, RGC-32, N-cadherin and vimentin, but suppressed the expression of E-cadherin and cytokeratins. The inhibition of HIF-1α by HIF-1α-Homo-1216 siRNA transfection or HIF-1α inhibitor repressed hypoxia-induced HIF-1α, RGC-32, N-cadherin and vimentin, but increased the expression of E-cadherin and cytokeratins. When RGC-32 was knocked down, hypoxia-induced vimentin was suppressed; however, hypoxia-suppressed N-cadherin was released. In conclusion, the present results demonstrated that hypoxia induced the expression of HIF-1α to activate the levels of RGC-32, in turn to regulate the expression EMT-associated proteins for EMT. These findings revealed the function of RGC-32 in hypoxia-induced EMT and may have identified a novel link between HIF-1α and EMT for pancreatic cancer therapy.

RGC32 induces epithelial-mesenchymal transition by activating the Smad/Sip1 signaling pathway in CRC

Response gene to complement 32 (RGC32) is a transcription factor that regulates the expression of multiple genes involved in cell growth, viability and tissue-specific differentiation. However, the role of RGC32 in tumorigenesis and tumor progression in colorectal cancer (CRC) has not been fully elucidated. Here, we showed that the expression of RGC32 was significantly up-regulated in human CRC tissues versus adjacent normal tissues. RGC32 expression was significantly correlated with invasive and aggressive characteristics of tumor cells, as well as poor survival of CRC patients. We also demonstrated that RGC32 overexpression promoted proliferation, migration and tumorigenic growth of human CRC cells in vitro and in vivo. Functionally, RGC32 facilitated epithelial-mesenchymal transition (EMT) in CRC via the Smad/Sip1 signaling pathway, as shown by decreasing E-cadherin expression and increasing vimentin expression. In conclusion, our findings suggested that overexpression of RGC32 facilitates EMT of CRC cells by activating Smad/Sip1 signaling.